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Unlabelled: Ciprofloxacin labeled with (99m)Tc specifically binds to various bacteria. Thus, it potentially constitutes a specific marker allowing discrimination between septic arthritis/osteomyelitis and aseptic osteoarticular diseases. The aim of this prospective study was to evaluate such properties in patients with skeletal diseases., Methods: We prospectively investigated 2 groups of patients: patients with suspected osteoarticular infections (G1, n = 16) and a control group of patients with a presentation of osteoarticular diseases and no sign suggestive of infection (G2, n = 11). All had clinical, biologic, and radiologic evaluations and had 1-, 4-, and 24-h images from (99m)Tc-ciprofloxacin scintigraphy (370 MBq) before planned biopsy or surgery. For 23 patients, the scintigraphic results were compared with histologic and bacteriologic analyses of biopsy tissue samples; for 4 patients, the scintigraphic results were compared with the findings from 23 +/- 5 mo of follow-up., Results: In G1, (99m)Tc-ciprofloxacin findings were true-positive in all 11 infected sites, true-negative in 2 cases, and false-positive in 3. In G2, (99m)Tc-ciprofloxacin was true-negative in 4 cases and false-positive in 7. Neither the location of (99m)Tc-ciprofloxacin activity nor its intensity or kinetics between 1, 4, and 24 h allowed discrimination between infection and aseptic diseases (sterile loosened joint replacement, pseudoarthrosis, or arthrosis). Sensitivity, specificity, and accuracy were 100%, 37.5%, and 63%., Conclusion: (99m)Tc-Ciprofloxacin scintigraphy showed good sensitivity and a high negative predictive value for the detection of bone and joint infection, but it did not discriminate between infected and aseptic osteoarticular diseases in symptomatic patients referred for surgery.

Unlabelled: Ciprofloxacin, a quinolone antibiotic drug, binds to DNA topoisomerase IV and DNA gyrase of various bacteria. Thus ciprofloxacin labeled with (99m)Tc could potentially act as a specific marker allowing discrimination between infection and sterile inflammation. We evaluated these properties on a rabbit model of prosthetic joint infection previously validated. We compared the images obtained in 2 groups of animals: rabbits with infected (G1; n = 6) and uninfected (G2; n = 7) prosthesis., Methods: Partial right-knee arthroplasty was performed on 13 New Zealand White female rabbits, with a tibial silicone-elastomer implant fitting into the intramedullary canal of the tibia. After the surgical wound was closed, 10(7) cfu of a clinical strain of methicillin-susceptible Staphylococcus aureus were injected into the joint in G1 rabbits. G2 rabbits were injected with sterile saline. No antibiotic therapy was given to the animals. (99m)Tc-ciprofloxacin planar imaging was performed on days 5, 12, and 19 after surgery, and after 3 mo in 1 uninfected rabbit. Images were obtained 1, 4, and 24 h after injection (147 +/- 13 MBq)., Results: In G1, increased right knee (99m)Tc-ciprofloxacin uptake was observed in 3 of 5 rabbits on day 5, and in all cases on days 12 and 19. Killing of the animals revealed purulent arthritis, osteitis, and tibial myelitis. In G2, significant right-knee uptake was found on days 12 and 19 in 5 of 6 rabbits, and after 3 mo in 1; all sets of images were negative in 1 animal. Bacteriologic studies after the animals were killed were negative in G2. Mean right/left knee uptake ratios on day 19 (4-h images) were 1.8 +/- 0.4 in G1 versus 1.4 +/- 0.3 in G2 (not significant). Late images did not discriminate between infected and uninfected arthroplasty., Conclusion: Results of (99m)Tc-ciprofloxacin imaging in rabbits with infected/uninfected knee prosthesis suggest good sensitivity but lack of specificity for the detection of S. aureus infection.

Objectives: The aim of this study was to assess the diagnosis of myocarditis in patients presenting with acute myocardial infarction (MI) and normal coronary angiograms., Background: Most often in these patients, the etiologic diagnosis remains unclear once they are found to have normal coronary arteries. The diagnosis of myocarditis mimicking MI is clinically relevant, because numerous arguments suggest a relation between myocarditis and dilated cardiomyopathy. Myocardial indium-111 (111In)-antimyosin antibody (AMA)/rest thallium-201 (201Tl) imaging allows noninvasive detection of myocarditis., Methods: Forty-five patients admitted to three intensive care units for suspicion of acute MI, with normal coronary angiograms, were investigated. Indium-111-AMA planar images and then a dual-isotope rest AMA/201Tl tomographic study were performed. Six-month echocardiographic follow-up was obtained in 80% of the patients with initial left ventricular (LV) wall motion abnormalities., Results: In eight patients, AMA and 201Tl scintigraphy were negative. In two patients, a matched 201Tl defect and focal AMA uptake suggested acute MI (due to prolonged vasospasm or spontaneously reperfused coronary occlusion). In 17 patients, diffuse AMA uptake over the whole LV suggested diffuse myocarditis. In 18 patients, focal AMA uptake with a normal 201Tl scan suggested diffuse but heterogeneous, or focal myocarditis. Complete functional recovery was observed in 81% of the patients with a pattern of myocarditis., Conclusions: Among 45 patients presenting with acute MI and normal coronary angiograms, 38% had diffuse myocarditis and 40% had a scintigraphic pattern of heterogeneous or focal myocarditis. Short-term follow-up showed complete LV functional recovery in 81% of these patients.

Penicillium cyclopium, grown in stationary culture, produces a type I lipase specific for triacylglycerols while, in shaken culture, it produces a type II lipase only active on partial acylglycerols. Lipase II has been purified by ammonium sulfate precipitation and chromatographies on Sephadex G-75 and DEAE-Sephadex. The enzyme exists in several glycosylated forms of 40-43 kDa, which can be converted to a single protein of 37 kDa by enzymatic deglycosylation. Activity of lipase II is maximal at pH 7.0 and 40 degrees C. The enzyme is stable from pH 4.5 to 7.0. Activity is rapidly lost at temperatures above 50 degrees C. The enzyme specifically hydrolyzes monoacylglycerols and diacylglycerols, especially of medium chain fatty acids. The sequence of the 20 first amino acid residues is similar to the N-terminal region of P. camembertii lipase and partially similar to lipases from Humicola lanuginosa and Aspergillus oryzae, but is different from Penicillium cyclopium lipase I. However, it can be observed that residues of valine and serine at positions 2 and 5 in Penicillium cyclopium lipase II are conserved in Penicillium expansum lipase, of which 16 out of the 20 first amino acid residues are similar to Penicillium cyclopium lipase I.

Unlabelled: Somatostatin receptor scintigraphy (SRS) has been used for the detection of gastroenteropancreatic (GEP) tumors. This study evaluates the clinical impact of SRS in GEP tumor detection and its therapeutic implications on patient management., Methods: We prospectively studied 160 patients with biologically and/or histologically proven GEP tumors. Before SRS, patients were classified into three groups: gastrointestinal (Group 1; n = 90) patients without known metastases; (Group 2; n = 59) patients with metastases limited to the liver; (Group 3; n = 11) patients with known extrahepatic metastases. The scintigraphic data were compared to the radiological findings., Results: In Group 1, without known metastases, conventional imaging detected 53 primary sites in 44 patients: SRS was positive in 68% of these sites and discovered 4 additional primary tumors in 3 patients and 16 metastases in 14 patients. Conventional imaging was negative in 46 patients: SRS discovered 47 new sites in 36 patients. In Group 2, SRS confirmed liver metastases in 95% of patients and discovered 45 new sites in 36 of these patients. In Group 3, SRS disclosed 11 new sites in 7 patients. These results modified patient classification in 38 cases (24%). Surgical therapeutic strategy was changed in 40 patients (25%)., Conclusion: Somatostatin receptor scintigraphy improves tumor detection, has major clinical significance and should be performed systematically for staging and therapeutic decision making in patients with GEP tumors.

We report a case of left ventricular (LV) myocardial uptake of a labeled somatostatin analog in a patient with a carcinoid tumor of the small bowel. The patient developed liver metastases and a carcinoid syndrome, including right carcinoid heart disease, without right-to-left shunt on contrast ultrasonography or left ventricular myocardial metastases. The basis for visualization of the LV myocardium is probable somatostatin receptor upregulation.

Nonpancreatic secretory phospholipase A2 (sPLA2) displays proinflammatory properties; however, its physiological substrate is not identified. Although inactive toward intact cells, sPLA2 hydrolyzed phospholipids in membrane microvesicles shed from Ca(2+)-loaded erythrocytes as well as from platelets and from whole blood cells challenged with inflammatory stimuli. sPLA2 was stimulated upon degradation of sphingomyelin (SPH) and produced lysophosphatidic acid (LPA), which induced platelet aggregation. Finally, lysophospholipid-containing vesicles and sPLA2 were detected in inflammatory fluids in relative proportions identical to those used in vitro. We conclude that upon loss of phospholipid asymmetry, cell-derived microvesicles provide a preferential substrate for sPLA2. SPH hydrolysis, which is provoked by various cytokines, regulates sPLA2 activity, and the novel lipid mediator LPA can be generated by this pathway.

Procolipase is the precursor of colipase, which acts as protein cofactor for the activity of pancreatic lipase. The solution structure of procolipase has been determined by 1H NMR using two- and three-dimensional measurements. The secondary structure determination identified two separate three-stranded beta-sheet regions with concomitant hydrogen bond patterns. The tertiary structure of the protein was determined using 863 non-trivial proton--proton distance constraints, 14 hydrogen bond distance constraints and 55 phi and 25 X1 dihedral constraints. The structure that was obtained from distance geometry and energy refinement contains three highly disordered loops as well as a disordered N- and C-terminal region. The remaining part of the structure is well defined with a root-mean-square deviation (rmsd) relative to the average of 0.09 +/- 0.02 nm for backbone atoms (residues 11-30, 37-50, 57-69, 83-89). The protein comprises two identical domains, each containing a three-strand beta-sheet and two disulfide bonds: a 15-residue region in each domain superimposes with 0.07 nm rmsd, measured on backbone atoms. The solution structure is nearly identical to the crystal structure. It is in agreement with previous NMR data and, in combination with these data, supports the current model of procolipase micelle interaction and the lipase activation by colipase.

Colipase (Mr 10 kDa) confers catalytic activity to pancreatic lipase under physiological conditions (high bile salt concentrations). Previously determined 3-A-resolution X-ray structures of lipase-colipase complexes have shown that, in the absence of substrate, colipase binds to the noncatalytic C-terminal domain of pancreatic lipase (van Tilbeurgh H, Sarda L, Verger R, Cambillau C, 1992, Nature 359:159-162; van Tilbeurgh et al., 1993a, Nature 362:814-820). Upon lipid binding, conformational changes at the active site of pancreatic lipase bring a surface loop (the lid) in contact with colipase, creating a second binding site for this cofactor. Covalent inhibition of the pancreatic lipase by a phosphonate inhibitor yields better diffracting crystals of the lipase-colipase complex. From the 2.4-A-resolution structure of this complex, we give an accurate description of the colipase. It confirms the previous proposed disulfide connections (van Tilbeurgh H, Sarda L, Verger R, Cambillau C, 1992, Nature 359:159-162; van Tilbeurgh et al., 1993a, Nature 362:814-820) that were in disagreement with the biochemical assignment (Chaillan C, Kerfelec B, Foglizzo E, Chapus C, 1992, Biochem Biophys Res Commun 184:206-211). Colipase lacks well-defined secondary structure elements. This small protein seems to be stabilized mainly by an extended network of five disulfide bridges that runs throughout the flatly shaped molecule, reticulating its four finger-like loops. The colipase surface can be divided into a rather hydrophilic part, interacting with lipase, and a more hydrophobic part, formed by the tips of the fingers. The interaction between colipase and the C-terminal domain of lipase is stabilized by eight hydrogen bonds and about 80 van der Waals contacts. Upon opening of the lid, three more hydrogen bonds and about 28 van der Waals contacts are added, explaining the higher apparent affinity in the presence of a lipid/water interface. The tips of the fingers are very mobile and constitute the lipid interaction surface. Two detergent molecules that interact with colipase were observed in the crystal, covering part of the hydrophobic surface.

The time-course effects of two diets containing raw (RSF) or heated (HSF) soya-bean flour on the digestive enzyme levels in the pancreas and in pancreatic juice were investigated in growing rats fed, alternately, on RSF or HSF diets for two 4-week periods. These values were compared with those obtained in a control group fed on a casein diet. RSF and HSF diets lowered N balance (84.8 (SE 0.9), 82.6 (SE 0.8) and 79.9 (SE 0.8)% with control, HSF and RSF diets respectively, at the third week). However, they increased protease activities compared with the control diet (3-fold for trypsin (EC 3.4.21.4) and chymotrypsin (EC 3.4.21.1) activities in pancreas contents and outputs with the RSF diet; 2-fold for trypsin in pancreas contents and outputs and by 60% for chymotrypsin contents with the HSF diet). The poorer nutritional N utilization might be attributable to soya-bean flour heat-stable (lectins) and heat-labile components (trypsin inhibitors). The decrease in lipid apparent digestibilities in RSF and HSF diets (97.0 (SE 0.8), 91.1 (SE 0.9) and 90.4 (SE 0.7)% with control, RSF and HSF diets at the seventh week) were correlated with a diminution in apparent lipase (EC 3.1.1.3; measured without addition of exogenous colipase), potential lipase (measured with addition of saturated amounts of exogenous colipase) and colipase activities. Compared with control values, gains in potential and apparent lipase outputs were diminished by nearly 40% and gain in colipase outputs by 60% with RSF and HSF diets. These results show clearly that heated or raw soya-bean flours have a significant inhibitory effect on lipase digestive enzyme activities in the pancreas and in its secretion, which might explain impaired lipid digestibility.

Five monoclonal antibodies (mAb) directed against human gastric lipase (HGL) have been produced by hybridization of myeloma cells with spleen cells of BALB/c immunized mice. All these mAb belong to the IgG1 class with a kappa light chain. The effects of these mAb on the enzymic activity of HGL were studied and used to define three classes of antibodies, depending upon their immunoinactivation properties. As determined by ELISA and immunoinactivation studies, four overlapping epitopes were found to be part of the functional sites of the enzyme. The mAb appear to be suitable probes for studying the lipid binding and catalytic domains of HGL. The results of the ELISA additivity test were used to describe tentatively the epitopes of HGL in terms of a schematic spatial map.

Interfacial adsorption of pancreatic lipase is strongly dependent on the physical chemical properties of the lipid surface. These properties are affected by amphiphiles such as phospholipids and bile salts. In the presence of such amphiphiles, lipase binding to the interface requires a protein cofactor, colipase. We obtained crystals of the pancreatic lipase-procolipase complex and solved the structure at 3.04 A resolution. Here we describe the structure of procolipase, which essentially consists of three 'fingers' and is topologically comparable to snake toxins. The tips of the fingers contain most of the hydrophobic amino acids and presumably form the interfacial binding site. Lipase binding occurs at the opposite side to this site and involves polar interactions. Determination of the three-dimensional structure of pancreatic lipase has revealed the presence of two domains: an amino-terminal domain, at residues 1-336 containing the active site and a carboxy-terminal domain at residues 337-449 (ref. 6). Procolipase binds exclusively to the C-terminal domain of lipase. No conformational change in the lipase molecule is induced by the binding of procolipase.

This study shows that human bile, at a concentration in the range of that found in the intestinal lumen, inhibited the hydrolysis of emulsified triolein by pancreatic lipase in vitro. The addition of colipase in excess to lipase failed to restore lipolytic activity. In contrast, a partially degraded emulsion of triolein containing diacylglycerol, monoacylglycerol and free fatty acid was hydrolysed by lipase and colipase in the presence of bile. The results presented in this paper allow to conclude that triacylglycerol digestive products associated with emulsified lipid played an important role in the intraluminal degradation of dietary fat by pancreatic lipase and colipase in the presence of bile.

Under optimal conditions, assay for pure human gastric lipase was carried out with short- and long-chain triacylglycerol emulsions. Maximal specific activities of 1160 and 620 U/mg were obtained with tributyrin and soybean emulsion, respectively. We observed that with a tributyrin substrate, bovine serum albumin or bile salts must be added before the addition of the enzyme in order to prevent its irreversible interfacial denaturation. With long-chain triacylglycerols as substrate, a decrease with time in the rate of hydrolysis was associated with release of protonated long-chain fatty acids. The inhibitory effect of protonated fatty acids was also observed using tributyrin at pH 3.0. These observations support the conclusion that human gastric lipase shows no intrinsic specificity for short-chain triacylglycerols and that its apparent specificity is modulated by pH and presence of amphiphile in the incubation medium. Our conclusions support the view that, in the human, gastric lipolysis may play an important role in long-chain fat digestion.

Studies on gastrointestinal lipolysis have underestimated several important points. In view of recent in vitro data obtained in our laboratories, this review focuses on the role of gastric lipolysis during fat digestion. Polyclonal antibodies generated from purified rat lingual lipase were used to screen a cDNA library prepared from mRNA isolated from the serous glands of rat tongue cloned in E. coli expression vectors. A cDNA clone was isolated and the nucleotide and predicted amino acid sequences obtained. Comparison with the N-terminal amino acid sequence of the purified enzyme confirmed the identity of the cDNA. The amino acid sequence of rat lingual lipase consisted of 377 residues and showed little homology with porcine pancreatic lipase, apart from a short region containing a serine residue at an analogous position to the Ser 152 of the porcine enzyme. Human gastric lipase activity on tributyrin emulsion was detected only in the presence of amphiphiles. This behaviour was in sharp contrast with the strong inhibitory effect of amphiphiles observed on pure pancreatic lipase. To reveal human gastric lipase activity, amphiphiles must be added to human gastric lipase in order to prevent irreversible interfacial denaturation. Human gastric lipase activity was found to be restricted to triacylglycerol/water surface tensions ranging from 8 to 13 dynes/cm. All amphiphiles which decrease interfacial tension to less than 8 dynes/cm act as irreversible inhibitors of human gastric lipase in the absence or presence of bile salts. Our results confirm that human gastric lipase is capable of hydrolysing triacylglycerol in the presence of the bile salts concentration prevailing in the upper small intestine and in the presence of alimentary proteins. These observations could explain the high dietary lipid absorption observed under pancreatic lipase deficiency.

Human gastric lipase (HGL) activity on tributyrin emulsion was detected only in the presence of amphiphiles such as bile salts, proteins (serum albumin, beta-lactoglobulin or ovalbumin) or phosphatidylcholine. These findings are contrary to the strong inhibitory effect of amphiphiles observed on pure pancreatic lipase. To reveal HGL activity, amphiphiles should be added prior to HGL. This may prevent irreversible interfacial denaturation. HGL activity was found to be restricted to a triacylglycerol/water surface tension ranging from 8 dyn/cm to 13 dyn/cm. All amphiphiles, which decrease the interfacial tension below 8 dyn/cm, act as irreversible inhibitors of HGL in the absence and in the presence of bile salts. Our results confirm that HGL is capable of hydrolysing triacylglycerol in the presence of the physiological concentration of bile salts prevailing in the upper small intestine and in the presence of alimentary proteins. These observations could explain the high dietary lipid absorption observed under pancreatic lipase deficiency.