bluuebunny/crossref_metadata_2025 huggingface dataset:
Hypothermia Pediatric Head Injury Trial: The Value of a Pretrial Clinical Evaluation Phase
Authors: Hutchison J. , Ward R. , Lacroix J. , Hébert P. , Skippen P. , Barnes M. , Meyer P. , Morris K. , Kirpalani H. , Singh R. , Dirks P. , Bohn D. , Moher D.
Published: August 2006

Background: The utility of a pretrial clinical evaluation or run-in phase prior to conducting trials of complex interventions such as hypothermia therapy following severe traumatic brain injury in children and adolescents has not been established. Methods: The primary objective of this study was to prospectively evaluate the ability of investigators to adhere to the clinical protocols of care including the cooling and rewarming procedures as well as management guidelines in patients with severe traumatic brain injury (Glasgow Coma Scale ≤8) treated with 24 h of hypothermia therapy. A secondary objective was to evaluate the ability of study research assistants to complete the study case report form using a procedures manual. The study was conducted at 18 sites in Canada, the United Kingdom and France prior to proceeding to a randomized controlled trial (RCT). After 2 patients were enrolled at each center, an independent clinical evaluation committee examined the process of care and the completeness of data collection. Centers were permitted to enroll patients in the RCT once they met pre-established adherence criteria. Results: Seventeen of the 18 centers completed the pretrial clinical evaluation phase demonstrating compliance with study procedures and proceeded to an RCT of hypothermia therapy. One center enrolled only 1 patient in the pretrial clinical evaluation phase due to small numbers of patients with traumatic brain injury, and therefore, did not proceed to the RCT. Three centers were required to enroll more than 2 patients in the pretrial clinical evaluation phase prior to proceeding to the RCT because of problems with adherence to the clinical protocols at two centers and the training of new study personnel at another center. Of the 39 patients enrolled during the pretrial clinical evaluation phase, 8 (20.5%) died and 22 (62.9%) had a good outcome defined as normal, mild or moderate disability assessed using the Pediatric Cerebral Performance Category score at 6 months following injury. Discussion: The pretrial clinical evaluation phase was useful to ensure compliance with complex hypothermia therapy and consensus-based clinical management guidelines of care successfully implemented across 17 of 18 centers. This study maneuver allowed us to complete a subsequent RCT in 225 children following severe traumatic brain injury.
Changes in White Matter Late after Severe Traumatic Brain Injury in Childhood
Authors: Tasker Robert C.
Published: August 2006

Severe traumatic brain injury in childhood, particularly that complicated by raised intracranial pressure, has significant long-term effects on the brain. Since magnetic resonance imaging provides a means of visualizing neuroanatomic structure in exquisite detail, the scope of this review is to revisit the pathology of traumatic brain injury described in recent clinical imaging studies. Acute imaging provides insight into the acute mechanism of focal and diffuse injury. There is some reduction in threshold for white matter pathology in the hemisphere ipsilateral to injury. After injury, there may be long-term effects on white matter architecture and the potential for brain growth. In this context, the pattern of hippocampal rather than parahippocampal gyrus tissue loss provides insight into the likely cause of white matter injury being cerebral hypoperfusion.
Mitochondrial Heat Shock Protein 60 Is Increased in Cerebrospinal Fluid following Pediatric Traumatic Brain Injury
Authors: Lai Yichen , Stange Christopher , Wisniewski Stephen R. , Adelson P. David , Janesko-Feldman Keri L. , Brown Danielle S. , Kochanek Patrick M. , Clark Robert S.B.
Published: August 2006

Mitochondrial dysfunction occurs after traumatic brain injury (TBI) and contributes significantly to subsequent cell death. Heat shock protein 60 (hsp60) is a predominantly mitochondrial protein with important homeostatic functions. Induction of hsp60 has been demonstrated in cerebral ischemia models, possibly reflecting mitochondrial stress. We measured hsp60 concentration in the cerebrospinal fluid (CSF) of 34 infants and children after severe TBI and of 7 control patients by ELISA. Peak CSF hsp60 concentration was increased in TBI patients versus controls (0.84 ng/ml, range 0–44.59, vs. 0.0 ng/ml, range 0–0.48; p < 0.05). Induction of hsp60 occurred early after the injury. Peak hsp60 concentration was independently associated with the severity of injury, defined as the admission Glasgow Coma Scale score. These data suggest that increased hsp60 in CSF might reflect the severity of early mitochondrial stress or damage after TBI.
Impaired Cerebral Autoregulation and 6-Month Outcome in Children with Severe Traumatic Brain Injury: Preliminary Findings
Authors: Vavilala M.S. , Muangman S. , Tontisirin Nuj , Fisk D. , Roscigno C. , Mitchell P. , Kirkness C. , Zimmerman J.J. , Chesnut Randall , Lam A.M.
Published: August 2006

The objective of this study was to describe the incidence of impaired cerebral autoregulation and to describe the relationship between impaired cerebral autoregulation and outcome after severe pediatric traumatic brain injury (TBI). We prospectively examined cerebral autoregulation in 28 children ≤17 (10 ± 5) years with a Glasgow coma scale score <9 within the first 72 h of pediatric intensive care unit admission. Children with isolated focal TBI were excluded. Glasgow outcome scores (GOS) were collected at hospital discharge, as well as 3 and 6 months after severe TBI. GOS <4 reflected poor outcome. Cerebral autoregulation was impaired in 12/28 children. An autoregulatory index <0.4 was associated with GOS <4 at 6 months (p = 0.005). Impaired cerebral autoregulation, early after severe pediatric TBI, was associated with a poor 6-month outcome.
Pediatric Posttraumatic Seizures: Epidemiology, Putative Mechanisms of Epileptogenesis and Promising Investigational Progress
Authors: Statler Kimberly D.
Published: August 2006

Posttraumatic seizures and epilepsy are common in children experiencing traumatic brain injury and portend worse functional outcome. Unfortunately, the pathogenesis of pediatric posttraumatic seizures and epilepsy remains poorly understood, and no efficacious preventive therapy for post- traumatic epilepsy has been identified. This article reviews the epidemiology of pediatric posttraumatic seizures, discusses prominent putative mechanisms of posttraumatic epileptogenesis and highlights recent promising progress in experimental investigations of posttraumatic seizures and epilepsy.
Is Being Plastic Fantastic? Mechanisms of Altered Plasticity after Developmental Traumatic Brain Injury
Authors: Giza Christopher C. , Prins Mayumi L.
Published: August 2006

Traumatic brain injury (TBI) is predominantly a clinical problem of young persons, resulting in chronic cognitive and behavioral deficits. Specifically, the physiological response to a diffuse biomechanical injury in a maturing brain can clearly alter normal neuroplasticity. To properly evaluate and investigate developmental TBI requires an understanding of normal principles of cerebral maturation, as well as a consideration of experience-dependent changes. Changes in neuroplasticity may occur through many age-specific processes, and our understanding of these responses at a basic neuroscience level is only beginning. In this article, we will particularly discuss mechanisms of TBI-induced altered developmental plasticity such as altered neurotransmission, distinct molecular responses, cell death, perturbations in neuronal connectivity, experience-dependent ‘good plasticity’ enhancements and chronic ‘bad plasticity’ sequelae. From this summary, we can conclude that ‘young is not always better’ and that the developing brain manifests several crucial vulnerabilities to TBI.
Large Animal Models of Traumatic Injury to the Immature Brain
Authors: Duhaime Ann-Christine
Published: August 2006

Large animal models have been used much less frequently than rodent models to study traumatic brain injury. However, large animal models offer distinct advantages in replicating specific mechanisms, morphology and maturational stages relevant to age-dependent injury responses. This paper reviews how each of these features is relevant in matching a model to a particular scientific question and discusses various scaling strategies, advantages and disadvantages of large animal models for studying traumatic brain injury in infants and children. Progress to date and future directions are outlined.
Traumatic Injury to the Immature Brain Results in Progressive Neuronal Loss, Hyperactivity and Delayed Cognitive Impairments
Authors: Pullela Ramadevi , Raber Jacob , Pfankuch Timothy , Ferriero Donna M. , Claus Catherine P. , Koh Seong-Eun , Yamauchi Toshihiro , Rola Radoslaw , Fike John R. , Noble-Haeusslein Linda J.
Published: August 2006

The immature brain may be particularly vulnerable to injury during critical periods of development. To address the biologic basis for this vulnerability, mice were subjected to traumatic brain injury at postnatal day 21, a time point that approximates that of the toddler-aged child. After motor and cognitive testing at either 2 weeks (juveniles) or 3 months (adults) after injury, animals were euthanized and the brains prepared for quantitative histologic assessment. Brain-injured mice exhibited hyperactivity and age-dependent anxiolysis. Cortical lesion volume and subcortical neuronal loss were greater in brain-injured adults than in juveniles. Importantly, cognitive decline was delayed in onset and coincided with loss of neurons in the hippocampus. Our findings demonstrate that trauma to the developing brain results in a prolonged period of pathogenesis in both cortical and subcortical structures. Behavioral changes are a likely consequence of regional-specific neuronal degeneration.
Gel-Based Hippocampal Proteomic Analysis 2 Weeks following Traumatic Brain Injury to Immature Rats Using Controlled Cortical Impact
Authors: Kochanek Ashley R. , Kline Anthony E. , Gao Wei-Min , Chadha Mandeep , Lai Yichen , Clark Robert S.B. , Dixon C. Edward , Jenkins Larry W.
Published: August 2006

Traumatic brain injury (TBI) to postnatal day 17 rats has been shown to produce acute changes in hippocampal global protein levels and spatial learning and memory deficits. The purpose of the present study was to analyze global hippocampal protein changes 2 weeks after a moderate ipsilateral controlled cortical impact in postnatal day 17 rats using 2-dimensional difference gel electrophoresis and mass spectrometry. Paired sham and ipsilateral injured hippocampal lysates were independently labeled with different fluorescent cyanine dyes and coseparated within the same immobilized pH gradient strips and slab gel based on isoelectric point and molecular mass. Significant changes in key proteins involved in glial and neuronal stress, oxidative metabolism, calcium uptake and neurotransmitter function were found 2 weeks after injury, and their potential roles in hippocampal plasticity and cognitive dysfunction were discussed.
The Potential Role of Mitochondria in Pediatric Traumatic Brain Injury
Authors: Robertson Courtney L. , Soane Lucian , Siegel Zachary T. , Fiskum Gary
Published: August 2006

Mitochondria play a central role in cerebral energy metabolism, intracellular calcium homeostasis and reactive oxygen species generation and detoxification. Following traumatic brain injury (TBI), the degree of mitochondrial injury or dysfunction can be an important determinant of cell survival or death. Literature would suggest that brain mitochondria from the developing brain are very different from those from mature animals. Therefore, aspects of developmental differences in the mitochondrial response to TBI can make the immature brain more vulnerable to traumatic injury. This review will focus on four main areas of secondary injury after pediatric TBI, including excitotoxicity, oxidative stress, alterations in energy metabolism and cell death pathways. Specifically, we will describe what is known about developmental differences in mitochondrial function in these areas, in both the normal, physiologic state and the pathologic state after pediatric TBI. The ability to identify and target aspects of mitochondrial dysfunction could lead to novel neuroprotective therapies for infants and children after severe TBI.
Induction of Monocarboxylate Transporter 2 Expression and Ketone Transport following Traumatic Brain Injury in Juvenile and Adult Rats
Authors: Prins M.L. , Giza C.C.
Published: August 2006

Based on recent work demonstrating age-dependent ketogenic neuroprotection after traumatic brain injury (TBI), it was hypothesized that the neuroprotection among early post-weaned animals was related to induced cerebral transport of ketones after injury. Regional changes in monocarboxylate transporter 2 (MCT2) were acutely examined with immunohistochemistry after sham surgery or controlled cortical impact injury among postnatal day 35 and adult rats. Both ages showed elevated MCT2 expression in the ipsilateral cerebral vasculature after TBI. Using Western blotting, MCT2 expression was 80–88% greater in microvessels isolated from postnatal day 35 rats at all time points relative to adults. The increased MCT2 expression was temporally correlated with an age-related increase in cerebral uptake of ketones, when ketones were made available after injury.
Multiplex-FISH (M-FISH): technique, developments and applications
Authors: Kearney L.
Published: August 2006

Multiplex FISH (M-FISH) represents one of the most significant developments in molecular cytogenetics of the past decade. Originally designed to generate 24 colour karyotyping, the technique has spawned many variations and an equally diverse range of applications. In tumour and leukaemia cytogenetics, the two groups that have been targeted represent both ends of the cytogenetic spectrum: those with an apparently normal karyotype (suspected of harbouring small rearrangements not detectable by conventional cytogenetics) and those with a complex aberrant karyotype (which are difficult to karyotype accurately due to the sheer number of aberrations). In research, mouse M-FISH provides a powerful tool to characterize mouse models of a disease. In addition, the ability to accurately karyotype single metaphases without selection makes M-FISH the perfect tool in chromosome breakage studies and for characterizing clonal evolution of tumours. Finally, M-FISH has emerged as the perfect partner for the developing genomic microarray (array CGH) technologies, providing a powerful approach to gene discovery.
COmbined Binary RAtio fluorescence in situ hybridiziation (COBRA-FISH): development and applications
Authors: Raap A.K. , Tanke H.J.
Published: August 2006

The ability to probe for the location of DNA sequences in morphologically preserved chromosomes and nuclei by fluorescence in situ hybridization (FISH) provided for cytogenetics a quantum leap forward in resolution and ease of detection of chromosomal aberrations. COBRA-FISH, an acronym for COmbined Binary RAtio-FISH is a multicolor FISH methodology, which enables recognition of all human chromosome arms on the basis of color, thus greatly facilitating cytogenetic analysis. It also permits gene and viral integration site mapping in the context of chromosome arm painting. Here we review the principle, practice and applications of COBRA-FISH.
Combined multicolor-FISH and immunostaining
Authors: Ye C.J. , Stevens J.B. , Liu G. , Ye K.J. , Yang F. , Bremer S.W. , Heng H.H.Q.
Published: August 2006

The combination of multicolor-FISH and immunostaining produces a powerful visual method to analyze in situ DNA-protein interactions and dynamics. Representing one of the major technical improvements of FISH technology, this method has been used extensively in the field of chromosome and genome research, as well as in clinical studies, and serves as an important tool to bridge molecular analysis and cytological description. In this short review, the development and significance of this method will be briefly summarized using a limited number of examples to illustrate the large body of literature. In addition to descriptions of technical considerations, future applications and perspectives have also been discussed focusing specifically on the areas of genome organization, gene expression and medical research. We anticipate that this versatile method will play an important role in the study of the structure and function of the dynamic genome and for the development of potential applications for medical research.
Proof of partial imbalances 6q and 11q due to maternal complex balanced translocation analyzed by microdissection of multicolor labeled chromosomes (FISH-MD) in a patient with Dandy-Walker variant
Authors: Weimer J. , Cohen M. , Wiedemann U. , Heinrich U. , Jonat W. , Arnold N.
Published: August 2006

We report on a family in which a daughter is described with mental retardation, as well as malformations of the heart, and of the brain (Dandy-Walker variant). The patient’s phenotype suggests a chromosomal rearrangement. However, her karyotype was unremarkable by conventional cytogenetic analysis. In order to detect chromosome rearrangements overseen by this method, the subtelomere regions of suspicious chromosomes were verified by fluorescence in situ hybridization (FISH). A rearranged derivative chromosome 6 was identified. Further examinations by FISH-microdissection (FISH-MD) revealed a maternal complex balanced translocation. The patient inherited the derivative chromosome 6 from her mother and therefore carries a partial monosomy 6q26→qter and a partial trisomy 11q23.3→qter.
Multicolor chromosome bar codes
Authors: Müller S. , Wienberg J.
Published: August 2006

Chromosome bar codes are multicolor banding patterns produced by fluorescence in situ hybridization (FISH) with differentially labeled and pooled sub-regional DNA probes. These molecular cytogenetic tools facilitate chromosome identification and the delineation of both inter- and intra-chromosomal rearrangements. We present an overview of the various conceptual approaches which can be largely divided into two classes: Simple bar codes designed for chromosome identification and complex bar codes for high resolution aberration screening of entire karyotypes. We address the issue of color redundancy and how to overcome this limitation by complementation of bar codes with whole chromosome painting probes.
Multicolor PRINS and multicolor PNA
Authors: Pellestor F. , Paulasova P. , Andréo B. , Lefort G. , Hamamah S.
Published: August 2006

Both PRimed IN Situ (PRINS) and Peptide Nucleic Acid (PNA) technologies have emerged as research techniques, but they have quickly evolved to applications in biological diagnosis assays. The two procedures now constitute efficient alternatives to the conventional fluorescence in situ hybridization (FISH) procedure for in situ chromosome identification and aneuploidy detection. They present several advantages (specificity, speed, discriminating ability) that make them very attractive for a number of cytogenetic purposes. Multicolor PRINS and PNA protocols have been described for the specific identification of human chromosomes. Various applications have already been developed in human genetics and new adaptations are ongoing.
Multicolor-FISH applied to resolve complex chromosomal changes in a case of T-ALL (FAB L2)
Authors: Mkrtchyan H. , Glaser M. , Gross M. , Wedding U. , Höffken K. , Liehr T. , Karst C. , Aroutiounian R.
Published: August 2006

We report on a patient with a clinically diagnosed acute lymphoblastic leukemia (ALL) with partial unrecorded complex translocation events especially involving chromosomes 5, 9 and 18. At the GTG-band level the karyotype was abnormal in 20% of the analyzed cells. The complex karyotype was studied in more detail by spectral karyotyping (SKY) and multicolor banding (MCB) to characterize it in more detail. Thus, the karyotype could be described very accurately and in summary three different clones were detected, reflecting a high rate of karyotypic evolution in this patient.
Characterization of chromosomal aberrations in diffuse large B-cell lymphoma (DLBL) by G-banding and spectral karyotyping (SKY)
Authors: Adam P. , Steinlein C. , Schmid M. , Haralambieva E. , Stöcklein H. , Leich E. , Rosenwald A. , Müller-Hermelink H.K. , Ott G.
Published: August 2006

Cytogenetic chromosome analysis by classical G-banding was supplemented by spectral karyotyping (SKY) in 12 cases of diffuse large B-cell lymphoma (DLBL). SKY is a fluorescence in-situ-based, genome-wide screening technique allowing identification of genetic material even in highly condensed metaphase chromosomes of poor morphology. By simultaneous hybridization of whole chromosome painting probes onto tumor chromosome spreads genetic rearrangements are visualized permitting the clarification of even complex karyotype alterations and the identification of genetic material of previously unknown origin, so-called marker chromosomes. Taking the SKY results into account, we reevaluated the G-banding karyotypes initially carried out, thus generating a more precise karyotype in ten of twelve (83%) cases investigated. In particular, thirteen chromosomal rearrangements not correctly recognized by classical cytogenetics were identified, the genetic origin of seven marker chromosomes was elucidated and three structural genetic rearrangements were redefined. We found SKY to be a valuable technique to establish a definite karyotype in addition to classical cytogenetics.
Molecular cytogenetic characterization of chromosome 9-derived material in a human thyroid cancer cell line
Authors: Weier H.-U.G. , Tuton T.B. , Ito Y. , Chu L.W. , Lu C.-M. , Baumgartner A. , Zitzelsberger H.F. , Weier J.F.
Published: August 2006

The incidence of papillary thyroid carcinoma (PTC) increases significantly after exposure of the head and neck region to ionizing radiation, yet we know neither the steps involved in malignant transformation of thyroid epithelium nor the specific carcinogenic mode of action of radiation. Such increased tumor frequency became most evident in children after the 1986 nuclear accident in Chernobyl, Ukraine. In the eight years following the accident, the average incidence of childhood PTCs (chPTC) increased 70-fold in Belarus, 200-fold in Gomel, 10-fold in the Ukraine and 50-fold in Tschnigov, Kiev, Rovno, Shitomyr and Tscherkassy compared to the rate of about 1 tumor incidence per 106 children per year prior to 1986 (Likhtarev et al., 1995; Sobolev et al., 1997; Jacob et al., 1998). To study the etiology of radiation-induced thyroid cancer, we formed an international consortium to investigate chromosomal changes and altered gene expression in cases of post-Chernobyl chPTC. Our approach is based on karyotyping of primary cultures established from chPTC specimens, establishment of cell lines and studies of genotype-phenotype relationships through high resolution chromosome analysis, DNA/cDNA micro-array studies, and mouse xenografts that test for tumorigenicity. Here, we report the application of fluorescence in situ hybridization (FISH)-based techniques for the molecular cytogenetic characterization of a highly tumorigenic chPTC cell line, S48TK, and its subclones. Using chromosome 9 rearrangements as an example, we describe a new approach termed ‘BAC-FISH’ to rapidly delineate chromosomal breakpoints, an important step towards a better understanding of the formation of translocations and their functional consequences.
Interphase M-FISH applications using commercial probes in prenatal and PGD diagnostics
Authors: Stumm M. , Wegner R.-D. , Bloechle M. , Eckel H.
Published: August 2006

Early, rapid and reliable diagnosis is of first priority in prenatal medicine. The combination of specific sonographic markers (e.g. nuchal translucency) and biochemical parameters in maternal serum (e.g. free beta-human chorionic gonadotropin, pregnancy-associated plasma protein A), has already dramatically improved the sensitivity of non-invasive first trimester risk screening in pregnancy. In invasive prenatal diagnosis, in addition to well-established chorionic villi short-term culture, interphase multi-colour-fluorescence in situ hybridisation (M-FISH) on uncultured amnion cells has become a reliable tool for the rapid detection of fetal aneuploidies. Interphase M-FISH applications have enabled the diagnosis of selected chromosomal abnormalities in single cells and, therefore, have also become an important diagnostic tool for preimplantation diagnosis (PGD). The development of commercially available probe sets, in particular, has led to a broad use of interphase M-FISH in prenatal and PGD diagnosis.
Molecular cytogenetic studies towards the full karyotype analysis of human blastocysts and cytotrophoblasts
Authors: Weier J.F. , Ferlatte C. , Baumgartner A. , Jung C.J. , Nguyen H.-N. , Chu L.W. , Pedersen R.A. , Fisher S.J. , Weier H.-U.G.
Published: August 2006

Numerical chromosome aberrations in gametes typically lead to failed fertilization, spontaneous abortion or a chromosomally abnormal fetus. By means of preimplantation genetic diagnosis (PGD), we now can screen human embryos in vitro for aneuploidy before transferring the embryos to the uterus. PGD allows us to select unaffected embryos for transfer and increases the implantation rate in in vitro fertilization programs. Molecular cytogenetic analyses using multi-color fluorescence in situ hybridization (FISH) of blastomeres have become the major tool for preimplantation genetic screening of aneuploidy. However, current FISH technology can test for only a small number of chromosome abnormalities and hitherto failed to increase the pregnancy rates as expected. We are in the process of developing multi-color FISH-based technologies to score all 24 chromosomes in single cells within a three-day time limit, which we believe is vital to the clinical setting. Also, human placental cytotrophoblasts (CTBs) at the fetal-maternal interface acquire aneuploidies as they differentiate to an invasive phenotype. About 20–50% of invasive CTB cells from uncomplicated pregnancies were found to be aneuploid, suggesting that the acquisition of aneuploidy is an important component of normal placentation, perhaps limiting the proliferative and invasive potential of CTBs. Since most invasive CTBs are interphase cells and possess extreme heterogeneity, we applied multi-color FISH and repeated hybridizations to investigate the feasibility of a full karyotype analysis of individual CTBs. In summary, this study demonstrates the strength of Spectral Imaging analysis and repeated hybridizations, which provides a basis for full karyotype analysis of single interphase cells.
Studying meiosis: a review of FISH and M-FISH techniques used in the analysis of meiotic processes in humans
Authors: Oliver-Bonet M. , Benet J. , Martin R.H.
Published: August 2006

It is well known that chromosome in situ hybridization allows the unequivocal identification of targeted human somatic chromosomes. Different fluorescent in situ hybridization (FISH) techniques have been developed throughout the years and, following the mitotic studies, meiotic analyses have been performed using these different techniques. The introduction of M-FISH techniques to the analysis of meiotic cells has allowed the study of meiotic processes for every individual human chromosome. In this paper, we review the different FISH and M-FISH techniques that have been used on human meiotic cells in both men and women.
Multicolor FISH used for the characterization of small supernumerary marker chromosomes (sSMC) in commercially available immortalized cell lines
Authors: Brecevic L. , Michel S. , Starke H. , Müller K. , Kosyakova N. , Mrasek K. , Weise A. , Liehr T.
Published: August 2006

There are only about 30 commercially available cell lines which include small supernumerary marker chromosomes (sSMC). As approximately 2.5 million people worldwide are carriers of an sSMC, this small number of immortalized cell lines is hard to understand. sSMC cell lines provide practically unlimited material for continuing studies e.g. to learn more about marker chromosome formation, or karyotypic evolution. To obtain information about their genetic content, in the present study we analyzed by FISH and multicolor-FISH approaches 19 sSMC cell lines obtained from the European Collection of Cell Cultures (ECACC). Microdissection and reverse painting, (sub-) centromere-specific multicolor-FISH (sub-)cenM-FISH, multicolor banding (MCB) and selected locus-specific FISH probes were applied. Thus, we were able to characterize comprehensively 14 out of 19 sSMC carrying cell lines; in the remaining five cases an sSMC could not be detected. Surprisingly, in six of the nine cell lines with sSMC previously characterized for their chromosomal origin by others, those results had to be revised. This has impact on the conclusions of previous studies, e.g. for uniparental disomy (UPD) in connection with sSMC.
Molecular cytogenetic identification and characterization of a de novo supernumerary neocentromeric derivative chromosome 13
Authors: Tönnies H. , Gerlach A. , Heineking B. , Starke H. , Neitzel H. , Neumann L.M.
Published: August 2006

We report a young girl with microphthalmia, conductive deafness, aortic isthmus stenosis, laryngomalacia, and laryngeal stenosis carrying a de novo supernumerary neocentromeric derivative chromosome 13. For the precise identification and characterization of the eu- and heterochromatic content of the marker chromosome, straightforward molecular cytogenetic analyses were performed, such as chromosome microdissection, FISH with different probes (e.g. wcp, alphoid centromeric probes, BAC), centromere-specific multicolor FISH (cenM-FISH), and multicolor banding (MCB). The analyses demonstrated that the marker consisted of an inverted duplication (partial tetrasomy) of the distal portion of chromosome 13 that was separated from the endogenous chromosome 13 centromere. Using an all-centromere probe and multicolor cenM-FISH, no alpha-satellite DNA hybridization signal was detectable on any portion of the derivative chromosome. The presence of a functional and active neocentromere on the derivative chromosome 13 was confirmed by positive immunofluorescence signals with CENP-C antibodies. BAC-FISH confirmed the cytogenetic localization of the neocentromere in band 13q31.3. Thus the patient had a mosaic conventional karyotype mos 47,XX,+inv dup(13)(qter→q21.3::q21.3→q31.3→neo→q31.3→qter)[6]/46,XX [49].
Supernumerary marker chromosome 5 diagnosed by M-FISH in a child with congenital heart defect and unusual face
Authors: Sarri C. , Gyftodimou Y. , Grigoriadou M. , Pandelia E. , Kalogirou S. , Kokotas H. , Mrasek K. , Weise A. , Petersen M.B.
Published: August 2006

We describe a female patient with a small supernumerary marker chromosome (sSMC) present in mosaic and characterized in detail by fluorescence in situ hybridization (FISH) using all 24 human whole chromosome painting probes, multicolor banding (MCB) and subcentromere specific multicolor FISH (subcenM-FISH). The sSMC was demonstrated to be derived from chromosome 5 and the karyotype of our patient was as follows: 47,XX,+mar.ish r(5)(::p13.2∼p13.3→q11.2::) [60%]/46,XX [40%]. Partial trisomy for the proximal 5p and q chromosomal regions is a rare event. A critical region exists at 5p13 for the phenotype associated with duplication 5p. As far as we know, eight similar cases have been published up to now. We describe a new case which, to our knowledge, is the first characterized in such detail. The role of uniparental disomy (UPD) in cases of SMC is also discussed.
Molecular cytogenetic characterization of a constitutional complex intrachromosomal 4q rearrangement in a patient with multiple congenital anomalies
Authors: Thienpont B. , Gewillig M. , Fryns J.-P. , Devriendt K. , Vermeesch J.
Published: August 2006

Constitutional Complex Chromosomal Rearrangements (CCRs) are very rare. While the vast majority of CCRs involve more than one chromosome, only seven cases describe CCRs with four or more breakpoints within a single chromosome. Here, we present a patient with multiple congenital anomalies and mental retardation. Array Comparative Genomic Hybridisation (array CGH), FISH and Multicolour Banding FISH revealed a de novo complex rearrangement with two deletions, a duplication and an inversion of 4q. This CCR involving at least seven breakpoints is one of the most complex rearrangements of a single chromosome reported thus far. Potential mechanisms generating such complex rearrangements are discussed.
Duplications of proximal 16q flanked by heterochromatin are not euchromatic variants and show no evidence of heterochromatic position effect
Authors: Barber J.C.K. , Zhang S. , Friend N. , Collins A.L. , Maloney V.K. , Hastings R. , Farren B. , Barnicoat A. , Polityko A.D. , Rumyantseva N.V. , Starke H. , Ye S.
Published: August 2006

Extra euchromatic material was found within the major heterochromatic block of chromosome 16 (16qh) in one de novo case and seven members of two families. In contrast to the euchromatic variants of chromosome 9 (9qh), which are derived from pericentromeric euchromatin, molecular cytogenetics confirmed that these duplications were of 16q11.2→q12.2 in the de novo case, of 16q11.2→q13 in three members of family 1 and 16q11.2→q12.1 in four members of family 2. The duplication had arisen as a post-zygotic mitotic event in the mother of family 1 and been transmitted paternally in family 2. An insertional mechanism of origin is proposed for the duplications in case 1 and family 1. Expression at the 16q13 matrix metalloproteinase-2 (MMP2)locus in families 1 and 2 was proportional to genomic copy number and not therefore consistent with position effect silencing due to the flanking blocks of heterochromatin. We conclude that proximal 16q duplications within 16qh are not novel euchromatic variants but associated with a variable phenotype including developmental delay, speech delay, learning difficulties and behavioural problems. The behavioural problems in families ascertained through affected children are much less severe than those encountered in previous patients ascertained as adults.
Towards many colors in FISH on 3D-preserved interphase nuclei
Authors: Walter J. , Joffe B. , Bolzer A. , Albiez H. , Benedetti P.A. , Müller S. , Speicher M.R. , Cremer T. , Cremer M. , Solovei I.
Published: August 2006

The article reviews the existing methods of multicolor FISH on nuclear targets, first of all, interphase chromosomes. FISH proper and image acquisition are considered as two related components of a single process. We discuss (1) M-FISH (combinatorial labeling + deconvolution + wide-field microscopy); (2) multicolor labeling + SIM (structured illumination microscopy); (3) the standard approach to multicolor FISH + CLSM (confocal laser scanning microscopy; one fluorochrome – one color channel); (4) combinatorial labeling + CLSM; (5) non-combinatorial labeling + CLSM + linear unmixing. Two related issues, deconvolution of images acquired with CLSM and correction of data for chromatic Z-shift, are also discussed. All methods are illustrated with practical examples. Finally, several rules of thumb helping to choose an optimal labeling + microscopy combination for the planned experiment are suggested.
Rearrangements of chromosome 18 illustrate the utility of array-based comparative genomic hybridization
Authors: Kashork C.D. , Theisen A. , Bejjani B.A. , Shaffer L.G.
Published: August 2006

Comprehensive and reliable testing is an important component of counseling and management in clinical genetics. Identification of imbalances of chromosomal segments has uncovered new genes and has established phenotype/genotype correlations for many syndromes with previously unidentified causes. Conventional cytogenetics has proven to be useful for the detection of large aberrations, but its resolution limits the identification of submicroscopic alterations. Comparative genomic hybridization (CGH) on a microarray-based platform has the potential to detect and characterize both microscopic and submicroscopic chromosomal abnormalities. Nine cases of aberrations involving chromosome 18 are used to illustrate the use and clinical potential of array CGH.
Effect of 5α-Dihydrotestosterone and Testosterone on Apoptosis in Human Dermal Papilla Cells
Authors: Winiarska A. , Mandt N. , Kamp H. , Hossini A. , Seltmann H. , Zouboulis C.C. , Blume-Peytavi U.
Published: August 2006

Pathogenetic mechanisms in androgenetic alopecia are not yet fully understood; however, it is commonly accepted that androgens like testosterone (T) and 5α-dihydrotestosterone (5α-DHT) inhibit hair follicle activity with early induction of the catagen. Thus, we investigated the influence of T and 5α-DHT on proliferation, cell death and bcl-2/bax expression in cultured dermal papilla cells (DPC) from nonbalding scalp regions of healthy volunteers. T and 5α-DHT induced apoptosis in DPC in a dose-dependent and time-related manner; in addition a necrotic effect due to T at 10–5M was found. Interestingly, bcl-2 protein expression was decreased in T- and 5α-DHT-treated cells, leading to an increase in the bax/bcl-2 ratio. In addition, T and 5α-DHT induced proteolytic cleavage of caspase 8 and inhibited proliferation of DPC at 10–5M. High concentrations of T and 5α-DHT were needed to induce apoptotic effects in DPC. These data suggest that DPC from nonbalding scalp regions do have the capacity to undergo apoptosis, but need a high androgen stimulus. The present study provides an interesting new pathogenetic approach in androgenetic alopecia.
Microtubules Regulate Expression of ICAM-1 in Epidermoid Cells (KB Cells)
Authors: Kaatz M. , Berod L. , Lagadari M. , Fluhr J.W. , Elsner P. , Norgauer J.
Published: August 2006

The intercellular adhesion molecule-1/CD54 (ICAM-1) functions as a counterreceptor for other adhesion molecules (e.g. the lymphocyte function-associated antigen-1/CD11a/CD18) required for the interaction of a large variety of cells with leucocytes. Constitutive expression of ICAM-1 in human epidermoid cells (KB cells) is low, but inducible by interferon-γ (IFN-γ). Treatment of KB cells with microtubule-disrupting agents, like colchicine, nocodazole and vinblastine, potentiated the constitutive and cytokine-induced ICAM-1 expression on the cell surface. Actinomycin D inhibited microtubule-disrupting agent-induced ICAM-1 surface expression. Increased steady-state levels of ICAM-1 transcripts were found after treatment of KB cells with microtubule-disrupting agents. However, microtubule-disrupting agents neither altered the glyceraldehyde-3-phosphate dehydrogenase mRNA levels nor the amount of expressed α2-, α3-and β1-integrins at the cell surface. In addition, they did not change the ICAM-1 mRNA half-life. These studies indicate a control function of the microtubule network on the expression of ICAM-1.
In vitro 5-Lipoxygenase and Anti-Oxidant Activities of South African Medicinal Plants Commonly Used Topically for Skin Diseases
Authors: Frum Y. , Viljoen A.M.
Published: August 2006

An investigation was undertaken to determine the possible mechanisms of action of medicinal plants used for dermatological pathologies. A total of 14 plant species were selected from the readily available ethnobotanical literature. 5-Lipoxygenase and DPPH (2,2-diphenyl-1-picrylhydrazyl) assays were used to determine the anti-inflammatory activity and the anti-oxidant activity of selected medicinal plants, respectively. Both aqueous and methanol extracts were tested. Among the plants screened, four species (Croton sylvaticus, Warburgia salutaris, Pentanisia prunelloides, and Melianthus comosus) displayed promising 5-lipoxygenase inhibitory activity with IC50 values <61 ppm. A large number of plants exhibited significant anti-oxidant activities with IC50 values between 5.27 and 83.36 ppm. Aqueous extracts of M. comosus exhibited the most potent anti-inflammatory and anti-oxidant activity.
Percutaneous Absorption of Steroids: Determination of in vitro Permeability and Tissue Reservoir Characteristics in Human Skin Layers
Authors: Magnusson B.M. , Cross S.E. , Winckle G. , Roberts M.S.
Published: August 2006

The skin localization of steroids following topical application is largely unknown. We determined the distribution of five steroids in human skin using excised epidermal, dermal, and full-thickness membranes in vitro. There was no significant difference in steroid maximum flux through epidermal and full-thickness membranes, other than significantly lower fluxes for the most polar steroid, aldosterone. Hydrocortisone had the highest dermal diffusivity and dermal penetration, and the accumulation of hydrocortisone and corticosterone was higher than that of the other steroids. Slower penetration and higher accumulation in the viable epidermis of progesterone in full-thickness skin were consistent with dermal penetration limitation effects associated with high lipophilicity.
Primary Prevention of Asthma: Age and Sex Influence Sensitivity to Allergen-Induced Airway Inflammation and Contribute to Asthma Heterogeneity in Guinea Pigs
Authors: Regal Jean F. , Regal Ronald R. , Meehan Jessica L. , Mohrman Margaret E.
Published: August 2006

Background: Limiting allergen exposure in the sensitization phase has been proposed as a means of primary prevention of asthma, but its effectiveness is debated. Hypothesis: Primary prevention of asthma is more effective in limiting asthma symptoms in young guinea pigs compared with adults, whether males or females. Methods: The following experimental groups were used: young/young, sensitized and challenged before sexual maturity; young/adult, sensitized young and challenged after sexual maturity; adult/adult, sensitized and challenged after sexual maturity. Males and females were sensitized intraperitoneally with varying doses of ovalbumin (OVA) and challenged intratracheally with a constant OVA dose. Cellular infiltration into lung and lavage fluid as well as airway hyperresponsiveness to intravenous methacholine was determined 24 h later. Results: In unsensitized animals, density of resident inflammatory cells as well as baseline pulmonary function differed with age and sex. Maximum OVA-induced eosinophilia in females occurred at a lower sensitizing dose of OVA than in males, and the slopes of the dose-response relationship differed significantly between sexes. Young females had more pronounced increases in eosinophils compared with some adult treatment groups. The concentrations of OVA-specific antibodies were not directly related to differences in cellular infiltration. Airway hyperresponsiveness to methacholine challenge was observed in all treatment groups. Conclusion: Young animals require major reductions in allergen exposure compared with adults to effectively limit airway inflammation in primary prevention. Heterogeneity of asthma symptoms seen with age and sex suggests that primary prevention by limiting allergen exposure or treatment with anti-inflammatory or bronchodilator drugs may be more effective strategies for specific age and gender populations.
Allelic Polymorphisms in the Interleukin-4 Promoter Regions and Their Association with Bronchial Asthma among the Russian Population
Authors: Gervaziev Y.V. , Kaznacheev V.A. , Gervazieva V.B.
Published: August 2006

Background: Susceptibility to the development of bronchial asthma (BA) and other atopic diseases is known to be associated with genetic components. Objective: To evaluate the possible role of the polymorphisms in IL-4 gene promoters (C-33T, C-590T and G-1098T) in modulating allergic response and asthma in the Russian population. Methods: The polymorphism analysis was carried out by PCR-RFLP; IL-4 and total IgE concentrations were determined by ELISA. Results: In the case group, the T allele was found at frequencies of 74% (C-33T), 51% (C-590T) and 5% (G-1098T); in the control group the frequencies were 22, 42 and 8%, respectively. Only the C-33T polymorphism was associated with BA. The concentrations of total IgE and serum IL-4 were raised in the case group, while in the control group they were normal. Serum IL-4 level depended on C-33T polymorphism both in the case and control groups, the mutant T allele promoting its increase. The dependence on C-590T polymorphism was detected only in the case group. As for the total IgE level, in both cases it depended on the polymorphism in the case group rather than the control. G-1098T polymorphism did not demonstrate any correlations with total IgE or serum IL-4 levels. All 3 polymorphisms did not affect the severity of BA in the case group. On the basis of the computer analysis, we propose that the T-33C region is the CREB-binding site. Conclusions: Our data suggest that IL-4 promoter polymorphism in the Russian population might play a role both conferring susceptibility to BA and modulating the levels of serum IL-4 and total IgE.
Nitration Enhances the Allergenic Potential of Proteins
Authors: Gruijthuijsen Y.K. , Grieshuber I. , Stöcklinger A. , Tischler U. , Fehrenbach T. , Weller M.G. , Vogel L. , Vieths S. , Pöschl U. , Duschl A.
Published: August 2006

Background: Recent investigations have shown that proteins, including Bet v 1a, are nitrated by exposure to polluted urban air. We have investigated immunogenic and allergenic properties of in vitro nitrated allergens in in vivo models. Methods: Untreated and nitrated samples of ovalbumin or Bet v 1a were compared for their ability to stimulate proliferation and cytokine secretion in splenocytes from DO11.10 or from sensitized BALB/c mice, and for their ability to induce specific immunoglobulin (Ig)G1, IgG2a and IgE in sensitized mice. Additionally, sera from birch pollen-allergic individuals were analysed for IgE and IgG specific for nitrated Bet v 1a. Results: Upon splenocyte stimulation with nitrated as compared with unmodified allergens, proliferation as well as interleukin 5 and interferon-γ production were enhanced. Sera of mice sensitized with nitrated allergens showed elevated levels of specific IgE, IgG1 and IgG2a, compared with sera from mice sensitized with unmodified allergens. Moreover, cross-reactivity of antibodies against unrelated, nitrated allergens was observed in mice. We also found higher amounts of functional, specific IgE against nitrated than against untreated Bet v 1a in sera from birch pollen-allergic patients. Conclusions: Our findings suggest that nitration enhances allergic responses, which may contribute to an increased prevalence of allergic diseases in polluted urban environments.