Brucellosis in a Singaporean with prolonged fever.

Brucellosis, a zoonotic disease of worldwide distribution, is common in many developing countries as well as in countries of the Mediterranean basin. We report brucellosis in a 52-year-old man, who had a recent travel history to Saudi Arabia, and who presented with prolonged fever and deranged liver enzymes. In view of the rarity of brucellosis and its potential life-threatening complications, patients returning from an endemic country need to be questioned for possible Brucella exposure, to ensure that diagnostic tests and treatment are carried out in a timely fashion. In addition to notifying the authorities, the clinician should also warn the laboratory early as cultures of brucellosis are highly transmissible and are one of the most common laboratory-acquired infections.

Western Australian cigarette smokers have fewer small lung nodules than North Americans on CT screening for lung cancer.

To determine the prevalence of small lung nodules on low-dose helical computed tomography (CT) in a Western Australian cohort of asymptomatic long-term cigarette smokers and to compare this with a large, similarly derived cohort of North Americans from the Mayo Clinic Lung Cancer Screening Trial. Forty-nine asymptomatic long-term cigarette smokers of minimum age 50 years underwent a low-dose 64-slice helical CT of the lungs. Images were viewed on a soft copy reporting station with thin section axial and coronal images, maximum intensity projection images, and advanced image manipulation tools. The prevalence of all nodules was 39%, significantly lower than the Mayo Clinic cohort prevalence of 51% (P < 0.01, Fisher's exact test), despite the use of more advanced imaging technology and image manipulation designed to increase the sensitivity for nodules. The prevalence of small nodules in asymptomatic long-term cigarette smokers in Western Australia is high, though significantly less than that found in a large study in North America. The authors postulate this is due to the relatively low rates of mycobacterium tuberculosis and soil-derived fungal pulmonary infections in Western Australia, as well as a lower degree of urban air pollution.

Emphysema in young adult survivors of moderate-to-severe bronchopulmonary dysplasia.

Improved survival following extreme preterm birth complicated by bronchopulmonary dysplasia (BPD) is resulting in an increasing number of affected infants surviving to adulthood. The aim of the present pilot study was to describe the functional and structural pulmonary sequelae of moderate and severe BPD in a population of adult survivors. All babies were cared for at one institution (King Edward Memorial Hospital, Subiaco, Australia). Subjects born between 1980 and 1987 with birthweight <1,500 g and requiring supplementary oxygen at 36 weeks post-menstrual age were identified from a complete neonatal database and recruited prospectively. Local physicians were concurrently asked to refer suitable patients. Demographics, respiratory symptoms and examination results, pulmonary function tests and computed tomography images were acquired. In total, 21 subjects were studied. Of these, 12 were female, the median (range) age was 19 (17-33) yrs and 15 (71%) had persistent respiratory symptoms. The median (range) forced expiratory volume in one second (FEV(1)) z-score was -0.77 (-8.20-1.37), the forced expiratory flow at 25-75% of forced vital capacity was -1.81 (-6.00-0.75) and the diffusing capacity of the lung for carbon monoxide was -5.04 (-13.17- -1.24). Computed tomography was carried out on 19 subjects and all had abnormal findings, with emphysema being the most common, present in 84% of subjects. The extent of radiological emphysema was inversely related to the FEV(1) z-score. Young adult survivors of moderate and severe bronchopulmonary dysplasia may be left with residual functional and characteristic structural pulmonary abnormalities, most notably emphysema.

The involvement of Ran GTPase in lipopolysaccharide endotoxin-induced responses.

By functional cloning, we have established that Ran GTPase is involved in LPS-induced signal transduction. This has been accomplished by several functional comparisons of the two cDNAs, Lps(n)/Ran (or RanT/n) and Lps(d)/Ran (or RanC/d), which were isolated from cDNA libraries of LPS responder and hyporesponder mice, respectively. The letter n refers to the "normal" phenotype and the letter d refers to the "deficient" phenotype. Consistent with our previous results, more animal studies indicated that adenoviral transduction of RanC/d cDNA, but not RanT/n cDNA, into sensitive mice conferred significant resistance against endotoxin challenge. Thus the incorporation of RanC/d cDNA into gene therapy protocols as a therapeutic sequence remains very attractive. At steady state, hematopoietic cells transduced with RanC/d cDNA led to about a 10-fold increase in exogenous Ran protein compared with RanT/n cDNA. Furthermore, our cumulative data suggest that a slight elevation of Ran protein in B cells enhances LPS responsiveness, but the same elevation of Ran in macrophages does not. On the other hand, a high level of overexpression of Ran in both macrophages and B cells down-regulates LPS signal transduction. Thus LPS-induced signal transduction in macrophages and B cells is likely to occur via different signaling pathways.

Specific association of Type I c-Abl with Ran GTPase in lipopolysaccharide-mediated differentiation.

Each of several isoforms of c-Abl may be involved in different biological functions. Type I c-Abl has been shown to be involved in LPS-induced differentiation and Type IV c-Abl, apoptosis. Ran has recently been shown to be involved in LPS endotoxin signal transduction. Here we show that Type I c-Abl associates with Ran. Formation of this complex is specific, as Ran did not associate with the highly homologous Type IV c-Abl isoform. In non-stimulated lymphoid B cells, Type I c-Abl tyrosine kinase is inactive, whereas Type IV kinase is active. Formation of Type I c-Abl/Ran complex and activation of Type I c-Abl kinase activity are LPS dose-dependent. This complex is detectable in B cells of endotoxin-sensitive inbred mice but absent in B cells of endotoxin-resistant mice. These findings therefore suggest that Type I c-Abl and Ran are important targets in lipopolysaccharide-induced biological responses of hematopoietic cells.

A single point mutation at the 3'-untranslated region of Ran mRNA leads to profound changes in lipopolysaccharide endotoxin-mediated responses.

By functional cDNA expression cloning, we have previously established that Ran is important in lipopolysaccharide (LPS) signaling. This was achieved by functional comparison between two cDNAs, differing by a single base substitution within the 3'-untranslated region of the cDNA. This point mutation results in a striking RNA conformational change. No dramatic difference in total RNA at steady state could be found between the two molecules. However, at the protein level, RanC/d (from 870C mRNA) was 5-10-fold higher than RanT/n (from 870T mRNA) and this difference was not observed in non-hematopoietic cells transduced with the same vectors. This tissue-specific difference correlated with a difference in LPS endotoxin responses in corresponding hematopoietic cells. Importantly, the amounts of Ran- C/d and RanT/n proteins were similar initially but the difference became obvious with time. Both Ran proteins migrated from the cytoplasm to the nucleus, but Ran from RanC/d migrated faster than that of RanT/n. RanT/n protein preferentially remained in the cytoplasm and its overall amount was reduced at steady state, consistent with its degradation by intracellular proteases known to be involved in LPS-mediated signal transduction. As the two proteins are identical, the faster RanC/d nuclear localization and a preferred initial cytoplasmic RanT/n distribution suggest a difference in mRNA intracellular localization between the two molecules, as dictated by their RNA structural difference. By pulse-chase experiments, RanC/d proteins are more resistant to degradation than RanT/n protein; there also appear to have two populations of RanT/n proteins, one may reside in the cytoplasm and the other, in the nucleus. More RanC/d GTPase accumulated in the nuclei would conceivably alter the potency of signal transduction and therefore down-modulate LPS-mediated biological responses.

Expression of a truncated retroviral envelope gene enhances expression of normal cellular phenotypes.

The envelope gene of Moloney murine leukemia virus (Mo-MLV) and its various functional domains have been studied extensively but not as much in terms of their biological effects on cell growth. In this study, we report the biological characterization of a truncated Mo-MLV envelope gene, LN11, which is devoid of a signal peptide. Its expression in various cell types, as compared to the control, enabled the transduced cells to assume a more normal phenotype, which is defined by an increase in contact inhibition and factor dependence, as well as reduced tumorigenicity. LN11-transduced fibroblasts exhibited a higher degree of contact inhibition, assumed a more flattened morphology and were more adherent compared to the control. In v-abl transformed hematopoietic cells, expression of LN11 resulted in slower cell growth, which was due to an enhanced dependence on exogenous growth factors. Enforced expression of LN11 also resulted in a slower rate of tumor development and a reduced tumor load. Thus, modification of a retroviral genome could have a significant impact on cell growth and development. This is one example where we need to consider the safety issue carefully when constructing retrovirus vectors for gene therapy.

Dominant negative down-regulation of endotoxin-induced tumor necrosis factor alpha production by Lps(d)/Ran.

We recently showed that adenoviral transfer and expression of the Lps(d)/Ran gene isolated from endotoxin-resistant C3H/HeJ mice could protect endotoxin-sensitive mice from endotoxic shock. Elevation of proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-alpha), is thought to be essential for the development of septic shock. To investigate the extent to which Lps(d)/Ran affects TNF-alpha production, we transduced primary macrophages from endotoxin-sensitive and -resistant mice with adenoviral vectors expressing the wild-type and the mutant Lps/Ran cDNAs and other control genes, and compared the amount of TNF-alpha produced by these various transduced macrophages. Successful transfer and expression of Lps(d)/Ran cDNA in endotoxin-sensitive C3H/HeOuJ macrophages reduced TNF-alpha production upon lipopolysaccharide (LPS) stimulation, as compared with macrophages transduced with vectors expressing the wild-type Lps(n)/Ran cDNA, the green fluorescent protein gene, or the lacZ gene. On the other hand, successful transfer and expression of the wild-type Lps(n)/Ran cDNA in primary macrophages from endotoxin-resistant C3H/HeJ mice failed to induce TNF-alpha production to any significant extent unless a very high LPS concentration was used. Given our previous demonstration that Lps(n)/Ran functions effectively in restoring LPS responsiveness in B cells from C3H/HeJ mice, we conclude that Lps/Ran is involved in a CD14-independent signal transduction pathway. This dominant negative down-regulation by Lps(d)/Ran on TNF-alpha production by macrophages and probably other innate immune responses may be key to the development of an effective gene therapy for endotoxic or septic shock.

Genes, receptors, signals and responses to lipopolysaccharide endotoxin.

C3H/HeJ inbred mice have been very useful for identifying genetic elements responsible for endotoxin mediated responses. Depending on the type of assays employed, Tlr-2, Tlr-4 and Lps/Ran have been shown to be important in lipopolysaccharide (LPS)-mediated responses. The concept of a single LPS gene being responsible for the genetic defect found in C3H/HeJ mice should therefore be re-examined more closely. Given the most recent discoveries, it is probable that more than one signal transduction pathway is involved. One is a CD14-dependent pathway, the other a CD14-independent pathway. Identification of the genetic elements involved in these pathways will be beneficial in designing therapeutic strategies for treating patients with endotoxic or septic shock.

Lps(d)/Ran of endotoxin-resistant C3H/HeJ mice is defective in mediating lipopolysaccharide endotoxin responses.

C3H/HeJ inbred mice are defective in that they are highly resistant to endotoxic shock as compared with normal responder mice. Their B cells and macrophages do not respond significantly when exposed to lipopolysaccharide (LPS), whereas cells from the responder mice do. Using a functional assay, we previously isolated a cDNA, which encodes for Ran/TC4 GTPase. We now show that this gene is mutated in C3H/HeJ mice, which accounts for their resistance to endotoxin stimulation. Sequence analysis of independent mutant Lps(d)/Ran cDNAs isolated from splenic B cells of C3H/HeJ mice reveals a consistent single base substitution at position 870, where a thymidine is replaced with a cytidine. In situ hybridization maps the Lps(d)/Ran cDNA to mouse chromosome 4. By retroviral gene transfer, the wild-type Lps(n)/Ran cDNA but not the mutant Lps(d)/Ran cDNA can restore LPS responsiveness of C3H/HeJ cells. Adenoviral gene transfer in vivo with the mutant Lps(d)/Ran cDNA but not the wild-type Lps(n)/Ran cDNA rescues endotoxin-sensitive mice from septic shock. Thus Lps/Ran is an important target for LPS-mediated signal transduction, and the Lps(d)/Ran gene may be useful as a therapeutic sequence in gene therapy for endotoxemia and septic shock.

Does platelet glycoprotein IIb/IIIa receptor antibody improve in-hospital outcome of coronary stenting in high-risk thrombus containing lesions?

Coronary stenting in acute coronary syndromes probably increases the risk of acute stent thrombosis. Recently, use of platelet glycoprotein IIb/IIIa receptor antibody has been shown to improve percutaneous transluminal coronary angioplasty (PTCA) outcomes in high risk lesions. The purpose of this analysis was to determine safety and efficacy of platelet glycoprotein IIb/IIIa receptor antibody administration in patients receiving coronary stents in high-risk lesions. Between October 1995 and November 1996, 282 patients with acute ischemic syndromes received coronary stents at our center: 73 had thrombus containing lesions--40 presented with AMI and 33 with unstable angina and make up the study population. The mean age of these patients was 61+/-13 years, 56 were male, 35 had a history of myocardial infarctions (MI), 21 had prior coronary artery bypass graft (CABG), and 21 had prior PTCA. Coronary stenting was used for suboptimal result in 46 patients (63%), threatened closure in 25 patients (34%), and acute closure in 2 patients (3%). Platelet glycoprotein IIb/IIIa receptor antibody was administered during the procedure in 74% and after the procedure in 26%. A total of 115 stents were deployed (Gianturco-Roubin 80, Palmaz-Schatz 29, and Wallstent 6) in 24 LAD, 21 RCA, 15 LCX, and 13 saphenous vein graft (SVG) lesions. Procedural success was 100%. The mean diameter stenosis before and after intervention was 60%+/-31% and 4%+/-14%, respectively. In-hospital events included 1 Q-wave MI (1.4%), 13 non-Q-wave MI (18%), and 1 death (1.4%). There was no subacute stent thrombosis, emergency CABG, or repeat PTCA. Significant in-hospital bleeding complications were noted in seven (10%) patients, with five patients (6.8%) requiring blood transfusions. In this series of patients with acute ischemic syndromes associated with angiographic evidence of thrombus, combined use of platelet glycoprotein IIb/IIIa receptor antibody and stenting resulted in a very low incidence of subacute stent thrombosis and emergency target lesion revascularization. However, bleeding complications were higher than expected with conventional antiplatelet therapy following routine stenting.

Efficacy of coronary stenting in the management of cardiac allograft vasculopathy.

We undertook a study to determine the efficacy of stents in reducing restenosis in cardiac allograft vasculopathy. The result shows that coronary stenting significantly reduces restenosis in cardiac allograft vasculopathy compared with balloon angioplasty alone.

Elective carotid artery stenting in the presence of contralateral occlusion.

Significant carotid stenosis in the presence of an occluded contralateral artery has a poor prognosis with medical therapy alone. Carotid cross clamping during surgical endarterectomy results in critical flow reductions in patients with inadequate collateral flow, and represents a significant risk for procedural strokes. Carotid stenting is being evaluated as an alternative to endarterectomy. We describe the immediate and late outcome of a series of 26 patients treated with carotid stenting in the presence of contralateral carotid occlusion. The mean age of the patients in this group was 65 +/- 9 years, 23 (89%) were men and 10 (39%) were symptomatic from the vessel treated. The procedural success of carotid stenting in this group of patients was 96%. The mean diameter stenosis was reduced from 76 +/- 15% to 2.8 +/- 5%. There was 1 (3.8%) minor stroke in a patient who developed air embolism during baseline angiography. At late follow-up there was no neurologic event in any patient at a mean of 16 +/- 9.5 months after the procedure. Thus, carotid stenting of lesions with contralateral occlusion can be performed successfully with a low incidence of procedural neurologic complications and late stroke.

Angioplasty of totally occluded old vein grafts with new interventional techniques: a long-term follow-up study.

The long-term patency of saphenous vein graft (SVG) lesions after intervention has been shown to be improved with new interventional techniques such as stents. Long-term outcome of patients undergoing successful angioplasty of totally occluded old SVGs with new devices is unknown. From July 1994 to June 1996, 19 patients with totally occluded old SVGs had successful angioplasty with new interventional techniques. Mean SVG age was 123 +/- 8 mo. Thrombolysis in myocardial infarction trial (TIMI) flow was 0 in all target lesions. TIMI 2 or 3 flow was restored after angioplasty in all patients. Intracoronary urokinase, transluminal extractional atherectomy, and stenting were used in 14, 12, and 6 patients, respectively. There was one in-hospital death due to ongoing myocardial infarction, no recurrent infarction, and no repeat angioplasty or bypass surgery in the hospital. At follow-up of 21 +/- 1 mo, there was one sudden death and one myocardial infarction. Five patients had repeat coronary bypass surgery, and 4 had repeat angioplasty. Thirteen patients remained asymptomatic, and 4 had angina. The long-term outcome of patients who had successful reopening of occluded old SVGs is encouraging in this small sample.

Involvement of C-Abl tyrosine kinase in lipopolysaccharide-induced macrophage activation.

LPS endotoxin-induced macrophage activation is recognized to be important in both nonspecific immunity and endotoxin-induced sepsis when excessive macrophage stimulation occurs. In this study, we showed that reduction of c-Abl in macrophages prevented LPS-induced growth arrest, nitric oxide production and TNF-alpha secretion by ANA-1 macrophages. These cells continued to grow but later underwent apoptosis. Reduction of c-Abl in these cells led to reduced c-Abl kinase activity associated with Ran, which recently has been shown to be an LPS-responsive gene product. Our data suggest that c-Abl tyrosine kinase is one of the intermediates downstream of the initial signal transduction event related to activation of macrophages by LPS.

Activation of quiescent ABL-transduced hemopoietic stem cells.

Chronic myelogenous leukemia (CML) is a hemopoietic stem cell disorder in which an activated ABL oncogene is expressed and has been shown to play an important role in disease pathogenesis. A mouse model has been established in which hemopoietic stem cells (HSCs) transduced with a retrovirus vector carrying an activated ABL oncogene can be analysed. Using this model, we now report that abl-transduced HSCs can be quiescent without causing a disease for an extended period of time. Recipient mice were able to survive more than one treatment of 5-fluorouracil (5-FU) at a dose that normally eliminates cycling hemopoietic progenitor cells; subsequently, transduced HSCs could become activated and undergo clonal expansion, resulting in abl-induced leukemic development. The disease developed in these mice was transplantable. Upon engraftment into secondary mice, previously unidentified abl-transduced HSC clones appeared. These data suggest the presence of an abl-suppressive mechanism in HSCs and have important implications to the pathogenesis of stem cell diseases and leukemic clonal evolution.