Does the type of bladder augmentation influence the resolution of pre-existing vesicoureteral reflux? Urodynamic studies.

AIMS: The type of bladder augmentation on pre-existing vesicoureteral reflux (VUR) was assessed. The effects of urodynamic changes on the resolution of VUR following augmentation cystoplasty performed with various gastrointestinal segments were examined. It was queried whether elimination of high-pressure bladder is sufficient to resolve pre-existing reflux. METHODS: A retrospective record review of patients who underwent bladder augmentation between 1987 and 2004. Patients were divided into two groups. Group I included patients who had a simultaneous augmentation and ureteral reimplantation. Group II included patients with reflux in whom only a bladder augmentation was performed. Pre-and post-augmentation urodynamic results were compared in both groups. The outcome of VUR and the role of various gastrointestinal (GI) segments on the resolution of VUR were studied. RESULTS: Sixty-three patients underwent bladder augmentation during the study period. Twenty-six of them had VUR before augmentation. There were 10 patients in Group I and 16 patients in Group II. In Group I, VUR ceased in all patients, while in group II, VUR resolved in 14 patients and persisted in two patients. Small and large bowel segments used for augmentation had no effect on the resolution of VUR but the results of gastrocystoplasties were less favorable. Urodynamically there was no significant difference between the various augmentation cystoplasties. CONCLUSIONS: Bladder augmentation alone without simultaneous antireflux repair is usually sufficient for the resolution of pre-existing reflux. The various GI segments used for augmentation have no effect on urodynamic results and the resolution of VUR.

Cutaneous vesicostomy revisited--the second 15 years.

UNLABELLED: The aim of this paper was to review the authors' experience with cutaneous vesicostomy (CV) over the last 15 years including indications, results, and complications of CV. MATERIALS AND METHODS: The records of 31 patients treated by CV between 1987 and 2002 were reviewed. There were 20 boys and 11 girls. The two main primary pathologies were neuropathic bladder (19 patients) and posterior urethral valve (PUV) (7 patients). All patients underwent a Blocksom-type operation at a mean age of 23 months (range 14 days-9 years). Pre- and postoperative conventional uromanometry was performed in 18 patients (58%) and bladder function was assessed. RESULTS: In 23 patients (74%) the CV provided a successful diversion with improvement of the upper urinary tract and/or stabilization of the renal function. In 5 patients (16%) with PUV, the improvement was temporary. In 3 patients (10%) the CV did not result in an improvement. Twenty-four patients underwent CV closure after a mean duration of 23 months (range, 1 month-7 years) of diversion. In 2 patients with myelomeningocele (MMC) and severe somato-mental developmental delay, CV was not closed and is being considered as a permanent treatment option. Urodynamic studies in 5 PUV patients showed impaired compliance and high intravesical pressure following a successful valve ablation and closure of CV. In the neuropathic bladder group the bladder function improved following closure of CV and commencement of anticholinergic medication and clean intermittent catheterization (CIC). Our augmentation ratio in the neuropathic bladder group was 22%. Complications of CV included: stenosis in 7 patients (22%), prolapse in 2 (6%), and cellulitis in 2 (6%). The revision rate was 16%. CONCLUSIONS: In young infants CV had a less favourable result in the PUV patients than in cases with high-pressure neuropathic bladder with upper tract dilatation and severe urinary tract infection (UTI), where CV provided decompression and prevented deterioration of the renal function. Cutaneous vesicostomy has stood the test of time in our changing paediatric urological practice and it remains a valuable weapon in the armoury of paediatric urologists in selected patients.

Precise control of end-tidal carbon dioxide levels using sequential rebreathing circuits.

Anaesthesiologists have traditionally been consulted to help design breathing circuits to attain and maintain target end-tidal carbon dioxide (P(ET)CO2). The methodology has recently been simplified by breathing circuits that sequentially deliver fresh gas (not containing carbon dioxide (CO2)) and reserve gas (containing CO2). Our aim was to determine the roles of fresh gas flow, reserve gas PCO2 and minute ventilation in the determination of P(ET)CO2. We first used a computer model of a non-rebreathing sequential breathing circuit to determine these relationships. We then tested our model by monitoring P(ET)CO2 in human volunteers who increased their minute ventilation from resting to five times resting levels. The optimal settings to maintain P(ET)CO2 independently of minute ventilation are 1) fresh gas flow equal to minute ventilation minus anatomical deadspace ventilation, and 2) reserve gas PCO2 equal to alveolar PCO2. We provide an equation to assist in identifying gas settings to attain a target PCO2. The ability to precisely attain and maintain a target PCO2 (isocapnia) using a sequential gas delivery circuit has multiple therapeutic and scientific applications.

Isocapnic hyperpnoea accelerates recovery from isoflurane anaesthesia.

BACKGROUND: Hyperventilation should speed up elimination of volatile anaesthetic agents from the body, but hyperventilation usually results in hypocapnia. We compared recovery from isoflurane anaesthesia in patients allowed to recover with assisted spontaneous ventilation (control) and those treated with isocapnic hyperpnoea. METHODS: Fourteen patients were studied after approximately 1 h of anaesthesia with isoflurane. Control patients were allowed to recover in the routine way. Isocapnic hyperpnoea patients received 2-3 times their intraoperative ventilation using a system to maintain end tidal PCO(2) at 45-50 mm Hg. We measured time to removal of the airway and rate of change of bispectral index (BIS) during recovery. RESULTS: With isocapnic hyperpnoea, the time to removal of the airway was markedly less (median and interquartile range values of 3.6 (2.7-3.7) vs 12.1 (6.8-17.2) min, P<0.001); mean (SD) BIS slopes during recovery were 11.8 (4.4) vs 4.3 (2.7) min(-1) (P<0.01) for isocapnic hyperpnoea and control groups, respectively. Isocapnic hyperpnoea was easily applied in the operating room. CONCLUSIONS: Isocapnic hyperpnoea at the end of surgery results in shorter and less variable time to removal of the airway after anaesthesia with isoflurane and nitrous oxide.

Transient expression of the Bcl-2 family member, A1-a, results in nuclear localization and resistance to staurosporine-induced apoptosis.

The Bcl-2 family of proteins has been characterized by either anti-apoptotic or pro-apoptotic activity. Insight into how Bcl-2 family members function has been gained by determining their intracellular localization. We have generated a monoclonal anti-A1-a antibody and used a COS-7 overexpression system to study the localization of the murine anti-apoptotic Bcl-2 family member, A1-a. A1-a overexpressed in COS-7 cells localized to the nucleus as determined by subcellular fractionation and immunofluorescent microscopy. A1-a in the COS-7 nucleus bound tightly to the nuclear matrix as evidenced by resistance to treatment with DNAse I and RNAse A and sequential extraction with 1.0% Triton X-100, 0.15 M NaCl, 0.25 M HCl, 0.5 M Tris pH 7.4 and 6 M urea. HPLC analysis of A1-a, subsequent to SDS extraction, produced fractions that gave multiple bands when analyzed by Western blot analysis suggesting a propensity to form multimers. COS-7 cells transfected with A1-a were protected from apoptotic induction by staurosporine treatment.

MRI mapping of cerebrovascular reactivity using square wave changes in end-tidal PCO2.

Cerebrovascular reactivity can be quantified by correlating blood oxygen level dependent (BOLD) signal intensity with changes in end-tidal partial pressure of carbon dioxide (PCO2). Four 3-min cycles of high and low PCO2 were induced in three subjects, each cycle containing a steady PCO2 level lasting at least 60 sec. The BOLD signal closely followed the end-tidal PCO2. The mean MRI signal intensity difference between high and low PCO2 (i.e., cerebrovascular reactivity) was 4.0 +/- 3.4% for gray matter and 0.0 +/- 2.0% for white matter. This is the first demonstration of the application of a controlled reproducible physiologic stimulus, i.e., alternating steady state levels of PCO2, to the quantification of cerebrovascular reactivity.

Requirement of A1-a for bacillus Calmette-Guérin-mediated protection of macrophages against nitric oxide-induced apoptosis.

The role of apoptosis in regulating the course of intracellular microbial infection is not well understood. We studied the relationship between apoptotic regulation and bacillus Calmette-Guérin (BCG) treatment in murine peritoneal exudate macrophages (PEM) and the J774 macrophage cell line. In both PEM and J774 cells, mRNA expression of the anti-apoptotic gene, A1, was selectively induced by BCG treatment as compared with other bcl2 family members (bcl-w, bcl-2, bcl-xl, bcl-xs, bax, bak, bad). In PEM, A1 expression was maximal by 8 h postinfection and was abrogated by the proteasomal inhibitor MG-132. The induction was independent of protein synthesis as well as the p38 mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways and did not require live organism. Three genes encoding closely related isoforms of A1 were all expressed; however, the A1-a isoform displayed the greatest fold induction in PEM. BCG-induced A1 expression was associated with protection of host macrophages from NO-mediated apoptosis in both PEM and J774 cells. BCG-mediated protection was abrogated in PEM derived from A1-a(-/-) mice, indicating a requirement of A1-a for survival of inflammatory macrophages.

A gene expression profile of Alzheimer's disease.

Postmortem analysis of brains of patients with Alzheimer's disease (AD) has led to diverse theories about the causes of the pathology, suggesting that this complex disease involves multiple physiological changes. In an effort to better understand the variety and integration of these changes, we generated a gene expression profile for AD brain. Comparing affected and unaffected brain regions in nine controls and six AD cases, we showed that 118 of the 7050 sequences on a broadly representative cDNA microarray were differentially expressed in the amygdala and cingulate cortex, two regions affected early in the disease. The identity of these genes suggests the most prominent upregulated physiological correlates of pathology involve chronic inflammation, cell adhesion, cell proliferation, and protein synthesis (31 upregulated genes). Conversely, downregulated correlates of pathology involve signal transduction, energy metabolism, stress response, synaptic vesicle synthesis and function, calcium binding, and cytoskeleton (87 downregulated genes). The results support several separate theories of the causes of AD pathology, as well as add to the list of genes associated with AD. In addition, approximately 10 genes of unknown function were found to correlate with the pathology.

Evidence for shared genetic programs from cluster analysis of hippocampal gene expression dynamics in development and response to injury.

Cluster analysis is a computational method that groups together similarly-shaped patterns. It may be applied to large-scale gene expression data to form new hypotheses regarding gene function. In the present study, we clustered the temporal expression patterns of genes expressed in the rat hippocampus during normal development and after a kainate-induced seizure injury at postnatal day 25. We found that two different methods, Euclidean hierarchical and K-means clustering, produced slightly different results, and concluded that different clustering methods may he used to complement one another. We also found that certain genes cluster together both during development and after seizure injury, consistent with the idea of sets of genes that act in concert under various conditions.

Genetic network inference: from co-expression clustering to reverse engineering.

MOTIVATION: Advances in molecular biological, analytical and computational technologies are enabling us to systematically investigate the complex molecular processes underlying biological systems. In particular, using high-throughput gene expression assays, we are able to measure the output of the gene regulatory network. We aim here to review datamining and modeling approaches for conceptualizing and unraveling the functional relationships implicit in these datasets. Clustering of co-expression profiles allows us to infer shared regulatory inputs and functional pathways. We discuss various aspects of clustering, ranging from distance measures to clustering algorithms and multiple-cluster memberships. More advanced analysis aims to infer causal connections between genes directly, i.e. who is regulating whom and how. We discuss several approaches to the problem of reverse engineering of genetic networks, from discrete Boolean networks, to continuous linear and non-linear models. We conclude that the combination of predictive modeling with systematic experimental verification will be required to gain a deeper insight into living organisms, therapeutic targeting and bioengineering.

Gene expression microarray data analysis for toxicology profiling.

When dealing with thousands of genes, all potentially interesting, it is desirable to rank the genes according to their degree of participation in a physiological process. Therefore, genes with the highest Shannon entropy and ERL can be selected as the best toxicity target candidates, permitting preclinical scientists to focus their research and resources on those genes.

Determination of temporal expression patterns for multiple genes in the rat carotid artery injury model.

Vascular injury induces extensive alteration to the extracellular matrix (ECM). These changes contribute to lesion formation and promote cell migration and proliferation. To elucidate ECM response to arterial injury, we used real-time polymerase chain reaction monitoring to quantitate the expression levels of 81 genes involved in the synthesis and breakdown of ECM as well as receptors and signaling proteins that communicate and respond to ECM molecules. The temporal regulation of gene expression in the carotid was measured at 1, 3, 5, 7, 9, 14, and 28 days postinjury. Among the 68 genes that showed detectable expression by our method, 47 (69%) were significantly induced or repressed over time, confirming the extensive ECM gene response in this model. More ECM-related genes (31) were regulated at day 1 than at any other time point, and the number of regulated genes decreased over time. However, 14 of the genes were still induced or repressed at day 28, indicating that return to preinjury expression patterns did not occur and no new steady state was achieved over 28 days. In spite of the large number of changes in gene expression, only a small number of expression patterns was observed, suggesting that ECM-related genes could potentially be coregulated.

Differential regulation of adult and embryonic glutamate decarboxylases in rat dentate granule cells after kainate-induced limbic seizures.

In adult brain, the inhibitory GABAergic neurons utilize two distinct molecular forms of the GABA-synthesizing enzyme glutamate decarboxylase (GAD), GAD65 and GAD67. During embryonic development, two truncated forms of GAD67 are also expressed (GAD25 and GAD44), which are translated from two embryonic-specific splice variants of GAD67 messenger RNA. It has recently been established that the excitatory dentate granule cells, in addition to the neurotransmitter glutamate, also contain low levels of GABA and GAD67, which are increased after limbic seizures. To study the seizure-induced activation of glutamate decarboxylase, we investigated the expression of both embryonic and adult glutamate decarboxylase messenger RNAs in the adult rat hippocampus after kainic acid administration by semi-quantitative reverse transcription-coupled polymerase chain reaction, in situ hybridization and immunoblotting. We observed a rapid induction of the embryonic glutamate decarboxylase messenger RNA in the granule cells of dentate gyrus. The expression of embryonic glutamate decarboxylase transcripts, identified here as the splice variant that contains exon 7/B, peaked at about 2h after kainic acid injection and gradually returned to nearly basal levels by 24h. Strikingly, this transient induction of embryonic glutamate decarboxylase messenger RNA was not accompanied by concomitant synthesis of its corresponding protein product GAD25. In contrast, the adult GAD67 messenger RNA and protein were both clearly up-regulated in granule cells, albeit with a certain delay, reaching a maximum around 4-6h after kainic acid injection and gradually returned to control levels by 24h. GAD65 remained unchanged at both messenger RNA and protein levels during the studied period. These characteristic and highly reproducible changes in the synthesis of glutamate decarboxylases indicate that GAD67 is the predominant form of glutamate decarboxylases involved in the elevated synthesis of GABA during seizures and suggest that the transient induction of the embryonic GAD67 messenger RNA that contains exon 7/B, but not GAD25 protein, may exert a role solely in the subsequent up-regulation of adult GAD67 transcription. Expression of the messenger RNA encoding for an alternatively spliced, truncated form of the GABA-synthesizing enzyme glutamate decarboxylase was detected in dentate granule cells briefly after kainic acid-induced seizures. Just as during embryonic development, expression of the alternatively spliced messenger RNA was transient and followed by transcription of its adult form, indicating a possible recapitulation of an embryonic program of gene expression in adult granule cells after epileptic seizures.

Large scale gene expression analysis of cholesterol-loaded macrophages.

We conducted large scale gene expression analysis of the response of macrophages to exposure to oxidized low density lipoprotein (Ox-LDL). Much of the vessel wall lesion of atherosclerosis is composed of macrophages that have become engorged with cholesterol. These resulting "foam cells" contribute to the progression of vascular disease through several pathways. As a potential model of foam cell formation, we treated THP-1 cells with 12-O-tetradecanoylphorbol 13-acetate to differentiate them into a macrophage-like phenotype and subsequently treated them with oxidized low density lipoprotein for various time periods. RNA from Ox-LDL treated and time-matched control untreated cells was hybridized to microarrays containing 9808 human genes. 268 genes were found to be at least 2-fold regulated at one or more time points. These regulation patterns were classified into seven clusters of expression profiles. The data is discussed in terms of the overall pattern of gene expression, the thematic classification of the responding genes, and the clustering of functional groups in distinct expression patterns. The magnitude and the temporal patterns of gene expression identified known and novel molecular components of the cellular response that are implicated in the growth, survival, migratory, inflammatory, and matrix remodeling activity of vessel wall macrophages. In particular, the role of nuclear receptors in mediating the gene expression modulation by Ox-LDL is highlighted.

Large-scale identification of differentially expressed genes during neurogenesis.

We report here a modified mRNA differential display method and its application for the analysis of differential gene expression in NGF-treated PC12 cells and in embryonic rat spinal cord. The optimized protocol is based on low fidelity priming of multiple cDNAs followed by high fidelity amplification. In PC12 cells induction by nerve growth factor (NGF) altered the expression of 4% of the 466 transcripts evaluated. During neurogenesis of the spinal cord we found that 30% of the 288 examined products changed. The differential expression of the characterized genes was confirmed by independent quantitative PCR. We conclude this method is suitable for the identification of increases and decreases of mRNA levels and allows the discovery of differentially expressed unknown transcripts.

The application of shannon entropy in the identification of putative drug targets.

A major challenge in the field of functional genomics is the development of computational techniques for organizing and interpreting large amounts of gene expression data. These methods will be critical for the discovery of new therapeutic drug targets. Here, we present a simple method for determining the most likely drug target candidates from temporal gene expression patterns assayed with reverse-transcription polymerase chain reaction (RT-PCR) and DNA microarrays.

The murine antiapoptotic protein A1 is induced in inflammatory macrophages and constitutively expressed in neutrophils.

Myeloid leukocytes are thought to regulate their susceptibility to apoptosis upon migration to a site of inflammation. However, factors that determine survival have not been well characterized in these cells. We have examined the expression of murine A1, an antiapoptotic Bcl-2 relative found in activated myeloid cells, during the course of an acute inflammatory response. Intraperitoneal infection of mice with the virulent RH strain of Toxoplasma gondii led to a 5- to 10-fold increase in A1 mRNA levels in peritoneal cells after several days. Bcl-2 expression was unchanged. The increase in A1 expression depended on the dose of the organism and coincided with a sharp increase in peritoneal cellularity. A1 protein levels were also increased as determined by Western blot analysis and immunohistochemical studies. All neutrophils and approximately half of the macrophages in the inflammatory exudate contained high levels of A1 in cytoplasm. A1 expression did not correlate with intracellular parasitization. Peripheral blood neutrophils from normal mice strongly expressed A1 protein, whereas normal monocytes showed only weak staining. Bax mRNA was induced in parallel with A1 in macrophages. Exudate macrophages and granulocytes that were apoptotic by TUNEL staining occasionally appeared to display A1 throughout the cell nucleus. These studies identify A1 as a potential regulator of apoptosis during acute inflammation.

Linear modeling of mRNA expression levels during CNS development and injury.

Large-scale gene expression data sets are revolutionizing the field of functional genomics. However, few data analysis techniques fully exploit this entirely new class of data. We present a linear modeling approach that allows one to infer interactions between all the genes included in the data set. The resulting model can be used to generate interesting hypotheses to direct further experiments.

GABAergic cells and signals in CNS development.

GABA is formed primarily from decarboxylation of glutamate by a family of cytosolic and membrane-bound GAD enzymes. In the adult, GAD-derived GABA sustains the vitality of the central nervous system (CNS), since blockage of GAD rapidly leads to convulsions and death. In plants, cytosolic GAD synthesizes GABA in response to hormones and environmental stress. Since decarboxylation involves protonation, secretion of GABA serves to buffer cytosolic pH in plant cells. Families of GAD and GABAA receptor/Cl- channel transcripts and encoded proteins emerge early and seemingly everywhere during CNS development, with their abundance closely paralleling neurogenesis and peaking before birth. Micromolar GABA acts at receptor/Cl-channels to depolarize progenitor cells in the cortical neuroepithelium; it also elevates their cytosolic Ca2+ (Cac2+) levels. In some way, these effects decrease proliferation. GABA directs the migration of postmitotic neuroblasts at femtomolar concentrations and stimulates their random motility at micromolar concentrations via Ca2+ signaling mechanisms. Activation of GABAA receptors by micromolar GABA may limit motility via membrane depolarization and elevated Cac2+. These results indicate that in vitro GABA can affect embryogenesis of the CNS through effects on cell proliferation and migration. As neurons differentiate postnatally, Cl(-)-dependent depolarization disappears together with GABAergic Cac2+ signals. Physiologically occurring GABAergic signals at Cl-channels exist in tonic and transient forms. Since the former are found on progenitor cells while both are present in postmitotic neurons, mechanisms to generate transients differentiate in the latter. Surprisingly, tonic and transient forms of GABAergic signaling at Cl-channels are rapidly and smoothly interconvertible and seem to be derived from online GABA synthesis in a surface-accessible compartment of the membrane.