Ex-vivo Data Research Articles

The effect of autoimmune arthritis treatment strategies on regulatory T-cell dynamics

Since their discovery over 15 years ago, intensive research has focused on the presence, phenotype and function of FOXP3(+) regulatory T cells (Treg) in autoimmune diseases such as rheumatoid arthritis (RA). The questions of whether Treg deficiencies underlie autoimmune pathology and whether or how Treg-related therapeutic approaches might be successful are still a subject of a vivid debate. In this review we give an overview of how current therapies influence Treg numbers and function in RA and juvenile idiopathic arthritis (JIA) and discuss these findings in the light of new Treg-based intervention strategies for autoimmune arthritis. The attempt to relate rheumatic diseases like rheumatoid arthritis and juvenile idiopathic arthritis to Treg has led to somewhat heterogeneous observations. So far, no clear defects in Treg numbers or function have been identified in autoimmune arthritis. The current standard therapies, that is methotrexate and biologicals, are generally effective, but the exact mechanism of action and their effect on Treg is not fully known. Nevertheless, the majority of in-vitro and ex-vivo data point towards a positive influence of these treatments on Treg number and function. These observations are not all consistent, however, and it is not known whether the observed effects on Treg are primary or secondary effects. To safely conduct targeted regulatory T-cell therapy in rheumatic diseases more knowledge about regulatory T-cell function in an inflammatory environment is needed that coincides with the initiative to elucidate the exact mechanism of current therapies.

An IVUS image-based approach for improvement of coronary plaque characterization

Virtual Histology-Intravascular Ultrasound (VH-IVUS) is widely used for studying atherosclerosis plaque composition. However, one of the main limitations of the VH-IVUS relates to its dependence to the Electrocardiogram (ECG)-gated acquisition. To overcome this limitation, this paper proposes a robust image-based approach for characterization of the plaques using IVUS images. The proposed method consists of three main steps of (1) shadow detection: as an efficient preprocessing step to identify and remove acoustic shadow regions; (2) feature extraction: a combination of gray-scale based features and textural descriptors; and (3) classification: to classify each pixel into one of the three classes (calcium, necrotic core and fibro-fatty). In order to evaluate the efficiency of the proposed algorithm two in-vivo and ex-vivo data sets are considered. The kappa values of 0.639 on in-vivo and 0.628 on ex-vivo tests with VH-IVUS and the histology images labeled by the experts respectively indicate the effectiveness of the proposed algorithm.

Tumour volume delineation in prostate cancer assessed by [11C]choline PET/CT: validation with surgical specimens

PET has been proven to be helpful in the delineation of gross tumour volume (GTV) for external radiation therapy in several tumour entities. The aim of this study was to determine if [(11)C]choline PET could be used to localize the carcinomatous tissue within the prostate in order to specifically target this area for example with high-precision radiation therapy. Included in this prospective study were 20 patients with histological proven prostate carcinoma who underwent [(11)C]choline PET/CT before radical prostatectomy. After surgical resection, specimens were fixed and cut into 5-mm step sections. In each section the area of the carcinoma was delineated manually by an experienced pathologist and digitalized, and the histopathological tumour volume was calculated. Shrinkage due to resection and fixation was corrected using in-vivo and ex-vivo CT data of the prostate. Histopathological tumour location and size were compared with the choline PET data. Different segmentation algorithms were applied to the PET data to segment the intraprostatic lesion volume. A total of 28 carcinomatous lesions were identified on histopathology. Only 13 (46 %) of these lesions had corresponding focal choline uptake. In the remaining lesions, no PET uptake (2 lesions) or diffuse uptake not corresponding to the area of the carcinoma (13 lesions) was found. In the patients with corresponding PET lesions, no suitable SUV threshold (neither absolute nor relative) was found for GTV segmentation to fit the volume to the histological tumour volume. The choline uptake pattern corresponded to the histological localization of prostate cancer in fewer than 50 % of lesions. Even when corresponding visual choline uptake was found, this uptake was highly variable between patients. Therefore SUV thresholding with standard algorithms did not lead to satisfying results with respect to defining tumour tissue in the prostate.

Harnessing Gene Expression Profiles for the Identification of Drug Resistance Genes in Pediatric AML

AbstractAbstract 283The vast majority of pediatric AML patients (>90%) achieve complete remission, however 30–40% relapse and face a dismal prognosis. Current therapy is insufficient as drug resistant cells survive chemotherapy; novel strategies are needed to overcome chemoresistance and improve outcome. The molecular basis underlying drug resistance in AML cells remains largely unknown. Based on the hypothesis that drug resistance in AML patients is largely due to intrinsic properties of leukemic blasts, we here correlated ex-vivo drug resistance data of primary patient samples to genome wide microarray gene expression profiles of AML blasts from diagnosis samples. Peripheral blood or bone marrow samples of 73 pediatric AML patients were enriched for leukemic blasts (median 89% blasts). Ex-vivo drug resistance towards cytarabine (ara-C, N=73), daunorubicin (DNR, N=69), etoposide (VP16, N=39) and cladribine (CDA, N=59) was assayed using the 4 days colorimetric MTT assay; median LC50 values are shown in Table 1. Genome wide expression profiling on the enriched samples was performed using the Affymetrix HGU 133 plus 2 platform (Balgobind et al, Hematologica, 2011).Spearman’s rank correlation analyses were used to correlate gene expression levels to the LC50 values, nominal p-values < 0.001 were considered significant. The number of significant probe sets for each drug is shown in Table 1. The strongest correlation of ex-vivo drug resistance and gene expression was found for VP16 (r2 ranged from −0.78 to 0.69 with p values ranging from 1×10−4 to 2×10−7 for the above mentioned 656 probes). The figure illustrates the correlation of ex-vivo DNR resistance with gene expression levels. We performed Gene Ontology (GO) enrichment analysis and Ingenuity Pathway Analysis (IPA) using expression values of the probe sets that were associated with ex-vivo resistance for each drug to gain insight in the possible cellular pathways involved. Chromatin remodeling, epigenetic regulation of gene expression and methyltransferase activity were among the top GO categories for ara-C resistance. For example, a high expression of MLL2, MLL4, ASXL1, and CARM1 was associated with high ara-C LC50 values. For DNR, GO and IPA indicated a role for response to growth factor stimuli and mitochondrial response to oxidative stress; examples of individual genes are shown in the Figure below. For VP16, a low expression of genes that are implicated in cell cycle, DNA replication and DNA damage response was associated with increased resistance. This included DNA polymerases, genes in BRCA1 signaling as well as the target of VP16, topoisomerase 2α. Upstream regulators that contribute to the gene expression profiles that were associated with ex-vivo drug resistance according to IPA are shown in Table 2. Interestingly, for DNR, VP16 and CDA the expression profiles in part explained by regulation via CD40L, a gene that has been associated with drug resistance in lymphatic leukemias. Targeted therapeutics are being developed to interfere in the CD40L mediated anti-apoptotic signaling and thus may offer alternative treatment options in drug resistant AML.Hence, we present novel data in which diagnosis samples of a relatively large group of pediatric AML patients were used to identify gene expression profiles that are associated with cellular drug resistance. These data may pave the way to the identification of genes that contribute to drug resistance, e.g. CD40L. Moreover, our findings may enhance the development of personalized treatment strategies by sensitizing patients to conventional chemotherapeutic drugs.Table 1Summary of ex-vivo drug resistance of primary AML blasts and its correlation with genome wide gene expression dataDrugLC50significant probe setsAra-C.360 (.182-.616)113DNR.172 (.093-.250)465VP162.65 (1.84-6.70)656CDA.020 (.004-.027)269LC50 = lethal concentration needed to kill 50% of the cells depicted as median ug/mL(p25-p75).Table 2Summary of pathway analysis of gene expression that correlated with ex-vivo drug sensitivityDrugtop 3 upstream regulatorsp range upstream regulatorsAra-CIL52.40×10−02DNRCD40L, IRF8, OSCAR7.4×10−4 to 4.4×10−5VP16CD40L, BRCA1, ACAT13.2×10−2 to 9.6×10−3CDACD40L, ASB2,IL10RB1.15×10−2 to 8.3×10−4Upstream regulators are ranked according to p-value. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

CDK1 Hyperphosphorylation Maintenance Drives the Time-course of G2-M Cell Cycle Arrest after Short Treatment with NAMI-A in Kb Cells

We investigated the molecular events of the ruthenium complex NAMI-A (0.1 mM for 1 h) on cell cycle G2-M arrest in KB carcinoma cells. Flow cytometry analysis showed a progressive accumulation of cells in S phase at 16 h, and in G2-M phase at 20 h after the end of treatment. NAMI-A pre-mitotic stop to cell proliferation was due to the maintenance of the phosphorylated, inactive, form of Cdk1, caused by the activation of the ATM/ATR checkpoint, as confirmed by the up-regulation and phosphorylation of Chk1. All these events are related to intracellular ruthenium accumulation, as confirmed by the lack of similar effects in cell lines unable to take the ruthenium compound up. Considering the dependence of NAMI-A cell cycle arrest on the dose and on the length of cell challenge, and considering the prolonged NAMI-A t1/2 in vivo in the lungs, we proved an even greater perturbation of the cell cycle regulating pathways in lung metastases of NAMI-A treated mice. The ex-vivo data confirm the interaction of the ruthenium compound NAMI-A with the ATM/ATR pathway, leading to the modulation of cell cycle regulating proteins, that can break the metastases cell cycle progression off.

Interaction of chandipura virus N and P proteins: identification of two mutually exclusive domains of N involved in interaction with P.

The nucleocapsid protein (N) and the phosphoprotein (P) of nonsegmented negative-strand (NNS) RNA viruses interact with each other to accomplish two crucial events necessary for the viral replication cycle. First, the P protein binds to the aggregation prone nascent N molecules maintaining them in a soluble monomeric (N0) form (N0-P complex). It is this form that is competent for specific encapsidation of the viral genome. Second, the P protein binds to oligomeric N in the nucleoprotein complex (N-RNA-P complex), and thereby facilitates the recruitment of the viral polymerase (L) onto its template. All previous attempts to study these complexes relied on co-expression of the two proteins in diverse systems. In this study, we have characterised these different modes of N-P interaction in detail and for the first time have been able to reconstitute these complexes individually in vitro in the chandipura virus (CHPV), a human pathogenic NNS RNA virus. Using a battery of truncated mutants of the N protein, we have been able to identify two mutually exclusive domains of N involved in differential interaction with the P protein. An unique N-terminal binding site, comprising of amino acids (aa) 1–180 form the N0-P interacting region, whereas, C-terminal residues spanning aa 320–390 is instrumental in N-RNA-P interactions. Significantly, the ex-vivo data also supports these observations. Based on these results, we suggest that the P protein acts as N-specific chaperone and thereby partially masking the N-N self-association region, which leads to the specific recognition of viral genome RNA by N0.

689 Bipolar electrogram signatures of therapeutic interest during human ventricular fibrillation

689 Bipolar electrogram signatures of therapeutic interest during human ventricular fibrillation

Split operator method for fluorescence diffuse optical tomography using anisotropic diffusion regularisation with prior anatomical information

Fluorescence diffuse optical tomography (fDOT) is an imaging modality that provides images of the fluorochrome distribution within the object of study. The image reconstruction problem is ill-posed and highly underdetermined and, therefore, regularisation techniques need to be used. In this paper we use a nonlinear anisotropic diffusion regularisation term that incorporates anatomical prior information. We introduce a split operator method that reduces the nonlinear inverse problem to two simpler problems, allowing fast and efficient solution of the fDOT problem. We tested our method using simulated, phantom and ex-vivo mouse data, and found that it provides reconstructions with better spatial localisation and size of fluorochrome inclusions than using the standard Tikhonov penalty term.

Controlling coagulation dysregulation in xenotransplantation.

Deletion of the α1,3-galactosyltransferase (GalT) gene in pigs has removed a major xenoantigen but has not eliminated the problem of dysregulated coagulation and vascular injury. Rejecting GalT knockout organ xenografts almost invariably show evidence of thrombosis and platelet sequestration, and primate recipients frequently develop consumptive coagulopathy. This review examines recent findings that illuminate potential mechanisms of this current barrier to successful xenotransplantation. The coagulation response to xenotransplantation differs depending on the type of organ and quite likely the distinct vasculatures. Renal xenografts appear more likely to initiate consumptive coagulopathy than cardiac xenografts, possibly reflecting differential transcriptional responses. Liver xenografts induce rapid and profound thrombocytopenia resulting in recipient death within days due to bleeding; ex-vivo data suggest that liver endothelial cells and hepatocytes are responsible for platelet consumption by a coagulation-independent process.It has been proposed that expression of recipient tissue factor on platelets and monocytes is an important trigger of consumptive coagulopathy. Finally, pigs transgenic for human anticoagulants and antithrombotics are slowly but surely coming on line, but have not yet been rigorously tested to date. Successful control of coagulation dysregulation in xenotransplantation may require different combinatorial pharmacological and genetic strategies for different organs.

The late phase of sepsis is characterized by an increased microbiological burden and death rate

IntroductionRecent models capturing the pathophysiology of sepsis and ex-vivo data from patients are speculating about immunosuppression in the so-called late phase of sepsis. Clinical data regarding survival and microbiological burden are missing. The aim of this study was to determine the clinical significance of the 'late phase' of sepsis with respect to overall survival and occurrence of microbiological findings.MethodsIn a retrospective trial, 16,041 patient charts from a university intensive care unit were screened, and 999 patients with severe sepsis or septic shock were identified. Three phases were established according to the mortality peaks which were separated by two distinct nadirs: phase I (days 1 to 5), phase II (days 6 to 15) and phase III (days 16 to 150). Patients were analyzed for outcome, SOFA scores, procalcitonin levels, antimicrobial treatment, dialysis, mechanical ventilation and results of blood cultures during their hospital stay.ResultsOut of 999 enrolled patients, 308 died during the course of sepsis presenting a characteristic mortality rate (30.8%) with three distinct mortality peaks (at days 2, 7 and 17). Overall 36.7% of all deaths occurred in the early phase (phase I) and 63.3% during the later phases (phase II + III). In total 2,117 blood cultures were drawn. In phase I, 882 blood cultures were drawn, representing a sampling rate of 88% with a positive rate of 14.9%. In phase II, 461 samples were taken, indicating a sampling rate of 52% and a positive rate of 11.3%. Within phase III, 524 samples were obtained representing a sampling rate of 66% with a positive rate of 15.3%, which was significantly higher compared to the positive rate of phase II and similar to phase I. In particular, the rate of typically opportunistic bacteria increased significantly from 9% in phase I up to 18% in phase III. The same is true for Candida spp. (phase I 13%, phase III 30%).ConclusionsThe later phase of sepsis is associated with a significant re-increase of positive blood culture results, especially regarding opportunistic bacteria and fungi. These observations warrant further studies focusing on the underlying mechanisms resulting in this outcome burden in the later phase of sepsis.

Comparative in vitro and ex-vivo myelotoxicity of aflatoxins B1 and M1 on haematopoietic progenitors (BFU-E, CFU-E, and CFU-GM): Species-related susceptibility

Haemato- and myelotoxicity are adverse effects caused by mycotoxins. Due to the relevance of aflatoxins to human health, the present study, employing CFU-GM-, BFU-E- and CFU-E-clonogenic assays, aimed at (i) comparing, in vitro, the sensitivity of human vs. murine haematopoietic progenitors to AFB1 and AFM1 (0.001–50 μg/ml), (ii) assessing whether a single AFB1 in vivo treatment (0.3–3 mg/kg b.w.) alters the ability of murine bone marrow cells to form myeloid and erythroid colonies, and (iii) comparing the in vitro with the in vitro ex-vivo data. We demonstrated (i) species-related sensitivity to AFB1, showing higher susceptibility of human myeloid and erythroid progenitors (IC 50 values: about 4 times lower in human than in murine cells), (ii) higher sensitivity of CFU-GM and BFU-E colonies, both more markedly affected, particularly by AFB1 (IC 50: 2.45 ± 1.08 and 1.82 ± 0.8 μM for humans, and 11.08 ± 2.92 and 1.81 ± 0.20 μM for mice, respectively), than the mature CFU-E (AFB1 IC 50: 12.58 ± 5.4 and 40.27 ± 6.05 μM), irrespectively of animal species, (iii) regarding AFM1, a species- and lineage-related susceptibility similar to that observed for AFB1 and (iv) lack of effects after AFB1 in vivo treatment on the proliferation of haematopoietic colonies.

The accuracy and precision of a micro computer tomography volumetric measurement technique for the analysis of in-vitro tested total disc replacements

Total disc replacements (TDRs) in the spine have been clinically successful in the short term, but there are concerns over long-term failure due to wear, as seen in other joint replacements. Simulators have been used to investigate the wear of TDRs, but only gravimetric measurements have been used to assess material loss. Micro computer tomography (microCT) has been used for volumetric measurement of explanted components but has yet to be used for in-vitro studies with the wear typically less than < 20 mm3 per 10(6) cycles. The aim of this study was to compare microCT volume measurements with gravimetric measurements and to assess whether microCT can quantify wear volumes of in-vitro tested TDRs. microCT measurements of TDR polyethylene cores were undertaken and the results compared with gravimetric assessments. The effects of repositioning, integration time, and scan resolution were investigated. The best volume measurement resolution was found to be +/- 3 mm3, at least three orders of magnitude greater than those determined for gravimetric measurements. In conclusion, the microCT measurement technique is suitable for quantifying in-vitro TDR polyethylene wear volumes and can provide qualitative data (e.g. wear location), and also further quantitative data (e.g. height loss), assisting comparisons with in-vivo and ex-vivo data. It is best used alongside gravimetric measurements to maintain the high level of precision that these measurements provide.

A β3 Asp217→Val substitution in a patient with variant Glanzmann Thrombasthenia severely affects integrin αIIBβ3 functions

A dysfunctional Glanzmann Thrombasthenia (variant) is a rare bleeding disorder due to qualitative abnormalities of platelets alphaIIBbeta3 heterodimers. Dynamically conformational change of alphaIIBbeta3 is a complex mechanism that is not fully understood. For these reasons, genotyping and functional analysis of variant Glanzmann Thrombasthenia is important to elucidate the molecular basis of alphaIIBbeta3 receptor functions. In this report, we have analyzed the molecular effects of an A>T substitution leading to an amino acid change, D217>V, in the beta3 integrin gene identified in patients with variant Glanzmann Thrombasthenia. As the D217 residue is highly conserved among all seven beta integrin subunits and among beta3 integrins of different species, we tested the effect on the phenotype of the D217V mutation by cotransfecting the beta3 mutant (V217) or wild-type beta3 (D217) construct with the wild-type alphaIIb into eukaryotic Chinese hamster ovary cells. Levels of mutant alphaIIBbeta3 heterodimers on Chinese hamster ovary cell surface were lightly reduced as compared with the wild type. Functional investigation of alphaIIBbeta3 V217 on Chinese hamster ovary cell surface was carried out, as fibrinogen binding, adhesion and aggregation tests showed a substantial reduction in respect to the control sample. Our results confirm ex-vivo data and suggest that the D217 amino acid is required for alphaIIBbeta3 receptor interactions with fibrinogen.

In-vitro evaluation of coronary stents and 64-detector-row computed tomography using a newly developed model of coronary artery stenosis

Stent implantation is the predominant therapy for non-surgical myocardial revascularization in patients with coronary artery disease. However, despite substantial advances in multidetector computed tomography (MDCT) coronary imaging, a reliable detection of coronary in-stent restenosis is currently not possible. To examine the ability of 64-detector-row CT to detect and to grade in-stent stenosis in coronary stents using a newly developed ex-vivo vessel phantom with a realistic CT density pattern, artificial stenosis, and a thorax phantom. Four different stents (Liberté and Lunar ROX, Boston Scientific; Driver, Medtronic; Multi-Link Vision, Guidant) were examined. The stents were placed on a polymer tube with a diameter of 2.5, 3.0, 3.5, or 4.0 mm. Different degrees of stenosis (0%, 30%, 50%, 70-80%) were created inside the tube. For quantitative analysis, attenuation values were measured in the non-stenotic vessel outside the stent, in the non-stenotic vessel inside the stent, and in the stenotic area inside the stent. The grade of stenosis was visually assessed by two observers. All stents led to artificial reduction of attenuation, the least degree of which was found in the Liberté stent (11.3+/-10.2 HU) and the Multi-Link Vision stent (17.6+/-17.9 HU; P = 0.25). Overall, the non-stenotic vessel was correctly diagnosed in 55.5%, the low-grade stenosis in 58.3%, the intermediate stenosis in 63.8%, and the high-grade stenosis in 80.5%. In the 3.0-, 3.5-, and 4.0-mm vessels, in none of the cases was a non-stenotic or low-grade stenotic vessel misdiagnosed as intermediate or high-grade stenosis. The average deviation from the real grade of stenosis was 0.40 for the Liberté stent, 0.46 for the Lunar ROX stent, 0.45 for the Driver stent, and 0.58 for the Multi-Link Vision stent. Our ex-vivo data show that non-stenotic stents and low-grade in-stent stenosis can be reliably differentiated from intermediate and high-grade in-stent stenosis in vessels with a diameter of 3 to 4 mm. With regard to artifacts and the grading of stenoses, the Liberté stent was best suited for CT coronary angiography.

A predictive mechano-biological model of the bone-implant healing

The quality of the fixation orthopaedic implant to its surrounding bone determines its clinical longevity. Up to 20% of hip replacement operations are currently revisions for aseptic loosening. While this fixation quality is determined primarily by the bone and tissue anchoring the implant, conditions influencing bone growth in the early post-operative period include the surgical technique and coupled mechanical and biochemical factors. The aim of the study was to propose an original mechano-biological formulation of the healing process of periprosthetic tissue. The multiphasic porous model involved the solid osseous matrix, the extracellular fluid phase, the osteoblastic cellular phase responsible from the bone formation and the growth factor phase promoting the cellular activity. To derive the non-linear convective-diffuse governing equations, mass balance was associated to cell active haptotactic and chemotactic migration, growth factor diffusion, cell proliferation (logistic law) and bone formation (reactive medium). The in-vivo application concerned a canine axisymmetric implant which was stable and mechanically unloaded. Predictive numerical results were compared to ex-vivo data from a histologic study. The generic healing pattern involving two main oscillations of the radial bone formation was well predicted. In the future, the model could assist in evaluating the role of growth factor concentrations and their temporal delivering as far as the role of pertinent sources such as bioactive coating or additional biomaterials.

Tyrolean Tensiometer: a new instrument for easy intraoperative tension measurement before vascular anastomosis

In reconstructive surgery microsurgical repair of dissected arteries sometimes has to be done under longitudinal tension. Guidelines to support an objective decision on whether tension associated with direct suture is acceptable or whether grafting is needed, do not exist. All experimental data found concerned the clinical outcome of a certain length defect treated in various animal models. The aim of this study was to show the feasibility of a new instrument for measuring the tension required to adapt arterial stumps, thereby allowing surgical outcome to be assessed before beginning anastomosis. A modified tension spring balance was used to measure the force applied to the arterial stumps before knotting. Twenty-four rat femoral arteries were dissected, segments of up to 9mm were resected, and the tension needed for approximation was measured. These ex-vivo data were combined with clinical outcome data of previous animal trials. The tension measured increased proportionally to the size of the arterial gap created. The correlation between tension and arterial gap was found to be almost linear. The average additional time required for tension measurement using the Tyrolean Tensiometer was 13 (+/-6) s. High anastomotic tension may cause critical or even poor clinical outcome. None of the tension measurement methods described so far allow intraoperative measurement at a time when changes in strategy are still possible. The Tyrolean Tensiometer for the first time allows fast and reliable measurement of the tension acting on the first suture of an anastomosis.

Styrene-7,8-Oxide Burden in Ventilated, Perfused Lungs of Mice and Rats Exposed to Vaporous Styrene

Styrene (ST) is an important industrial chemical. In long-term inhalation studies, ST-induced lung tumors in mice but not in rats. To test the hypothesis that the lung burden by the reactive metabolite styrene-7,8-oxide (SO) would be most relevant for the species-specific tumorigenicity, we investigated the SO burden in isolated lungs of male Sprague-Dawley rats and in-situ prepared lungs of male B6C3F1 mice ventilated with air containing vaporous ST and perfused with a modified Krebs-Henseleit buffer (37 degrees C). Styrene vapor concentrations were determined in air samples collected in the immediate vicinity of the trachea. They were almost constant during each experiment. Styrene exposures ranged from 50 to 980 ppm (rats) and from 40 to 410 ppm (mice). SO was quantified from the effluent perfusate. Lungs of both species metabolized ST to SO. After a mathematical translation of the ex-vivo data to ventilation and perfusion conditions as they are occurring in vivo, a species comparison was carried out. At ST concentrations of up to 410 ppm, mean SO levels in mouse lungs ranged up to 0.45 nmol/g lung, about 2 times higher than in rat lungs at equal conditions of ST exposure. We conclude that the species difference in the SO lung burden is too small to consider the genotoxicity of SO as sufficient for explaining the fact that only mice developed lung tumors when exposed to ST. Another cause is considered as driving force for lung tumor development in the mouse.

Clinical involvement of the tonsillar immune system in IgA nephropathy

The temporal association of tonsillitis and hematuria or proteinuria in IgA nephropathy suggests that there might be a link between the physiological properties of the secondary lymphoid organ that tonsils represent and the mesangial deposition of IgA characteristic of this nephropathy. A number of clinical and ex-vivo data support this hypothesis. One of the earliest was the demonstration of the dimeric nature of mesangial IgA, composed of IgA monomers linked by a J chain, yet lacking the polyIg receptor acquired by secretory IgA during transcytosis through epithelial cells. This molecular structure is that of IgA synthesized in human tonsils, the epithelium of which lacks polyIg receptor. Moreover, tonsils from patients with IgA nephropathy display an abnormal partition of IgG and IgA producing plasma cells associated with a significantly developed web of high endothelial venules. IgA nephropathy could thus be in part related to an alteration of IgA precursors homing in tonsils. Tonsillectomy thus would present the advantage of removing an abnormally functioning source of dimeric IgA. Performed early enough in the course of the renal disease, tonsillectomy could suffice to halt the development of the nephropathy and restore the kidneys to health.

Measurement of the activated clotting time during cardiopulmonary bypass: differences between Hemotec® ACT and Hemochron® Jr apparatus

Measurement of the activated clotting time (ACT) represents a standard method for coagulatory assessments. The test employs specific agents to trigger the coagulation process. The present study aimed to compare kaolin (Hemotec) versus a combination of silica, kaolin and phospholipid (Hemochron Jr) ACTs. Hemotec and Hemochron Jr ACT monitors were compared by simultaneous measurement of paired arterial blood samples (n = 114) with respect to precision and bias during clinical conditions of cardiopulmonary bypass (CPB). The influence of haemodilution on the ACT was tested in an ex-vivo model. The precision of Hemotec and Hemochron Jr ACT measurements attained 21 +/- 2.6 s versus 27.0 +/- 2.6 s (p = 0.126) during CPB and 2.5 +/- 2.2 s versus 9.4 +/- 6.9 s (p = 0.000) after protamine administration, respectively. The Hemochron Jr monitor was associated with a bias of -102 +/- 13.7 s compared to the Hemotec ACT monitor (p = 0.000) during CPB and -6.9 +/- 2.9 s after protamine (p = 0.025). Linear regression analysis of ACT readings between monitors reached r = 0.526 (p = 0.000). Hemochron Jr ACT values correlated with the erythrocyte volume fraction r = 0.379 (p = 0.000). Ex-vivo data indicated that the Hemotec ACT monitor was associated with relatively higher ACT readings after haemodilution. The ACT is not a standardized measure. Test results are strongly associated with the specific compounds used to initiate the coagulation process.

NON-INVASIVE IDENTIFICATION OF PHYSIOLOGICALLY SIGNIFICANT PUMPING STATES IN IMPLANTABLE ROTARY BLOOD PUMPS USED AS LEFT VENTRICULAR ASSIST DEVICES

Non-invasive detection of dangerous pumping states is important for the control of rotary blood pumps. The VentrAssistTM LVAD, is an implantable centrifugal rotary blood pump (iRBP), the impeller supported by a hydrodynamic bearing. In-vitro (pulsatile mock-loop) and ex-vivo (acute sheep) experiments (N=3) were conducted. By perturbing system parameters of the pulsatile mock loop and by physiological intervention through pharmacological or mechanical methods in the ovine experiments, and by varying iRBP impeller speed, it was possible to transition through five physiologically significant pumping states. These states were defined as regurgitant pump flow, ventricular ejection, continuous aortic valve closure, partial sucking of the ventricle, and total ventricular collapse, states confirmed in the experiments through observation and through implanting of additional flow and pressure sensors. Using instantaneous impeller speed and pump input power, we investigated the detection of these states without the use of additional sensors. Indices based on pump flow, flow amplitude and flow waveform symmetry were analyzed using classification and regression tree methods. The classification tree derived from the in-vitro experiments was applied to the ex-vivo data with perfect accuracy (0% false positives and negatives in predicting the pumping states). These data indicate that the state detection methods may be a valuable mechanism in optimal LVAD control and operational diagnosis