Gene Expression Changes in a Prefinal Health Stage of Lethally Irradiated Male and Female Rhesus Macaques.

Radiation-induced gene expression (GE) changes can be used for early and high-throughput biodosimetry within the first three days postirradiation. However, is the method applicable in situations such as the Alexander Litvinenko case or the Goiania accident, where diagnosis occurred in a prefinal health stage? We aimed to characterize gene expression changes in a prefinal health stage of lethally irradiated male and female rhesus macaques. Peripheral blood was drawn pre-exposure and at the prefinal stage of male and female animals, which did not survive whole-body exposure with 700 cGy (LD66/60). RNA samples originated from a blinded randomized Good Laboratory Practice study comprising altogether 142 irradiated rhesus macaques of whom 60 animals and blood samples (15 samples for both time points and sexes) were used for this analysis. We evaluated GE on 34 genes widely used in biodosimetry and prediction of the hematological acute radiation syndrome severity (H-ARS) employing quantitative real-time polymerase chain reaction (qRT-PCR). These genes were run in duplicate and triplicate and altogether 96 measurements per time point and sex could be performed. In addition, 18S ribosomal RNA (rRNA) was measured to depict the ribosome/transcriptome status as well as for normalization purposes and 16S rRNA was evaluated as a surrogate for bacteremia. Mean differential gene expression (DGE) was calculated for each gene and sex including all replicate measurements and using pre-exposure samples as the reference. From 34 genes, altogether 27 genes appeared expressed. Pre-exposure samples revealed no signs of bacteremia and 18S rRNA GE was in the normal range in all 30 samples. Regarding prefinal samples, 46.7% and 40% of animals appeared infected in females and males, respectively, and for almost all males this was associated with out of normal range 18S rRNA values. The total number of detectable GE measurements was sixfold (females) and 15-fold (males) reduced in prefinal relative to pre-exposure samples and about tenfold lower in 80% of prefinal compared to pre-exposure samples (P < 0.0001). An overall 11-fold (median) downregulation in prefinal compared to pre-exposure samples was identified for most of the 27 genes and even FDXR appeared 4-14-fold downregulated in contrast to a pronounced up-regulation according to cited work. This pattern of overall downregulation of almost all genes and the rapid reduction of detectable genes at a prefinal stage was found in uninfected animals with normal range 18S rRNA as well. In conclusion, in a prefinal stage after lethal radiation exposure, the ribosome/transcriptome status remains present (based on normal range 18S rRNA values) in 60-67% of animals, but the whole transcriptome activity in general appears silenced and cannot be used for biodosimetry purposes, but probably as an indicator for an emerging prefinal health stage.

Predicting the Radiation Sensitivity of Male and Female Rhesus Macaques Using Gene Expression.

Radiosensitivity differs in humans and likely among closely-related primates. Reasons for variation in radiosensitivity are not well known. We examined preirradiation gene expression in peripheral blood among male and female rhesus macaques which did or did not survive (up to 60 days) after whole-body irradiation with 700 cGy (LD66/60). RNA samples originated from a blinded randomized Good Laboratory Practice study in 142 irradiated rhesus macaques. Animals were untreated (placebo), or treated using recombinant human IL-12, G-CSF or combination of the two. We evaluated gene expression in a two-phase study design where phase I was a whole genome screen [next generation sequencing (NGS)] for mRNAs (RNA-seq) using five RNA samples from untreated male and female animals per group of survivor and non-survivor (total n = 20). Differential gene expression (DGE) was defined as a statistically significant and ≥2-fold up- or downregulation of mRNA species and was calculated between groups of survivors and non-survivors (reference) and by gender. Altogether 659 genes were identified, but the overlapping number of differentially expressed genes (DGE) observed in both genders was small (n = 36). Fifty-eight candidate mRNAs were chosen for independent validation in phase II using the remaining samples (n = 122) evaluated with qRT-PCR. Among the 58 candidates, 16 were of significance or borderline significance (t test) by DGE. Univariate and multivariate logistic regression analysis and receiver operating characteristic (ROC) curve analysis further refined and identified the most outstanding validated genes and gene combinations. For untreated male macaques, we identified EPX (P = 0.005, ROC=1.0), IGF2BP1 (P = 0.05, ROC=0.74) and the combination of EPX with SLC22A4 (P = 0.03, ROC=0.85) which appeared most predictive for the clinical outcome for treated and combined (untreated and treated) male macaque groups, respectively. For untreated, treated and both combined female macaque groups the same gene (MBOAT4, P = 0.0004, ROC = 0.81) was most predictive. Based on the probability function of the ROC curves, up to 74% of preirradiation RNA measurements predicted survival with a positive and negative predictive value ranging between 85-100% and associated odds ratios reflecting a 2-3-fold elevated risk for surviving per unit change (cycle threshold value) in gene expression. In conclusion, we identified gender-dependent genes and gene combinations in preirradiation blood samples for survival prediction after irradiation in rhesus macaques.

Mathematical model of radiation effects on thrombopoiesis in rhesus macaques and humans.

A mathematical model that describes the effects of acute radiation exposure on thrombopoiesis in primates and humans is presented. Thrombopoiesis is a complex multistage dynamic process with potential differences between species. Due to known differences in cellular radiosensitivities, nadir times, and cytopenia durations, direct extrapolation from rhesus to human platelet dynamics is unrealistic. Developing mathematical models of thrombopoiesis for both humans and primates allows for the comparison of the system's response across species. Thus, data obtained in primate experiments can be extrapolated to predictions in humans. Parameter values for rhesus macaques and humans were obtained either from direct experimental measurements or through optimization procedures using dynamic data on platelet counts following radiation exposure. Model simulations accurately predict trends observed in platelet dynamics: at low radiation doses platelet counts decline after a time lag, and nadir depth is dose dependent. The models were validated using data that was not used during the parameterization process. In particular, additional experimental data was used for rhesus, and accident and platelet donor data was used for humans. The model aims to simulate the average response in rhesus and humans following irradiation. Variation in platelet dynamics due to individual variability can be modeled using Monte Carlo simulations in which parameter values are sampled from distributions. This model provides insight into the time course of the physiological effects of radiation exposure, information which could be valuable for disaster planning and survivability analysis and help in drug development of radiation medical countermeasures.

Genotypic and phenotypic characterization of Bordetella pertussis strains used in different vaccine formulations in Latin America.

AIM: To characterize Bordetella pertussis vaccine strains in comparison with current circulating bacteria. METHODS AND RESULTS: Genomic and proteomic analyses of Bp137 were performed in comparison with other vaccine strains used in Latin America (Bp509 and Bp10536) and with the clinical Argentinean isolate Bp106. Tohama I strain was used as reference strain. Pulse-field gel electrophoresis (PFGE) and pertussis toxin promoter (ptxP) sequence analysis revealed that Bp137 groups with Bp509 in PFGE group III and contains ptxP2 sequence. Tohama I (group II) and Bp10536 (group I) contain ptxP1 sequence, while Bp106 belongs to a different PFGE cluster and contains ptxP3. Surface protein profiles diverged in at least 24 peptide subunits among the studied strains. From these 24 differential proteins, Bp10536 shared the expression of ten proteins with Tohama I and Bp509, but only three with Bp137. In contrast, seven proteins were detected exclusively in Bp137 and Bp106. CONCLUSIONS: Bp137 showed more features in common with the clinical isolate Bp106 than the other vaccine strains here included. SIGNIFICANCE AND IMPACT OF THE STUDY: The results presented show that the old strains included in vaccines are not all equal among them. These findings together with the data of circulating bacteria should be taken into account to select the best vaccine to be included in a national immunization programme.

Laboratory adaptation of Bordetella pertussis is associated with the loss of type three secretion system functionality.

Although Bordetella pertussis contains and transcribes loci encoding type III secretion system (TTSS) homologues, expression of TTSS-associated proteins has been reported only for non-laboratory-adapted Irish clinical isolates. Here we confirm such a result for clinical isolates obtained from patients treated in Argentinean hospitals. Moreover, we demonstrate that the expression of TTSS-associated proteins is independent both of the year in which the isolate was obtained and of the types of polymorphic alleles for other virulence factors but is dependent on environmental growth conditions. Interestingly, we observed that TTSS-associated protein expression is lost after successive in vitro passages but becomes operative again when bacteria come into contact with the host. This in vivo activation of TTSS expression was observed not only for clinical isolates previously adapted to the laboratory after successive in vitro passages but also for vaccine strains that did not express the system in vitro. The reversibility of TTSS expression, demonstrated by its switching off-on when the bacterium comes into contact with the host, appears to be an adaptive response of this pathogen.

Neonatal vitamin D status at birth at latitude 32 degrees 72': evidence of deficiency.

OBJECTIVE: With vitamin D deficiency as a serious public health problem, vitamin D status at birth was measured in neonates at latitude 32 degrees 72' (southeastern United States). STUDY DESIGN: In umbilical cord blood, vitamin D status, demonstrated by circulating 25-hydroxyvitamin D, was measured and related to race and season of birth. RESULT: The mean+/-standard deviation of 25-hydroxyvitamin D in 100 cord blood samples was 13.5+/-8.3 ng/ml for the cohort. African-American infants, with a mean+/-standard deviation of 10.5+/-6.0 ng/ml, demonstrated significantly lower vitamin D status than Caucasian infants, with a mean+/-standard deviation of 19.5+/-9.6 ng/ml (P<0.0001). By season, the mean 25-hydroxyvitamin D level at birth in November-March compared to April-October was 11.3 ng/ml lower in Caucasian infants (from 29.0 to 17.7 ng/ml) and 3 ng/ml lower in African-American infants (from 13.1 to 10.1 ng/ml). CONCLUSION: The prevalence of vitamin D insufficiency is high in this cohort. African-American infants demonstrate significantly lower vitamin D status at birth than Caucasian infants. Seasonality, while significant in both groups, had a greater impact on the vitamin D status of Caucasian newborns.

Incidence of obsessive-compulsive disorder in a community sample of young adolescents.

OBJECTIVE: To investigate the incidence, transition probabilities, and risk factors for obsessive-compulsive disorder (OCD) and subclinical OCD in adolescents. METHOD: A two-stage epidemiological study originally designed to investigate depression was conducted between 1987 and 1989 in the southeastern United States. For the screening, a self-report depressive symptom questionnaire was administered to a community sample of 3,283 adolescents. In the diagnostic stage, the Schedule for Affective Disorders and Schizophrenia for School-Age Children was administered to 488 mother-child pairs. Baseline screening and diagnostic data from the first year the subject completed an interview and follow-up diagnostic data from subsequent years were used. RESULTS: The 1-year incidence rates of OCD and subclinical OCD were found to be 0.7% and 8.4%, respectively. Transition probabilities demonstrated a pattern of moving from more severe to less severe categories. Of those with baseline OCD, 17% had the diagnosis of OCD at follow-up; 62% moved to the referent group. Of those with baseline subclinical OCD, 1.5% had OCD at follow-up and 75% moved to the referent group. Black race (odds ratio [OR] = 23.38), age (OR = 4.02), desirable life events (OR = 0.78), undesirable life events (OR = 1.21), and socioeconomic status (OR not estimable) were significant predictors of incident OCD. Age (OR = 2.30), desirable life events (OR = 0.92), and undesirable life events (OR = 1.13) were significantly associated with incident subclinical OCD. CONCLUSION: An initial diagnosis of subclinical OCD was not significantly predictive of a diagnosis of OCD at 1-year follow-up. The overall morbidity remained higher at follow-up in the baseline OCD group than in the baseline subclinical OCD group. The baseline subclinical OCD group was more dysfunctional at follow-up than was the baseline referent group. Further research concerning differences in symptomatology and impairment between OCD and subclinical OCD is warranted.

[New drugs in the treatment of childhood epilepsy: vigabatrin (study of 61 subjects)]. / Nuovi farmaci nella terapia dell'epilessia dell'infanzia: vigabatrin (studio di 61 soggetti).

This study was conducted to determine the efficacy of vigabatrin in children with epilepsy. Sixty-one children with various types of severe epilepsy were studied. In 12 children vigabatrin was introduced as monotherapy, while in 49 it was added to other antiepileptic drugs. Following the introduction of vigabatrin, 17 patients became seizure free, and 19 responded with a greater than 50% reduction in seizure frequency. The following types of epilepsy responded favorably to treatment (in order of decreasing efficacy): West syndrome, especially if secondary to Tuberous Sclerosis; cryptogenic and symptomatic partial epilepsy; Lennox-Gastaut syndrome, and other symptomatic generalized epilepsy. Optimal responses were found with vigabatrin doses of 30 to 50 mg/kg/day. Forty-one responders continued on vigabatrin, with generally good efficacy. Vigabatrin tolerability was good; in only 6 children was treatment discontinued because of side-effects such as somnolence, irritability, weight gain, and cutaneous rash. This study shows that vigabatrin is effective in the treatment of refractory epilepsy, especially in West syndrome secondary to Tuberous Sclerosis, partial epilepsy, and Lennox-Gastaut syndrome. Further studies are needed to analyze the increased frequency of seizures and the appearance of new seizure types associated with vigabatrin treatment.

Frequency of obsessive-compulsive disorder in a community sample of young adolescents.

OBJECTIVE: To investigate the frequency and phenomenology of obsessive-compulsive disorder (OCD) and subclinical OCD in young adolescents. METHOD: A two-stage epidemiological study originally designed to investigate adolescent depression was conducted between 1986 and 1988 in the southeastern United States. In the first stage, a self-report depressive symptom questionnaire was administered to a community sample of 3,283 adolescents. In the diagnostic stage, the Schedule for Affective Disorders and Schizophrenia for School-Age Children and the Children's Global Assessment Scale were administered to 488 mother-child pairs. RESULTS: The prevalences of OCD and subclinical OCD were found to be 3% and 19%, respectively. Prevalences were similar in males and females. Females reported more symptoms of compulsions although males reported more obsessions. About 55% of adolescents with OCD reported both obsessions and compulsions. The most common compulsions were arranging (56%), counting (41%), collecting (38%), and washing (17%). Major depressive disorder (45%), separation anxiety (34%), dysthymia (29%), suicidal ideation (15%), and phobia (8%) were the diagnoses most frequently comorbid with OCD. CONCLUSIONS: Findings suggest that OCD is not infrequent among adolescents and that the characteristic comorbidity and symptomatology of OCD may facilitate earlier identification and treatment by clinicians.

Benign epilepsy of childhood with centrotemporal spikes and unilateral developmental opercular dysplasia.

The case of a 15-year-old right-handed girl with developmental delay, mild retardation, astereognosis, and tactile discrimination impairment in the left arm, is reported. During sleep, right partial simple motor seizures with sialorrhea and saliva pooling were present, sometimes followed by secondary generalization. Absences and drop attacks were also observed. Electroencephalography revealed left centrotemporal spike waves spreading to the homologous contralateral region, generalized 3-Hz spike-and-wave complexes enhanced by hyperventilation, and generalized polyspike-and-wave discharges during drowsiness. Magnetic resonance imaging showed right perisylvian (opercular) malformation. Benign epilepsy of childhood with centrotemporal spikes (BECCTS) is a focal idiopathic epilepsy presumed to be of genetic origin. Although brain damage is not expected, structural lesions including opercular macrogyria have been reported. This coexistence has been considered mainly causal and only exceptionally causal. The Foix-Chavany-Marie syndrome or operculum syndrome is the result of bilateral opercular damage. In our child the right structural and the left functional lesions appeared mutually activated, yielding a transient opercular syndrome. The concomitance of BECCTS and developmental opercular dysplasia is suggestive of a common genetic substratum.

Neuronal migrational disorders: diffuse cortical dysplasia or the "double cortex" syndrome.

We report a 10-year-old girl who presented with a single episode of partial complex seizure. Neurological examination and psychological evaluation were normal. Electroencephalogram showed abnormalities in the left temporo-occipital region. Magnetic resonance imaging revealed a bilateral layer of tissue iso-intense with gray matter, between the wall of the lateral ventricle and the cortex, extending from the frontal to the occipital region, and enlarged left lateral ventricle. The images indicated diffuse cortical dysplasia, also known as band heterotopia or "double cortex", and left cortical hemiatrophy.

Gelastic epilepsy. A clinical contribution.

Gelastic (laughing) epilepsy, relatively uncommon, is usually associated with hypothalamic hamartomas, pituitary tumors, astrocytomas of the mammillary bodies, and dysraphic conditions. Cases of unknown etiology are rare. In three of the four cases reported here, the diagnoses were hamartoma of the tuber cinereum; lobar holoprosencephaly; and lissencephaly type I, grade 2. In the fourth, radiographic investigation gave a normal result; a genetic etiology was suggested because of bilateral familial idiopathic epilepsy. In all patients, EEGs showed both focal spikes and generalized spike-and-wave discharges. The primary underlying neurophysiologic disorder may be provoked by the diffuse hyperexcitability of the cortex and subsequent firing of the thalamocortical networks with which the cortical brain is reciprocally interlinked.

Optical activity associated with the sulfur to metal charge transfer bands of Zn and Cd GAL4.

The N-terminal DNA-binding domain of the GAL4 transcription factor, isolated as a proteolytic fragment of 63 amino acid residues, GAL4(63), or cloned as a 62-residue subdomain, GAL4(62*), binds 2 Cd(II) ions. Both Cd derivatives show intense ellipticity bands associated with -S(-)-->Cd charge transfer bands at 250 nm (+), 233 nm (-), and 218 nm (+). Molar ellipticity values are (5-7) x 10(4) deg.cm2.dmol-1. The energies and pattern (+,-,+) of these ellipticity bands are similar to those observed for the Cd(II) clusters of metallothionein. The latter are believed to be due to the presence of bridging cysteine ligands (Willner, H.W., Vasak, M., & Kägi, J.H.R., 1987, Biochemistry 26, 6287-6292). Circular dichroism provides further evidence that the GAL4 DNA-binding domain forms a binuclear Cd cluster with the amino acid sequence -C11-X2-C14-X6-C21-X6-C28-X2-C31-X6-C38- found in GAL4(62*). Zn2GAL4(62*) has much weaker ellipticity associated with the higher energy -S(-)--> Zn charge transfer bands. Removal of the metal ions from GAL4(62*) results in substantial changes in the near UV circular dichroism due to both loss of the charge transfer bands and changes in the conformation of the peptide backbone. Binding of the UASG enhancer DNA element to either Zn or Cd GAL4(62*) alters the ellipticity of the -S(-)--> metal charge transfer bands suggesting that DNA binding is associated with some changes in conformation around the metal cluster.

Chiral probes for the handedness of DNA helices: enantiomers of tris(4,7-diphenylphenanthroline)ruthenium(II).

The chiral complexes tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) (RuDIP) are shown to be specific chemical probes with which to distinguish right- and left-handed DNA helices in solution. In spectrophotometric titrations of racemic RuDIP with both B-form calf thymus DNA and Z-form poly[d(G-C)], hypochromicity in the intense metal-to-ligand charge-transfer band is found and enhancement in luminescence is observed. The spectrophotometric assay of DNA binding to the well-resolved enantiomers of RuDIP provides a means to determine the helical conformation. Strong chiral specificity is seen in binding experiments with right-handed B-DNA and, on this basis, the absolute configurations are assigned. Although delta-RuDIP can bind by intercalation into the right-handed helix, steric constraints imposed by the helix asymmetry preclude completely binding by the lambda enantiomer. Both isomers, however, are found to bind equally to Z-DNA. Left-handed helices that are more similar structurally to B-DNA would be predicted to display a stereospecific preference for this lambda isomer.

The presence of zinc in the restriction enzyme Eco RI.

We have determined that the restriction endonuclease Eco RI contains 1.0 +/- 0.1 eq of zinc/monomeric enzyme. DNA cleavage by Eco RI is inhibited by ortho-phenanthroline after preincubation of the enzyme with the chelating agent. A similar inhibition by the nonchelating meta-phenanthroline is not seen. The sensitivity of the inhibition by the neutral ligand ortho-phenanthroline to preincubation is consistent with the tightly bound and inaccessible nature of the metal site. Extensive dialysis against the ortho-phenanthroline inhibitor leads to the release of the bound metal with the concomitant loss of enzyme activity. The tightly bound Zn2+ cation, then, appears to be necessary for enzyme function. The finding of zinc in Eco RI further illustrates the ubiquity of Zn2+ to DNA-protein complexes.