Imaging epilepsy in larval zebrafish.

Our understanding of the genetic aetiology of paediatric epilepsies has grown substantially over the last decade. However, in order to translate improved diagnostics to personalised treatments, there is an urgent need to link molecular pathophysiology in epilepsy to whole-brain dynamics in seizures. Zebrafish have emerged as a promising new animal model for epileptic seizure disorders, with particular relevance for genetic and developmental epilepsies. As a novel model organism for epilepsy research they combine key advantages: the small size of larval zebrafish allows high throughput in vivo experiments; the availability of advanced genetic tools allows targeted modification to model specific human genetic disorders (including genetic epilepsies) in a vertebrate system; and optical access to the entire central nervous system has provided the basis for advanced microscopy technologies to image structure and function in the intact larval zebrafish brain. There is a growing body of literature describing and characterising features of epileptic seizures and epilepsy in larval zebrafish. Recently genetically encoded calcium indicators have been used to investigate the neurobiological basis of these seizures with light microscopy. This approach offers a unique window into the multiscale dynamics of epileptic seizures, capturing both whole-brain dynamics and single-cell behaviour concurrently. At the same time, linking observations made using calcium imaging in the larval zebrafish brain back to an understanding of epileptic seizures largely derived from cortical electrophysiological recordings in human patients and mammalian animal models is non-trivial. In this review we briefly illustrate the state of the art of epilepsy research in zebrafish with particular focus on calcium imaging of epileptic seizures in the larval zebrafish. We illustrate the utility of a dynamic systems perspective on the epileptic brain for providing a principled approach to linking observations across species and identifying those features of brain dynamics that are most relevant to epilepsy. In the following section we survey the literature for imaging features associated with epilepsy and epileptic seizures and link these to observations made from humans and other more traditional animal models. We conclude by identifying the key challenges still facing epilepsy research in the larval zebrafish and indicate strategies for future research to address these and integrate more directly with the themes and questions that emerge from investigating epilepsy in other model systems and human patients.

A comparison of two nasal continuous positive airway pressure interfaces--a randomized crossover study.

BACKGROUND: In spite of widespread use of nasal CPAP there are comparatively few studies to guide the choice of nasal prongs. OBJECTIVES: To determine whether the Fisher & Paykel Healthcare (FPH) neonatal continuous positive airway pressure (CPAP) interface was effective in providing bubble CPAP when compared to the Hudson prong interface. METHODS: The study was a randomized cross-over study of twenty newborn infants 500 g or more requiring CPAP for respiratory support at birth. Infants were randomized to either the Fisher & Paykel Healthcare or Hudson CPAP interface for twenty four hours. Crossover between interfaces occurred after subsequent twenty four hour periods. The primary outcome was the provision of desired CPAP pressures, defined as provision of CPAP within ± one cm H2O of set pressure. RESULTS: The percentage time CPAP was within ± one cm H2O of set pressure was 66.5% for the Hudson and 71.8% for the FPH interface (p = 0.66). Oxygen saturations for the Hudson interface were in target range for a median of 97.8% of the time, and, with the FPH interface, for a median of 98.2% of the time (p = 0.76). Clinically significant differences in primary or secondary outcomes between the two groups were not detected. CONCLUSIONS: The nasal CPAP interfaces studied were equally effective in achieving desired bubble CPAP pressures and target saturations.

Brain imaging: how stable are synaptic connections?

Cortical circuits can undergo experience-dependent remodeling, while retaining the capacity for long-term information storage. The stability of individual synaptic connections is fundamental to both processes, but poorly understood; two studies using new in vivo imaging techniques have finally shed some light on this important issue.

Recombinant erythropoietin and blood transfusion in selected preterm infants.

OBJECTIVES: To comprehensively identify preterm infants likely to require blood transfusion and to investigate the effectiveness of recombinant erythropoietin in this high risk subgroup. DESIGN: Double blind randomised controlled trial. SETTING: Neonatal Intensive Care Unit, Middlemore Hospital, Auckland, New Zealand. PATIENTS: Preterm infants < 33 weeks gestation and < 1700 g birth weight meeting specific criteria indicating a high possibility of requiring blood transfusion. INTERVENTIONS: Predictors of blood transfusion were determined by analysis of preterm infants admitted to a neonatal intensive care unit over a two year period. Using the criteria developed, high risk infants entered the study and received erythropoietin or sham treatment until 34 weeks completed gestation. The sample size was calculated to detect a reduction of one blood transfusion per infant (significance level 5%, power 80%). RESULTS: The selection criteria had a positive predictive value for transfusion of 91% and a negative predictive value of 94%. Mean birth weights and gestational ages were similar in the two groups. Absolute reticulocyte counts and haemoglobin values were higher in the group receiving erythropoietin. There was no significant difference in the number of blood transfusions received in the treatment and control groups. However, comparing transfusions given to < 1000 g infants after 30 days of age, there were significantly fewer transfusions in the erythropoietin group (mean (SD) 0.5 (0.7) in those receiving erythropoietin and 1.6 (1.1) in the controls). No adverse effects were noted. CONCLUSIONS: The selection criteria for the study were highly predictive of subsequent transfusion. In the group receiving erythropoietin, a reduction in transfusion requirements was apparent only in the < 1000 g birthweight group after 1 month of age.

The extracellular ATP receptor, cP2Y(1), inhibits cartilage formation in micromass cultures of chick limb mesenchyme.

We have investigated the function of the G protein-coupled receptor for extracellular ATP, chick P2Y(1) (cP2Y(1)) during development of the chick limb. cP2Y(1) is strongly expressed in undifferentiated limb mesenchyme cells but appears to be lost from cells as they differentiate, raising the possibility that the function of this receptor may be to inhibit cell differentiation. This pattern of expression was particularly striking surrounding areas of cartilage formation. We tested whether cP2Y(1) was able to regulate cartilage formation by using an in-vitro micromass model of chondrogenesis. Because limb cells in micromass culture lose expression of cP2Y(1), we have used a gain-of-function approach to demonstrate that cP2Y(1) expression can inhibit cartilage differentiation. We also demonstrate that early limb mesenchyme cells release ATP into the extracellular medium and have mechanisms to breakdown extracellular ATP. These results suggest that extracellular ATP, signaling through cP2Y(1), can modulate the differentiation of limb mesenchyme cells in vitro, and the expression pattern of cP2Y(1) suggests that this type of signaling could play a similar role in ovo.

A clinical comparison of radiant warmer and incubator care for preterm infants from birth to 1800 grams.

OBJECTIVE: The objective of this study was to compare radiant warmer and incubator care for preterm infants from birth with respect to temperature control and weight gain. METHODS: Sixty preterm infants <33 weeks' gestation were randomized at birth to radiant warmer or incubator care. The initial goal was to maintain abdominal temperature at 36.8 degrees C in both groups and axillary temperature at 36.8 to 37.3 degrees C; air servocontrol was used for incubator infants. Infants in both groups received added humidity for 5 days if their weight was <1000 g and for 3 days if they weighed between 1000 and 1249 g. During a 3-hour period on days 1 to 7, recordings of abdominal, forehead, and foot temperatures were obtained. The percentage of the recording time during which the abdominal temperature was in the target range of between 36 degrees C and 37.5 degrees C was determined as an indicator of temperature control. Weight gain from birth to 1800 g was compared. Secondary outcomes included fluid balance and clinical events. RESULTS: There were 30 infants in each group; 48 were <1500 g (of whom 17 were <1000 g). There were no significant differences in birth weight, gestation, gender, or illness severity scores in the 2 groups. Significant differences in temperature control were noted on day 1. Although admission temperatures were similar, lower abdominal temperatures were noted in the first 2 hours of life in the incubator group (medians were 36.6 degrees C and 35.9 degrees C in the radiant warmer and incubator groups, respectively). Similarly, mean abdominal temperatures during the 3-hour recording on day 1 were lower in the incubator group, and infants in this group spent a significantly greater percentage of the recording time with temperatures outside the target range (17.3% compared with 0.88%). Other temperature recordings from the forehead and foot were not significantly different in the groups. Fluid intakes were higher for infants under radiant warmer on days 2, 3, and 4, and the difference amounted to a mean of 12.8 mL/kg/d. Maximum sodium levels in the first week were similar in the 2 groups. Mean weight gain was 17.4 g/kg/d for the radiant warmer group and 17.1 g/kg/d for the incubator group; days to regain birth weight and length of hospital stay were not significantly different. Greater numbers of infants in the radiant warmer group required phototherapy, and adverse events (which included death, necrotizing enterocolitis, chronic lung disease, grade 3 or 4 intraventricular hemorrhage, periventricular leukomalacia, or retinopathy requiring laser treatment) were less frequent in the radiant warmer group (1 infant compared with 8 in the incubator group; relative risk 0.1; 95% confidence intervals: 0.01-0.82). CONCLUSIONS: This study has shown differences in abdominal temperatures on day 1 and outcome, although the latter finding should be viewed with caution because of the sample size. The results indicate benefits for the initial use of the radiant warmer after birth. Although fluid requirements were higher in the radiant warmer group for days 2 through 4, the increased fluid volumes were given without apparent adverse effect.

Whole-blood hemagglutination inhibition test for venereal disease research laboratory (VDRL) antibodies.

Nontreponemal antibody tests such as the Venereal Disease Research Laboratory (VDRL) test are carried out on serum and widely used as screening tests for syphilis. The aim of the present study was to develop a screening test for syphilis making use of whole blood and VDRL liposomes. Antibody to human red blood cells was conjugated to VDRL liposomes and reacted with a diluted sample of patient whole blood. A total of 951 samples were tested by the new test and the VDRL tube test. All 49 VDRL samples positive by the VDRL test showed inhibition of hemagglutination in the whole-blood test (sensitivity, 100%). Of 902 samples with negative results by the VDRL test, 901 caused hemagglutination when tested with the liposomes (specificity, 99.9%). The hemagglutination inhibition method tests for syphilis in a simple one-step procedure in which whole blood is added to a tube containing liposomes. The new test has potential for point-of-care testing in developing countries.

Optimal Fungicide Management of Purple Spot of Asparagus and Impact on Yield.

Purple spot disease of asparagus, caused by the fungus Stemphylium vesicarium, results in lesions on spears and ferns and defoliation of ferns. In two newly established commercial asparagus fields (cvs. Jersey Giant and Jersey Knight), chlorothalonil or mancozeb was applied every 7, 10, or 14 days or according to Tom-Cast with a threshold of 15 disease severity values, and not applied to the control. Tom-Cast prompted four sprays, resulting in a 60% reduction in the number of fungicide applications when compared with the 7-day-interval treatment. When disease pressure was severe, lesions on ferns were significantly less for both cultivars when fungicides were applied according to Tom-Cast or every 7 days compared with spray intervals of 10 or 14 days. Applying fungicides according to Tom-Cast or every 7 days resulted in an increased Jersey Giant fern stand compared with applying fungicides every 10 or 14 days. Unsprayed control plots yielded 77 to 83% (depending on cultivar) of those plots treated according to Tom-Cast using chlorothalonil. Significantly higher yields of Jersey Knight were obtained for chlorothalonil versus mancozeb. When mancozeb was used, Jersey Knight yield was significantly increased with a 7-day versus Tom-Cast application regime. Using chlorothalonil in a Tom-Cast program provided a benefit per hectare (BPH) of $1,005.24 (Jersey Knight) to $2,057.69 (Jersey Giant). In comparison, using mancozeb in a Tom-Cast program provided a BPH of -$484.27 (Jersey Knight) to $1,030.55 (Jersey Giant) over a 2-year period.

Selective expression of purinoceptor cP2Y1 suggests a role for nucleotide signalling in development of the chick embryo.

Responses to extracellular nucleotides (e.g., ATP, ADP, etc.) have been demonstrated in a number of embryonic cell types suggesting they may be important signalling molecules during embryonic development. Here the authors describe for the first time the expression of a G-protein-coupled receptor for extracellular ATP, chick P2Y1 (cP2Y1), during embryonic development of the chick. During the first 10 days of embryonic development, cP2Y1 is expressed in a developmentally regulated manner in the limb buds, mesonephros, brain, somites, and facial primordia, suggesting that this receptor may have a role in the development of each of these systems.

Expression of two ATP-gated ion channels, P2X5 and P2X6, in developing chick skeletal muscle.

Physiological and pharmacological studies have shown that ATP has potent effects on developing chick skeletal muscle. These effects have previously been shown to be developmentally regulated, and the responses were characteristic of activation of the P2X ligand-gated ion-channel family of ATP receptors. Here, using immunohistochemistry, we describe the expression patterns of two members of the P2X receptor family, P2X5 and P2X6, during development of skeletal muscle in the chick embryo. These receptors were first expressed at early stages of skeletal muscle development, and expression disappeared immediately before the stage at which fusion of myoblasts to form myotubes occurs. P2X5 was also demonstrated in nerves supplying developing skeletal muscle, in some dorsal root ganglion cells, and in dorsal and ventral spinal cord. No expression of the other five members of the P2X family were demonstrated in developing skeletal muscle.

Mechanical activity is necessary for the elimination of polyneuronal innervation of developing rat soleus muscles.

During early development, rat soleus muscle fibres are innervated by several axons. Neuromuscular activity is involved in the elimination of all but one terminal, but it is not clear whether electrical or mechanical activity is important. Here, we reduced mechanical activity only, by interfering with excitation-contraction coupling. Muscles treated with dantrolene sodium at 9 days produced significantly less force at 13 days of age than normal muscles, and their sensitivity to ACh was greater than that of controls. The elimination of polyneuronal innervation occurs between days 9-12, but in muscles treated with dantrolene, the loss of synapses was slower. Thus, reducing mechanical activity by interfering with excitation-contraction coupling, (a) delays muscle development and (b) reduces the rate of elimination of polyneuronal innervation.

Anaemia of prematurity. Epidemiology, management and costs.

Recombinant human erythropoietin (rHuEpo) has been increasingly used in preterm infants in the last 3 to 4 years. Recent studies have indicated a reduction in blood transfusion requirements in infants receiving rHuEpo. No significant adverse effects have emerged, apart from iron deficiency (if iron supplementation is inadequate), and the risk of transfusion-related infection is decreased. Nevertheless, rHuEpo is relatively expensive (a 6-week course costs approximately the same as 2 blood transfusions), so its use requires careful consideration; it is logical to target rHuEpo therapy to those babies who are most likely to be transfused. Using this strategy, 1 study involving stable growing preterm infants has shown that direct costs of blood transfusion and rHuEpo were similar, and the use of rHuEpo was recommended. In addition, use of high-dosage rHuEpo early in the course of management on the neonatal intensive care unit has been shown to reduce direct treatment costs in ill preterm infants. Further studies will continue to identify infants who are likely to benefit from rHuEpo therapy and to define its cost effectiveness in more detail.

Symptomatic HIV infection in infancy--clinical and laboratory markers of infection.

OBJECTIVE: To investigate the usefulness of immunological tests in the diagnosis of HIV infection in young symptomatic children (< 15 months of age). DESIGN: Tests were evaluated in HIV-infected (HIV antibody- and PCR-positive) patients and non-infected individuals. SETTING: Hospitalised patients in a referral centre (Red Cross War Memorial Children's Hospital, Cape Town). PATIENTS: All admissions under 15 months of age who had HIV antibody requested were eligible, provided there was sufficient serum (150 microliter) for further study. Overall, there were 201 symptomatic cases and 49 healthy controls. Twenty of the symptomatic cases were HIV antibody-positive and 19 of these were HIV-infected on the basis of a positive PCR for HIV viral product. RESULTS: Of the tests we evaluated (total IgG, IgM, IgA and rheumatoid factors of the same classes), raised total IgG level (cut-off 18 g/I or above) was the most useful. We used a commercial radial immunodiffusion plate which was found to have excellent reproducibility (inter-assay coefficient of variation 3.2%). The test detected 16 of 19 infected infants (sensitivity 84%, negative predictive value 98%). With the exception of the finding of oral thrush (odds ratio 7; P < 0.001), the clinical signs at presentation did not distinguish those who were HIV antibody-positive from those who were negative. CONCLUSIONS: In our study of hospital admissions, the finding of elevated IgG and HIV antibody was diagnostic of HIV infection. (The positive predictive value of the combination was 100%.) Likewise, the presence of raised IgG levels and oral candidosis had a high specificity for HIV infection (98%) but the sensitivity was low (37%). Measurement of total IgG levels by radial immunodiffusion is simple, relatively inexpensive (< 10% of the cost of PCR), helpful in diagnosing HIV infection in symptomatic infants and able to be performed in areas with minimal laboratory back-up.

A comparison of oral and intravenous iron supplementation in preterm infants receiving recombinant erythropoietin.

OBJECTIVE: To determine whether intravenously administered iron supplements would improve the hematologic response to recombinant erythropoietin in stable preterm infants. METHODS: Forty-two preterm infants (<33 weeks' gestation, birth weight < 1500 gm, hematocrit <38%) were treated with recombinant human erythropoietin (Eprex), 600 U/kg per week, and randomly assigned to receive either an oral preparation of ferrous lactate (elemental iron, 12 mg/kg per day) or an intravenous preparation of iron sucrose (6 mg/kg per week). RESULTS: Hematocrits, reticulocyte counts, and transfusions were similar in the oral group (OG) and the intravenous group (IVG). However, markedly higher serum ferritin concentrations were noted in the IVG (p <0.001), and by completion of the study the arithmetic mean values were 265 +/- 127 microg/L versus 137 +/- 65 microg/L in the IVG and the OG, respectively. The numbers of hypochromic erythrocytes increased in both groups during the study but were significantly higher in the OG (p = 0.04). Mean daily weight gain in the IVG (27 +/- 6.4 gm/day) was greater than in the OG (22.9 +/- 4.78 gm/day; p = 0.04). CONCLUSIONS: High doses of both orally administered iron and intravenously administered iron sucrose appear to supply sufficient iron for erythropoiesis in stable infants. Storage iron may become depleted after oral supplementation. The intravenous preparation appears to be safe and maintains serum ferritin concentrations, and it may be indicated for patients with low ferritin levels and for those not established on enteral feedings.

Is the use of recombinant human erythropoietin in anaemia of prematurity cost-effective?

In a double-blind placebo-controlled study we showed a 3-fold decrease in blood transfusions (BTFs) given to preterm infants with anaemia of prematurity who received recombinant erythropoietin. However, only 50% of placebo recipients required a BTF. Data from the placebo group indicated that either mean daily weight gain < or = 7.5 g/day before study entry or haematocrit < or = 50% at birth was associated with BTFs (P < 0.001). We calculated that giving erythropoietin to patients in the treatment group with either of these variables prevented 24 of 28 BTFs and that it would cost R184 to prevent 1 BTF. The cost of each BTF was R187 (blood filtered to remove white cells and reduce cytomegalovirus transmission). Therefore, the costs of the two treatments were similar, but as the risk of transmitting infection is lower with erythropoietin, we recommend its use in selected preterm infants.

Comparison of a modified DNA hybridization assay with standard culture enrichment for detecting group B streptococci in obstetric patients.

Infection with group B streptococcus (GBS) results in 12,000 to 15,000 cases of neonatal sepsis annually in the United States. GBS is transmitted vertically in up to 70% of infants born to colonized women. Early-onset GBS disease (septicemia, pneumonia, or meningitis occurring within 7 days of life) has a mortality rate of up to 50%, with permanent neurologic sequelae occurring in 15 to 50% of infants surviving meningeal infection. Because of the fulminant nature of neonatal infection, it would be useful to have a rapid assay for determining the GBS status of laboring women. This study illustrated how a commercially available DNA probe-based test was modified to achieve this goal. Modifications included the use of mixed cultures rather than pure isolates for detecting GBS, along with a shorter culture enrichment time and a sample concentration step. To this end, vaginal and rectal swabs from 402 pregnant women during their third trimester were cocultured and tested for GBS rRNA. The 8-h enrichment protocol resulted in an assay with a sensitivity of 95% and specificity of 98%, while the 3-h enrichment protocol revealed a sensitivity of 73% and specificity of 99%. In summary, GBS was detected in the majority of colonized women in less than 4 h. This study illustrated the usefulness of the approach in identifying the most heavily colonized women, who are at the highest risk of transmitting GBS to their neonates. The modified test would have a significant impact on both the medical management and antibiotic therapy for these women and their newborns.

Determination of lymphocyte immunophenotypic values for normal full-term cord blood.

Normal reference values for flow cytometric immunophenotypic lymphocyte markers for cord blood (CB) were determined using sufficient numbers of subjects for clinical laboratory use. Samples from 202 normal gestations were processed by whole blood lysis and analyzed in the following combinations: CD14/45, CD4/3, CD8/3, CD45RA/4, CD29/4, CD56/3, Cd19/3, CD19/10. Thirty-five adult laboratory volunteers were analyzed as controls. When compared to adults, CB showed increased relative percentages of naive T-helper cells, B cells, immature B cells, and CD8+3-cells and decreased T cells, cytotoxic T cells, activated T-helper cells, and large granular lymphocytes (CD56+3+). Significant differences were also found when CB samples were stratified by sex and race. These results provide clinical laboratory normal reference values for lymphocyte markers for CB, demonstrate the need for determining separate standard reference values for significantly different patient populations, provide the basis for future investigation of pathologic gestations and for clinical laboratory applications, and provide insight into early immunologic development.

p53, retinoblastoma gene product, and cyclin protein expression in human papillomavirus virus DNA-positive cervical intraepithelial neoplasia and invasive cancer.

Human papillomavirus interacts with cyclin protein and tumor suppressor genes, p53, and retinoblastoma gene (Rb). Expression of these gene products was examined in 69 formalin-fixed, paraffin-embedded cervical biopsies by immunohistochemistry utilizing antibodies against p53, Rb, and proliferating cell nuclear antigen (PCNA) and by human papillomavirus DNA in-situ hybridization assays. Samples selected for this study included 27 normal/reactive atypia cases that were all human papillomavirus DNA in-situ hybridization negative, 37 cervical intraepithelial neoplasia (CIN) lesions, and 5 invasive carcinomas. The CIN and invasive carcinoma cases were all human papillomavirus DNA in-situ hybridization positive. p53 protein expression was detected in approximately one-third of the reactive atypia and CIN lesions and in 60% of invasive cancers. Neither the amount or the location of p53 staining was correlated with the histologic diagnosis. Rb staining was more frequently found in the CIN/invasive carcinoma cases compared to the normal/reactive atypia samples (39/42 [93%] versus 21/27 [78%], respectively; P < 0.05 by chi 2. PCNA staining was detected in virtually all samples tested. However, the location of both PCNA and Rb staining differed when the normal/reactive atypia cases were compared to the CIN cases. Only 10% of the former group demonstrated Rb staining throughout the basal two-thirds layer or full thickness of the epithelium compared with 65% of the latter group (P < 0.001 by chi2). Likewise, PCNA staining of the basal two-thirds or full-thickness of the epithelium was found in only 58% of normal/reactive atypia cases, but in 97% of the CIN group (P < 0.001). Our results suggest that the location of Rb and PCNA staining is quite different between normal/reactive atypia cervical biopsies and CIN lesions.