Factor VIII/von Willebrand factor protein: sensitivity of periodic acid Schiff stain to carbohydrate deficiency

We have investigated the periodic acid Schiff (PAS) Coomassie staining ratio of the human factor VIII/von Willebrand factor (fVIII/vWf) protein. The PAS-Coomassie staining ratio is consistent over 8 days. The PAS-Coomassie ratio of fVIII/vWf protein purified from different starting materials does not appear to be significantly different. The PAS stain can detect as little as 300 ng of carbohydrate in the fVIII/vWf protein. Desialation did not affect the PAS-Coomassie ratio, while removal of penultimate galactose resulted in a marked reduction in the PAS-Coomassie ratio. This reduction was further accentuated with the removal of N-acetylglucosamine. The smaller multimers of the fVIII/vWf protein have a reduced sialic acid and PAS-Coomassie staining ratio. This difference does not appear to be related to the sialic acid deficiency but may be related to the distribution or organization of the carbohydrate moieties on the smaller fVIII/vWf multimers.     

Pompe's disease in childhood: A metabolic myopathy

Background: Myopathy of metabolic origin in childhood occurs due to a variety of conditions. Pompe's Disease also known as Glycogen storage disease Type II, is a rare storage disorder with clinical presentation akin to spinal muscular atrophy.     

Interaction of benzylidene-anabaseine analogues with agonist and allosteric sites on muscle nicotinic acetylcholine receptors

Background and purpose

Benzylidene-anabaseines (BAs) are partial agonists of the α7 nicotinic acetylcholine receptor (nAChR) but their mechanism(s) of action are unknown. Our study explores several possibilities, including direct interactions of BAs with the nAChR channel.

Experimental approach

Functional and radioligand-binding assays were used to examine the interaction of two BA analogues, 3-(2,4-dimethoxybenzylidene)-anabaseine (DMXBA) and its primary metabolite 3-(4-hydroxy-2-methoxybenzylidene)-anabaseine (4OH-DMXBA) with both agonist and non-competitive antagonist (NCA)-binding sites on muscle-type nAChRs.

Key results

Both BAs non-competitively inhibited ACh activation of human fetal muscle nAChRs and sterically inhibited the specific binding of the NCAs [piperidyl-3,4-3H(N)]-(N-(1-(2-thienyl)cyclohexyl)-3,4-piperidine ([³H]TCP) and [³H]dizocilpine to Torpedo nAChRs in the desensitized state. These compounds modulated [³H]tetracaine, [¹⁴C]amobarbital and [³H]TCP binding to resting nAChRs by allosteric mechanisms. Both BAs enhanced [³H]TCP binding when the nAChR was initially in the resting but activatable state, suggesting that both compounds desensitized the Torpedo nAChR. Although DMXBA failed to activate human fetal muscle nAChRs, 4OH-DMXBA was found to be a partial agonist. [³H]Nicotine competition-binding experiments confirmed that 4OH-DMXBA has higher affinity than DMXBA for the agonist sites, and that DMXBA is also a competitive antagonist.

Conclusions and implications

3-(4-hydroxy-2-methoxybenzylidene)-anabaseine is a partial agonist for human fetal muscle nAChRs, whereas DMXBA only has competitive and NCA activities. The NCA-binding site for BAs overlaps both the phencyclidine-and dizocilpine-binding sites in the desensitized Torpedo nAChR ion channel. The desensitizing property of BAs suggests another possible mode of non-competitive inhibition in addition to direct channel-blocking mechanisms.     

The safety of intravenous fluorescein for confocal laser endomicroscopy in the gastrointestinal tract

Aliment Pharmacol Ther 31 , 548–552

Summary

Background Confocal laser endomicroscopy (CLE) is rapidly emerging as a valuable tool for gastrointestinal endoscopic imaging. Fluorescent contrast agents are used to optimize imaging with CLE, and intravenous fluorescein is the most widely used contrast agent. Fluorescein is FDA‐cleared for diagnostic angiography of the retina. For these indications, the safety profile of fluorescein has been well‐documented; however, to date, fluorescein is not cleared for use with CLE. Aims To estimate the rate of serious and total adverse events attributable to intravenous fluorescein when used for gastrointestinal CLE. Methods We performed a cross sectional survey of 16 International Academic Medical Centres with active research protocols in CLE that involved intravenous fluorescein. Centres using i.v. fluorescein for CLE who were actively monitored for adverse events were included. Results Sixteen centres performed 2272 gastrointestinal CLE procedures. The most common dose of contrast agent was 2.5–5 mL of 10% sodium fluorescein. No serious adverse events were reported. Mild adverse events occurred in 1.4% of individuals, including nausea/vomiting, transient hypotension without shock, injection site erythema, diffuse rash and mild epigastric pain. The limitation is that only immediate post procedure events were actively monitored. Conclusions Use of intravenous fluorescein for gastrointestinal CLE appears to be safe with few acute complications.     

The cytoprotective drug amifostine modifies both expression and activity of the pro-angiogenic factor VEGF-A

Background

Amifostine (WR-2721, delivered as Ethyol^®) is a phosphorylated aminothiol compound clinically used in addition to cis-platinum to reduce the toxic side effects of therapeutic treatment on normal cells without reducing their efficacy on tumour cells. Its mechanism of action is attributed to the free radical scavenging properties of its active dephosphorylated metabolite WR-1065. However, amifostine has also been described as a potent hypoxia-mimetic compound and as a strong p53 inducer; both effects are known to potently modulate vascular endothelial growth factor (VEGF-A) expression. The angiogenic properties of this drug have not been clearly defined.

Methods

Cancer cell lines and endothelial cells were used in culture and treated with Amifostine in order to study (i) the expression of angiogenesis related genes and proteins and (ii) the effects of the drug on VEGF-A induced in vitro angiogenesis.

Results

We demonstrated that the treatment of several human cancer cell lines with therapeutical doses of WR-1065 led to a strong induction of different VEGF-A mRNA isoforms independently of HIF-1α. VEGF-A induction by WR-1065 depends on the activation of the eIF2alpha/ATF4 pathway. This up-regulation of VEGF-A mRNA was accompanied by an increased secretion of VEGF-A proteins fully active in stimulating vascular endothelial cells (EC). Nevertheless, direct treatment of EC with amifostine impaired their ability to respond to exogenous VEGF-A, an effect that correlated to the down-regulation of VEGFR-2 expression, to the reduction in cell surface binding of VEGF-A and to the decreased phosphorylation of the downstream p42/44 kinases.

Conclusions

Taken together, our results indicate that amifostine treatment modulates tumour angiogenesis by two apparently opposite mechanisms - the increased VEGF-A expression by tumour cells and the inhibition of EC capacity to respond to VEGF-A stimulation.

     

Hemodynamic effects of dobutamine and dopexamine after cardiopulmonary bypass in pediatric cardiac surgery*

Background: After surgical repair of congenital heart disease, inotropic support is sometimes necessary to wean from cardiopulmonary bypass. In pediatric cardiac surgery, dobutamine and dopamine are often used as inotropic support. Dopexamine is a synthetic catecholamine, which has positive inotropic and vasodilating properties. Because the hemodynamic effects of catecholamines are modified after cardiopulmonary bypass, the aim of this study was to investigate the effects of dobutamine and dopexamine on cardiac index and systemic vascular resistance index after cardiopulmonary bypass in pediatric cardiac surgery. Methods: The study was performed in a prospective, randomized, and double‐blinded cross‐over design. The investigation included 11 children for elective, noncomplex congenital heart surgery. After weaning from cardiopulmonary bypass and a 20‐min period of steady state, children received either 2.5 μg·kg^?1·min^?1 dobutamine or 1 μg·kg^?1·min^?1 dopexamine for 20 min. Cardiac index (transpulmonary thermodilution), mean arterial pressure, central venous pressure, stroke volume, systemic vascular resistance, and central venous oxygen saturation were determined. The primary outcome variable was cardiac index. Results: No difference in cardiac index was observed between the two groups (P = 0.594). Both drugs increased cardiac index, dopexamine from 3.9 ± 0.6 to 4.7 ± 0.8 l·min^?1·m^?2 (P = 0.003) and dobutamine from 4.1 ± 0.7 to 4.8 ± 0.7 l·min^?1·m^?2 (P = 0.004). During treatment with dobutamine, children presented with significantly higher mean arterial pressure (P = 0.003) and systemic vascular resistance index (P = 0.026). Conclusions: This trial demonstrates that low‐dose dobutamine and dopexamine both increase cardiac index during pediatric cardiac surgery but with different hemodynamic effects.     

Hypothermie bei Traumapatienten

Coincidental hypothermia after trauma can aggravate the severity of injury and it therefore makes sense to monitor the body temperature in the preclinical setting. In a prospective study, incidence and degree of severity of hypothermia were analysed as a function of injury pattern and preclinical care. A main factor of hypothermia was severity of injury measured by the injury severity score (ISS 24.0±15.5 versus 8.3±7.8) and the revised trauma score (RTS 5.6±1.8 versus 7.3±1.1). Of the patients with multiple trauma, 88.2% were hypothermic when administered to hospital, but no reason was found concerning the rescue methods (e.g. rescue time, whole duration, weather). An increase in the rate of hypothermia was seen only after long lasting incarceration. A multivariate data analysis showed, that in addition to ISS and RTS patient age (p=0.014) was primarily responsible for hypothermia after trauma, whereas no dependency was demonstrated for other factors. Thus, parts of this analysis are in contrast with other studies. Overall approximately 50% of trauma patients in the emergency unit suffer from hypothermia, but at this time point changes in physiological control mechanisms are not yet detectable. Nevertheless prevention of hypothermia is very important in the preclinical setting.     

The hospital record of the injured child and the need for external cause-of-injury codes. American Academy of Pediatrics. Committee on Injury and Poison Prevention, 1998-1999

Proper record-keeping of emergency department visits and hospitalizations of injured children is vital for appropriate patient management. Determination and documentation of the circumstances surrounding the injury event are essential. This information not only is the basis for preventive counseling, but also provides clues about how similar injuries in other youth can be avoided. The hospital records have an important secondary purpose; namely, if sufficient information about the cause and mechanism of injury is documented, it can be subsequently coded, electronically compiled, and retrieved later to provide an epidemiologic profile of the injury, the first step in prevention at the population level. To be of greatest use, hospital records should indicate the "who, what, when, where, why, and how" of the injury occurrence and whether protective equipment (eg, a seat belt) was used. The pediatrician has two important roles in this area: to document fully the injury event and to advocate the use of standardized external cause-of-injury codes, which allow such data to be compiled and analyzed.     

ANAL TONOMETRY A Diagnostic Help in Hirschsprung's Disease1

Manometrical measurements of reflex responses in the internal anal sphincter were made in 93 patients. In normal controls, a relaxation was obtained within the internal sphincter after distension of the rectum. Patients suffering from Hirschsprung's disease showed either a contraction in the muscle or an absence of sphincteric response. In 17 cases, where the diagnosis of Hirschsprung's disease was established by conventional means, the diagnosis was confirmed by anal tonometry in 13 cases and disproved by this method in 4 cases. The subsequent clinical course and histological findings have verified the diagnosis obtained by tonometry. In one patient it was not possible to make the diagnosis by means of the usual methods, anal tonometry showed Hirschsprung's disease and this was subsequently confirmed by biopsy.