Spiral CT optimization for measurement of bronchial lumen diameter using an experimental model.

The aim of the study was to determine optimal parameters for demonstrating sublobar bronchi on spiral CT. Measurements were obtained from five parallel polyethylene tubes embedded in foam matrix with similar radiographic characteristics to segmental and subsegmental bronchi and to lung parenchyma, respectively. Collimation widths of 1.5, 2, 3 and 4 mm were used, with a pitch of 1 or 1.5 and a reconstruction interval of 1 mm or 2 mm. Various slice planes were used. Images acquired orthogonally were viewed normally. Images acquired in planes oblique or parallel to the long axes of the tubes were reformatted into a plane orthogonal to the long axes of the tubes to be comparable with the directly acquired orthogonal images. Tube diameters were measured at lung window settings (L, -400; W, 1300) and compared with known true inner and outer tube diameters. Measurements from images acquired orthogonal to the tube long axes were accurate regardless of slice thickness. Images acquired obliquely or parallel only produced accurate measurements at the lowest slice thickness (1.5 mm). Pitch and reconstruction interval had no effect on measurement error in any scan plane. It is concluded that a slice thickness of 1.5 mm or less, with a pitch of 1.5, should be used when acquiring images at angles other than orthogonal to the long axes of experimental tubes equivalent to the segmental and subsegmental bronchi. It is suggested that similar parameters should be used in vivo and that the examination should be targeted to the area of the bronchial tree in question to reduce patient dose and length of breath-hold.

Synthesis and enzymatic evaluation of a P1 arginine aminocoumarin substrate library for trypsin-like serine proteases.

A method for the solid-phase synthesis of P1 arginine containing peptides via attachment of the arginine side-chain guanidine group is described. This procedure is applied to the preparation of a tetrapeptide, P1 arginine aminocoumarin PS-SCL. This library was validated by using it to determine the P4-P2 specificity for thrombin and comparing the results to the known thrombin subsite specificity. This is the first reported example of a PS-SCL library containing a P1 arginine.

The discovery of non-basic atrial natriuretic peptide clearance receptor antagonists. Part 1.

The cyclic peptide ANP 4-23 and the linear peptide analogue AP-811 have been shown to be selective ANP-CR antagonists. Via alanine scanning and truncation studies we sought to determine which residues in these molecules were important in their binding to the clearance receptor and the relationship between these two molecules. These studies show that several modifications to these compounds are possible which improve physical properties of these molecules while retaining high affinity for the ANP-CR.

C3 activation is inhibited by analogs of compstatin but not by serine protease inhibitors or peptidyl alpha-ketoheterocycles.

C3 convertase is a key enzyme in the complement cascade and is an attractive therapeutic target for drug design. Recent studies have demonstrated that this enzyme is inhibited by compstatin (Morikis, D. , Assa-Munt, N., Sahu, A., Lambris, J.D., 1998. Solution structure of Compstatin, a potent complement inhibitor. Protein Sci. (7) 619-627; Sahu, A., Kay, B.K., Lambris, J.D., 1996. Inhibition of human complement by a C3-binding peptide isolated from a phage-displayed random peptide library. J. Immunol. (157) 884-891), a 13 amino acid cyclic peptide that binds to C3. Since the enzyme exhibits some homology to serine proteases, substrate-based design could be another avenue for drug design. In this study, we confirm the activity of compstatin using different sources of enzyme and different assay systems. We also tested the activity of substituted compstatin analogs and compared the selectivity and toxicity of these compounds to peptidyl alpha-ketoheterocyclic compounds. Our work confirms the activity of compstatin in both alternative and classical complement pathways, describes 11 new active analogs of this cyclic peptide, and provides evidence for key segments of the peptide for activity. Compstatin and related active analogs showed little or no inhibition of clotting or key enzymes in the clotting cascade nor did they appear to have significant cytotoxicity. The characteristics of compstatin suggest that this peptide and its analogs could be attractive candidates for further clinical development. By contrast, known serine protease inhibitors, including peptidyl alpha-ketoheterocycles, did not inhibit C3 convertase illustrating the atypical nature of this enzyme.

Bone age assessment: a large scale comparison of the Greulich and Pyle, and Tanner and Whitehouse (TW2) methods.

PURPOSE: Comparison of bone age assessed using either the "atlas matching" method of Greulich and Pyle or the "point scoring system" of Tanner and Whitehouse (TW2). MATERIALS AND METHODS: 362 consecutive "bone age" radiographs of the left hand and distal radius performed in a large provincial teaching hospital. Data were analysed using the "method comparison" statistical technique. Ten per cent of the radiographs were re-analysed to assess intra-observer variation. RESULTS: The 95% confidence interval for the difference between the two methods was 2.28 to -1.52 years. Intra-observer variation was greater for the Greulich and Pyle method than for the TW2 method (95% confidence limit, -2.46 to 2.18 v -1.41 to 1.43). CONCLUSION: The two methods of bone age assessment as used in clinical practice do not give equivalent estimates of bone age and we suggest that one method only (preferably the TW2) should be used when performing serial measurements on an individual patient.

Application of gene therapy to acute inflammatory diseases.

The application of gene therapy to acute inflammation has not received as much research attention as has the treatment of genetically-based diseases, cancer, and viral infections. However, gene therapy as a drug delivery system offers several theoretical and practical advantages over current protein delivery systems. These include the ability to target therapies to individual tissues or cell types, to locally produce proteins that can act intracellularly or in an autocrine, juxtacrine, or paracrine fashion, and to sustain new protein synthesis for periods up to several weeks after a single administration. Although retrovirus, herpes simplex, and adeno-associated virus have been proposed for gene therapy in cancer and in genetic diseases, nonviral and adenovirus approaches appear most applicable as drug delivery systems due to their rapid onset and short duration of transgene expression. The relative modest transduction efficiencies obtained at present with nonviral approaches, and the inherent inflammatory properties of first-generation adenovirus constructs, however, have limited their usefulness to date. The present review discusses the theoretical and practical benefits of specific gene therapy approaches for the treatment of acute inflammatory diseases, as well as our experiences with liposome:plasmid DNA and adenovirus-based approaches. Although a number of technical and theoretical hurdles remain before it can be evaluated in humans with acute inflammation, gene therapy offers a novel approach for the treatment of acute inflammation, and will likely enter the armamentarium of critical care physicians in the near future.

A new non-invasive test for the detection of compartment syndromes.

Chronic exertional compartment syndrome (CECS) is currently diagnosed using invasive pressure measurements. We report the use of 99Tcm-methoxyisobutyl isonitrile (99Tcm-MIBI) scintigraphy as a new non-invasive method of diagnosis. Forty-six patients with suspected chronic compartment syndrome underwent graded treadmill exercise to reproduce the presenting symptoms. At peak exercise, 300 MBq of 99Tcm-MIBI were injected intravenously. Subsequent cross-sectional imaging provided by emission tomography demonstrated regional abnormalities in muscle perfusion in the calf. A repeat study was performed at rest the following day. All patients in whom there was a strong clinical suspicion of CECS were considered for invasive pressure measurements. Statistical analysis of the results for investigation of CECS using 99Tcm-MIBI versus pressure studies gave P = 0.06. A comparison of 99Tcm-MIBI versus outcome gave P < 0.0001. The sensitivity was 80% and the specificity 97% for 99Tcm-MIBI studies based on outcome. The positive predictive value was 89% and the negative predictive value 94%. Thus 99Tcm-MIBI can detect compartment syndromes with good positive and negative predictive values. It is relatively simple, cheap and less invasive than pressure measurements. This technique shows promise in the diagnosis of CECS.

Molecular and mechanistic validation of delayed healing rat wounds as a model for human chronic wounds.

The purpose of this study was to provide molecular and mechanistic evaluation of an ischemic wound model in rats to determine if it is a valid model for human chronic wounds. Compared to acute wounds, human chronic wounds contain markedly elevated levels of proinflammatory cytokines and matrix metalloproteinases, while matrix metalloproteinase inhibitors and growth factor activity are diminished. Accordingly, tissue from ischemic and normal rat wounds were analyzed for cytokine, proteases and growth factor levels. Dorsal full thickness punch wounds were created in rats using a reproducible template. The ischemic wound group (n = 10) had six uniformly placed wounds within a bipedicled dorsal flap. The control group (n = 10) had the same wounds created without elevation of a flap. On postwound days 3, 6 and 13 wounds were excised and analyzed. Protein levels for tumor necrosis factor-alpha were determined with a rat-specific enzyme-linked immunosorbent assay, while mRNA was determined by RNase protection assay. Matrix metalloproteinases and serine protease detection was done using gelatin and casein zymography, respectively. Significant delay in healing was achieved in the ischemic group: 50% healing for control wounds was at 7 days and 11 days for ischemic wounds (p < 0.001). No significant differences between wound groups were found for interleukin-1beta, and mRNA for tumor necrosis factor-alpha and interleukin-1beta. However, at day 13 ischemic wounds contained significantly more tumor necrosis factor-alpha than controls and normal skin (586 +/- 106 pg/biopsy vs. 79 +/- 7 pg/biopsy vs. 52 +/- 2 pg/biopsy; p < 0. 001). Zymography showed substantially greater quantities of matrix metalloproteinase-2, matrix metalloproteinase-9, and serine proteases in ischemic wounds. This model of delayed healing in rats shares many of the key biochemical, molecular and mechanistic characteristics found in human chronic wounds, namely elevated tumor necrosis factor-alpha and protease levels. As such, this model will likely prove to be useful in chronic wound research, particularly in developing novel therapeutics.

Pharmacokinetics, immunogenicity, and efficacy of dimeric TNFR binding proteins in healthy and bacteremic baboon.

Immunogenicity, pharmacokinetics, and therapeutic efficacy of three novel dimeric soluble tumor necrosis factor (TNF)-receptor I constructs [TNF-binding protein (bp)] were evaluated in 28 baboons, 12 of which were healthy and 16 were challenged with a lethal Escherichia coli bacteremia. The three constructs differed only in the number of extracellular domains of the TNF receptor I and were dimerized with polyethylene glycol. Although all three constructs had generally similar pharmacokinetics when administered to a naive animal, they differed quantitatively in their immunogenicity. Antibodies were detected more frequently, and titers were significantly higher (P < 0.05) in both healthy and septic baboons that received the 4.0-domain TNF-bp construct, compared with animals receiving the 2.6-domain construct. When the TNF-bp constructs were administered a second time (21 days later), the half-lives of the three constructs were significantly shorter in animals that had an antibody response after the first injection. In contrast, all three TNF-bp constructs were equally effective at improving outcome, blocking a systemic TNF-alpha response, and attenuating the cytokine responses when administered at a dose of 1.0 mg/kg body wt 1 h before a lethal E. coli infusion. The findings suggest that immunogenicity of TNF-bp constructs can be altered by changing the number of functional domains, without affecting their capacity to neutralize TNF-alpha and to abrogate TNF-mediated pathology.

Role of cytokines in the metabolic response to stress.

Clinical trials with cytokine inhibitors have failed to show efficacy and confirm preclinical findings in sepsis and systemic inflammatory response syndromes. However, a better understanding of the pathogenesis of sepsis syndrome, and of proinflammatory cytokine signal transduction pathways, including the role of nuclear factor kappa B and inflammation-induced apoptosis, have provided new therapeutic opportunities for cytokine and anticytokine therapies.

CT dimensions of the normal pericardium.

Previous studies have suggested that the upper limit of the thinnest portion of the pericardium is 3-4 mm using 10 mm CT slices. However, these studies suffered from small sample sizes, long data acquisition times and unconventional viewing parameters. We have measured the width of the thinnest portion of the normal pericardium using 10 mm (100 patients) and 1 mm (100 patients) high resolution CT (HRCT) slices with modern CT equipment and fixed mediastinal window settings (400/20). The pericardium was identified in all patients and was best seen anterior to the heart. The pericardium is exceptionally well seen using 1 mm HRCT slices and this may be the optimal technique for visualization of the pericardium. The upper limit of the thinnest portion of the normal pericardium (mean value + 2 SD) was 1.2 mm (10 mm CT slices) and 0.7 mm (1 mm HRCT slices). These values are substantially lower than those previously reported and in line with anatomical findings.

CT measurement of main pulmonary artery diameter.

The aim of this study was to determine the upper limit of the normal main pulmonary artery diameter using a modern CT system. This was measured at the level of the pulmonary artery bifurcation in 100 normal subjects using unenhanced contiguous 10 mm CT slices viewed at fixed mediastinal window settings (400/20). These normal subjects were then compared with similar unenhanced 10 mm images from 12 patients with proven pulmonary arterial hypertension (mean pulmonary artery pressure > 20 mmHg). The main pulmonary artery diameter in normal subjects was 2.72 cm (SD = 0.3). Main pulmonary artery diameter in patients with pulmonary arterial hypertension was significantly greater (p < 0.01) at 3.47 cm (SD = 0.33). A pulmonary artery diameter of 3.32 cm (main pulmonary artery diameter + 2 SD) had a 58% sensitivity and 95% specificity for the presence of pulmonary arterial hypertension. It is concluded that, using unenhanced axial 10 mm CT sections, the upper limit of normal main pulmonary artery diameter is 3.32 cm. Pulmonary arterial hypertension should be considered in patients with values above this level.

Orally active trifluoromethyl ketone inhibitors of human leukocyte elastase.

This paper describes the development a series of peptidyl trifluoromethyl ketone inhibitors of human leukocyte elastase which are found to have excellent pharmacological profiles. Methods have been developed that allow for the synthesis of these inhibitors in stereochemically pure form. Two of these compounds, 1k and 1l, have high levels of oral bioavailability in several species. Compound 1l has entered development as ZD8321 and is presently undergoing clinical evaluation. These compounds demonstrate that peptidyl trifluoromethyl ketone inhibitors can achieve high levels of oral activity and bioavailability, and therefore they may prove useful as therapeutic agents in the treatment of diseases in which elastase is implicated.

Gastric cancer in the bypassed segment after operation for morbid obesity.

A case is reported in which two separate adenocarcinomas were detected in the bypassed distal stomach 13 years after gastric stapling with loop gastro-enterostomy was performed for the treatment of morbid obesity. Retrograde endoscopy via the afferent loop was used to establish the diagnosis. Although gastritis and metaplasia have been described in the bypassed stomach, only one case of carcinoma in this area has previously been reported.

Exogenously administered interleukin-10 decreases pulmonary neutrophil infiltration in a tumor necrosis factor-dependent murine model of acute visceral ischemia.

INTRODUCTION: Visceral ischemia and reperfusion associated with thoracoabdominal aortic aneurysm (TAAA) repair results in lung injury, which appears to be mediated in part by proinflammatory cytokines. The purpose of this study was to determine the effect of exogenous administration of the antiinflammatory cytokine, recombinant human IL-10 (rhIL-10), on proinflammatory cytokine production (IL-6 and TNF alpha) and pulmonary neutrophil infiltration after acute visceral ischemia-reperfusion. METHODS: Two hours before 25 minutes of supraceliac aortic occlusion, 80 C57BL/6 mice (20 to 22 g) received an intraperitoneal injection of rhIL-10 (0.2 microgram [n = 20], 2 micrograms [n = 20], 5 micrograms [n = 25], or 20 micrograms [n = 15]), and 16 mice received murine anti-IL-10 IgM 200 micrograms. Twenty-five additional mice underwent visceral ischemia-reperfusion without treatment (controls), and 16 mice underwent laparotomy without aortic occlusion (sham). RESULTS: Pretreatment with exogenous rhIL-10 resulted in significant reductions in lung neutrophil infiltration with 0.2 microgram, 2 micrograms, and 5 micrograms per mouse of rhIL-10 compared with lung neutrophil levels in control mice that underwent acute visceral ischemia-reperfusion alone (p < 0.05). In addition, serum TNF alpha was detected in 50% of control mice and in 75% of mice that received murine anti-IL-10, but in none of the mice that received rhIL-10 (2 micrograms per mouse) or the mice that underwent sham operative procedures (p < 0.05 by chi 2 analysis). CONCLUSION: Exogenous IL-10 limits pulmonary neutrophil recruitment and the appearance of TNF alpha in this model of visceral ischemia-reperfusion injury. Thus the use of exogenous IL-10 may offer a novel therapeutic approach to decrease the complications that are associated with TAAA repair.

Arginine-enhanced enteral nutrition augments the growth of a nitric oxide-producing tumor.

BACKGROUND: Arginine-enhanced diets have been shown to be beneficial in tumor-bearing hosts, but no data exist regarding their effects in hosts bearing nitric oxide (NO)-producting tumors. OBJECTIVE: To examine the effect of arginine supplementation on the growth of a NO-producing murine breast cancer cell line. METHODS: EMT-6 cells were grown in various concentrations of arginine in the presence or absence of the inducible nitric oxide synthase (iNOS) inhibitor, aminoguanidine (1 mmol/L). Forty-eight hours later, nitrite accumulation and viable cell number were assessed. BALB/c mice were then pair-fed basal purified diets (n = 10), 4% casein diets (isonitrogenous control, n = 5), or 4% arginine-enhanced diets (n = 10). One week later, 10(5) EMT-6 cells were implanted subcutaneously into the dorsal flank. After tumor implantation, five mice fed basal purified diets and five mice fed arginine-enhanced diets also received aminoguanidine (100 mg/kg subcutaneously twice daily). Two weeks after tumor cell implantation, tumor size (mean diameter), animal weight, serum and tumor nitrite and nitrate levels were measured. RESULTS: There was minimal nitrite accumulation in arginine-free media, while increasing the arginine concentration increased nitrite levels. Viable cell number did not increase in arginine-free media, but increased nearly twofold in 100 and 1000 mumol/L arginine. In 5000 and 10,000 mumol/L arginine, the difference in viable cell number was not statistically different than that seen in arginine-free media, whereas the addition of aminoguanidine blocked nitrite accumulation and increased viable cell number at these arginine concentrations. Arginine-enhanced diets stimulated tumor growth in vivo more than twofold over tumor growth in mice fed isonitrogenous control or basal purified enteral diets. Mice fed arginine-enhanced diets also had increased serum nitrite and nitrate levels over mice fed basal purified enteral diets, whereas tumors from mice fed arginine-enhanced diets had nitrite and nitrate levels similar to mice fed basal purified enteral diets. Aminoguanidine blocked the increase in serum nitrite and nitrate, but failed to block the increased tumor growth in mice receiving the arginine-supplemented diets. CONCLUSIONS: Arginine concentration influences the growth of EMT-6 tumor cells in vitro and dietary arginine supplementation augments tumor growth in vivo. The mechanism of the growth modulation in vitro is NO-dependent whereas the enhanced tumor growth in vivo is NO-independent.

Discovery and biological activity of orally active peptidyl trifluoromethyl ketone inhibitors of human neutrophil elastase.

Previously we had shown that tripeptidyl trifluoromethyl ketones (TFMKs) possessing an N-terminal diarylacylsulfonamide, such as ICI 200,880 and ICI 200,355, displayed unparalleled protection against the lung damage induced by human neutrophil elastase (HNE) when the inhibitors were administered intratracheally. Since the diarylacylsulfonamides were designed specifically to afford a long residence time in the lung, it was not unexpected that inhibitors from this class of TFMKs were not active when administered orally. Upon evaluating a large number of peptidyl TFMKs possessing a variety of N-terminal groups, several compounds were identified which demonstrated oral activity. Compounds were evaluated for their oral activity by measuring their ability to inhibit the increase in lung weight relative to body weight (Lw/Bw), the increase in red blood cells, and the increase in white blood cells induced by intratracheally administered HNE (100 micrograms/hamster). A number of tripeptidyl trifluoromethyl ketones containing neutral N-terminal groups displayed good oral activity, while those containing basic, acidic, or polar groups did not. Compound 50, possessing an N-terminal 4-(CH3O)C6H4CO group, was particularly effective, reducing Lw/Bw by 77%, red cells by 89%, and white cells by 91% when dosed at 37.5 mg/kg orally. Thus, by modifying the N-terminal group of tripeptidyl TFMKs, inhibitors can be designed which are effective in vivo when administered either orally or intratracheally.