Endothelial cell protection and complement inhibition in xenotransplantation: a novel in vitro model using whole blood

BACKGROUND: Studying the interactions between xenoreactive antibodies, complement and coagulation factors with the endothelium in hyperacute and acute vascular rejection usually necessitates the use of in vivo models. Conventional in vitro or ex vivo systems require either serum, plasma or anti-coagulated whole blood, making analysis of coagulation-mediated effects difficult. Here a novel in vitro microcarrier-based system for the study of endothelial cell (EC) activation and damage, using non-anticoagulated whole blood is described. Once established, the model was used to study the effect of the characterized complement- and coagulation inhibitor dextran sulfate (DXS, MW 5000) for its EC protective properties in a xenotransplantation setting. METHODS: Porcine aortic endothelial cells (PAEC), grown to confluence on microcarrier beads, were incubated with non-anticoagulated whole human blood until coagulation occurred or for a maximum of 90 min. PAEC-beads were either pre- or co-incubated with DXS. Phosphate buffered saline (PBS) experiments served as controls. Fluid phase and surface activation markers for complement and coagulation were analyzed as well as binding of DXS to PAEC-beads. RESULTS: Co- as well as pre-incubation of DXS, followed by washing of the beads, significantly prolonged time to coagulation from 39 +/- 12 min (PBS control) to 74 +/- 23 and 77 +/- 20 min, respectively (P < 0.005 vs. PBS). DXS treatment attenuated surface deposition of C1q, C4b/c, C3b/c and C5b-9 without affecting IgG or IgM deposition. Endothelial integrity, expressed by positivity for von Willebrand Factor, was maintained longer with DXS treatment. Compared with PBS controls, both pre- and co-incubation with DXS significantly prolonged activated partial thromboplastin time (>300 s, P < 0.05) and reduced production of thrombin-antithrombin complexes and fibrinopeptide A. Whilst DXS co-incubation completely blocked classical pathway complement activity (CH50 test) DXS pre-incubation or PBS control experiments showed no inhibition. DXS bound to PAEC-beads as visualized using fluorescein-labeled DXS. CONCLUSIONS: This novel in vitro microcarrier model can be used to study EC damage and the complex interactions with whole blood as well as screen ''endothelial protective'' substances in a xenotransplantation setting. DXS provides EC protection in this in vitro setting, attenuating damage of ECs as seen in hyperacute xenograft rejection.

Design of the blood group AB glycotope

Although the nature of the blood groups A and B has been comprehensively studied for a long time, it is still unclear as to what exactly is the epitope that is recognized by antibodies having AB specificity, i.e. monoclonal and polyclonal antibodies which are capable of interacting equally well with the antigens GalNAcalpha 1-3(Fucalpha 1-2)Gal (A trisaccharide) and Galalpha 1-3(Fucalpha 1-2)Gal (B trisaccharide), but do not react with their common fragment Fucalpha 1-2Gal. We have supposed that besides Fucalpha 1-2Gal, A and B antigens have one more shared epitope. The trisaccharides A and B are practically identical from the conformational point of view, the only difference being situated at position 2 of Galalpha residue, i.e. trisaccharide A has a NHAc group, whereas trisaccharide B has a hydroxyl group (see formulas). We have hypothesized that the AB-epitope should be situated in the part of the molecule that is opposite to the NHAc group of GalNAc residue. In order to test this hypothesis we have synthesized a polymeric conjugate in such a way that de-N-acetylated A-trisaccharide is attached to a polymer via the nitrogen in position C-2 of the galactosamine residue. In this conjugate the supposed AB-epitope should be maximally accessible for antibodies from the solution, whereas the discrimination site of antigens A and B by the antibodies should be maximally hidden due to the close proximity of the polymer. Interaction with several anti-AB monoclonal antibodies revealed that a part of them really interacted with the synthetic AB-glycotope, thus confirming our hypothesis. Moreover, similar antibodies were revealed in the blood of healthy blood group 0 donors. Analysis of spatial models was performed in addition to identify the hydroxyl groups of Fuc, Galalpha, and Galbeta residues, which are particularly involved in the composition of the AB-glycotope.

Assessment of a real-time PCR test to diagnose syphilis from diverse biological samples

OBJECTIVES: To investigate the contribution of a real-time PCR assay for the detection of Treponema pallidum in various biological specimens with the secondary objective of comparing its value according to HIV status. METHODS: Prospective cohort of incident syphilis cases from three Swiss hospitals (Geneva and Bern University Hospitals, Outpatient Clinic for Dermatology of Triemli, Zurich) diagnosed between January 2006 and September 2008. A case-control study was nested into the cohort. Biological specimens (blood, lesion swab or urine) were taken at diagnosis (as clinical information) and analysed by real-time PCR using the T pallidum 47 kDa gene. RESULTS: 126 specimens were collected from 74 patients with primary (n = 26), secondary (n = 40) and latent (n = 8) syphilis. Among primary syphilis, sensitivity was 80% in lesion swabs, 28% in whole blood, 55% in serum and 29% in urine, whereas among secondary syphilis, it was 20%, 36%, 47% and 44%, respectively. Among secondary syphilis, plasma and cerebrospinal fluid were also tested and provided a sensitivity of 100% and 50%, respectively. The global sensitivity of T pallidum by PCR (irrespective of the compartment tested) was 65% during primary, 53% during secondary and null during latent syphilis. No difference regarding serology or PCR results was observed among HIV-infected patients. Specificity was 100%. CONCLUSIONS: Syphilis PCR provides better sensitivity in lesion swabs from primary syphilis and displays only moderate sensitivity in blood from primary and secondary syphilis. HIV status did not modify the internal validity of PCR for the diagnosis of primary or secondary syphilis.

Laser-Doppler measurements of spinal cord blood flow changes during hemilaminectomy in chondrodystrophic dogs with disk extrusion

OBJECTIVES: (1) To assess spinal cord blood flow (SCBF) during surgical treatment of disk extrusion in dogs and (2) to investigate associations between SCBF, clinical signs, presurgical MRI images, and 24-hour surgical outcome. STUDY DESIGN: Cohort study. ANIMALS: Chondrodystrophic dogs with thoracolumbar disk extrusion (n=12). METHODS: Diagnosis was based on clinical signs and MRI findings, and confirmed at surgery. Regional SCBF was measured intraoperatively by laser-Doppler flowmetry before, immediately after surgical spinal cord decompression, and after 15 minutes of lavaging the lesion. Care was taken to ensure a standardized surgical procedure to minimize factors that could influence measurement readings. RESULTS: A significant increase in intraoperative SCBF was found in all dogs (Wilcoxon's signed-rank test; P=.05) immediately after spinal cord decompression and after 15 minutes. Changes in SCBF were not associated with duration of clinical signs; initial or 24-hour neurologic status; or degree of spinal cord compression assessed by MRI. CONCLUSION: SCBF increases immediately after spinal cord decompression in dogs with disk herniation; however, increased SCBF was not associated with a diminished 24-hour neurologic status. CLINICAL RELEVANCE: An increase in SCBF does not appear to be either associated with the degree of spinal cord compression or of a magnitude sufficient to outweigh the benefit of surgical decompression by resulting in clinically relevant changes in 24-hour outcome.

Comparison of arterial blood pressure measurements and hypertension scores obtained by use of three indirect measurement devices in hospitalized dogs

OBJECTIVE: To evaluate the agreement of blood pressure measurements and hypertension scores obtained by use of 3 indirect arterial blood pressure measurement devices in hospitalized dogs. Design-Diagnostic test evaluation. ANIMALS: 29 client-owned dogs. PROCEDURES: 5 to 7 consecutive blood pressure readings were obtained from each dog on each of 3 occasions with a Doppler ultrasonic flow detector, a standard oscillometric device (STO), and a high-definition oscillometric device (HDO). RESULTS: When the individual sets of 5 to 7 readings were evaluated, the coefficient of variation for systolic arterial blood pressure (SAP) exceeded 20% for 0% (Doppler), 11 % (STO), and 28% (HDO) of the sets of readings. After readings that exceeded a 20% coefficient of variation were discarded, repeatability was within 25 (Doppler), 37 (STO), and 39 (HDO) mm Hg for SAP. Correlation of mean values among the devices was between 0.47 and 0.63. Compared with Doppler readings, STO underestimated and HDO overestimated SAP. Limits of agreement between mean readings of any 2 devices were wide. With the hypertension scale used to score SAP, the intraclass correlation of scores was 0.48. Linear-weighted inter-rater reliability between scores was 0.40 (Doppler vs STO), 0.38 (Doppler vs HDO), and 0.29 (STO vs HDO). CONCLUSIONS AND CLINICAL RELEVANCE: Results of this study suggested that no meaningful clinical comparison can be made between blood pressure readings obtained from the same dog with different indirect blood pressure measurement devices.

In vitro drug causality assessment in Stevens-Johnson syndrome - alternatives for lymphocyte transformation test

BACKGROUND Patients with Stevens-Johnson syndrome (SJS) or toxic epidermal necrolysis (TEN) are often exposed simultaneously to a few potentially culprit drugs. However, both the standard lymphocyte transformation tests (LTT) with proliferation as the assay end-point as well as skin tests, if done, are often negative. OBJECTIVE As provocation tests are considered too dangerous, there is an urgent need to identify the relevant drug in SJS/TEN and to improve sensitivity of tests able to identify the causative drug. METHODS Fifteen patients with SJS/TEN with the ALDEN score ≥ 6 and 18 drug-exposed controls were included. Peripheral blood mononuclear cells (PBMC) were isolated and cultured under defined conditions with drugs. LTT was compared to the following end-points: cytokine levels in cell culture supernatant, number of granzyme B secreting cells by ELISpot and intracellular staining for granulysin and IFNγ in CD3(+) CD4(+), CD3(+) CD8(+) and NKp46(+) cells. To further enhance sensitivity, the effect of IL-7/IL-15 pre-incubation of PBMC was evaluated. RESULTS Lymphocyte transformation tests was positive in only 4/15 patients (sensitivity 27%, CI: 8-55%). Similarly, with granzyme B-ELISpot culprit drugs were positive in 5/15 patients (sensitivity 33%, CI: 12-62%). The expression of granulysin was significantly induced in NKp46(+) and CD3(+) CD4(+) cells (sensitivity 40%, CI: 16-68% and 53%, CI: 27-79% respectively). Cytokine production could be demonstrated in 38%, CI: 14-68% and 43%, CI: 18-71% of patients for IL-2 and IL-5, respectively, and in 55%, CI: 23-83% for IFNγ. Pre-incubation with IL-7/IL-15 enhanced drug-specific response only in a few patients. Specificities of tested assays were in the range of 95 (CI: 80-99%)-100% (CI: 90-100%). CONCLUSIONS AND CLINICAL RELEVANCE Granulysin expression in CD3(+) CD4(+) , Granzyme B-ELISpot and IFNγ production considered together provided a sensitivity of 80% (CI: 52-96%) and specificity of 95% (80-99%). Thus, this study demonstrated that combining different assays may be a feasible approach to identify the causative drug of SJS/TEN reactions; however, confirmation on another group of patients is necessary.

Determination of Ethyl glucuronide and Ethyl sulfate from dried blood spots

BACKGROUND: Ethyl glucuronide (EtG) and ethyl sulfate (EtS) are non-oxidative minor metabolites of ethanol. They are detectable in various body fluids shortly after initial consumption of ethanol and have a longer detection time frame than the parent compound. They are regarded highly sensitive and specific markers of recent alcohol uptake. This study evaluates the determination of EtG and EtS from dried blood spots (DBS), a simple and cost-effective sampling method that would shorten the time gap between offense and blood sampling and lead to a better reflectance of the actual impairment. METHODS: For method validation, EtG and EtS standard and quality control samples were prepared in fresh human heparinized blood and spotted on DBS cards, then extracted and measured by an LC-ESI-MS/MS method. Additionally, 76 heparinized blood samples from traffic offense cases were analyzed for EtG and EtS as whole blood and as DBS specimens. The results from these measurements were then compared by calculating the respective mean values, by a matched-paired t test, by a Wilcoxon test, and by Bland-Altman and Mountain plots. RESULTS AND DISCUSSION: Calibrations for EtG and EtS in DBS were linear over the studied calibration range. The precision and accuracy of the method met the requirements of the validation guidelines that were employed in the study. The stability of the biomarkers stored as DBS was demonstrated under different storage conditions. The t test showed no significant difference between whole blood and DBS in the determination of EtG and EtS. In addition, the Bland-Altman analysis and Mountain plot confirmed that the concentration differences that were measured in DBS specimens were not relevant.

Delayed diagnosis of cryptococcal meningoencephalitis due to negative cryptococcal antigen test

Cryptococcus spp. commonly causes infection in immunocompromised hosts. Clinical presentation of cryptococcal meningoencephalitis (CM) is variable, but headache, fever and a high intracranial pressure should suggest the diagnosis. The cryptococcal antigen test is a specific and sensitive rapid test that can be performed on blood or cerebrospinal fluid. We report a case of CM in a patient with previously undetected lymphocytopenia. Because cryptococcal antigen test results were negative, diagnosis and treatment were delayed.

Erythropoietin neuroprotection is enhanced by direct cortical application following subdural blood evacuation in a rat model of acute subdural hematoma

Recombinant human erythropoietin (EPO) has been successfully tested as neuroprotectant in brain injury models. The first large clinical trial with stroke patients, however, revealed negative results. Reasons are manifold and may include side-effects such as thrombotic complications or interactions with other medication, EPO concentration, penetration of the blood-brain-barrier and/or route of application. The latter is restricted to systemic application. Here we hypothesize that EPO is neuroprotective in a rat model of acute subdural hemorrhage (ASDH) and that direct cortical application is a feasible route of application in this injury type. The subdural hematoma was surgically evacuated and EPO was applied directly onto the surface of the brain. We injected NaCl, 200, 2000 or 20,000IU EPO per rat i.v. at 15min post-ASDH (400μl autologous venous blood) or NaCl, 0.02, 0.2 or 2IU per rat onto the cortical surface after removal of the subdurally infused blood t at 70min post-ASDH. Arterial blood pressure (MAP), blood chemistry, intracranial pressure (ICP), cerebral blood flow (CBF) and brain tissue oxygen (ptiO2) were assessed during the first hour and lesion volume at 2days after ASDH. EPO 20,000IU/rat (i.v.) elevated ICP significantly. EPO at 200 and 2000IU reduced lesion volume from 38.2±0.6mm(3) (NaCl-treated group) to 28.5±0.9 and 22.2±1.3mm(3) (all p<0.05 vs. NaCl). Cortical application of 0.02IU EPO after ASDH evacuation reduced injury from 36.0±5.2 to 11.2±2.1mm(3) (p=0.007), whereas 0.2IU had no effect (38.0±9.0mm(3)). The highest dose of both application routes (i.v. 20,000IU; cortical 2IU) enlarged the ASDH-induced damage significantly to 46.5±1.7 and 67.9±10.4mm(3) (all p<0.05 vs. NaCl). In order to test whether Tween-20, a solvent of EPO formulation 'NeoRecomon®' was responsible for adverse effects two groups were treated with NaCl or Tween-20 after the evacuation of ASDH, but no difference in lesion volume was detected. In conclusion, EPO is neuroprotective in a model of ASDH in rats and was most efficacious at a very low dose in combination with subdural blood removal. High systemic and topically applied concentrations caused adverse effects on lesion size which were partially due to increased ICP. Thus, patients with traumatic ASDH could be treated with cortically applied EPO but with caution concerning concentration.

A case of distinct difference between the measured blood Ethanol concentration and the concentration estimated by Widmark's equation

In the last century, several mathematical models have been developed to calculate blood ethanol concentrations (BAC) from the amount of ingested ethanol and vice versa. The most common one in the field of forensic sciences is Widmark's equation. A drinking experiment with 10 voluntary test persons was performed with a target BAC of 1.2 g/kg estimated using Widmark's equation as well as Watson's factor. The ethanol concentrations in the blood were measured using headspace gas chromatography/flame ionization and additionally with an alcohol Dehydrogenase (ADH)-based method. In a healthy 75-year-old man a distinct discrepancy between the intended and the determined blood ethanol concentration was observed. A blood ethanol concentration of 1.83 g/kg was measured and the man showed signs of intoxication. A possible explanation for the discrepancy is a reduction of the total body water content in older people. The incident showed that caution is advised when using the different mathematical models in aged people. When estimating ethanol concentrations, caution is recommended with calculated results due to potential discrepancies between mathematical models and biological systems

Wnt activation of immortalized brain endothelial cells as a tool for generating a standardized model of the blood brain barrier in vitro

Reproducing the characteristics and the functional responses of the blood-brain barrier (BBB) in vitro represents an important task for the research community, and would be a critical biotechnological breakthrough. Pharmaceutical and biotechnology industries provide strong demand for inexpensive and easy-to-handle in vitro BBB models to screen novel drug candidates. Recently, it was shown that canonical Wnt signaling is responsible for the induction of the BBB properties in the neonatal brain microvasculature in vivo. In the present study, following on from earlier observations, we have developed a novel model of the BBB in vitro that may be suitable for large scale screening assays. This model is based on immortalized endothelial cell lines derived from murine and human brain, with no need for co-culture with astrocytes. To maintain the BBB endothelial cell properties, the cell lines are cultured in the presence of Wnt3a or drugs that stabilize β-catenin, or they are infected with a transcriptionally active form of β-catenin. Upon these treatments, the cell lines maintain expression of BBB-specific markers, which results in elevated transendothelial electrical resistance and reduced cell permeability. Importantly, these properties are retained for several passages in culture, and they can be reproduced and maintained in different laboratories over time. We conclude that the brain-derived endothelial cell lines that we have investigated gain their specialized characteristics upon activation of the canonical Wnt pathway. This model may be thus suitable to test the BBB permeability to chemicals or large molecular weight proteins, transmigration of inflammatory cells, treatments with cytokines, and genetic manipulation.

Static and dynamic characteristics of cerebral blood flow during the resting state in schizophrenia

Background: The cerebral network that is active during rest and is deactivated during goal-oriented activity is called the default mode network (DMN). It appears to be involved in self-referential mental activity. Atypical functional connectivity in the DMN has been observed in schizophrenia. One hypothesis suggests that pathologically increased DMN connectivity in schizophrenia is linked with a main symptom of psychosis, namely, misattribution of thoughts. Methods: A resting-state pseudocontinuous arterial spin labeling (ASL) study was conducted to measure absolute cerebral blood flow (CBF) in 34 schizophrenia patients and 27 healthy controls. Using independent component analysis (ICA), the DMN was extracted from ASL data. Mean CBF and DMN connectivity were compared between groups using a 2-sample t test. Results: Schizophrenia patients showed decreased mean CBF in the frontal and temporal regions (P < .001). ICA demonstrated significantly increased DMN connectivity in the precuneus (x/y/z = -16/-64/38) in patients than in controls (P < .001). CBF was not elevated in the respective regions. DMN connectivity in the precuneus was significantly correlated with the Positive and Negative Syndrome Scale scores (P < .01). Conclusions: In schizophrenia patients, the posterior hub-which is considered the strongest part of the DMN-showed increased DMN connectivity. We hypothesize that this increase hinders the deactivation of the DMN and, thus, the translation of cognitive processes from an internal to an external focus. This might explain symptoms related to defective self-monitoring, such as auditory verbal hallucinations or ego disturbances.

Humoral immunity in tuberculin skin test anergy and its role in high-risk persons exposed to active tuberculosis.

The most common test to identify latent tuberculosis is the tuberculin skin test that detects T cell responses of delayed type hypersensitivity type IV. Since it produces false negative reactions in active tuberculosis or in high-risk persons exposed to tuberculosis patients as shown in this report, we studied antibody profiles to explain the anergy of such responses in high-risk individuals without active infection. Our results showed that humoral immunity against tuberculin, regardless of the result of the tuberculin skin test is important for protection from active tuberculosis and that the presence of high antibody titers is a more reliable indicator of infection latency suggesting that latency can be based on the levels of antibodies together with in vitro proliferation of peripheral blood mononuclear cells in the presence of the purified protein derivative. Importantly, anti-tuberculin IgG antibody levels mediate the anergy described herein, which could also prevent reactivation of disease in high-risk individuals with high antibody titers. Such anti-tuberculin IgG antibodies were also found associated with blocking and/or stimulation of in vitro cultures of PBMC with tuberculin. In this regard, future studies need to establish if immune responses to Mycobacterium tuberculosis can generate a broad spectrum of reactions either toward Th1 responses favoring stimulation by cytokines or by antibodies and those toward diminished responses by Th2 cytokines or blocking by antibodies; possibly involving mechanisms of antibody dependent protection from Mtb by different subclasses of IgG.

Influence of blood contamination on bond strength of a self-etching system.

OBJECTIVES: To detect the influence of blood contamination (BC) on the bond strength (BS) of a self-etching bonding system (SES) to enamel and dentine. METHODS: 25 human molars were longitudinally sectioned on the mesio-distal axis in order to obtain 50 specimens, which were embedded in acrylic resin. At first, the specimens were ground to expose a flat surface of enamel, and a bond strength test was performed. Afterwards, the samples were ground again in order to obtain a flat surface of dentine. Ten groups (total: n=100) were assigned according to substrate (enamel and dentine), step in the bonding sequence when contamination occurred (before the acidic primer and after the bonding resin), and contamination treatment (dry or rinse and dry procedure). Fresh human blood was introduced either before or after SES application (Clearfil SE Bond) and treated with air drying, or by rinsing and drying following application. Composite resin (Filtek Z-250,3M ESPE) was applied as inverted, truncated cured cones that were debonded in tension. RESULTS: The mean tensile BS values (MPa) for enamel/dentine were 19.4/23.0 and 17.1/10.0 for rinse-and-dry treatment (contamination before and after SES, respectively); while the measurements for the dry treatment, 16.2/23.3 and 0.0/0.0 contamination before and after SES, respectively. CONCLUSIONS: It was determined that blood contamination impaired adhesion to enamel and dentine when it occurred after bond light curing. Among the tested contamination treatments, the rinse-and-dry treatment produced the highest bond strength with BC after SES application, but it was not sufficient to recover the BS in the contamination-free group.

General anesthesia with sevoflurane decreases myocardial blood volume and hyperemic blood flow in healthy humans

BACKGROUND Preservation of myocardial perfusion during general anesthesia is likely important in patients at risk for perioperative cardiac complications. Data related to the influence of general anesthesia on the normal myocardial circulation are limited. In this study, we investigated myocardial microcirculatory responses to pharmacological vasodilation and sympathetic stimulation during general anesthesia with sevoflurane in healthy humans immediately before surgical stimulation. METHODS Six female and 7 male subjects (mean age 43 years, range 28-61) were studied at baseline while awake and during the administration of 1 minimum alveolar concentration sevoflurane. Using myocardial contrast echocardiography, myocardial blood flow (MBF) and microcirculatory variables were assessed at rest, during adenosine-induced hyperemia, and after cold pressor test-induced sympathetic stimulation. MBF was calculated from the relative myocardial blood volume multiplied by its exchange frequency (β) divided by myocardial tissue density (ρT), which was set at 1.05 g·mL(-1). RESULTS During sevoflurane anesthesia, MBF at rest was similar to baseline values (1.05 ± 0.28 vs 1.05 ± 0.32 mL·min(-1)·g(-1); P = 0.98; 95% confidence interval [CI], -0.18 to 0.18). Myocardial blood volume decreased (P = 0.0044; 95% CI, 0.01-0.04) while its exchange frequency (β) increased under sevoflurane anesthesia when compared with baseline. In contrast, hyperemic MBF was reduced during anesthesia compared with baseline (2.25 ± 0.5 vs 3.53 ± 0.7 mL·min(-1)·g(-1); P = 0.0003; 95% CI, 0.72-1.84). Sympathetic stimulation during sevoflurane anesthesia resulted in a similar MBF compared to baseline (1.53 ± 0.53 and 1.55 ± 0.49 mL·min(-1)·g(-1); P = 0.74; 95% CI, -0.47 to 0.35). CONCLUSIONS In otherwise healthy subjects who are not subjected to surgical stimulation, MBF at rest and after sympathetic stimulation is preserved during sevoflurane anesthesia despite a decrease in myocardial blood volume. However, sevoflurane anesthesia reduces hyperemic MBF, and thus MBF reserve, in these subjects.