Testing the Efficacy of Contrast-Enhanced Ultrasound in Detecting Transplant Rejection Using a Murine Model of Heart Transplantation.

One of the key unmet needs to improve long-term outcomes of heart transplantation is to develop accurate, noninvasive, and practical diagnostic tools to detect transplant rejection. Early intragraft inflammation and endothelial cell injuries occur prior to advanced transplant rejection. We developed a novel diagnostic imaging platform to detect early declines in microvascular perfusion (MP) of cardiac transplants using contrast-enhanced ultrasonography (CEUS). The efficacy of CEUS in detecting transplant rejection was tested in a murine model of heart transplants, a standard preclinical model of solid organ transplant. As compared to the syngeneic groups, a progressive decline in MP was demonstrated in the allografts undergoing acute transplant rejection (40%, 64%, and 92% on days 4, 6, and 8 posttransplantation, respectively) and chronic rejection (33%, 33%, and 92% on days 5, 14, and 30 posttransplantation, respectively). Our perfusion studies showed restoration of MP following antirejection therapy, highlighting its potential to help monitor efficacy of antirejection therapy. Our data suggest that early endothelial cell injury and platelet aggregation contributed to the early MP decline observed in the allografts. High-resolution MP mapping may allow for noninvasive detection of heart transplant rejection. The data presented have the potential to help in the development of next-generation imaging approaches to diagnose transplant rejection.

The effect of beta blocker withdrawal on myocardial SPECT modeled from adenosine 13N-ammonia PET.

AIM: The effect of beta blockers (BB) on myocardial imaging has been studied in several SPECT and PET studies with divergent results concerning perfusion and impact on diagnostic accuracy. The present study evaluated the effect of BB withdrawal on virtual SPECT studies modeled from quantitative PET perfusion scans. PATIENTS, METHODS: Data from 20 CAD patients scheduled for adenosine 13N-ammonia imaging with and without BB were considered. Modeling the uptake characteristics of 99mTc-MIBI, all parametric stress PET polarmaps were transferred to virtual 20-segment SPECT polarmaps. The SPECT studies were categorized with a 5-point score and read to assess the effect of the BB withdrawal on scan result and interpretation. RESULTS: The SPECT analysis revealed a mean score of 6.0 ± 4.7 with, and of 5.9 ± 4.5 without BB (p = 0.84). In 260 (74.9%) segments the scores were equal in both conditions. Without BB a downstaging was recorded in 44 segments (12.7%), an upstaging in 43 segments (12.4%). An essentially different interpretation (shift from medical therapy recommendation to angiography) was recorded in one patient. In six cases the interpretation differed mildly. CONCLUSION: In the majority of patients studied, scan results and interpretation remain unchanged after discontinuation of the BB. Nevertheless, the segmental scan results are not uniformly affected. The recommendation to stop BBs prior to stress testing in order to ensure the highest MBF remains advisable. If temporary BB withdrawal is unfeasible due to contraindications, a tight clinical schedule, or because a patient forgot to withhold the BB, it is appropriate to perform adenosine stress testing according to the results of this study.

The role of lectins and glycans in platelet clearance.

In recent years, it has become increasingly apparent that the life span of transfused platelets in circulation is regulated, at least in part, by glycan-lectin mediated mechanisms. There is clear evidence that refrigerated platelets are cleared by glycan-lectin mediated clearance mechanisms. Acute platelet cooling clusters glycoprotein (GP) Ibα receptors bearing uncovered N-acetylglucosamine (GlcNAc), and α(M) ß(2) integrins on hepatic macrophages recognise clustered GlcNAc to rapidly clear these platelets from circulation. With prolonged refrigeration GPIbα clustering bearing uncovered galactose increases, which mediates the removal of long-term refrigerated platelets via hepatic Ashwell-Morell receptors (AMR), originally named as asialoglycoprotein receptors. In contrast, little is known about the molecular mechanisms of transfused room temperature platelet clearance. This review examines the role of glycan-lectin mediated clearance of exogenous, that is transfused chilled platelet clearance and briefly addresses the current knowledge of stored platelet function, degradation and its relation to platelet clearance.

Mechanisms of cold-induced platelet actin assembly.

Various agonists but also chilling cause blood platelets to increase cytosolic calcium, polymerize actin, and change shape. We report that cold increases barbed end nucleation sites in octyl glucoside-permeabilized platelets by 3-fold, enabling analysis of the intermediates of this response. Although chilling does not change polyphosphoinositide (ppI) levels, a ppI-binding peptide completely inhibits cold-induced nucleation. The C terminus of N-WASp, which inhibits the Arp2/3 complex, blocks nucleation by 40%; GDPbetaS, N17Rac and N17Cdc42 have no effects. Some gelsolin translocates to the detergent-insoluble cytoskeleton after cooling. Chilled platelets from gelsolin-deficient mice have approximately 50% fewer new actin nuclei compared with platelets from wild-type mice. EGTA completely inhibits gelsolin translocation into the cytoskeleton, and the small amount of gelsolin initially there becomes soluble. Chilling releases adducin from the detergent-resistant cytoskeleton. We conclude that platelet actin filament assembly induced by cooling involves ppI-mediated actin filament barbed end uncapping and de novo nucleation independently of surface receptors or downstream signaling intermediates besides calcium. The actin-related changes occur in platelets at temperatures below 37 degrees C, suggesting that the platelet may be more activable at temperatures at the body surface than at core temperature, thereby favoring superficial hemostasis over internal thrombosis.

Autosomal dominant macrothrombocytopenia with leukocyte inclusions (May-Hegglin anomaly) is linked to chromosome 22q12-13.

Macrothrombocytopenia with leukocyte inclusions (May-Hegglin anomaly) is a rare autosomal dominant disorder characterized by thrombocytopenia, giant platelets, and Döhle body-like inclusions in leukocytes. To determine the genetic basis of this disorder, we performed a genome-wide screen for linkage in three families with May-Hegglin anomaly. For the pooled analysis of the three families, three markers on chromosome 22 had two-point logarithm-of-difference (lod) scores greater than 3, with a maximum lod score of 3.91 at a recombination fraction (theta) of 0.076 for marker D22S683. Within the largest family (MHA-1), the maximum lod score was 5.36 at theta=0 at marker D22S445. Fine mapping of recombination events using eight adjacent markers indicated that the minimal disease region of family MHA-1 alone is in the approximately 26 cM region from D22S683 to the telomere. The maximum lod score for the three families combined was 5.84 at theta=0 for marker IL2RB. With the assumption of locus homogeneity, haplotype analysis of family MHA-4 indicated the disease region is centromeric to marker D22S1045. These data best support a minimal disease region from D22S683 to D22S1045, a span of about 1 Mb of DNA that contains 17 known genes and 4 predicted genes. Further analysis of this region will identify the genetic basis of May-Hegglin anomaly, facilitating subsequent characterization of the biochemical role of the disease gene in platelet formation.

Development of a new biodegradable intravascular polymer stent with simultaneous incorporation of bioactive substances.

OBJECTIVE: Due to the thrombogenicity and permanent implant nature of metallic stents, bioresorable synthetic polymers have been proposed for stents and local drug delivery systems. Bioresorbable polyesters like poly(D,L-lactide) demonstrated excellent biocompatibility in various tissues. This paper describes a novel method for the molding of these polymers. The specific CESP-process (Controlled Expansion of Saturated Polymers) is characterised by the use of the plasticizer carbon dioxide and allows the incorporation of bioactive substances at physiologic temperatures into the polymer bulk and the production of complex designed implants. METHODS: The CESP-process is characterised by the exposure of an amorphous polymer to an inert gas at high pressure with a significant lower glass transition point. The plasticizing effect makes it possible to process polylactides at a temperature close to room temperature. The low process temperature constitutes a key advantage for thermally sensitive polymers and allows the incorporation of thermally sensitive pharmaceutical additives. To obtain some preliminary information on the biocompatibility, in vitro cell toxicity testing as well as drug release assessment was performed. RESULTS: Different polymer sheets were produced using the CESP-process. Cytotoxicity was not observed in any molded polymer material. According to the mechanical and biocompatibility results Poly(D,L-lactide) (P-DL-LA) was investigated in the CESP-process. Finite element analysis was used to test the possible geometry of an adequate stent. A helical design was chosen and a stent-prototype was produced using the CESP-process. Peroxidase activity as an incorporated marker enzyme could be measured over 6 weeks. Different drug release profiles were obtained due to various pore sizes of the polymer. CONCLUSIONS: The new CESP-process can be used to process biodegradable polymers and to mold different stent geometries without inducing cytotoxic effects to the material. Furthermore, this procedure permits the simultaneous incorporation of bioactive substances during the molding process. Drug release kinetics can be regulated by different pore sizes of the material.

T-cell-rich B-cell lymphoma and Epstein-Barr virus infection of the uterus in a postmenopausal patient with an intrauterine contraceptive device in place for over 20 years.

Although secondary involvement of the female genital tract occurs in up to 40% of cases of disseminated lymphomas, lymphomas presenting with primary female genital tract symptomatology are very unusual. We report a case of T-cell-rich B-cell lymphoma (TCRBCL) arising in the uterine corpus of a 57-year-old female who carried an intrauterine contraceptive device (IUD) for over 20 years. Malignant lymphoid cells expressed the Epstein-Barr virus (EBV) late membrane protein (LMP), a feature described in TCRBCL but not previously reported in primary uterine lymphomas. To our knowledge, this is the first reported case of a TCRBCL of the uterus.

Transforming growth factor beta 1, a major stimulator of hyaluronan synthesis in human synovial lining cells.

OBJECTIVE: To investigate the role of cytokines and growth factors in the regulation of hyaluronan synthesis in human synovial lining cells. METHODS: Synovial lining cells were obtained from human knee joints, isolated by the explant method, and characterized by immunocytochemistry using monoclonal antibodies against monocyte/macrophage markers as well as antibodies against hyaluronan synthase. After stimulation by cytokines and growth factors, hyaluronan was measured by radiometric assay. The molecular weight distribution of the hyaluronan synthesized was determined by high-performance gel-permeation liquid chromatography. To test the effect of oxygen-derived free radicals, the concentration and molecular weight distribution of hyaluronan were determined in the presence and absence of catalase and superoxide dismutase. RESULTS: Hyaluronan synthesis was stimulated in synovial lining cells by transforming growth factor beta 1 (TGF beta 1), interleukin-1 beta (IL-1 beta), and to a lesser extent by tumor necrosis factor alpha (TNF alpha). Analysis of the molecular weight distribution of hyaluronan after stimulation of synovial lining cells with TGF beta 1, IL-1 beta, and TNF alpha indicated that hyaluronan is synthesized in a high molecular weight form and might be degraded in the course of inflammatory processes by oxygen-derived free radicals. CONCLUSION: Our findings suggest that TGF beta 1 is a major stimulator of hyaluronan synthesis in human synovial lining cells and might be involved in the pathogenic mechanisms of joint swelling in inflammatory and degenerative joint diseases.

Risk of ventricular dysrhythmias during 1-hour infusions of amphotericin B in patients with preserved renal function.

In order to assess the safety of 1-h infusions of amphotericin B (AMB), we prospectively monitored 213 1-h infusions of AMB (dose range, 0.27 to 0.89 mg/kg of body weight) in 27 patients with creatinine clearances of > 25 ml/min. Holter monitor tracings during 1-h infusions were compared with those during a 4-h baseline period of monitoring. There were no ventricular dysrhythmias during 1-h infusions of AMB that were not present during baseline monitoring. Nausea and/or rigors were noted for 32 (15%) infusions in six (22%) patients. No patient exhibited a temperature rise of > 1 degree C. We conclude that, in doses of up to 0.9 mg/kg, AMB does not appear to induce asymptomatic ventricular dysrhythmias when administered over 1 h to patients with creatinine clearances of > 25 ml/min.

[EBV in malignant lymphomas]. / EBV bei malignen Lymphomen.

PCR and in-situ-Hybridization were used to detect EBV-DNA within Hodgkin- and Non-Hodgkin-Lymphomas. By the use of primers for the Bam H1 W fragment we could show EBV-DNA to be associated with 45% Hodgkin- and 25% T-Lymphomas, whereas we could not find EBV-DNA within B-Lymphomas. In about one third of the PCR-positive cases we could localize EBV-DNA mostly in the tumor cell nuclei by in-situ-hybridization.