Identification of T cell epitopes occurring in a meningococcal class 1 outer membrane protein using overlapping peptides assembled with simultaneous multiple peptide synthesis.

The meningococcal class 1 outer membrane protein (OMP) plays an important role in the development of protective immunity against meningococcal infection, and is therefore considered to be a promising candidate antigen (Ag) for a meningococcal vaccine. The induction of an effective antibody response entirely depends upon T helper cells. To identify T cell epitopes of the OMP, we prepared 45 overlapping synthetic peptides representing the entire sequence of the class 1 protein of reference strain H44/76. Fully automated simultaneous multiple peptide synthesis (SMPS) was used to assemble the 45 twenty mer which overlapped by 12 amino acid residues on a 12 mumol scale. The peptides were tested for recognition by peripheral blood mononuclear cells (PBMC) obtained from 34 volunteers. Surprisingly, all synthetic peptides induced proliferative responses of PBMC isolated from one or more human histocompatibility leukocyte antigen (HLA)-typed immune adults. With PBMC from seven nonimmune donors, no proliferative response was observed. Immunodominant regions were found, recognized by PBMC from many volunteers, irrespective of their HLA type. Most of the immunodominant T cell epitopes are located outside the variable regions and, thus, will be conserved among different meningococcal (and gonococcal) strains. Furthermore, the overlapping peptides could be used to identify the epitopes recognized by OMP-specific T cell clones with known HLA restriction. It is interesting that the epitopes defined with the clones occur in highly conserved areas, shared by all neisserial porin proteins. In summary, this analysis of the T cell response to the meningococcal class 1 OMP constitutes a complete study of reactivity to a foreign protein, and illustrates some important features of Ag recognition by T cells. Our data demonstrate unexpected diversity in the T cell recognition of the OMP, and imply that the T cell repertoire against foreign Ag may be greater than previously assumed. This observation is supported by recent data on the interaction of peptide and major histocompatibility complex (MHC) class II, the latter being much less selective than MHC class I. Finally, a comparative analysis pointed out the limitations of algorithms predicting T cell determinants, and the importance of the empirical methodology provided by SMPS.

Thyrotropin dependent and independent thyroid cell lines selected from FRTL-5 derived tumors grown in nude mice.

FRTL-5 cells were used to set up a thyroid tumor model system in C3H nu/nu mice. FRTL-5 tumors could be grown in nude mice provided serum TSH levels were elevated. Persistent TSH elevation was obtained by administration of Na131I, rendering the mice hypothyroid. After 4 weeks FRTL-5 cells were injected sc resulting in tumor growth within 2 weeks in eight out of eight mice. Although the tumors showed an apparently undifferentiated histology, lacking normal follicular structures, they were functional since the tumors were capable of concentrating [131]iodine, as demonstrated by nuclear imaging. From one of the tumors a new cell line was isolated (FRTL-5/T) that, like the parental FRTL-5 cell line, was TSH dependent for growth. In a control group of six euthyroid nude mice FRTL-5 tumor growth could not be obtained with one exception. After 3 months one animal developed a small tumor that grew rapidly thereafter. This tumor was easily transplantable in other euthyroid nude mice, showed an undifferentiated histology, and was nonfunctional, as it could not concentrate [131]iodine. From this tumor two cell lines were derived: one cultured in the presence of TSH (FRTL-5/TP) and one in the absence of TSH (FRTL-5/TA). Both cell lines were found to be TSH independent for growth. The cell lines were analyzed for TSH responsive functions and TSH receptor expression. Responsiveness to TSH in FRTL-5/T and the parental FRTL-5 cell line were similar for most thyroid specific functions tested. However, FRTL-5/T was less sensitive than FRTL-5 for TSH induced [3H]thymidine incorporation. Both cell lines had two classes of TSH binding sites with high and low affinity respectively, as determined by Scatchard analysis. FRTL-5/TP and FRTL-5/TA were both able to grow in TSH free medium and were nonresponsive to TSH in vitro, as tested for [3H]thymidine and [3H]uridine incorporation, iodine uptake, thyroglobulin iodination, and thyroglobulin secretion. This correlated with an approximately 100-fold decreased number of TSH binding sites compared to FRTL-5. The latter was caused by a complete absence of low affinity binding sites, whereas high affinity receptors were still detectable. The FRTL-5/TA cell line was the least differentiated one as thyroglobulin mRNA was detectable in only minute amounts and thyroid peroxidase expression could not be measured. These in vivo selected FRTL-5 cell lines offer a suitable model to investigate several aspects of TSH responsiveness, including signal transduction and postreceptor events, thyroid differentiation, and thyroid tumorigenesis.

Efficient selection of high-affinity B cell hybridomas using antigen-coated magnetic beads.

A method for the selection of antigen-specific B cell hybridomas using antigen-coated magnetic beads is described. Stable B cell hybridoma cell lines directed against human thyroglobulin were incubated with thyroglobulin-coated beads. 2 h of incubation at 4 degrees C using bead-to-cell ratios of at least 3:1 were found to be the optimal conditions for rosette formation. Rosettes were efficiently isolated with a strong magnet. Rosette formation was antigen-specific since irrelevant hybridoma cell lines could not form rosettes, nor could BSA-coated or uncoated beads form rosettes. Free antibodies produced by the hybridoma cells were able to block rosette formation. Blocking of rosette formation permitted the identification of different and overlapping epitopes recognized by four different hybridomas. Using six stable hybridoma cell lines with different affinities for thyroglobulin, rosette formation appeared to be dependent on the affinity of the immunoglobulin membrane receptor for antigen. A correlation was observed between the affinity of the secreted antibodies and the capacity of the hybridomas to form rosettes, suggesting that this method is suitable for the selection of hybridomas producing antibodies with a high affinity for the antigen. Antigen-coated magnetic beads were found to be suitable for the efficient selection of thyroglobulin-specific hybridoma cells from bulk cultures shortly after fusion. A 300-fold enrichment of thyroglobulin-specific cells was obtained using this method.

Iodination of newly synthesized thyroglobulin by FRTL-5 cells is selective and thyrotropin dependent.

This study shows that the Fisher rat thyroidal cell line (FRTL-5) can iodinate newly synthesized thyroglobulin. Iodinated thyroglobulin was found intra- and extracellularly. Both the synthesis of thyroglobulin and its subsequent iodination were found to be thyrotropin (TSH) dependent, with optimal activity at 10-100 microU TSH/ml. Thyroglobulin was the only protein in the culture medium, that was iodinated with high specificity and in a TSH-dependent fashion. Albumin, which was abundantly present in the culture medium, was only weakly iodinated. Various proteins, including thyroglobulin, were found to be iodinated intracellularly. Of these iodoproteins only thyroglobulin appeared in the medium suggesting selective secretion of iodinated thyroglobulin. It was shown that the other intracellular iodoproteins were no thyroglobulin breakdown products. Their function is as yet unknown.

Dissolution of calcified gallstones. Part I. Correlation of in vitro dissolution kinetics in methyl tert-butyl ether with patterns of calcification by computed tomography.

RATIONALE AND OBJECTIVES: The authors evaluated the relationship between stone computed tomography (CT) attenuation patterns and the kinetics of dissolution with methyl tertbutyl ether (MTBE). METHODS: Single moderately and heavily calcified gallstones from 40 patients were selected from a gallstone library and classified for pattern of calcification by in vitro CT scan (dense, rim, core, and laminated). Each stone was placed in a 10-mL aliquot of MTBE for 24 hours. Stone residue was blotted dry and weighted at 8, 16, and 24 hours. Results were normalized with respect to stone size. RESULTS: Only 1 of 40 (4%) specimens dissolved to particulate matter that was smaller than 2 mm. All (6 of 6) stones that were densely calcified showed virtually no dissolution. The rate of gallstone dissolution varied temporally within the rim, core, and laminated stone categories and was related to the composition of the layer exposed to the solvent at any given time. CONCLUSION: The success and rate of dissolution may be predicted by the pattern of calcification as determined by computed tomography (CT).

Dissolution of calcified gallstones. Part II. Evaluation of edetic acid preparations for dissolution of residue after in vitro methyl tert-butyl ether treatment.

RATIONALE AND OBJECTIVES: The authors assessed the potential of edetic acid (EDTA) preparations to dissolve the residue of calcified gallstones partially treated with methyl tert-butyl ether (MTBE). METHODS: Nineteen triplets (57 gallstones) were submitted to dissolution in EDTA, urea-EDTA, and an MTBE control for 48 hours after initial partial dissolution in MTBE for 24 hours. Results were compared with findings at specimen computed tomography and crystallographic analysis. All data were corrected for differences in stone size. RESULTS: In all three treatment groups (EDTA, urea-EDTA, MTBE), almost identical dissolution outcomes were observed within each triplet. Most triplets that dissolved displayed a laminated or a core-calcification pattern and consisted primarily of cholesterol. Specimens that dissolved poorly in all three groups displayed dense calcifications or thick calcified rims and were classified as pigment stones. CONCLUSION: Because no statistically significant differences in dissolution were found among the EDTA, urea-EDTA, and MTBE treatments, we conclude that EDTA preparations are not superior to the continued use of MTBE for dissolution of residue after initial MTBE treatment.

Quantitative assessment of burn injury in porcine skin with high-frequency ultrasonic imaging.

Early excision and grafting of full thickness burns has been shown to decrease morbidity and mortality. Errors made in assessing acute burn depth are common and result in prolonged hospitalization in expectant healing of full-thickness burns and in unnecessary excision and grafting of potentially regenerative partial-thickness burns. High-frequency ultrasonic imaging may be a noninvasive, convenient means of quantitating burn depth. A depth analysis system for imaging burned skin was developed using a high-frequency 18.5 MHz (nominal 25 MHz) pulse-echo ultrasound system with a longitudinal resolution of 86 mu. Five adult mini-swine (15 kg) were burned with a temperature-(190 degrees C) and pressure-controlled (236 g/cm2) burning iron. A series of burn durations (1-45 seconds) was used to inflict partial- and full-thickness burns of various depths. Ultrasonic scans of the acutely excised burns were performed across the lateral margin of the burn, including adjacent normal skin to serve as control. Direct histologic comparison was made with each scan plane. Average burn and normal skin depth measurements were made by independent observers for 34 scans and corresponding histologic sections. A significant correlation was achieved between burn depth and percent burn (burn depth/adjacent normal skin depth) as measured by ultrasound and histology (R = 0.90, t = 11.2, P less than .001).

Technical aspects of helical (spiral) CT.

Care must be taken to optimally select settings for collimation, table feed, and reconstruction interval for successful helical CT and associated special application such as CT angiography. Thus, prospective planning is critical for optimal 3-D imaging. Generally, one wishes to minimize all of these parameters to maximize longitudinal resolution; however, one must be aware of the tradeoffs associated with these choices. Pixel noise increases with decreasing collimation. Limiting the table feed to a value equal to the collimation (pitch of 1:1) limits broadening of the effective slice thickness associated with the table motion but also limits the scan coverage. Generally, one may accept a table feed up to twice the collimation (pitch of 2:1) on modern equipment with current software. Reconstruction intervals should also be selected to satisfy the clinical need. When multiplanar and 3-D imaging is desired, the reconstruction interval should be selected such that at least two slices are generated per table increment.

Percutaneous balloon dilatation of benign biliary strictures.

Percutaneous biliary dilatation is an effective alternative to surgical management of benign biliary strictures that has low morbidity and no reported mortality. Reported success rates for this procedure range from 40% to 90% depending on the size of the series, the type of patient being treated, and the length of follow-up period. The procedure is done in the fluoroscopy suite with an angioplasty balloon catheter. Transhepatic access is most common, but the procedure may be done via existing T-tube tracts or specially created jejunal loops. As the frequency of radical liver surgery such as liver transplant and radical trisegmentectomy rises, so too, the rate of biliary stricture is likely to rise, making percutaneous balloon dilatation an increasingly important tool in the interventional radiologist's armamentarium.

Can we improve trauma mortality in a state with a voluntary trauma system.

BACKGROUND: Arizona has no organized statewide trauma system. We looked at the 1997 and the 1998 Uniform Hospital Discharge Data Set (UHDDS) for the State of Arizona, and examined the trauma mortality data at both trauma hospitals and nontrauma hospitals. METHODS: All qualifying mortalities based on hospital data from 1997 through 1998 were reviewed for the State of Arizona. Trauma deaths from 32 nontrauma hospitals were examined and compared with that of 7 level 1 trauma centers within the state. RESULTS: In this time period, there were 375 qualified mortalities from nontrauma centers and 761 qualified mortalities from level 1 trauma facilities. Only 29 (8%) of nontrauma hospital deaths were found to be due to motor vehicle accidents. Only 8 (4%) mortalities at nontrauma centers were due to firearms. CONCLUSION: The data suggest that patients are arriving at the appropriate facility for definitive care despite the absence of a formal statewide trauma system.

Modified methods for the enumeration of spores of mesophilic Clostridium species in dried foods.

Clostridia constitute markers of limited though definite importance for the microbiological integrity of particular foods processed for safety, provided their application and the results obtained are meticulously considered and guided by proper ecological awareness. Their selective diagnostic enumeration in food specimens relies on their ability to reduce sulphite in agar media, visualised by the presence of ferrous cations leading to the production of black colonies. The composition of the medium used substantially affects the productivity of the procedure. We established that (1) the sulphite activity and the ferrous ion should be rigorously standardised; (2) tryptose is one of the appropriate nitrogen sources for a limited number of clostridia; (3) the basal medium should be free of added acetate and lactate. Black colonies obtained in the newly elaborated medium, termed Differential clostridial agar (DCA) should be further examined for morphology and metronidazole sensitivity, since some bacilli might mimic clostridia under the conditions of the procedure. An elegant variant of the technique relies on using a bottom-layer of mannitol/egg yolk/polymyxin/bromocresol purple agar, inoculated with macerates of food in buffered cysteine hydrochloride peptone saline, immediately liberally overlayered with freshly prepared DCA. Plates are incubated and read in tightly closed bags of plastic with a low oxygen permeability coefficient, which eliminates the need for using anaerobic jars. Colony identification is relying on assessment of sulphite reduction, egg yolk dissimilation, the mode of attack on mannitol and when required to be supported by classical other physiological traits. The mandatory precautions to be observed in this procedure call for extreme caution when introducing reference ranges ("standards") for clostridial spores in foods, particularly in the international commerce.

Spiral computed tomographic colonography: determination of the central axis and digital unraveling of the colon.

RATIONALE AND OBJECTIVES: The authors developed and tested automated and semiautomated bowel-lumen tracking and colon-unraveling techniques for determining the central axis of the bowel. METHODS: A computer-simulated gastrointestinal tract phantom was used to test the accuracy of an automated algorithm for central axis determination and bowel unraveling. Variations in cross-sectional features between straight and unraveled formats were compared in a canine bowel segment in vitro and a human colon in vivo by using spiral computed tomography. Three readers each performed three semiautomated evaluations. RESULTS: Accuracy of the automated algorithm was confirmed by the high degree of correlation in the cross-sectional feature measurements (length error, < 1%). For the canine colon segment, accuracy of the semiautomated algorithm was confirmed by comparison with the automated tracing. For the human colon, readings were reproducible with 3.3% (+/- 1.9 standard deviation) mean variation in length. CONCLUSION: An automated algorithm for central axis deterioration and unraveling the colon has been validated in a gastrointestinal tract phantom. A semiautomated algorithm has been shown to be reproducible and time-efficient.

Imaging for ureteropelvic junction obstruction in adults.

The three goals of imaging for ureteropelvic junction (UPJ) obstruction are to determine the presence and degree of renal obstruction, to assess renal function, and to determine the cause of the problem. The diagnostic techniques are intravenous urography, which is unreliable; ultrasonography, with the addition of pulsed Doppler technology; diuretic scintigraphy; and pressure-flow studies (Whitaker test), which is the only direct clinical test of renal outflow resistance. Voiding cystography, retrograde ureteropyelography, and CT have utility in some patients. In determining the cause of obstruction, the patient often is evaluated for crossing vessels using angiography, endoluminal ultrasonography, or spiral CT. It is not possible to formulate a single algorithm for radiographic evaluation of suspected UPJ obstruction, but in the average adult patient, urography and diuretic scintigraphy are sufficient.

MR imaging of the biliary system.

MR imaging has a distinct role to play in two areas of hepatobiliary imaging that continue to challenge the radiologist--evaluation of the gallbladder and evaluation of the jaundiced patient. The distinction between primary gallbladder carcinoma and chronic cholecystitis remains a diagnostic dilemma for all cross-sectional imaging modalities. MR imaging may be useful in detection of local invasion or metastatic disease. Gallstones are commonly seen coincidentally on cross-sectional imaging studies; the imaging characteristics of gallstones must be well-understood for the interpretation of routine abdominal MR examinations. The evaluation of jaundice is a multimodality process, often requiring three or four separate imaging techniques to determine the cause of biliary obstruction. MR may supplant more invasive techniques for anatomic depiction prior to therapeutic intervention.

Spiral CT angiography of the abdomen and pelvis: interventional applications.

Spiral CT angiography may be a useful adjunct for interventional radiologists in imaging vessels within the abdomen and pelvis. Specifically, the potential to assess abdominal aortic aneurysms preoperatively is within the realm of spiral CT angiography so long as scanners can quickly change collimation and table feed during the examination. The technique is also useful for imaging metal and nonmetal vascular grafts. The splanchnic arteries may be imaged in instances in which proximal vascular patency and course is questioned. The portal vein and its branches may be imaged prospectively with spiral CT angiography or retrospectively by postprocessing routine abdominal CT image data. The normal renal vasculature including accessory vessels may be imaged as part of preoperative evaluation for renal transplantation or for repair of ureteropelvic junction obstruction. Renal artery aneurysms may also be followed with spiral CT angiograms. Hemodynamically significant renal arterial stenoses may be detected with spiral CT angiography; grading the severity of those stenoses is within the realm of spiral CTA so long as careful attention is paid to technique.

Spiral (helical) CT.

Spiral (helical) computed tomography (CT) involves continuous patient translation during x-ray source rotation and data acquisition. As a result, a volume data set is obtained in a relatively short period of time. For chest or abdominal scanning, an entire examination can be completed in a single breath hold of the patient or in several successive short breath holds. The data volume may be viewed as conventional transaxial images or with multiplanar and three-dimensional methods. The authors review the technologic aspects of spiral CT, as well as its advantages, limitations, and current clinical applications.

Nonsurgical treatment of cholelithiasis. An analysis of clinical opportunity.

This article examines the potential impact of recently developed nonsurgical treatments for gallstones on patient care and resource utilization. Using epidemiological and efficacy data from the literature and current patient selection criteria, the authors evaluate UDCA, extracorporeal shock-wave lithotripsy, and direct instillation of methyltertbutyl ether in terms of short-term clinical results, health policy, and economic implications.