Functional MRI at 3T using intermolecular double-quantum coherence (iDQC) with spin-echo (SE) acquisitions.

OBJECT: To reinvestigate the dependence of the signal and contrast on sequence parameters and tissue relaxation times for intermolecular double-quantum coherence (iDQC) signals, and to explore the possibility to use a spin-echo (SE)-iDQC sequence for detecting activation signals at 3T. MATERIALS AND METHODS: Brain activations were detected in five human volunteers in a visual simulation study using a SE-iDQC sequence, in addition to a GE-iDQC and a conventional single-quantum coherence (SQC) blood-oxygenation-level-dependent (BOLD) sequence. A brain phantom was also used for some quantitative measurements. RESULTS: By choosing an optimal echo time TE (approximately T2) and iDQC evolution time tau(approximately 20 ms), robust brain activations were detected using the SE-iDQC sequence, in addition to the GE-iDQC and a conventional single-quantum coherence (SQC) BOLD sequence. A higher percentage signal change due to activation was observed for both the iDQC-based measurements in comparison to the conventional SQC acquisition. CONCLUSION: Even though a phenomenological analysis consistent with the experimental results was provided, a detailed model is still needed for the contrast mechanism at microscopic level to guide potential applications of brain functional imaging based on the SE-iDQC.

Quantitation of intermolecular dipolar effects in NMR spectroscopy and high order MSE MR imaging.

An analytical expression for intermolecular dipolar effects was derived for the CRAZED sequence with an arbitrary flip angle of the second RF pulse and time-varying gradients. A combination of the demagnetizing field theory and product operator formalism was utilized in the derivation. It is demonstrated that the time-averaged, not instantaneous, orientation of the applied gradients determines the contributions of long-range intermolecular dipole effects. An imaging sequence to detect intermolecular dipolar effects was designed. The second- and third-order multiple spin echo (MSE) NMR signals of swine muscle were observed and were found to be in good agreement with the theoretical predictions. MSE images of a water phantom with nth orders (n = 2, - 3, - 4, and - 5) were also obtained, and their relative signal intensities and optimized TE values were elucidated and compared with the theoretical prediction.

Persistence of external chloride and DIDS binding after chemical modification of Glu-681 in human band 3.

Although its primary function is monovalent anion exchange, the band 3 protein also cotransports divalent anions together with protons at low pH. The putative proton binding site, Glu-681 in human erythrocyte band 3, is conserved throughout the anion exchanger family (AE family). To determine whether or not the monovalent anion binding site is located near Glu-681, we modified this residue with Woodward's reagent K (N-ethyl-5-phenylisoxazolium-3'-sulfonate; WRK). Measurements of Cl(-) binding by (35)Cl-NMR show that external Cl(-) binds to band 3 even when Cl(-) transport is inhibited approximately 95% by WRK modification of Glu-681. This indicates that the external Cl(-) binding site is not located near Glu-681 and thus presumably is distant from the proton binding site. DIDS inhibits Cl(-) binding even when WRK is bound to Glu-681, indicating that the DIDS binding site is also distant from Glu-681. Our data suggest that the DIDS site and probably also the externally facing Cl(-) transport site are located nearer to the external surface of the membrane than Glu-681.

Identification of a human heavy chain antibody fragment directed against human platelet alloantigen 1a by phage display library.

The human platelet alloantigen HPA-1a (PlA1) is responsible for most cases of post-transfusion purpura and neonatal alloimmune thrombocytopenia in the Caucasian population. HPA-1a and HPA-1b are two allelic forms of the platelet membrane glycoprotein IIIa (GPIIIa) gene that differ by a single amino acid. In this report, we describe the development of a recombinant heavy chain antibody fragment capable of distinguishing between the homozygous forms of HPA-1a and HPA-1b. This antibody fragment was isolated from the lymphocytes of an immunized individual through the use of a phage display library system. The recombinant antibody fragment reacted with human platelet lysates from HPA-1a homozygous donors, the HPA-1a form of recombinant N-terminal GPIIIa and intact HPA-1a platelets, but did not react with platelet lysate from HPA-1b homozygous donors, reduced HPA-1a form of platelet GPIIIa or other platelet glycoproteins. This HPA-1a specific human antibody fragment works well in common laboratory assays such as ELISA and flow cytometry, which can assist in identifying HPA-1b homozygous individuals who are known to have a higher risk for developing neonatal alloimmmune thrombocytopenia and post-transfusion purpura. Thus, selection of recombinant antibody fragment using phage display offers a promising alternative to hybridoma technology for the production of human antibodies against human alloantigens and holds potential as a technique in therapeutic applications.

Detection of human immunodeficiency virus type 1 (HIV-1) DNA and RNA sequences in HIV-1 antibody-positive blood donors in Uganda by the Roche AMPLICOR assay.

The ability of commercially available PCR-based assays to accurately detect or quantitate human immunodeficiency virus type 1 (HIV-1) DNA or RNA in individuals predominantly infected with HIV-1 subtypes A and D is not known. Therefore, peripheral leukocytes from 43 individuals in Kampala, Uganda, positive for HIV by the Western blot (immunoblot) assay were tested by using the Roche AMPLICOR HIV-1 assay for the detection of DNA gag sequences. Plasma from these same individuals was tested by using the Roche HIV-1 AMPLICOR MONITOR HIV-1 assay for the quantitation of HIV-1 RNA gag sequences. In addition, peripheral leukocytes were tested for HIV-1 DNA by using a lower annealing temperature or a different primer pair for the HIV-1 pol region. The proportions of individuals with detectable HIV-1 DNA and RNA gag sequences by the Roche assays were 74 and 90%, respectively. The proportions positive for HIV-1 DNA sequences by using a 50 degrees C annealing temperature or the pol primer pair were 71 and 98%, respectively. In summary, the standard Roche assay did not detect HIV-1 DNA sequences in a significant number of HIV-1-infected individuals in Uganda. However, use of a pol primer pair increased the sensitivity of the assay to 98%. The sensitivity of the Roche AMPLICOR MONITOR assay for the detection and quantitation of HIV-1 RNA sequences was significantly higher than that of the DNA-based assay, but the efficiency of the assay, and hence, the accuracy of the values obtained with RNA, is not known. Modifications to existing assays are needed to enhance the sensitivities and accuracies of these commercially available assays for use in developing countries where non-B HIV-1 subtypes predominate.

Measurement of in vitro P-selectin expression by flow cytometry.

Measurement of in vivo platelet activation is difficult after phlebotomy and during blood processing for analysis. We used flow cytometry to measure platelet surface expression of P-selectin in the presence and absence of trimethylsphingosine (a platelet activation inhibitor) and compared the results with those from the standard methods of preventing in vitro P-selectin expression. Percent activation was calculated as a ratio of mean sample fluorescence to 100% mean fluorescence after phorbol myristate acetate treatment. Twenty-five micromoles per liter of trimethylsphingosine kept in vitro platelet activation below 5% up to 6 hours after collection and below 10% at 24 hours after collection. Trimethylsphingosine failed to prevent platelet activation caused by centrifugation, storage at 4 degrees C, or stimulation with common agonists. Addition of trimethylsphingosine to whole blood was valuable in preventing in vitro platelet activation. This compound promises to be a useful preservative for diagnostic testing of platelet activation.

Source of transport site asymmetry in the band 3 anion exchange protein determined by NMR measurements of external Cl- affinity.

Flux measurements indicate that a far greater number of unloaded band 3 anion transport sites face the cytoplasm than face the external medium, but the reason for this striking asymmetry has remained obscure. To resolve this question, we have measured the apparent Cl- affinity of the transport site of human red blood cell band 3 protein under various conditions by analyzing the 35Cl NMR free induction decay (FID). The [Cl-] that half-saturates the transport sites with [Cli] = [Clo] (K1/2) in RBC membranes (ghosts) is 46 +/- 5 mM at 0 degree C, while the Ko1/2 (for half-saturation with [Clo] at constant [Cli]) of intact cells is 3.2 +/- 2.1 mM. When cells were pretreated with EM, an inhibitor of band 3 anion exchange that does not prevent Cl- binding to the external transport site, K1/2 and Ko1/2 are 41 +/- 14 and 46 +/- 12 mM, respectively. The EM-induced increase in Ko1/2 with little change in K1/2 can be most simply interpreted as meaning that EM abolishes the effects of the translocation rate constants on Ko1/2 so that Ko1/2 and K1/2 of EM-treated cells now both reflect the true dissociation constant for binding of Cl- to the external transport site, Ko. The fact that Ko for a slowly transported anion, iodide, is nearly the same in EM-treated as in control cells indicates that EM does not significantly affect Ko for chloride. Our results indicate that the true dissociation constants for Cl- at the inside and outside are very similar but that the rate constant for inward translocation is much larger than that for outward translocation. For this reason, both unloaded and Cl-loaded transport sites are asymmetrically oriented toward the inside, and Ko1/2 (in untreated cells) is much lower than Ko.

Cytokine induction of platelet activation.

Recent studies have focused on the accumulation of cytokines in stored platelet concentrates and the role that these cytokines play in mediating transfusion reactions. To elucidate any additional adverse effects that may be associated with cytokine accumulation, the authors examined whether cytokines, which normally accumulate during routine platelet storage, can cause platelet activation in vitro. Concentrations of IL-6 and IL-8 were first determined for random donor platelet concentrates on days 1 through 4 of storage. Fresh platelets were then incubated with these levels of exogenous cytokines, and activation measured by flow cytometry using a monoclonal antibody directed against p-Selectin. Significant platelet activation was observed with concentrations of cytokines which are normally present in days 3 and 4 of shelf life. The study data demonstrate that levels of cytokines that routinely accumulate in stored platelet products can affect platelet biology. Strategies to reduce cytokine generation during platelet storage may be a method to improve the function and viability of stored platelets used for transfusion.

The role of HLA antibodies in neonatal thrombocytopenia: a prospective study.

The role of HLA antibodies in neonatal alloimmune thrombocytopenia is controversial. We prospectively studied the sera of obstetric patients at delivery for HLA antibodies and correlated their presence with umbilical cord blood platelet counts. We studied 493 births at The Johns Hopkins Hospital comprising of 357 African American, 115 Caucasian, and 21 babies of other racial groups. One hundred and thirty nine mothers had HLA antibodies. Of these HLA alloimmunized mothers, only ten infants had platelet counts of 150,000/ microL or less. Three hundred and eight mothers with no detectable antibodies gave birth to 27 infants with platelet counts of 150,000/microL or less. Yates corrected Chi square analysis showed no significant relationship between maternal HLA alloimmunization and baby platelet count (p = 0.709). Only 8 of sixty cord sera from babies of HLA alloimmunized mothers were positive for HLA antibodies. The HLA cord blood antibody results were then correlated with the neonatal platelet counts. The Fisher's exact test showed no significant relationship between the presence of HLA antibodies in cord blood samples and neonatal platelet counts (p = 0.232). Although one third (31%) of mothers have HLA antibodies, neonatal thrombocytopenia is rarely associated with this finding. However, HLA antibodies can cross the placenta, and in these unusual cases, may be associated with a higher risk of neonatal thrombocytopenia.

Detection of Cl- binding to band 3 by double-quantum-filtered 35Cl nuclear magnetic resonance.

We have applied double-quantum-filtered (DQF) NMR of 35Cl to study binding of Cl- to external sites on intact red blood cells, including the outward-facing anion transport sites of band 3, an integral membrane protein. A DQF 35Cl NMR signal was observed in cell suspensions containing 150 mM KCl, but the DQF signal can be totally eliminated by adding 500 microM 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS), an inhibitor that interferes with Cl- binding to the band 3 transport site. Therefore, it seems that only the binding of Cl- to transport sites of band 3 can give rise to a 35Cl DQF signal from red blood cell suspensions. In accordance with this concept, analysis of the single quantum free induction decay (FID) revealed that signals from buffer and DNDS-treated cells were fitted with a single exponential function, whereas the FID signals of untreated control cells were biexponential. The DQF signal remained after the cells were treated with eosin-5-maleimide (EM), a noncompetitive inhibitor of chloride exchange. This result supports previous reports that EM does not block the external chloride binding site. The band 3-dependent DQF signal is shown to be caused at least in part by nonisotropic motions of Cl- in the transport site, resulting in incompletely averaged quadrupolar couplings.

Studies using immobilized platelet glycoproteins for detection of platelet alloantibodies.

The utility of an enzyme linked immunoassay for the detection of platelet alloantibodies to isolated platelet glycoproteins employing commercially available monoclonal antibodies was studied. The authors established the usefulness of the assay for antibodies to platelet alloantigens (APAA) by testing its sensitivity and specificity under different conditions and using a variety of alloantibodies from patients with immune mediated thrombocytopenia. The authors were able to specifically detect a variety of antibodies to platelet alloantigens on different platelet glycoproteins. These antibodies had specificity for HPA-1a (PIA1), HPA-3 (Bak) and HPA-5 (Br). These results show that the APAA is easily adaptable for routine diagnostic testing in hospital laboratories using commercially available monoclonal antibodies and for the diagnosis of alloimmune mediated thrombocytopenic conditions.

35Cl nuclear magnetic resonance line broadening shows that eosin-5-maleimide does not block the external anion access channel of band 3.

It has been suggested that Lys-430 of band 3, with which eosin-5-maleimide (EM) reacts, is located in the external channel through which anions gain access to the external transport site, and that EM inhibits anion exchange by blocking this channel. To test this, we have used 35Cl nuclear magnetic resonance (NMR) to measure Cl- binding to the external transport site in control and EM-treated human red blood cells. Intact cells were used rather than ghosts, because in this case all line broadening (LB) results from binding to external sites. In an NMR spectrometer with a 9.4-T magnetic field, red blood cells at 50% concentration (v/v) in 150 mM Cl- medium at 3 degrees C caused 19.0 +/- 1.2 Hz LB. Of this, 7.9 +/- 0.7 Hz was due to Cl- binding to the high affinity band 3 transport sites, because it was prevented by an apparently competitive inhibitor of anion exchange, 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS). The LB was not due to hemoglobin released from the cells, as little LB remained in the supernatant after cells were removed by centrifugation. Saturable Cl- binding remained in EM-treated cells, although the binding was no longer DNDS-sensitive, because EM prevents binding of DNDS. The lower limit for the rate at which Cl- goes from the binding site to the external medium is 2.15 x 10(5) s-1 for control cells and 1.10 x 10(5) s-1 for EM-treated cells, far higher than the Cl- translocation rate at 3 degrees C (about 400 s-1). Thus, EM does not inhibit Cl- exchange by blocking the external access channel. EM may therefore be useful for fixing band 3 in one conformation for studies of Cl- binding to the external transport site.

Quantitative MRI of Gd-DTPA uptake in tumors: response to photodynamic therapy.

A partial saturation method is described for obtaining rapid images of tissue 1H spin-lattice relaxation rates following administration of the paramagnetic contrast agent gadolinium-diethylenetriaminepentaacetate. The paramagnetic contribution to the relaxation rates is proportional to the concentration of contrast agent, making possible quantitative studies of paramagnetic contrast agent uptake or vessel leakage. Snapshot imaging capabilities are not required. Maps of contrast agent uptake rates are made in rat borne tumors before and following photodynamic therapy, which is known to cause vascular damage. Uptake efficiency is spatially heterogeneous before and after therapy. Decreases in uptake rate are observed after two photo-irradiation protocols, which differ by a factor of four in fluence rate but deliver the same total fluence. There is no apparent fluence rate dependence for changes in the uptake rates within 5 h after therapy. Whole tumor measurements of nucleotide triphosphates, inorganic phosphate, pH, and lactate made with NMR spectroscopy indicate that, while net ATP production is inhibited, lactate concentrations are not strongly affected by photodynamic therapy. The ratio of nucleotide triphosphates to inorganic phosphate falls to 0.21 +/- 0.02 of initial values 5 h after tumors are treated with the lower fluence rate protocol, and falls to 0.40 +/- 0.06 in tumors treated with the higher fluence rate.

Flow through clots determines the rate and pattern of fibrinolysis.

Thrombolytic therapy depends on penetration of plasminogen activator into clots which occurs through diffusion and flow. An in vitro system has been developed to characterize the rate and pattern of fibrinolysis in relation to flow through occlusive clots exposed to a pressure gradient. Whole blood clots formed in plastic tubes were perfused with plasma containing 1 microgram/ml tissue plasminogen activator (t-PA) and 0.5 or 1 mmol/l gadolinium-diethylentriamine pentaacetic acid (Gd-DTPA), a paramagnetic substance used as a contrast enhancer for magnetic resonance (MR) imaging. T1-weighted spin echo MR images were obtained during clot perfusion at 3-5 min intervals for 45 min. Characteristic signal intensities allowed identification of non-perfused, perfused but non-lysed, and completely lysed areas of clot. A spatially resolved time course of perfusion and subsequent lysis was constructed for 10 clots. Plasma flowed non-uniformly through clots forming asymmetric channels that left some areas non-perfused. The longitudinal velocity of flow through the dominant channel was 1.6 +/- 0.7 mm/min. The flow rate during the first five minutes was 7.5 +/- 6.5 microliters/min and 15.3 +/- 10 microliters/min between min 26-30 in clots that had not completely recanalized by that time. A sharp increase in flow was noted at the time of recanalization that occurred at 37 +/- 11 min. Clot lysis followed the pattern of perfusion through the dominant channel after a lag time of 13 +/- 4 min, representing the time required for enzymatic processes. The delay time between perfusion and lysis was longer in regions with slower flow indicating that the rate of t-PA delivery influenced the rate of fibrinolysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Solid-state and solution conformations of isotiazofurin: crystallographic, computational and 1H NMR studies.

Isotiazofurin (C9H12N2O5S, NSC363223) is a thiazole nucleoside analogue of the antitumour agent tiazofurin. The conformation of this analogue has been studied using a variety of experimental and computational techniques. The crystal and molecular structure of isotiazofurin has been determined using single-crystal X-ray diffraction techniques and refined to a conventional R value of 0.030 for all data. Conformational features conserved in other thiazole nucleoside structures are also observed in the solid-state structure of isotiazofurin. The C-glycosidic torsion angle remains in the anti conformation and the carboxamide amino group remains cis-planar to the ring nitrogen. Ab initio calculations at the RHF/321G*@321G* level and natural bond orbital analysis of the results suggest that the carboxamide cis-planar conformation observed in the solid state is maintained in solution. However, semiempirical calculations suggest that a syn conformation about the C-glycosidic bond is energetically favored. This is supported by 1H nuclear Overhauser enhancement (NOE) studies. Analyses of NOE results using both slow- and rapid-exchange models indicate a preference for the syn conformation in solution. Thus, the anti conformation observed in the crystal structures of isotiazofurin is not maintained by isotiazofurin in solution.

Physicians' utilization and charges for outpatient diagnostic imaging in a Medicare population.

OBJECTIVES AND RATIONALE: For 10 common clinical presentations, we assessed differences in physicians' utilization of and charges for diagnostic imaging, depending on whether they performed imaging examinations in their offices (self-referral) or referred their patients to radiologists (radiologist-referral). METHODS: Using previously developed methodologies, we generated episodes of medical care from an insurance claims database. Within each episode, we determined whether diagnostic imaging had been performed, and if so, whether by a self-referring physician or a radiologist. For each of the 10 clinical presentations, we compared the mean imaging frequency, mean imaging charges per episode of care, and mean imaging charges for diagnostic imaging attributable to self- and radiologist-referral. RESULTS: Depending on the clinical presentation, self-referral resulted in 1.7 to 7.7 times more frequent performance of imaging examinations than radiologist-referral (P < .01, all presentations). Within all physician specialties, self-referral uniformly led to significantly greater utilization of diagnostic imaging than radiologist-referral. Mean imaging charges per episode of medical care (calculated as the product of the frequency of utilization and mean imaging charges) were 1.6 to 6.2 times greater for self-referral than for radiologist-referral (P < .01, all presentations). When imaging examinations were performed--including those performed in both physicians' offices and hospital outpatient departments--mean imaging charges were significantly greater for radiologists than for self-referring physicians in seven of the clinical presentations (P < .01). This result is related to the high technical charges of hospital outpatient departments; in office practice, radiologists' mean charges for imaging examinations were significantly less than those of self-referring physicians for seven clinical presentations (P < .01). CONCLUSIONS: Nonradiologist physicians who operate diagnostic imaging equipment in their offices perform imaging examinations more frequently, resulting in higher imaging charges per episode of medical care. These results extend our previous research on this subject by their focus on a broader range of clinical presentations; a mostly elderly, retired population; and the inclusion of higher-technology imaging examinations.

Hetergeneous tumour response to photodynamic therapy assessed by in vivo localised 31P NMR spectroscopy.

Photodynamic therapy (PDT) is efficacious in the treatment of small malignant lesions when all cells in the tumour receive sufficient drug, oxygen and light to induce a photodynamic effect capable of complete cytotoxicity. In large tumours, only partial effectiveness is observed presumably because of insufficient light penetration into the tissue. The heterogeneity of the metabolic response in mammary tumours following PDT has been followed in vivo using localised phosphorus NMR spectroscopy. Alterations in nucleoside triphosphates (NTP), inorganic phosphate (Pi) and pH within localised regions of the tumour were monitored over 24-48 h following PDT irradiation of the tumour. Reduction of NTP and increases in Pi were observed at 4-6 h after PDT irradiation in all regions of treated tumours. The uppermost regions of the tumours (those nearest the skin surface and exposed to the greatest light fluence) displayed the greatest and most prolonged reduction of NTP and concomitant increase in Pi resulting in necrosis. The metabolite concentrations in tumour regions located towards the base of the tumour returned a near pre-treatment levels by 24-48 h after irradiation. The ability to follow heterogeneous metabolic responses in situ provides one means to assess the degree of metabolic inhibition which subsequently leads to tumour necrosis.

Comparison of agarose and cross-linked protein gels as magnetic resonance imaging phantoms.

Measurements of the magnetic field dependence of spin-lattice relaxation rates and the response of the water-proton signal intensity to off-resonance radio frequency fields show that the commonly used agarose phantom provides a less faithful representation for the magnetic response of tissue than does a cross-linked protein system. The origin of these differences lies in the structure and intramolecular dynamics of the macromolecular system used to make the gel. These distinctions will also cause differences in the magnetic response of the water spin system when paramagnetic relaxation agents or contrast agents are incorporated. Use of a thermally cross-linked bovine serum albumin phantom is suggested.

Frequency and costs of diagnostic imaging in office practice--a comparison of self-referring and radiologist-referring physicians.

BACKGROUND: To assess possible differences in physicians' practices with respect to diagnostic imaging, we compared the frequency and costs of imaging examinations as performed by primary physicians who used imaging equipment in their offices (self-referring) and as ordered by physicians who always referred patients to radiologists (radiologist-referring). METHODS: Using a large, private insurance-claims data base, we analyzed 65,517 episodes of outpatient care by 6419 physicians for acute upper respiratory symptoms, pregnancy, low back pain, or (in men) difficulty urinating. The respective imaging procedures studied were chest radiography, obstetrical ultrasonography, radiography of the lumbar spine, and excretory urography, cystography, or ultrasonography. RESULTS: For all four clinical presentations, the self-referring physicians obtained imaging examinations 4.0 to 4.5 times more often than the radiologist-referring physicians (P less than 0.0001 for all four). For chest radiography, obstetrical ultrasonography, and lumbar spine radiography, the self-referring physicians charged significantly more than the radiologists for imaging examinations of similar complexity (P less than 0.0001 for all three). The combination of more frequent imaging and higher charges resulted in mean imaging charges per episode of care that were 4.4 to 7.5 times higher for the self-referring physicians (P less than 0.0001). These results were confirmed in a separate analysis that controlled for the specialty of the physician. CONCLUSIONS: Physicians who do not refer their patients to radiologists for medical imaging use imaging examinations more frequently than do physicians who refer their patients to radiologists, and the charges are usually higher when the imaging is done by the self-referring physician. From our results it is not possible to determine which group of physicians uses imaging more appropriately.

Structural effects of hydration: studies of lysozyme by 13C solids NMR.

13C-nmr spectra of lysozyme obtained at 50.3 MHz using both static and magic-angle-spinning-cross-polarization methods are reported at several water contents. The line widths and consequent resolution in the hydrated material is substantially improved over that in the lyophilized protein. The line narrowing is not commensurate with loss of a proton-carbon dipole-dipole coupling or dramatic changes in the relaxation parameters characterizing magnetization transfer from protons to carbon in the Hartmann-Hahn cross-polarization experiment. We interpret these data in terms of the water inducing a decrease in the distribution of local conformations sampled by the protein, although the magnitude of the conformational reorientations required to account for the data are not necessarily large nor do they imply a major unfolding of the protein on dehydration.