Gefitinib targets ZAP-70-expressing chronic lymphocytic leukemia cells and inhibits B-cell receptor signaling.

Chronic lymphocytic leukemia (CLL) can be divided into groups based on biomarkers of poor prognosis. The expression of the tyrosine kinase ZAP-70 (member of the Syk tyrosine kinase family) in CLL cells is associated with shorter overall survival in CLL patients. Currently, there is a lack of targeted therapies for patients with ZAP-70 expression in CLL cells. The tyrosine kinase inhibitor gefitinib has been shown to be effective at induce apoptosis in acute myeloid leukemia through inhibition of Syk. In this study, we sought to test the efficacy of gefitinib in primary human ZAP-70+ CLL cells. We demonstrate that gefitinib preferentially induces cell death in ZAP-70-expressing CLL cells with a median IC50 of 4.5 µM. In addition, gefitinib decreases the viability of ZAP-70+ Jurkat T leukemia cells but fails to affect T cells from CLL patients. Western blot analysis shows gefitinib reduces both basal and B-cell receptor (BCR)-stimulated phosphorylation of Syk/ZAP-70, ERK, and Akt in ZAP-70+ CLL cells. Moreover, gefitinib inhibits the pro-survival response from BCR stimulation and decreases pro-survival proteins such as Mcl-1. Finally, ZAP-70 expression sensitizes Raji cells to gefitinib treatment. These results demonstrate that gefitinib specifically targets ZAP-70+ CLL cells and inhibits the BCR cell survival pathway leading to apoptosis. This represents the likelihood of tyrosine kinase inhibitors being effective targeted treatments for ZAP-70+ CLL cells.

Epidermal and dermal integration into sphere-templated porous poly(2-hydroxyethyl methacrylate) implants in mice.

Percutaneous medical devices remain susceptible to infection and failure. We hypothesize that healing of the skin into the percutaneous device will provide a seal, preventing bacterial attachment, biofilm formation, and subsequent device failure. Porous poly(2-hydroxyethyl methacrylate) [poly(HEMA)] with sphere-templated pores (40 microm) and interconnecting throats (16 microm) were implanted in normal C57BL/6 mice for 7, 14, and 28 days. Poly(HEMA) was either untreated, keeping the surface nonadhesive for cells and proteins, or modified with carbonyldiimidazole (CDI) or CDI reacted with laminin 332 to enhance adhesion. No clinical signs of infection were observed. Epidermal and dermal response within the poly(HEMA) pores was evaluated using light and transmission electron microscopy. Cells (keratinocytes, fibroblasts, endothelial cells, inflammatory cells) and basement membrane proteins (laminin 332, beta4 integrin, type VII collagen) could be demonstrated within the poly(HEMA) pores of all implants. Blood vessels and dermal collagen bundles were evident in all of the 14- and 28-day implants. Fibrous capsule formation and permigration were not observed. Sphere-templated polymers with 40 microm pores demonstrate an ability to recapitulate key elements of both the dermal and the epidermal layers of skin. Our morphological findings indicate that the implant model can be used to study the effects of biomaterial pore size, pore interconnect (throat) size, and surface treatments on cutaneous biointegration. Further, this model may be used for bacterial challenge studies.

16-detector row computed tomographic coronary angiography in patients undergoing evaluation for aortic valve replacement: comparison with catheter angiography.

AIM: To evaluate the diagnostic accuracy of 16-detector row computed tomography (CT) in assessing haemodynamically significant coronary artery stenoses in patients under evaluation for aortic stenosis pre-aortic valve replacement. SUBJECTS AND METHODS: Forty consecutive patients under evaluation for severe aortic stenosis and listed for cardiac catheterization before potential aortic valve replacement underwent coronary artery calcium (CAC) scoring and retrospective electrocardiogram (ECG)-gated multi-detector row computed tomographic coronary angiography (MDCTA) using a GE Lightspeed 16-detector row CT within 1 month of invasive coronary angiography (ICA) for comparative purposes. All 13 major coronary artery segments of the American Heart Association model were evaluated for the presence of > or =50% stenosis and compared to the reference standard. Data were analysed on a segment-by-segment basis and also in "whole patient" terms. RESULTS: A total of 412/450 segments from 35 patients were suitable for analysis. The overall accuracy of MDCTA for detection of segments with > or =50% stenosis was high, with a sensitivity of 81.3%, specificity 95.0%, positive predictive value (PPV) 57.8%, and negative predictive value (NPV) 98.4%. On a "whole-patient" basis, 100% (19/19) of patients with significant coronary disease were correctly identified and there were no false-negatives. Excluding patients with CAC >1000 from the analysis improved the accuracy of MDCTA to: sensitivity 90%, specificity 98.1%, PPV 60%, NPV 99.7%. CONCLUSION: Non-invasive 16-detector row MDCTA accurately excludes significant coronary disease in patients with severe aortic stenosis undergoing evaluation before aortic valve replacement and in whom ICA can therefore be avoided. Its segment-by-segment accuracy is improved further if CAC>1000 is used as a gatekeeper to MDCTA.

Highly accurate coronary angiography with submillimetre, 16 slice computed tomography.

OBJECTIVE: To assess submillimetre coronary computed tomographic angiography (CTA) in comparison with invasive quantitative coronary angiography as the gold standard and to examine the effect of significant coronary artery calcification (CAC), which is known to impede lumen visualisation, on the accuracy of the examination. METHODS: After invasive coronary angiography, 58 patients underwent coronary imaging with a GE Lightspeed 16 computed tomography (CT) system. CAC was quantified after an ECG triggered acquisition with a low tube current. Coronary CTA was performed with retrospective ECG gating and a 16 x 0.63 mm collimation and was reconstructed with an effective 65-250 ms temporal resolution. All 13 major coronary artery segments were evaluated for the presence of > or = 50% stenosis, and compared with the gold standard. RESULTS: One patient moved and could not be evaluated. All segments (except occluded segments) were evaluated for 57 patients. Overall the accuracy of coronary CTA for detection of > or = 50% stenosis was: sensitivity 83%, specificity 97%, positive predictive value 80%, and negative predictive value 97%. The number of diseased coronary arteries was correctly diagnosed in 34 of 38 (89%) patients overall. Altogether 21 of 57 (37%) patients had a CAC score > or = 400, which was predefined as representing significant CAC. Excluding these patients from the analysis improved the accuracy of coronary CTA to a sensitivity of 89%, specificity 98%, positive predictive value 79%, and negative predictive value 99%. CONCLUSIONS: Non-invasive coronary angiography with submillimetre CT is reliable and accurate. It appears that a subgroup of patients may be selected based on CAC score in whom the investigation has even higher accuracy. Coronary CTA has reached the stage where it should be considered for a clinical role. Further research is required to define this role.

Three dimensional volume quantification of aortic valve calcification using multislice computed tomography.

OBJECTIVE: To assess a new multislice computed tomography (CT) technique for three dimensional quantification of aortic valve calcification volume (3D AVCV) and to study the relation between stenosis and calcification of the aortic valve. METHODS: 50 patients with echocardiographic calcification of the aortic valve underwent two separate ECG triggered multislice CT for quantification of 3D AVCV. The agreement between the two 3D AVCV scores was assessed and 3D AVCV was compared with echocardiographic markers of severity of aortic stenosis. RESULTS: Overall the level of agreement between the two 3D AVCV scores was excellent (median interscan variability 7.9% (interquartile range 10.1); correlation coefficient, r = 0.99; repeatability coefficient 237.8 mm3 (limits of agreement -393 to 559 mm3)). However, the magnitude of the 3D AVCV did influence the interscan variability. The 3D AVCV correlated closely with the maximal predicted transvalvar gradient (r2 = 0.77) and aortic valve area (r2 = 0.73). CONCLUSIONS: Multislice CT provides a technique for quantifying 3D AVCV that has good reproducibility. There is a close non-linear relation between echocardiographic parameters of severity of valve stenosis and 3D AVCV scores.

Multislice computed tomography cardiac imaging: current status.

Non-invasive CT coronary artery imaging has previously had little relevance to most UK radiologists due to the limited availability of electron beam CT scanners. Major advances in CT technology have promoted new applications for helical CT, which include cardiac imaging. Widespread installation of 'multislice' helical CT scanners will make CT coronary artery imaging available for the first time in many UK hospitals. The technical advances and early clinical trial data are reviewed and multislice helical CT cardiac imaging in general is discussed.

New insights into onset mechanisms of atrial fibrillation and flutter after coronary artery bypass graft surgery.

OBJECTIVE: To determine by means of a monitoring study the onset mechanisms of atrial fibrillation (AF) after coronary artery bypass graft surgery (CABG). PATIENTS AND DESIGN: During elective CABG, 81 patients had one bipolar atrial and one ventricular epicardial electrode attached. These were connected to a Vitatron 900E external pacing device, which monitored the patients for four consecutive days. 12 lead ECGs were obtained if AF was clinically detected and Holter ECGs were obtained in 8 (33%) of these patients. RESULTS: 24 patients (30%) developed paroxysmal AF (50%), atrial flutter (17%), or both (33%). The number of AF episodes varied from 1-169 a day (median 5) and were usually of short duration (median 2.25 minutes). Pacemaker diagnostics showed much intrapatient and interpatient variability in onset mechanisms but the majority of AF onsets (71%) were preceded by either short runs of AF or multiple atrial extrasystoles. The final trigger was a conducted atrial extrasystole in 72% of cases. There were no bradycardic triggers. The Holter ECGs confirmed the device's data. CONCLUSIONS: The onset mechanisms of post-CABG AF are dominated by atrial extrasystoles with multiple atrial extrasystoles and short runs of AF preceding the main AF onset in the majority of cases. These results have major implications for the development of new preventive pacing algorithms.

Aortic valve imaging with computed tomography: a review.

The finding of aortic valve calcification is of clinical relevance. Thickening and calcification of the aortic valve ('aortic sclerosis') may progress over time to calcific aortic stenosis, and calcification of the aortic valve has prognostic importance even in the absence of valve obstruction. Aortic valve calcification may also have effects on the conduction system. There is progressive awareness of the need for an imaging technique that can accurately and reproducibly quantify calcification of native and prosthetic aortic valves. Through adaptation of techniques from electron beam computed tomography (CT) coronary calcium scoring, CT has been proposed as the appropriate imaging modality. Although originally described as a method of comparing the calcification of different aortic valve bioprostheses, the major role suggested for CT aortic valve calcium quantification is now in the field of preventive medicine. This has stemmed from the recognition that traditional vascular risk factors also have a role in the etiology of calcific aortic stenosis. Subsequently, the realization that pharmacological modification of lipid profiles may result in slowing of progression or even regression of aortic valve calcification has led to a need to quantify aortic valve calcification for follow up purposes. Echocardiography has been used to estimate aortic valve calcification in studies of the natural history of aortic stenosis, but it does not accurately quantify calcium. CT appears able to fulfil this requirement, though the technique is still relatively novel. This review examines the need for aortic valve calcium quantification and the evolution of imaging to the current status. Future directions and the promise of new helical CT technologies with respect to cardiac imaging are explored.

Churg-Strauss syndrome with critical endomyocardial fibrosis: 10 year survival after combined surgical and medical management.

A case is presented of the Churg-Strauss syndrome with hypereosinophilia and severe cardiac involvement, namely biventricular endomyocardial fibrosis and gross encroachment of the right ventricular cavity. The clinical picture was similar to Loeffler's syndrome and the idiopathic hypereosinophilic syndrome. Combined aggressive surgical and medical management led to full recovery and survival at 10 years. The good long term outcome is attributed to strict control of peripheral eosinophil count by oral corticosteroids. This case illustrates the damaging effects of hypereosinophilia on the heart.

Osteoprotegerin, a crucial regulator of bone metabolism, also regulates B cell development and function.

Osteoprotegerin (OPG) is a CD40-regulated gene in B cells and dendritic cells (DCs). We investigated the role of OPG in the immune system by generating opg(-/-) mice. Like its role as a regulator of bone metabolism, OPG also influences processes in the immune system, notably in B cell development. Ex vivo, opg(-/-) pro-B cells have enhanced proliferation to IL-7, and in opg(-/-) spleen, there is an accumulation of type 1 transitional B cells. Furthermore, opg(-/-) bone marrow-derived DCs are more effective in stimulating allogeneic T cells than control DCs. When challenged with a T-dependent Ag, opg(-/-) mice had a compromised ability to sustain an IgG3 Ag-specific response. Thus, in the immune system, OPG regulates B cell maturation and development of efficient Ab responses.

Anthracycline-induced cardiomyopathy.

Anthracycline cardiomyopathy is less frequently encountered nowadays, due to the well-recognised dose limitations and cardiac monitoring protocols used by chemotherapy centres. However, it is a condition that will persist due to the sensitivity of some patients to these drugs and the necessity for large doses to be used for certain individuals. We have demonstrated the benefit of angiotensin-converting enzyme inhibitor therapy and would consider introducing these compounds at the earliest opportunity. The use of probucol and vitamins as antioxidants capable of preventing the onset of cardiomyopathy in humans appears to require further investigation but may significantly reduce the incidence of this condition in the future.

Toxoplasma gondii and Schistosoma mansoni synergize to promote hepatocyte dysfunction associated with high levels of plasma TNF-alpha and early death in C57BL/6 mice.

To address the question of how the murine host responds to a prototypic type 1 cytokine inducer while concurrently undergoing a helminth-induced type 2 cytokine response, C57BL/6 strain animals with patent schistosomiasis mansoni were orally infected with the cystogenic Toxoplasma gondii strain ME49. Schistosoma mansoni infection resulted in a significantly higher mortality rate when mice were subsequently orally infected with ME49, and these animals displayed a defective IFN-gamma and NO response relative to animals infected with T. gondii alone. Plasma levels of TNF-alpha and aspartate transaminase in double-infected mice were greatly elevated relative to mice infected with either parasite alone. Consistent with the latter observation, these animals exhibited severe liver pathology, with regions of coagulative necrosis and hepatocyte vacuolization unapparent in mice carrying either infection alone. Interestingly, mean egg granuloma size was approximately 50% of that in mice with S. mansoni infection alone. The exacerbated liver pathology in coinfected mice did not appear to be a result of uncontrolled tachyzoite replication, because both parasite-specific RT-PCR analysis and immunohistochemical staining demonstrated a low number of tachyzoites in the liver. We hypothesize that mortality in these animals results from the high level of systemic TNF-alpha, which mediates a severe liver pathology culminating in death of the animal.

Human polymorphonuclear leukocytes produce IL-12, TNF-alpha, and the chemokines macrophage-inflammatory protein-1 alpha and -1 beta in response to Toxoplasma gondii antigens.

The induction of a type 1 inflammatory cytokine response is a key event in the initiation of immunity to Toxoplasma gondii. Because polymorphonuclear leukocytes rapidly respond to infection by exiting the peripheral blood and accumulating at a site of infection, we sought to determine whether these cells produce cytokines in response to T. gondii. When human peripheral blood neutrophils were stimulated with parasite Ag, they produced both IL-12 (p70) and TNF-alpha. Similarly, up-regulated expression of macrophage-inflammatory protein-1 alpha (MIP-1 alpha) and MIP-1 beta gene transcripts was induced. Kinetic analysis of IL-12 and TNF-alpha production revealed distinct patterns following stimulation by T. gondii or LPS. Exogenous TNF-alpha alone also provided a potent stimulus of MIP-1 alpha and MIP-1 beta expression, and when neutralizing anti-TNF-alpha antiserum was included in cultures of parasite-stimulated cells, expression of these CC-family chemokines was partially blocked. These results establish that T. gondii possesses the ability of driving neutrophil proinflammatory cytokine production, and they suggest that parasite-induced MIP-1 alpha and MIP-1 beta partly results from autocrine stimulation through TNF-alpha.

GrpL, a Grb2-related adaptor protein, interacts with SLP-76 to regulate nuclear factor of activated T cell activation.

Propagation of signals from the T cell antigen receptor (TCR) involves a number of adaptor molecules. SH2 domain-containing protein 76 (SLP-76) interacts with the guanine nucleotide exchange factor Vav to activate the nuclear factor of activated cells (NF-AT), and its expression is required for normal T cell development. We report the cloning and characterization of a novel Grb2-like adaptor molecule designated as Grb2-related protein of the lymphoid system (GrpL). Expression of GrpL is restricted to hematopoietic tissues, and it is distinguished from Grb2 by having a proline-rich region. GrpL can be coimmunoprecipitated with SLP-76 but not with Sos1 or Sos2 from Jurkat cell lysates. In contrast, Grb2 can be coimmunoprecipitated with Sos1 and Sos2 but not with SLP-76. Moreover, tyrosine-phosphorylated LAT/pp36/38 in detergent lysates prepared from anti-CD3 stimulated T cells associated with Grb2 but not GrpL. These data reveal the presence of distinct complexes involving GrpL and Grb2 in T cells. A functional role of the GrpL-SLP-76 complex is suggested by the ability of GrpL to act alone or in concert with SLP-76 to augment NF-AT activation in Jurkat T cells.

Diagnostic cardiac catheterisation in a hospital without on-site cardiac surgery.

OBJECTIVE: To assess the feasibility, safety, and clinical impact of diagnostic cardiac catheterisation in a multipurpose laboratory in a district general hospital without cardiac surgery. METHODS: A prospective audit of the first 2000 consecutive cases between September 1992 and March 1997. Unstable patients were referred to a surgical centre for investigation, in line with subsequently published British Cardiac Society (BCS) guidelines, but all other patients requiring cardiac catheterisation were investigated locally and are included in this report. The function of the laboratory was also compatible with the BCS guidelines regarding staffing, operators, equipment, number of cases, and locally available vascular surgery. RESULTS: Of the 2000 cases, 1988 studies were completed (99%), 1985 (99%) included coronary angiography, and 1798 (90%) were performed as day cases. Left main stem disease was present in 157 (8%), three vessel disease in 683 (34%), two vessel disease in 387 (19%), single vessel disease in 424 (21%), and normal coronary arteries in 494 (25%). Of the latter, 284 (14% of the total) had another cardiac diagnosis for which they were investigated (for example, valvar heart disease). Referral for cardiac intervention following catheterisation was made in 1172 of the 2000 cases (intervention rate 59%; catheter:intervention ratio 1. 7:1). The interventions performed were coronary artery bypass grafting (CABG) in 736 of the 1172 cases (63%), other types of cardiac surgery in 122 (10%), combined CABG and other cardiac surgery in 71 (6%), and percutaneous transluminal coronary angioplasty in 243 (21%). There were two catheter related deaths (0. 1%), both of which occurred within 24 hours of the procedure, and a further nine major cardiovascular complications with residual morbidity (0.45%). These were myocardial infarction in two (0.1%), cerebrovascular events in two (0.1%), and surgical vascular complications in five (0.25%). In addition, there were eight successfully treated, life threatening arrhythmias (0.4%). CONCLUSIONS: Diagnostic cardiac catheterisation can be performed safely and successfully in a local hospital. When BCS guidelines are followed, the mortality is similar to published pooled data from regional centres (0.1% v 0.12%). The high intervention rate indicates a persistent unmet demand in the districts, which will continue to affect surgical and interventional services.

Modulation of the IL-7 dose-response threshold during pro-B cell differentiation is dependent on pre-B cell receptor expression.

The IL-7R and the pre-B cell receptor (pre-BCR) each provide critical signals during differentiation of B cell precursors. In this study we examine the interplay between signals dependent upon these receptors. We demonstrate that pre-BCR-deficient pro-B cells differ significantly from controls in their ability to use the IL-7R. We show that this difference, characterized by a failure to proliferate in response to IL-7, is narrowly restricted to IL-7 concentrations in the picogram per milliliter range and can be overcome with increasing amounts of IL-7. Restoration of Ig heavy chain to recombinase-activating gene-2-deficient pro-B cells leads to a restored response to picogram per milliliter levels of IL-7, providing strong evidence that modulation of the IL-7 dose-response threshold is dependent on pre-BCR. Culture of normal pro-B cells under low IL-7 conditions leads to selective outgrowth of cells expressing mu heavy chain, suggesting that modulation of IL-7 dose-response thresholds can allow for selective expansion of pre-BCR+ cells under conditions where IL-7 is limiting. We also provide evidence that expression of pre-BCR on pro-B cells limits the duration of IL-7 responsiveness by causing differentiation to an IL-7-unresponsive pre-B cell stage. Thus, the pre-BCR-dependent modulation of IL-7 responsiveness affects both the dose-response threshold and the duration of IL-7-induced clonal expansion. Our results suggest that positive selection of pre-BCR+ pro-B cells may be achieved through the fine tuning of IL-7 responses.