Immunolocalization of factor V in adult and fetal rat liver

Immunolocalization of the factor V was performed in adult and fetal rat liver by means of an indirect peroxidase labelling. This could be done owing to the production in our laboratory of a monospecific antiserum anti rat factor V. In all the cases (perfused and non-perfused adult liver and fetal one) the observation of the sections has revealed an intense circular or granular labelling into all the hepatocytes whatever was their localization in the hepatic lobule. Hepatic endot helial cells seemed to be negative for factor V and this aspect of our results was discussed.     

Selective in vivo fluorescence labelling of cholinergic neurons containing p75 in the rat basal forebrain

The cholinergic system of the rat basal forebrain is used as a model for the homologous region in humans which is highly susceptible to neuropathological alterations as in Alzheimer's disease. Cholinergic cells in the basal forebrain express the low-affinity neurotrophin receptor p75^NTR. This has been utilized for selective immunolesioning of cholinergic neurons after internalization of an immunotoxin composed of anti-p75^NTR and the ribosome-inactivating toxin saporin. However, the goal of many studies may be not the lesion, but the identification of cholinergic cells after other experimentally induced alterations in the basal forebrain. Therefore, a novel cholinergic marker was prepared by conjugating the monoclonal antibody 192IgG directed against p75^NTR with the bright red fluorochrome carbocyanine 3 (Cy3). Three days after intraventricular injection of Cy3-192IgG the fluorescence microscopic analysis revealed a pattern of Cy3-labelled cells matching the distribution of cholinergic neurons. Apparently the marker was internalized within complexes of p75^NTR and Cy3-192IgG which were then retrogradely transported to the cholinergic perikarya of the basal forebrain. In addition to the even labelling of somata, a strong punctate-like Cy3-immunofluorescence was seen in structures resembling lysosomes. The specificity of the in vivo staining was proven by subsequent immunolabelling of choline acetyltransferase (ChAT) with green fluorescent Cy2-tagged secondary antibodies. In the medial septum, the diagonal band and the nucleus basalis only cholinergic neurons were marked by Cy3-192IgG. In parallel experiments, digoxigenylated 192IgG was not detectable within cholinergic basal forebrain neurons after intraventricular injection. Presumably, this modified antibody could not be internalized. On the other hand, digoxigenylated 192IgG was found to be an excellent immunocytochemical marker for p75^NTR as shown by double labelling including highly sensitive mouse antibodies directed against ChAT. Based on the present findings, future applications of the apparently non-toxic Cy3-192IgG and other antibodies for fluorescent in vivo and in vitro labelling are discussed.     

Helsinki Declaration on patient safety in anaesthesiology: putting words into practice - experience in Germany

For years now, the German Society of Anaesthesiology and Intensive Care Medicine and the Professional Association of German Anaesthesiologists have been actively involved in efforts to improve patient safety. To this end, a whole range of activities have been initiated in recent years and, since February 2011, collected together on our home page 'PATSI' (www.patientensicherheit-ains.de). Further, the implementation of syringe labelling (ISO 26825) with additional information on drugs frequently used in intensive care was carried out. Under the item Helsinki Declaration, all decisions and recommendations so far worked out by our speciality have, in structured form, been assigned to individual points and saved as PDF files. This has made it possible for every anaesthesiological department in Germany to integrate all the relevant instructions and conditions of the Helsinki Declaration into their own individual work structures. These systematic solutions represent a major contribution towards reducing the possibility of errors at the workplace. We are certainly still in the early stages of our efforts to achieve a nationwide integration of a cultural change in the way we deal with mistakes in medicine. We have incorporated the item 'learning from mistakes' in our project 'critical incident reporting system for anaesthesia, intensive care medicine, emergency care, and pain therapy, CIRS-AINS', and have brought out a range of relevant illustrative publications. Accepting these 'mistakes' as an opportunity to critically examine ourselves and our work with a view to learning from them and further improving our speciality service is, we believe, a great challenge for future developments in anaesthesia.     

Identification of evolutionary conserved mouse sperm surface antigens by human antisperm antibodies (ASA) from infertile patients

PROBLEM: The presence of antisperm antibodies (ASA) in semen may impair sperm function leading to immunological infertility. The aim of the study was to identify the evolutionary conserved antigens on mouse sperm surface that react with human ASA in order to study the mechanism of autoimmune infertility. METHODS OF STUDY: The binding of human ASA to mouse sperm was investigated by means of indirect immunofluorescence. 2D-electrophoresis was applied to separate the biotin-labelled mouse membrane proteins using isoelectric focusing followed by polyacrylamide gel electrophoresis. Cognate antigens of ASA from seminal plasma of infertile patients were analysed by Western blotting. Performing avidin-blots it was detected which of the proteins recognized were sperm surface proteins. The spots of interest were analysed by means of mass spectrometry. RESULTS: ASA bound most frequently (36%) to the post-acrosomal region and to the midpiece of mouse spermatozoa. About 30% of ASA recognized apo lactate dehydrogenase (LDHC4) as a cognate antigen, 30% voltage-dependent anion channel (VDAC2). ASA of 20% bound to outer dense fibre protein and 20% of samples recognized glutathione S-transferase mu5. CONCLUSIONS: Human ASA bound to specific cognate antigens of mouse spermatozoa, offering the possibility to study their functional relevance in the mouse model.     

Fluorine‐18 labelling of a series of potential EGFRvIII targeting peptides with a parallel labelling approach using [18F]FPyME

There is growing interest in the use of radiolabelled peptides as receptor targeting agents for diagnostic imaging of various cancer types using positron emission tomography. In this work, 1‐[3‐(2‐[¹⁸F]fluoropyridin‐3‐yloxy)propyl]pyrrole‐2,5‐dione ([¹⁸F]FPyME) has been used for parallel fluorine‐18 labelling of PEPHC1, a peptide selective towards the cancer‐specific mutation of the epidermal growth factor receptor (EGFRvIII), and a number of truncated and mutated analogues. Conjugation of the peptides with [¹⁸F]FPyME was achieved within 10 min in non‐decay‐corrected radiochemical yields of 30–50%. The high yield of the conjugation reaction combined with its short synthesis time allows the labelling of several peptides from a single batch of [¹⁸F]FPyME. Copyright © 2010 John Wiley & Sons, Ltd.     

Synthesis of [15N]t‐butylamine hydrochloride

This report presents an efficient synthesis of [¹⁵N]t‐butylamine hydrochloride. Acylation of [¹⁵N]ammonia with pivaloyl chloride provided [¹⁵N]pivalamide, this was converted to benzyl [¹⁵N]N‐t‐butylcarbamate through a Hofmann rearrangement. Hydrogenolysis of benzyl [¹⁵N]N‐t‐buylcarbamate and acidification afforded [¹⁵N]t‐butylamine hydrochloride in an overall yield of 79.2% in four steps. Copyright © 2010 John Wiley & Sons, Ltd.     

Significance of adrenoceptor-mediated atrial natriuretic factor release in normal humans: Erratum

Abstract

The orosomucoid content of serum has been determined in healthy infants and children. Analyses of haptoglobin and of electrophoretic protein fractions have also been made.

The mean umbilical cord serum level of orosomucoid was significantly lower than the level in the maternal serum. Apart from a transient increase during the first week of life, low serum levels were found up to the age of three months.

The alpha₁, alpha₂ and beta globulins showed essentially the same changes with age as the orosomucoid.

The serum content of haptoglobin was very low at birth and rose slowly to the level found in adults.     

Mapping of the cerebral vascular response to hypoxia and hypercapnia using quantitative perfusion MRI at 3 T

Changes in breathing change the concentration of oxygen and carbon dioxide in arterial blood resulting in changes in cerebral blood flow (CBF). This mechanism can be described by the cerebral vascular response (CVR), which has been shown to be altered in different physiological and pathophysiological states. CBF maps of grey matter (GM) were determined with a pulsed arterial spin labelling technique at 3 T in a group of 19 subjects under baseline conditions, hypoxia, and hypercapnia. Experimental conditions allowed a change in either arterial oxygen (hypoxia) or carbon dioxide (hypercapnia) concentration compared with the baseline, leaving the other variable constant, in order to separate the effects of these two variables. From these results, maps were calculated showing the regional distribution of the CVR to hypoxia and hypercapnia in GM. Maps of CVR to hypoxia showed very high intra-subject variations, with some GM regions exhibiting a positive response and others a negative response. Per 10% decrease in arterial oxygen saturation, there was a statistically significant 7.0 +/- 2.9% (mean +/- SEM) increase in GM-CBF for the group. However, 70% of subjects showed an overall positive CVR (positive responders), and the remaining 30% an overall negative CVR (negative responders). Maps of CVR to hypercapnia showed less intra-subject variation. Per 1 mm Hg increase in partial pressure of end-tidal carbon dioxide, there was a statistically significant 5.8 +/- 0.9% increase in GM-CBF, all subjects showing an overall positive CVR. As the brain is particularly vulnerable to hypoxia, a condition associated with cardiorespiratory diseases, CVR maps may help in the clinic to identify the areas most prone to damage because of a reduced CVR.     

Imaging the neural correlates of neuropathic pain and pleasurable relief associated with inherited erythromelalgia in a single subject with quantitative arterial spin labelling

We identified a patient with severe inherited erythromelalgia secondary to an L858F mutation in the voltage-gated sodium channel Na(v)1.7. The patient reported severe ongoing foot pain, which was exquisitely sensitive to limb cooling. We confirmed this heat hypersensitivity using quantitative sensory testing. Additionally, we employed a novel perfusion imaging technique in a simple block design to assess her baseline erythromelalgia pain vs cooling relief. Robust activations of key pain, pain-affect, and reward-related centres were observed. This combined approach allowed us to confirm the presence of a temperature-sensitive channelopathy of peripheral neurons and to investigate the neural correlates of tonic neuropathic pain and relief in a single subject.