Echocardiography as an indication of continuous-time cardiac quiescence.

Cardiac computed tomography (CT) angiography using prospective gating requires that data be acquired during intervals of minimal cardiac motion to obtain diagnostic images of the coronary vessels free of motion artifacts. This work is intended to assess B-mode echocardiography as a continuous-time indication of these quiescent periods to determine if echocardiography can be used as a cost-efficient, non-ionizing modality to develop new prospective gating techniques for cardiac CT. These new prospective gating approaches will not be based on echocardiography itself but on CT-compatible modalities derived from the mechanics of the heart (e.g. seismocardiography and impedance cardiography), unlike the current standard electrocardiogram. To this end, echocardiography and retrospectively-gated CT data were obtained from ten patients with varied cardiac conditions. CT reconstructions were made throughout the cardiac cycle. Motion of the interventricular septum (IVS) was calculated from both echocardiography and CT reconstructions using correlation-based, deviation techniques. The IVS was chosen because it (1) is visible in echocardiography images, whereas the coronary vessels generally are not, and (2) has been shown to be a suitable indicator of cardiac quiescence. Quiescent phases were calculated as the minima of IVS motion and CT volumes were reconstructed for these phases. The diagnostic quality of the CT reconstructions from phases calculated from echocardiography and CT data was graded on a four-point Likert scale by a board-certified radiologist fellowship-trained in cardiothoracic radiology. Using a Wilcoxon signed-rank test, no significant difference in the diagnostic quality of the coronary vessels was found between CT volumes reconstructed from echocardiography- and CT-selected phases. Additionally, there was a correlation of 0.956 between the echocardiography- and CT-selected phases. This initial work suggests that B-mode echocardiography can be used as a tool to develop CT-compatible gating techniques based on modalities derived from cardiac mechanics rather than relying on the ECG alone.

Gut modulator effects of Conyza bonariensis explain its traditional use in constipation and diarrhea.

BACKGROUND AND OBJECTIVES: The current investigation was carried out to explore the pharmacological basis of the crude extract of Conyza bonariensis (Cb.Cr) for its use in constipation and diarrhea. MATERIALS AND METHODS: The plant extract of Conyza bonariensis (C. bonariensis) was prepared, isolated guinea-pig ileum and rabbit jejunum preparations were used to evaluate its gut modulator effects. RESULTS: The Cb.Cr (0.3-10 mg/mL) exhibited spasmogenic effect in isolated guinea-pig ileum preparation, which was about 19-84% of the acetylcholine maximum. Pretreatment of the tissues with atropine (0.1 µM) abolished the contractile effect, similar to acetylcholine. Among the fractions, only the butanol fraction exhibited atropine sensitive contractile effect. In isolated rabbit jejunum preparations, Cb.Cr produced appreciable atropine-sensitive spasmogenic effect at lower concentrations (0.03-0.3 mg/mL) followed by spasmolytic effect at next higher concentration (1.0 and 3.0 mg/mL). Cb.Cr caused an inhibition of the high K+ induced contraction in isolated rabbit jejunum preparation with EC50 value of 0.62 mg/mL. Similarly, verapamil, a standard calcium blocker, inhibited high K+ induced contraction in isolated rabbit jejunum preparations. Cb.Cr caused a right ward shift in the Ca++ concentration response curve, similar to verapamil. Among various fractions of C. bonariensis, only hexane and ethylacetate fractions showed spasmolytic effects. CONCLUSIONS: The crude extract of C. bonariensis contains spasmogenic and spasmolytic constituents, which explains its medicinal use in constipation and diarrhea.

The application of discriminant analysis and Machine Learning methods as tools to identify and classify compounds with potential as transdermal enhancers.

Discriminant analysis (DA) has previously been shown to allow the proposal of simple guidelines for the classification of 73 chemical enhancers of percutaneous absorption. Pugh et al. employed DA to classify such enhancers into simple categories, based on the physicochemical properties of the enhancer molecules (Pugh et al., 2005). While this approach provided a reasonable accuracy of classification it was unable to provide a consistently reliable estimate of enhancement ratio (ER, defined as the amount of hydrocortisone transferred after 24h, relative to control). Machine Learning methods, including Gaussian process (GP) regression, have recently been employed in the prediction of percutaneous absorption of exogenous chemicals (Moss et al., 2009; Lam et al., 2010; Sun et al., 2011). They have shown that they provide more accurate predictions of these phenomena. In this study several Machine Learning methods, including the K-nearest-neighbour (KNN) regression, single layer networks, radial basis function networks and the SVM classifier were applied to an enhancer dataset reported previously. The SMOTE sampling method was used to oversample chemical compounds with ER>10 in each training set in order to improve estimation of GP and KNN. Results show that models using five physicochemical descriptors exhibit better performance than those with three features. The best classification result was obtained by using the SVM method without dealing with imbalanced data. Following over-sampling, GP gives the best result. It correctly assigned 8 of the 12 "good" (ER>10) enhancers and 56 of the 59 "poor" enhancers (ER<10). Overall success rates were similar. However, the pharmaceutical advantages of the Machine Learning methods are that they can provide more accurate classification of enhancer type with fewer false-positive results and that, unlike discriminant analysis, they are able to make predictions of enhancer ability.

Alternative transplant procedures for acute liver failure.

Acute liver failure (ALF) is associated with significant morbidity and mortality. The outcome is highly unpredictable and recovery depends on several factors. Patients can deteriorate with increasing encephalopathy, coagulopathy and progress to multiorgan failure (MOF). In such patients, liver transplantation (LT) is the only current potential cure. Orthotopic liver transplantation remains the standard procedure for LT in ALF, however, other surgical options have been explored. This review summarises the use of a variety of alternative transplant procures for the treatment of acute liver failure including: Two stage OLT, Auxiliary liver transplant, Living donor liver transplantation (LDLT), and ABO incompatible liver transplant.

Risk factors for litigation following major transectional bile duct injury sustained at laparoscopic cholecystectomy.

BACKGROUND: Bile duct injuries after laparoscopic cholecystectomy often cause long-term morbidity, with a number of patients resorting to litigation. The present study aimed to analyze risk factors for litigation and to quantify the subsequent medicolegal burden. METHODS: A total of 67/106 patients (26 male) with major laparoscopic cholecystectomy bile duct injuries (LCBDI) and a minimum 2-year follow-up, replied to a questionnaire covering patient perception toward the complication, physical/psychological recovery, and subsequent litigation. These data were collated with prospectively collected data related to the LCBDI and subsequent management, and a multivariate regression model was designed to identify potential risk factors associated with litigation. RESULTS: Most patients felt they had been inadequately informed prior to surgery [47/67 (70%)] and after the LCBDI [50/67 (75%)], and a majority remained psychologically traumatized at the time of evaluation [50/67 (75%)]. Of these, 22 patients had started litigation by means of a "letter of demand" (LOD; n = 10) or prosecution (n = 12). Nineteen (19/22%) cases have been closed in favor of the plaintiff. There was no difference between the awards for LOD versus prosecution cases, and average compensation was £40,800 versus £89,875, respectively (p = n.s). On multivariate analysis, age < 52 years (p = 0.03), associated vascular injury (p = 0.014), immediate nonspecialist repair (p = 0.009), and perceived incomplete recovery following LCBDI (p = 0.017) were identified as independent predictors for possible litigation. CONCLUSIONS: On the basis of the present study, nearly one third of patients with major transectional LCBDI are likely to resort to litigation. Younger patients and those in whom repair is attempted prior to specialist referral are likely to initiate litigation.

Clinical risk score can be used to select patients for staging laparoscopy and laparoscopic ultrasound for colorectal liver metastases.

BACKGROUND: Despite advanced staging investigations, some patients with potentially resectable colorectal liver metastases (CLM) are unresectable at laparotomy. Staging laparoscopy and laparoscopic ultrasound (Lap + LUS) detects a subset of these unresectable patients before a major laparotomy. Clinical risk scoring may be helpful to identify this subgroup. The goal of our study was to evaluate the role of Lap + LUS and to assess the value of the Memorial Sloan Kettering clinical risk score (CRS) in identifying this subset. METHODS: Patients were identified from the regional multidisciplinary team (MDT) cancer database and operative records for a 5-year period. All patients whose tumors were deemed resectable proceeded to Lap + LUS. LUS findings were recorded and any change in MDT plan was noted. LUS findings were compared with resectability at open surgery. The CRS (Memorial Sloan-Kettering) based on five factors was calculated. RESULTS: A total of 79 patients were identified. In 15 of 74 patients, LUS prevented an unnecessary laparotomy by predicting the benign nature of lesions or demonstrating unresectability. The CRS ranged from 0 to 4. Lap + LUS prevented an operation in only 7% of patients with a CRS of < or =2. However in patients with a CRS > 2, Lap + LUS prevented an operation in 24% of patients. CONCLUSIONS: LUS prevented an unnecessary laparotomy in 20% of patients. This may reduce inpatient stay, morbidity, and mortality, allowing some patients to proceed to palliative treatments earlier. The benefit of Lap + LUS is limited in patients with a CRS of < or =2. It is worth considering selective use of Lap + LUS for the staging of CLM.

Rapid high-throughput assay for the measurement of amino acids from microdialysates and brain tissue using monolithic C18-bonded reversed-phase columns.

A rapid precolumn high-performance liquid chromatography method based on fluorescence detection has been developed for the measurement of multiple amino acids from both ex vivo and in vivo biological samples using monolithic C18 columns. A mixture of 18 primary amino acids were derivatised with napthalene-2,3-dicarboxaldehyde (NDA) in the presence of cyanide. The resulting isoindole derivatives were resolved within 10 min using a linear binary gradient elution profile with Rs values in the range 1.2-9.0. The limit of detection (LOD) was found to be between 6.0 and 60 fmol for 5 microl injection with a signal to noise ratio of 3:1. The NDA derivatives were found to be stable for 9 h at 4 degrees C. This assay has been employed for the rapid analysis of amino acids from brain tissue and microdialysis samples. Examples of application of the method are given.

Nanomanipulation and characterization of structural proteins.

A methodology is presented for simultaneous mechanical testing and atomic force microscopy imaging of single collagen fibrils under load. This method holds the promise for determining single-fibril modulus and strength in various experimental preparations. Examples of this utility include characterization of deformation and failure modes of naturally occurring and engineered structural proteins. Additional promise of this technique is robotic surgery at the submicron scale for repairing neuronal tracts and capillaries with structural proteins. A series of algorithms for tying knots at the nanoscale in single fibrils is also presented.

Development and application of a sensitive high performance ion-exchange chromatography method for the simultaneous measurement of dopamine, 5-hydroxytryptamine and norepinephrine in microdialysates from the rat brain.

A high performance liquid chromatography (HPLC) method based on cation exchange separation has been developed for the measurement of dopamine (DA), 5-hydroxytryptamine (5-HT) and norepinephrine (NE) in microdialysates. The separation conditions have been optimised for using electrochemical detection. All three bioamines were resolved in less than 22 min using isocratic conditions. The optimum oxidation potential for the three bioamines was found to be +0.4 V vs. in situ Ag/AgCl reference electrode. Linear regression analysis of HPLC-peak area as a function of concentrations in the range 1-50 ng x ml(-1) gave coefficients of correlation between 0.998 and 0.999. The limit of detection for DA, 5-HT and NE was found to be between 50 and 100 pg x ml(-1) with a signal to noise ratio of 3:1. The method has been applied to the simultaneous measurement of the three monoamines in microdialysates from the medial prefrontal cortex under basal conditions and following the administration of the antipsychotic drug clozapine (10 mg x kg(-1) s.c.).

Increased responsiveness of dopamine to atypical, but not typical antipsychotics in the medial prefrontal cortex of rats reared in isolation.

Dopaminergic hypofunction in the medial prefrontal cortex (mPFC) has been associated with the aetiology of negative symptoms and cognitive dysfunction of schizophrenia, which are both alleviated by clozapine and other atypical antipsychotics such as olanzapine. In rodents, early life exposure to stressful experiences such as social isolation produces a spectrum of symptoms emerging in adult life, which can be restored by antipsychotic drugs. The present series of experiments sought to investigate the effect of clozapine (5-10 mg/kg s.c.), olanzapine (5 mg/kg s.c.), and haloperidol (0.5 mg/kg s.c.) on dopamine (DA) and amino acids in the prelimbic/infralimbic subregion of the mPFC in group- and isolation-reared rats. Rats reared in isolation showed significant and robust deficits in prepulse inhibition of the acoustic startle. In group-reared animals, both clozapine and olanzapine produced a significant increase in DA outflow in the mPFC. Isolation-reared rats showed a significant increase in responsiveness to both atypical antipsychotics compared with group-reared animals. In contrast, the administration of haloperidol failed to modify dialysate DA levels in mPFC in either group- or isolation-reared animals. The results also show a positive relationship between the potency of the tested antipsychotics to increase the release of DA in the mPFC and their respective affinities for 5-HT1A relative to DA D2 or D3 receptors. Finally, isolation-reared rats showed enhanced neurochemical responses to the highest dose of clozapine as indexed by alanine, aspartate, GABA, glutamine, glutamate, histidine, and tyrosine. The increased DA responsiveness to the atypical antipsychotic drugs clozapine and olanzapine may explain, at least in part, clozapine- and olanzapine-induced reversal of some of the major behavioral components of the social isolation syndrome, namely hyperactivity and attention deficit.

Adenoviral infections and a prospective trial of cidofovir in pediatric hematopoietic stem cell transplantation.

Adenoviral (ADV) infections are increasingly recognized as a cause of morbidity and mortality in pediatric hematopoietic stem cell transplantation (HSCT). We reviewed our experience with ADV infections in HSCT patients hospitalized for transplantation at Childrens Hospital Los Angeles January 1998 through December 1998. ADV was detected in 47% of patients, with recipients of HSCT from alternative donors (matched unrelated, unrelated cord, and mismatched related donors) being more frequently culture positive than recipients of HSCT from matched siblings (62% versus 27%, P = .04). Detection of ADV from 2 or more sites was associated with organ injury, eg, hemorrhagic cystitis, enteritis, and hepatitis. Because of the high incidence of ADV culture-positive patients and the lack of effective anti-ADV therapy, we initiated a prospective trial to evaluate cidofovir (CDV) in the treatment of ADV infections in HSCT recipients. Eight patients were enrolled on a dosage schedule of 1 mg/kg 3 times weekly. AD of these patients eventually achieved long-term viral suppression and clinical improvement, although 6 patients needed prolonged CDV therapy for up to 8 months before CDV could be stopped without ADV recurrence. We did not observe dose-limiting nephrotoxicity, and the discontinuation of the drug was not required in any patients. Prospective controlled trials to further define the role of CDV in the treatment of ADV infections in HSCT patients are warranted.

In vivo characterization of basal amino acid levels in subregions of the rat nucleus accumbens: effect of a dopamine D(3)/D(2) agonist.

Recent evidence demonstrates that two subdivisions of the nucleus accumbens, the dorsolateral core and the ventromedial shell can be distinguished by morphological, immunohistochemical and chemoarchitectural differences. In the present study, we measured basal levels of amino acids in microdialysates from both the shell and core subterritories of the nucleus accumbens in freely moving rats using HPLC with fluorescence detection. The effect of the dopamine D(3)/D(2) receptor agonist quinelorane (30 microg/kg s.c.) was then investigated in both subregions. With the exception of glutamate, histidine, and serine, which showed similar levels in both subterritories, alanine, arginine, aspartate, gamma-aminobutyric acid, glutamine, and tyrosine were significantly higher in the shell compared with the core. In contrast, taurine levels were significantly lower in the shell than in the core. A particularly striking difference across subregions of the nucleus accumbens was observed for basal GABA levels with a shell/core ratio of 18.5. Among all the amino acids investigated in the present study, quinelorane selectively decreased dialysate GABA levels in the core subregion of the nucleus accumbens. The results of the present study point to specific profiles of both shell and core in terms of: (1) basal chemical neuroanatomical markers for amino acids; and (2) GABAergic response to the DA D(3)/D(2) agonist quinelorane.

Detection of leukemic cells in the CD34(+)CD38(-) bone marrow progenitor population in children with acute lymphoblastic leukemia.

Successful autologous hematopoietic stem cell (HSC) transplantation in childhood acute lymphoblastic leukemia (ALL) requires the ability to either selectively kill the leukemia cells or separate normal from leukemic HSC. Based on previous studies showing that more than 95% of childhood B-lineage ALL express CD38, this study evaluated whether normal CD34(+)CD38(-) progenitors from children with B-lineage ALL could be isolated by flow cytometry. CD34(+) cells from bone marrow samples from 10 children with B-lineage ALL were isolated at day 28 of treatment, when clinical remission had been attained. The CD34(+) progenitor cells were flow cytometrically sorted into CD34(+)CD38(+) and CD34(+)CD38(-) populations. The absolute numbers of CD34(+)CD38(-) cells that could be isolated ranged from 401 to 6245. The cells were then analyzed for the presence of clonotypic rearrangements of the T-cell receptor (TCR) Vdelta2-Ddelta3 locus. Only patients whose diagnostic marrow had an informative TCR Vdelta2-Ddelta3 rearrangement were included in this study. Detection thresholds were typically 10(-4) to 10(-5) leukemic cells in normal marrow. In 6 of 10 samples analyzed, the sorted CD34(+)CD38(-) cells had no detectable Vdelta2-Ddelta3 rearrangements. In 4 cases, the clonotypic leukemic Vdelta2-Ddelta3 rearrangement was detected in the CD34(+)CD38(-) population, indicating that the putative normal HSC population also contained leukemic cells. The data indicate that although most childhood ALL cells express CD34 and CD38, leukemic cells are also frequently present in the CD34(+)CD38(-) population. Therefore, strategies to isolate and transplant normal HSC from children with ALL will require a more stringent definition of the normal HSC than the CD34(+)CD38(-) phenotype. (Blood. 2001;97:3925-3930)

Hematopoietic stem cell transplantation for severe combined immune deficiency.

Hematopoietic stem cell transplantation (HSCT) has been the definitive therapy for severe combined immune deficiency (SCID) since the first successful transplant for SCID in 1968. Improvements in the use of HSCT to treat patients with SCID are continuing. For example, during the last 5 years, the first successful in-utero HSCT, and the first success with gene therapy have occurred in patients with SCID. Debate still continues about the role of pretransplantation therapy for SCID patients, and the biology of post-HSCT immune reconstitution is under investigation.

Development of a protocol for the automated analysis of amino acids in brain tissue samples and microdialysates.

An automated precolumn derivatisation method has been developed for the measurement of fourteen amino acids in brain tissue and microdialysate samples. The method involves labelling amino acids with naphthalene-2,3-dicarboxaldehyde (NDA) in the presence of cyanide (CN-). The resulting highly stable N-substituted 1-cyanobenz[f]isoindole (CBI) derivatives were separated using a binary gradient elution profile and detected fluorometrically. The order of elution of the derivatised amino acids was confirmed by using liquid chromatography with fluorescence and mass spectrometric detection in tandem. Linear calibration plots were obtained for all amino acids in the range studied (0.2-12.5 microM). The limit of detection for CBI derivatives of amino acids was in the range 5-20 fmol (S/N=2) using a 5 microl injection volume. The method has been used for the measurement of amino acids in microdialysates from rat brain and tissue homogenates from different regions of mouse brain.

Engraftment and retroviral marking of CD34+ and CD34+CD38- human hematopoietic progenitors assessed in immune-deficient mice.

Retroviral-mediated transduction of human hematopoietic stem cells to provide a lifelong supply of corrected progeny remains the most daunting challenge to the success of human gene therapy. The paucity of assays to examine transduction of pluripotent human stem cells hampers progress toward this goal. By using the beige/nude/xid (bnx)/hu immune-deficient mouse xenograft system, we compared the transduction and engraftment of human CD34+ progenitors with that of a more primitive and quiescent subpopulation, the CD34+CD38- cells. Comparable extents of human engraftment and lineage development were obtained from 5 x 10(5) CD34+ cells and 2,000 CD34+CD38- cells. Retroviral marking of long-lived progenitors from the CD34+ populations was readily accomplished, but CD34+CD38- cells capable of reconstituting bnx mice were resistant to transduction. Extending the duration of transduction from 3 to 7 days resulted in low levels of transduction of CD34+CD38- cells. Flt3 ligand was required during the 7-day ex vivo culture to maintain the ability of the cells to sustain long-term engraftment and hematopoiesis in the mice.