Inhibition of human mitochondrial peptide deformylase causes apoptosis in c-myc-overexpressing hematopoietic cancers.

Inhibition of human mitochondrial peptide deformylase (HsPDF) depolarizes the mitochondrial membrane, reduces mitochondrial protein translation and causes apoptosis in Burkitt's lymphoma. We showed that HsPDF mRNA and protein levels were overexpressed in cancer cells and primary acute myeloid leukemia samples. Myc regulates mitochondria and metabolism; we also demonstrated c-myc regulated the expression of HsPDF, likely indirectly. Inhibition of HsPDF by actinonin blocked mitochondrial protein translation and caused apoptotic death of myc-positive Burkitt's lymphoma, but not myc-negative B cells. Inhibition of mitochondrial translation by chloramphenicol or tetracycline, structurally different inhibitors of the mitochondrial ribosome, which is upstream of deformylase activity, followed by treatment with actinonin, resulted in reversal of the biochemical events and abrogation of the apoptosis induced by actinonin. This reversal was specific to inhibitors of HsPDF. Inhibition of HsPDF resulted in a mitochondrial unfolded protein response (increased transcription factors CHOP and CEB/P and the mitochondrial protease Lon), which may be a mechanism mediating cell death. Therefore, HsPDF may be a therapeutic target for these hematopoietic cancers, acting via a new mechanism.

Indications for and efficacy of splenectomy for haematological disorders.

BACKGROUND: Splenectomy is performed for a variety of indications in haematological disorders. This study was undertaken to analyse outcomes, and morbidity and mortality rates associated with this procedure. METHODS: Patients undergoing splenectomy for the treatment or diagnosis of haematological disease were included. Indications for operation, preoperative risk, intraoperative variables and short-term outcomes were evaluated. RESULTS: From January 1997 to December 2010, 381 patients underwent splenectomy for diagnosis or treatment of haematological disease. Some 288 operations were performed by an open approach, 83 laparoscopically, and there were ten conversions. Overall 136 patients (35·7 per cent) experienced complications. Postoperative morbidity was predicted by age more than 65 years (odds ratio (OR) 1·63, 95 per cent confidence interval 1·05 to 2·55), a Karnofsky performance status (KPS) score lower than 60 (OR 2·74, 1·35 to 5·57) and a haemoglobin level of 9 g/dl or less (OR 1·74, 1·09 to 2·77). Twenty-four patients (6·3 per cent) died within 30 days of surgery. Postoperative mortality was predicted by a KPS score lower than 60 (OR 16·20, 6·10 to 42·92) and a platelet count of 50,000/µl or less (OR 3·34, 1·25 to 8·86). The objective of the operation was achieved in 309 patients (81·1 per cent). The success rate varied for each indication: diagnosis (106 of 110 patients, 96·4 per cent), thrombocytopenia (76 of 115, 66·1 per cent), anaemia (10 of 16, 63 per cent), to allow further treatment (46 of 59, 78 per cent) and primary treatment (16 of 18, 89 per cent). CONCLUSION: Splenectomy is an effective procedure in the diagnosis and treatment of haematological disease in selected patients.

Effects of sequential changes from conventional ventilation to high-frequency oscillatory ventilation at increasing mean airway pressures in an ovine model of combined lung and head injury.

BACKGROUND: The objective of this study was to determine the intracranial, cardiovascular and respiratory changes induced by conversion to high-frequency oscillator ventilation from conventional mechanical ventilation at increasing airway pressures. METHODS: In this study, 11 anaesthetized sheep had invasive cardiovascular and intracranial monitors placed. Lung injury was induced by saline lavage and head injury was induced by inflation of an intracranial balloon catheter. All animals were sequentially converted from conventional mechanical ventilation to high-frequency oscillator ventilation at target mean airway pressures of 16, 22, 28, 34 and 40 cm H(2)O. The mean airway pressure was achieved by adjusting positive end expiratory pressure while on conventional mechanical ventilation, and continuous distending pressures while on high-frequency oscillator ventilation. Cerebral lactate production, oxygen consumption and venous oximetry were measured and analysed in relation to changes in transcranial Doppler flow velocity. Transcranial Doppler profiles together with other physiological parameters were measured at each airway pressure. RESULTS: Cerebral perfusion pressure was significantly lower during high-frequency oscillator ventilation than during conventional mechanical ventilation (CMV: 45, 34, 22, 6, 9 mmHg vs. HFOV: 33, 20, 19, 5, 5 mmHg at airway pressures mentioned above, P = 0.02). Intracranial pressure and cerebrovascular resistance increased with increasing intrathoracic pressures (P = 0.001). Cerebral metabolic indices demonstrated an initial increase in anaerobic metabolism followed by a decrease in cerebral oxygen consumption progressing to cerebral infarction as intrathoracic pressures were further increased in a stepwise fashion. Arterial PaCO(2) increased significantly after converting from conventional mechanical ventilation to high-frequency oscillator ventilation (P = 0.001). However, no difference was observed between conventional mechanical ventilation and high-frequency oscillator ventilation when intracranial pressure, metabolic and transcranial Doppler indices were compared at equivalent mean airway pressures. CONCLUSIONS: The use of high positive end expiratory pressure with conventional mechanical ventilation or high continuous distending pressure with high-frequency oscillator ventilation increased intracranial pressure and adversely affected cerebral metabolic indices in this ovine model. Transcranial Doppler is a useful adjunct to intracranial pressure and intracranial venous saturation monitoring when major changes in ventilation strategy are adopted.

Safe and cost effective use of alteplase for the clearance of occluded central venous access devices.

PURPOSE: To determine whether cryopreserved solutions of the thrombolytic agent alteplase could be used as a safe, effective, and economically reasonable alternative to urokinase in patients presenting with occluded central venous access devices (CVADs). MATERIALS AND METHODS: Alteplase has been reported as an efficacious alternative to urokinase for treatment of occluded CVADs. However, the practicality of using alteplase as the thrombolytic of choice for this indication remained conjectural. To make this approach economically feasible, alteplase was diluted to 1 mg/mL and 2.5-mL aliquots were stored at -20 degrees C until use. A need to confirm that the cryopreserving and thawing of the reconstituted solution did not compromise the safety and efficacy reported from prior trials was recognized. A quality assessment initiative was undertaken to concurrently monitor the safety and efficacy of this approach. Patients presenting with occluded CVADs received a sufficient volume of the thawed alteplase solution to fill the occluded catheter(s). Data, including efficacy, adverse reactions, dwell time, and catheter type, were collected over a 5-month period. RESULTS: One hundred twenty-one patients accounting for 168 attempted clearances were assessable for safety and efficacy. One hundred thirty-six (81%) of the 168 catheter clearance attempts resulted in successful catheter clearance (95% confidence interval, 74% to 86%). No adverse events were reported. CONCLUSION: Cryopreserved 1-mg/mL aliquots of alteplase are safe and effective in the clearance of occluded CVADs when stored at -20 degrees C for 30 days. The ability to cryopreserve alteplase aliquots makes it an economically reasonable alternative to urokinase in the setting of CVAD occlusion.

Renal involvement by Rosai-Dorfman disease: CT findings.

Rosai-Dorfman disease is a rare disease characterized histologically by proliferation of histiocytes and has clinical features suggestive of a lymphomalike disease. Lymph nodes and extranodal sites might be involved, but renal involvement is rare. We present computed tomographic findings in three cases of renal involvement by Rosai-Dorfman disease. Two cases showed renal hilar masses and one case showed subcapsular hypodense infiltration. Renal involvement by Rosai-Dorfman disease has a characteristic appearance and should be included in the differential diagnosis of renal hilar masses or subcapsular hypodense infiltration.

Critical evaluation of the World Health Organization classification of myelodysplasia and acute myeloid leukemia.

The separation of myelodysplastic syndromes from acute myeloid leukemia has been problematic because, as clinical entities, they represent different points in the spectrum of the same disease process. A new classification by the World Health Organization has incorporated recent prognostic findings in myelodysplastic syndrome and distinct genetic subtypes of acute myelogenous leukemia to provide an improved conceptual framework, if not a simpler nomenclature. Here, we review the new classification system and discuss its impact on diagnosis and treatment.

Molecular characterization of a granulocyte macrophage-colony-stimulating factor receptor alpha subunit-associated protein, GRAP.

The granulocyte macrophage-colony-stimulating factor receptor (GM-CSF-R) is a heterodimer composed of 2 subunits, alpha and beta, and ligand binding to the high-affinity receptor leads to signalling for the multiple actions of GM-CSF on target cells. In order to explore the role of the alpha subunit in signalling, we used a yeast-2-hybrid system to identify proteins interacting with the intracellular domain of the GMR-alpha. A cDNA encoding a predicted protein of 198 amino acids, designated GRAP (GM-CSF receptor alpha subunit-associated protein), was isolated in experiments using the intracellular portion of GMR-alpha as bait. The interaction between GRAP and GMR-alpha was confirmed by coimmunoprecipitation in mammalian cells. GRAP mRNA is widely expressed in normal human and mouse tissues and in neoplastic human cell lines, but it is not restricted to cells or tissues that express GM-CSF receptors. Three discrete GRAP mRNA species were detected in human tissues and cells, with estimated sizes of 3.3, 3.1, and 1.3 kb. GRAP is highly conserved throughout evolution, and homologues are found in yeast. The GRAP locus in Saccharomyces cerevisiae was disrupted, and mutant yeast cells showed an inappropriate stress response under normal culture conditions, manifested by early accumulation of glycogen during the logarithmic growth phase. GRAP is, therefore, a highly conserved and widely expressed protein that binds to the intracellular domain of GMR-alpha, and it appears to play an important role in cellular metabolism.

High-affinity binding to the GM-CSF receptor requires intact N-glycosylation sites in the extracellular domain of the beta subunit.

The human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor consists of 2 glycoprotein subunits, GMRalpha and GMRbeta. GMRalpha in isolation binds to GM-CSF with low affinity. GMRbeta does not bind GM-CSF by itself, but forms a high-affinity receptor in association with GMRalpha. Previously, it was found that N-glycosylation of GMRalpha is essential for ligand binding. The present study investigated the role of N-glycosylation of the beta subunit on GM-CSF receptor function. GMRbeta has 3 potential N-glycosylation sites in the extracellular domain at Asn58, Asn191, and Asn346. Single mutants and triple mutants were constructed, converting asparagine in the target sites to aspartic acid or alanine. A single mutation at any of the 3 consensus N-glycosylation sites abolished high-affinity GM-CSF binding in transfected COS cells. Immunofluorescence and subcellular fractionation studies demonstrated that all of the GMRbeta mutants were faithfully expressed on the cell surface. Reduction of apparent molecular weight of the triple mutant proteins was consistent with loss of N-glycosylation. Intact N-glycosylation sites of GMRbeta in the extracellular domain are not required for cell surface targeting but are essential for high-affinity GM-CSF binding.

Kinetic resolution of two mechanisms for high-affinity granulocyte-macrophage colony-stimulating factor binding to its receptor.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is an important hematopoietic cytokine that exerts its effects by interaction with the GM-CSF receptor (GMR) on the surface of responsive cells. The GM-CSF receptor consists of two subunits: GMRalpha, which binds GM-CSF with low affinity, and GMRbeta, which lacks intrinsic ligand-binding capability but complexes with GMRalpha to form a high-affinity receptor (GMRalpha/beta). We conducted dynamic kinetic analyses of GM-CSF receptors to define the role of GMRbeta in the interaction of ligand and receptor. Our data show that GMRalpha/beta exhibits a higher k(on) than GMRalpha, indicating that GMRbeta facilitates ligand acquisition to the binding pocket. Heterogeneity with regard to GM-CSF dissociation from GMRalpha/beta points to the presence of loose and tight ligand-receptor complexes in high-affinity binding. Although the loose complex has a k(off) similar to GMRalpha, the lower k(off) indicates that GMRbeta inhibits GM-CSF release from the tight receptor complex. The two rates of ligand dissociation may provide for discrete mechanisms of interaction between GM-CSF and its high-affinity receptor. These results show that the beta subunit functions to stabilize ligand binding as well as to facilitate ligand acquisition.

Using care pathways in pressure area management: a pilot study.

Care pathways are a method of managing, delivering and documenting care. A pilot study of a care pathway system in pressure area management was conducted in two clinical areas. Results from this pilot study indicate that the pathway approach to pressure area management is both useful and valid as a means to enhance clinical decision-making and to facilitate comprehensive pressure area management and the completeness of care documentation. Implementation of the pathway requires a facilitator to provide support and education and to monitor and maintain the change process.

Primary antibody deficiency and Crohn's disease.

Five patients with primary antibody deficiency were investigated because of intermittent but persistent diarrhoea of several years duration despite immunoglobulin replacement therapy. We found no evidence of Giardia lambia or other intestinal pathogens to explain their gastrointestinal symptoms. All five had definite radiological evidence of small bowel Crohn's disease and three had histological specimens available with abnormalities consistent with Crohn's disease. One patient had a non-caseating granuloma in an oral ulcer. A second patient with stricturing disease in the small bowel had a mucosal inflammatory infiltrate with non-caseating granulomas. A third had transmural inflammation but no granulomas. All five patents were diagnosed as having Crohn's disease and have responded symptomatically to steroid therapy.

Orthotopic neobladder reconstruction: findings on excretory urography and CT.

OBJECTIVE: The excretory urographic and CT appearance of orthotopic ileal neobladder reconstruction after cystectomy and its complications are described. MATERIALS AND METHODS: We retrospectively reviewed the excretory urograms and CT scans of 32 patients (29 men and three women, 35-76 years old) with transitional cell carcinoma of the bladder who underwent orthotopic neobladder reconstruction with anastomosis to the native urethra after cystectomy. The radiologic review consisted of 25 excretory urograms in 20 patients and 37 CT scans in 21 patients. RESULTS: On excretory urography, the afferent segment of the neobladder was identified as a contrast-filled structure in all 20 patients, and was located in the right lower quadrant in 18 (90%). On CT, the afferent segment and the neobladder were identified in all 21 patients. Delayed imaging performed after initial scanning in 12 (57%) of 21 patients was helpful for revealing detailed anatomy such as the ureteral-afferent limb anastomoses. Complications occurred in two patients and were caused by a lymphocele in one and a urine leak from the neobladder in the other. In six other patients we found evidence of recurrent or metastatic tumor or both: two had local pelvic recurrence and pelvic nodal metastases, two other patients had metastatic nodal disease, one patient had a malignant distal ureteral stricture, and the sixth patient had distant osseous metastases. CONCLUSION: Orthotopic neobladder reconstruction after cystectomy has a characteristic appearance on both excretory urography and CT. Knowledge of this appearance and the altered anatomy is useful to recognize complications and tumor recurrence. Delayed images during excretory urography and CT are useful to define the ureteral-afferent limb anastomosis with the neobladder and also to differentiate between postoperative collections.

Expression of the human GM-CSF receptor alpha subunit in Saccharomyces cerevisiae.

The alpha subunit of the receptor for human granulocyte-macrophage colony-stimulating factor (GM-CSF) is a 45 kDa membrane protein with a higher apparent molecular weight of 50-85 kDa due to glycosylation. Previously, we had demonstrated that N-glycosylation plays a critical role in the GM-CSF receptor-ligand interaction. To assess the activity of the alpha subunit of the human GM-CSF receptor (GMRalpha) in a lower eukaryote, we expressed GMRalpha in the yeast S. cerevisiae and found that the protein has a lower apparent molecular weight compared with that expressed in mammalian cells. Using indirect immunofluorescence microscopy, we showed that GMRalpha protein expressed in yeast localizes to the plasma membrane. Although the yeast-expressed GMRalpha is able to interact with anti-GMRalpha antibody, the heterologously expressed receptor does not bind GM-CSF. Our results indicate that specific sites and/or forms of glycosylation of the GM-CSF receptor are crucial for ligand binding.

Soluble receptors in human disease.

Soluble cytokine receptors naturally arise from genes encoding membrane-bound receptors or are direct derivatives of the receptors themselves. There is mounting evidence that soluble receptors play important roles in human disease states. In many cases, soluble receptors appear to play an integral part in the dynamic interaction between ligands and their membrane-bound receptors in maintaining and restoring health after a pathological insult but, in some instances, dysregulated expression of soluble receptors can contribute to disease pathology. Nonetheless, an appreciation of the biological actions of soluble receptors, particularly as cytokine inhibitors, has led to their therapeutic use in human diseases. Although early clinical trails of soluble receptors have had unexpected toxicities, their application in medicine continues to advance and it is likely that soluble receptors will join hormones, cytokines, and growth factors as established biological therapies.

Expression of granulocyte-macrophage colony-stimulating factor receptors in human prostate cancer.

We studied the expression and function of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor in the human prostate carcinoma cell line LNCaP and looked for its presence in normal and neoplastic human prostatic tissue. The GM-CSF receptor is composed of two subunits, alpha and beta. While the isolated alpha subunit binds GM-CSF at low-affinity, the isolated beta subunit does not bind GM-CSF by itself; but complexes with the alpha subunit to form a high-affinity receptor. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) showed expression of mRNAs encoding the alpha and beta subunits of the GM-CSF receptor in LNCaP cells, and the presence of the alpha and beta proteins was confirmed by immunolocalization with anti-alpha and anti-beta antibodies. Receptor binding studies using radiolabeled GM-CSF showed that LNCaP cells have about 150 high-affinity sites with a kd of 40 pmol/L and approximately 750 low-affinity sites with a kd of 2 nmol/L. GM-CSF signaled, in a time- and dose-dependent manner, for protein tyrosine phosphorylation and induced the proliferation of the LNCaP cells. Immunolocalization studies showed low level expression of GM-CSF alpha and beta subunits in normal prostate tissue, with substantial expression in benign prostatic hyperplasia and prominent expression in neoplastic prostate tissue. Maximal expression of both subunits was observed in prostatic carcinomas metastatic to lymph node and bone. Tumor cells that stained positively with anti-alpha subunit antibodies were also reactive with anti-beta subunit antibodies, indicating that they express high-affinity GM-CSF receptors. Our data show that the LNCaP cells express functional GM-CSF receptors and that prostatic carcinomas have prominent GM-CSF receptor expression. These findings imply that both hyperplastic and neoplastic prostatic tissues may be responsive to GM-CSF.

Maternally reported fetal activity levels and developmental diagnoses.

Retrospective maternal report of fetal activity level was compared with developmental diagnosis in 608 consecutively referred children. Maternal history of fetal activity level was also obtained from 140 unmatched well children in a general pediatric clinic. Fetal hyperactivity was positively associated with a diagnosis of child hyperactivity, and fetal hypoactivity was positively associated with a diagnosis of mental retardation in the children. Maternal histories of fetal activity level in the control group weakened the strength of the association between fetal hyperactivity and child hyperactivity but did not affect the association between fetal hypoactivity and mental retardation in children.

N-glycosylation of the human granulocyte-macrophage colony-stimulating factor receptor alpha subunit is essential for ligand binding and signal transduction.

The alpha subunit of the receptor for human granulocyte-macrophage colony-stimulating factor (GM-CSF) is a glycoprotein containing 11 potential N-glycosylation sites in the extracellular domain. We examined the role of N-glycosylation on alpha subunit membrane localization and function. Tunicamycin, an N-glycosylation inhibitor, markedly inhibited GM-CSF binding, GM-CSF-induced deoxyglucose uptake, and protein tyrosine phosphorylation in HL-60(eos) cells but did not affect cell surface expression of the alpha subunit as detected by an anti-alpha subunit monoclonal antibody. In COS cells expressing the alpha subunit and treated with tunicamycin, N-unglycosylated alpha subunit was expressed and transported to the cell surface but was not capable of binding GM-CSF. High affinity binding in COS cells expressing both alpha and beta subunits was also blocked by tunicamycin treatment. These studies indicate that N-linked oligosaccharides are essential for alpha subunit ligand binding and signaling by the human GM-CSF receptor.