Sci-Fri AM: Imaging - 04: SPECT-based functional lung imaging in the prediction of radiation pneumonitis: A retrospective clinical and dosimetric correlation.

PURPOSE: To investigate whether functionally-weighted dose-volume histogram (DVH) parameters are more predictive of radiation-induced pneumonitis (RP) than standard parameters such as V20 and mean lung dose (MLD). MATERIALS AND METHODS: A retrospective chart review identified 26 patients who received curative-intent radiation therapy for primary carcinoma of the lung. Prior to treatment, all patients received single photon emission computed tomography (SPECT) to assess both lung ventilation and lung perfusion. Patients were assessed for clinical RP using standard criteria and were separated into a non-RP group (RP grade < 2) and an RP-group (RP grade ≥ 2). Standard DVH parameters (V10, V20, V30, MLD) and their function-weighted counterparts (for perfusion: pF10, pF20, pF30, pMLD; for ventilation: vF10, vF20, vF30, vMLD) were evaluated for each group. Receiver operating characteristics (ROC) curves were created and the area under the curve (AUC) computed. RESULTS: 7 of 26 patients had grade ≥ 2 pneumonitis. Both pF20 (p=0.022) and vF20 (p=0.036) were significantly different between the 2 groups; V20 was not (p=0.06). Both pF30 (p=0.008) and vF30 (p=0.025) were significantly different between groups while V30 failed to reach significance (p=0.072). Standard MLD (p=0.011), pMLD (p=0.001), and vMLD (p=0.011) were all significantly different. The ROC curves indicated that both the perfusion-weighted parameters and the ventilation-weighted parameters outperformed the standard DVH parameters as predictors of RP grade ≥2. CONCLUSIONS: SPECT-based, function-weighted DVH parameters appear to be useful as predictors of RP.

Palliation of pain associated with metastatic bone cancer using samarium-153 lexidronam: a double-blind placebo-controlled clinical trial.

PURPOSE: To evaluate the effectiveness and safety of samarium-153 (153Sm) lexidronam (EDTMP) in a double-blind, placebo-controlled study. PATIENTS AND METHODS: Patients with painful bone metastases secondary to a variety of primary malignancies were randomized to receive 153Sm-EDTMP 0.5 or 1.0 mCi/kg, or placebo. Treatment was unblinded for patients who did not respond by week 4, with those who had received placebo eligible to receive 1.0 mCi/kg of active drug in an open-label manner. Patient and physician evaluations were used to assess pain relief, as was concurrent change in opioid analgesia. RESULTS: One hundred eighteen patients were enrolled onto the study. Patients who received 1.0 mCi/kg of active drug had significant reductions in pain during each of the first 4 weeks in both patient-rated and physician-rated evaluations. Pain relief was observed in 62% to 72% of those who received the 1.O-mCi/kg dose during the first 4 weeks, with marked or complete relief noted in 31% by week 4. Persistence of pain relief was seen through week 16 in 43% of patients who received 1.0 mCi/kg, of active drug. A significant correlation (P = .01) was observed between reductions in opioid analgesic use and pain scores only for those patients who received 1.0 mCi/kg 153Sm-EDTMP. Bone marrow suppression was mild, reversible, and not associated with grade 4 toxicity. CONCLUSION: A single dose of 1.0 mCi/kg of 153Sm-EDTMP provided relief from pain associated with bone metastases. Pain relief was observed within 1 week of administration and persisted until at least week 16 in the majority of patients who responded.

Pulmonary uptake of technetium-99m-sestamibi induced by dipyridamole-based stress or exercise.

UNLABELLED: On poststress images with 99mTc-sestamibi (MIBI), increased lung uptake of the radiotracer may reflect severe or multivessel coronary artery disease. METHODS: We measured pulmonary/myocardial ratios of MIBI at standardized times on immediate poststress acquisitions and on delayed tomographic acquisitions. In 1500 sequential patients referred for rest and stress myocardial tomography, ancillary planar images were obtained 4 min postinjection at peak stress with exercise, either alone (exercise, n = 674), or after intravenous dipyridamole (dipyridamole, n = 826). RESULTS: Based on 95% confidence limits in the angiographic normals, high values for immediate acquisitions were found in 17% of dipyridamole studies and 15% of exercise studies. High values for delayed acquisitions were found in 10% of dipyridamole studies and 9% of exercise studies. For both stress modes, increased values were related (p < 0.001) to ischemic perfusion defects for immediate images, to fixed defects for delayed images, and to ventricular dilation in both cases. By logistic regression analysis, body weight and history of infarction were also minor independent determinants (p < 0.01) of delayed acquisitions. In a subset of 250 cases with angiographic correlation (163 with dipyridamole; 87 with exercise), immediate lung uptake was highly correlated with ventricular dysfunction and with coronary stenoses (p < 0.0001). Relationships were similar to those in a historic control series imaged with 201TI. Values for delayed poststress images, and for corresponding rest images, showed strong relationships to ventricular dysfunction but not to stenosis severity. CONCLUSION: The relationships of immediate lung uptake to scintigraphic and angiographic disease patterns suggest its possible diagnostic use as an indicator of stress-induced ventricular decompensation.

Linear epitopes of colonization factor antigen I and peptide vaccine approach to enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) cause diarrhea in infants and in travelers to developing countries. The bacteria utilize colonization factors (CF) for adherence to intestinal epithelia, then release toxins causing diarrhea. CF are strong immunogens as well as protective antigens. While 20 ETEC CF have been described in the literature, 11 CF are prominent enough to be considered for vaccine targeting. Of this group, six of the members fall into the CFA/I family of CF. Geysen pin (peptide) linear epitope analysis demonstrated that three regions containing linear epitopes exist in CFA/I, and that both B- and T-cell linear epitopes of CFA/I were concentrated at the N-terminus of the protein. We have determined N-terminal sequence of the CFA/I family members not previously sequenced. Comparison of the protein sequence of the six members of the family showed a strong homology up to residue 36. A peptide of 36 amino acids representing a consensus of the six sequences was synthesized and used to immunize animals. The antibody induced to the peptide was reactive to the peptide as well as cross-reactive to each member of the CFA/I family in Western blots. In addition, this antibody agglutinated three of the six members of the CFA/I family when added to whole cells expressing the native CF. We are currently evaluating different carriers and conjugation methods to maximize production of high titer, agglutinating antibody. It is hoped that this and related research will result in an effective and inexpensive cross-reactive and cross-protective ETEC vaccine.

The cortical representation of somatosensory evoked potentials of the phrenic nerve.

Respiratory electrophysiological studies are of essential value in diagnosing and managing patients with respiratory failure, but assessment of the sensory phrenic nerve fibres has been neglected. We recorded phrenic nerve somatosensory evoked potentials (SSEPs) by combining neurophysiological and neuroimaging techniques in three healthy subjects. Evoked potentials of the phrenic nerve showed the highest amplitude at CP3, determined by the modified 10-20 EEG system, and occurred at a constant latency, PI at 12.0 +/- 0.6 ms, and NI at 17.3 +/- 0.8 ms. Single photon emission computer tomography (SPECT) performed during phrenic nerve stimulation revealed focal neuronal activation in the somatosensory pathways. Intravenously administered Tc-99m Ethyl Cysteinate Dimer (ECD) was used as a blood flow tracer to obtain baseline and activated images. After image registration, baseline images were compared voxel-by-voxel with the activation images. The mean inter-subject summation image of the activated state was compared with that of the baseline state using ten normal subjects. The extent of the total voxel volume increase on the mean images of the 3 activated SPECT images was 0.7%, and a mean signal increase of 22%. For further anatomic localization of regional increases in signal, the magnetic resonance image (MRI) scan of each subject was registered and superimposed on the activated stage SPECT image. This method may be used clinically to study the pathophysiology of impaired central respiratory drive.

Brain mapping of median nerve somatosensory evoked potentials with combined 99mTc-ECD single-photon emission tomography and magnetic resonance imaging.

Single-photon emission tomography (SPET) was performed during electrical median nerve stimulation and used to detect focal neuronal activation in the somatosensory pathways. Intravenously administered technetium-99m ethyl cysteinate dimer (ECD) was used as a blood flow tracer to obtain baseline and activated images in each of three subjects. After image registration, baseline images were compared voxel by voxel with the activation images. In addition, the mean summation of the activated-state images of the subjects was compared with the mean summation of the baseline-state images of ten normal subjects. Discrete brain regions occupying 0.9%-1.6% of total brain volume showed an increase in signal from 33.6% to 35.0%. For further anatomical localization of regional increases in signal, the MRI scan of each subject was registered and superimposed on the activated-state SPET image. This method may be used to localize lesions in various disorders of the central nervous system.

Declaring pediatric brain death: current practice in a Canadian pediatric critical care unit.

OBJECTIVE: To document the criteria used to declare brain death in a pediatric critical care unit (PCCU). DESIGN: Retrospective chart review. SETTING: Regional PCCU in southwestern Ontario. PATIENTS: Sixty patients 16 years of age or less declared brain dead from January 1987 through December 1992. OUTCOME MEASURES: Presence or absence of documentation of irreversible deep coma, nonresponsive cranial nerves, absent brain-stem reflexes, persistent apnea after removal from ventilator, presence or absence of blood flow detected by radioisotope scanning, presence or absence of electroencephalographic evidence of electrocerebral activity. RESULTS: The 60 patients accounted for 1.5% of all PCCU admissions; 17 were under 1 year of age. In 39 cases brain death was diagnosed using clinical criteria ("certified brain death"), which could not be fully applied in the remaining 21 cases ("uncertifiable but suspected brain death"). Electroencephalography and cerebral blood-flow studies with technetium-99m hexamethyl-propyleneamine oxime were used as ancillary tests in 16 patients with certified brain death and in 17 with uncertifiable but suspected brain death who survived long enough to be tested. Electrocerebral silence was demonstrated in all nine patients who underwent electroencephalography. Cerebral blood flow was undetectable in 26 of the 30 patients tested, and an abnormal pattern of blood flow was seen in the remaining 4, all of whom received a diagnosis of certified brain death. CONCLUSIONS: Pediatricians in this large tertiary care referral centre are using clinical criteria based on the 1987 guidelines of the CMA to diagnose brain death in pediatric patients, including neonates. When clinical criteria cannot be fully applied, ancillary methods of investigation are consistently used. Although the soundness of this pattern of practice is established for adults and older children, its applicability to neonates and infants still needs to be validated.

Accuracy of a structural homology model for a class II histocompatibility protein, HLA-DR1: comparison to the crystal structure.

Structural homology modeling is used to test the accuracy by which a Class I major histocompatibility complex (MHC) could be used to model a Class II MHC. The crystal structure of HLA-aw68 served as a reference molecule to model HLA-DR1. The resulting model was compared to the recently released crystal structure by Brown et al. (Nature, Vol. 364, p. 33-39 (1993)). The overall tertiary structure motif (two alpha-helices and a beta-sheet forming a peptide binding cleft) was maintained. However, significant deviations in the secondary structure elements were found between the model and the DR1 crystal structure. These deviations were consistent with the differences between Class I and Class II crystal structures. In regions where the model and DR1 crystals structures are most similar, side chain orientations are also similar. Specific peptide-MHC interactions are discussed and compared with the crystal structure results.

Biomechanics for preoperative planning and surgical simulations in orthopaedics.

Surgical simulations are particularly appropriate for the large volume and expense of joint replacement procedures in orthopaedics. A first generation surgical simulator has been developed to model the implantation procedure for cementless acetabular and femoral components in total hip replacement surgery. The simulator is based upon finite element analysis and predicts the early postoperative mechanical environment that results from a proposed surgery. Since the short- and long-term clinical success of cementless hip replacement components is very dependent upon the initial mechanics of the bone-implant system, such simulations can help orthopaedic surgeons to develop better preoperative plans.

Enteral immunization and challenge of volunteers given enterotoxigenic E. coli CFA/II encapsulated in biodegradable microspheres.

The development of a safe and effective vaccine against enterotoxigenic Escherichia coli (ETEC) would be useful for travellers and for young children in endemic areas. A feasibility study of an enteral ETEC vaccine prototype consisting of colonization factor antigen II (CFA/II), containing two component antigens CS1 and CS3, encapsulated in biodegradable polymer microspheres (BPM) was conducted in healthy volunteers. Ten adult volunteers swallowed intestinal tubes on days 0, 7, 14 and 28; after collection of jejunal fluid samples, 1 mg of CFA/II in BPM was administered via the tube. Volunteers kept a diary of symptoms after each dose. Secretory IgA in jejunal fluids, serum responses and circulating antibody-secreting cells (ASC) were measured before and after vaccination. The vaccine was well tolerated. Five of ten volunteers developed IgA anti-CFA/II ASC by 7 days after the last dose of vaccine; these same five vaccinees had IgA anti-CS3 ASC, and three of these five vaccinees had IgA anti-CS1 ASC. Five of ten vaccinees developed rises in jejunal fluid sIgA anti-CFA/II with peak GMT of 1:42. About 8 weeks after the first dose of vaccine, ten vaccinees and ten unvaccinated control volunteers underwent challenge with 10(9) c.f.u. ETEC E24377A (O139:H28 LT+ST+CS1+CS3+). Ten of ten controls and seven of ten vaccinees developed diarrhoea (p = 0.11, 30% vaccine efficacy). Two of the three protected vaccinees had the highest numbers of ASC and highest sIgA titres during the course of immunization, suggesting that these responses were protective and that this vaccine development strategy has merit. Future studies with higher dosages and a different dosing schedule are planned.

Usefulness of (Tc 99m) HM-PAO scan in supporting clinical brain death in children: uncoupling flow and function.

OBJECTIVE: To determine the usefulness of (Tc 99m) HM-PAO scan in supporting the clinical diagnosis of brain death. DESIGN: Retrospective review. SETTING: Paediatric Intensive Care Unit. SUBJECTS: A total of 39 paediatric patients had HM-PAO scans conducted to confirm the presence of brain death or to assess the degree of brain injury. INTERVENTIONS: All patients had (Tc 99m) HM-PAO injected before the scan was conducted. MEASUREMENTS AND MAIN RESULTS: Fifty-four scans were done in 39 patients. The majority of cerebral injury was as a result of closed head injury or asphyxia/anoxia. There were 20 scans which demonstrated no cerebral blood flow (CBF); however, in 26 situations patients were clinically brain dead. All of the patients who continued to have CBF in the presence of clinical brain death sustained asphyxial/anoxic injuries. CONCLUSIONS: The HM-PAO scan is a useful non-invasive portable tool for supporting the diagnosis of brain death when there is absent CBF. However, continued flow may be present in asphyxial/anoxic injuries in the presence of clinical brain death.

Preclinical evaluation of microencapsulated CFA/II oral vaccine against enterotoxigenic E. coli.

Colonization Factor Antigen (CFA/II) from enterotoxigenic Escherichia coli (ETEC) prepared under good manufacturing practices (GMP) was successfully incorporated into biodegradable poly(D,L-lactide-co-glycolide) (PLGA) polymer microspheres (BPM) under GMP and found to be safe and immunogenic when administered intraduodenally to rabbits. Following vaccination, Peyer's patch cells responded by lymphocyte proliferation to in vitro challenge with CFA/II. Also, B cells secreting specific anti-CFA/II antibodies were found in spleens following vaccination. No pathological changes were found following total necropsies of ten rabbits vaccinated with CFA/II BPM. Sixty-three per cent of the CFA/II BPM were between 5 and 10 microns diameter by volume particle size distribution; 1.17% protein content; 2.15% moisture; < 0.01% acetonitrile; 1.6% heptane; 22 non-pathogenic bacteria and three fungi per 1 mg protein dose; and passed the general safety test. We conclude that the CFA/II BPM oral vaccine is immunogenic and safe to begin a Phase I clinical safety study following Investigational New Drug approval.

Strontium-89 and low-dose infusion cisplatin for patients with hormone refractory prostate carcinoma metastatic to bone: a preliminary report.

Strontium-89 has been used for the treatment of painful bony metastases in patients suffering from disseminated adenocarcinoma of the prostate, with a variable proportion of patients obtaining clinically significant reductions in analgesic requirements. Based on data revealing enhancement of continuous low-dose rate irradiation by low-dose cisplatin in murine models, a protocol using 148 MBq (4 mCi) of 89Sr and 35 mg/m2 of cisplatin infused over 2 days, 1 and 4 wk after administration of the radioisotope was undertaken. Preliminary data suggest good pain relief with 55% of 18 patients entered thus far obtaining at least a 50% reduction in analgesic requirements. Improvements in total alkaline phosphatase and serum lactate dehydrogenase have consistently been seen, with some patients exhibiting improvements in hemoglobin, tumor markers and bone scans. Toxicity appears to be mild, with no life-threatening complications. In particular, myelosuppression after one course of treatment was modest, but retreatments in two patients has resulted in grade 3 hematologic toxicity. Two patients developed a "pain flare" after administration of cisplatin. Further accrual to this study will allow more accurate determination of pain response rate, and improved evaluation of parameters of objective response.

Analysis of Escherichia coli colonization factor antigen I linear B-cell epitopes, as determined by primate responses, following protein sequence verification.

Colonization factor antigen I (CFA/I)-bearing strains of enterotoxigenic Escherichia coli (ETEC) are responsible for a significant percentage of ETEC diarrheal disease worldwide whether the disease presents as infant diarrhea with high mortality or as traveler's diarrhea. CFA/I pili (fimbriae) are virulence determinants that consist of repeating protein subunits (pilin), are found in several ETEC serogroups, and promote attachment to human intestinal mucosa. While CFA/I pili are highly immunogenic, the antigenic determinants of CFA/I have not been defined. We wished to identify the linear B-cell epitopes within the CFA/I molecule as determined by primate response to the immunizing protein. To do this, we (i) resolved the discrepancies in the literature on the complete amino acid sequence of CFA/I by N-terminal and internal protein sequencing of purified and selected proteolytic fragments of CFA/I, (ii) utilized this sequence to synthesize 140 overlapping octapeptides covalently attached to polyethylene pins which represented the entire CFA/I protein, (iii) immunized three rhesus monkeys with multiple intramuscular injections of purified CFA/I subunit in Freund's adjuvant, and (iv) tested serum from each monkey for its ability to recognize the octapeptides in a capture enzyme-linked immunosorbent assay. Eight linear B-cell epitopes were identified; the region containing an epitope at amino acids 11 to 21 was strongly recognized by all three individual rhesus monkeys, while the amino acid stretches 22 to 29, 66 to 74, 93 to 101, and 124 to 136 each contained an epitope that was recognized by two of the three rhesus monkeys. The three other regions containing epitopes were recognized by one of the three individuals. The monkey antiserum to pilus subunits recognized native intact pili by immunogold labeling of CFA/I pili present on whole H10407 cells. Therefore, immunization with pilus subunits induces antibody that clearly recognizes both synthetic linear epitopes and intact pili. We are currently studying the importance of these defined epitope-containing regions as vaccine candidates.

Structure-function analysis of human transforming growth factor-alpha by site-directed mutagenesis.

Site-directed mutants of transforming growth factor-alpha (TGF-alpha) were expressed in an Escherichia coli outer membrane protein A (ompA) expression/secretion vector under the transcriptional control of the lambda PL promoter. TGF-alpha mutant proteins were isolated from cell pellets using alkaline extraction with 0.1 M-Tris (pH 10.5). The levels of protein expression of 23 TGF-alpha mutants were comparable with those of wild-type TGF-alpha, as determined by immunoblotting and radioimmunoassay. An analysis of biological activity using as assays radioreceptor binding competition and colony formation in soft agar showed that the following mutations destroy the activity of TGF-alpha: Gly-19 to Val, Val-33 to Pro and Gly-40 to Val. Mutations of Arg-42 to Lys, Leu-48 to Ala, Tyr-38 to Trp or Phe-17 to Tyr significantly decrease, but do not destroy, biological activity when compared with the wild-type. Mutations in 14 other residues did not significantly alter receptor binding or colony-forming activity. These studies suggest that two domains localized at the surface of TGF-alpha are important in receptor binding and colony-forming activity. Domain I involves amino acid residues which include Tyr-38 and Leu-48; domain II includes residues Phe-15, Phe-17 and Arg-42.

Differential renal uptake of 201Tl: requirements for acquisition, display and quantification.

Renal uptake of 201Tl may have a role in screening for renal asymmetry in hypertensive patients (HP) who are referred for myocardial scintigraphy. The qualitative aspects of digitized planar images, and quantified differential renal uptake (DRU) of 201Tl were rated by comparing a simple technique (S) for outlining each kidney with an interpolative background subtracted technique (IB). These parameters were assessed in an initial series of patients by varying the length of acquisition (from 1 to 5 min), delay in acquisition (from 10 to 210 min after injection), and image preparation (nine-point smoothing). Six blinded observers rated the quality of coded images. Image quality was improved (P less than 0.01) by increasing the length of acquisition to at least 2 min, by smoothing of the images and by imaging within 2 h of 201Tl injection. Variability in quantification of DRU was suboptimal with acquisition for only 1 min and was more adversely affected with S than with IB. Clinical application of the quantitative technique was assessed in 180 HP and 32 normotensive controls. With IB, the normal range for DRU was slightly greater than for S. The two techniques were comparable in identifying abnormal cases and found 21 +/- 3% (S) and 19 +/- 3% (IB) of HP as lying outside the normal 99% confidence interval. Both quantitative techniques showed excellent agreement with renal angiography (n = 24). Furthermore, preliminary experience with surface markers and with 180 degrees tomography suggests the potential for simultaneous correction for renal depth. These data justify the use of adjunctive renal imaging during myocardial scintigraphy with 201Tl.

Cerebral perfusion imaging with technetium-99m HMPAO following cerebral trauma. Initial experience.

Thirteen patients with cerebral trauma were studied for cerebral perfusion by the use of Tc-99m HMPAO scanning. CT imaging was performed on nine patients. Because of their clinical condition, four patients were scanned only in the planar mode to help establish the diagnosis of brain death. Other indications for study included gunshot wound and blunt or sharp object trauma with or without skull fracture. In all cases, HMPAO scans showed defects with a quality equivalent to or greater than that demonstrated by CT. Our initial results suggest that HMPAO may predict the degree of permanent damage and which patients may develop post-traumatic headache. A diagnosis of brain death can be established without the withdrawal of medical therapy.

Radiopharmaceutical factors in the variable quality of [99mTc]HM-PAO images of the brain.

Variability in the quality of [99mTc]HM-PAO images of the brain has been attributed to differences between kits and the addition of excessive amounts of pertechnetate to the kits. A retrospective study showed no significant differences between batches of kits with respect to radiochemical purity (RCP) or image quality. Up to 3000 MBq (81 mCi) pertechnetate could be added to the kit without adversely affecting RCP or image quality. It was shown that image quality was directly affected by RCP at time of injection and dropped sharply when RCP fell below 85%. Interstitial injection and mixing with blood prior to injection also resulted in poor image quality. Pretreatment with perchlorate reduced uptake of activity in the parotid glands; this improved image quality and reduced the influence of RCP.