Simultaneous R2*, R2, and R2' quantification by combining S0 estimation of the free induction decay with a single spin echo: A single acquisition method for R2 insensitive quantification of holmium-166-loaded microspheres.

PURPOSE: To present a new method, S0 estimation of the free induction decay combined with a single spin echo measurement (SOFIDSE), that enables simultaneous measurements of R2*, R2, and R2' in order to quantify the local concentration of holmium microspheres (Ho-MS) for radioembolization. THEORY AND METHODS: SOFIDSE estimates R2* and the signal magnitude at time point 0, S0, from a multigradient echo readout of the free induction decay and subsequently estimates R2 using S0 and a single spin echo, from which R2' is deducted. The method was evaluated by comparing SOFIDSE R2 values with values obtained from shifted spin echo (SSE) measurements on a phantom setup containing Ho-MS and from dual spin echo measurements on a healthy volunteer. RESULTS: On average, SOFIDSE showed a small overestimation of R2 values compared with SSE independent of the microsphere concentration. R2' values determined by subtraction of either SOFIDSE R2 or SSE R2 from R2* showed excellent agreement (correlation coefficient = 1; P = 9 · 10(-11)). The Ho-MS-induced R2' values obtained by SOFIDSE were insensitive to the R2 value of the tissue in which they resided. CONCLUSION: SOFIDSE enables quantification of Ho-MS, in media with spatially or temporally varying R2 values, in a single acquisition.

Center-out radial sampling with off-resonant reconstruction for efficient and accurate localization of punctate and elongated paramagnetic structures.

Accurate localization of interventional devices, for example, needles and brachytherapy seeds, is desired for interventional procedures. MRI is usually considered unsuitable for this purpose, as the induced signal voids and signal pile-ups do not necessarily represent the exact location of the devices. Center-out radial sampling with off-resonance reception (co-RASOR) has been shown to solve this problem by repositioning the signal pile-up into the geometrical center of the interventional devices. However, the multiple acquisitions required for co-RASOR resulted in a low efficiency and unsuitability for near real-time interventional purposes. Herein, we aim to increase the efficiency of co-RASOR by relying on multiple off-resonance reconstructions of a single acquisition rather than on multiple acquisitions. The soundness of this approach is shown by demonstrating the equivalence of acquisition co-RASOR and reconstruction co-RASOR, both theoretically and experimentally. An algorithm is proposed and evaluated to obtain the geometric centers of the devices, while suppressing the background. This procedure is shown to be effective, in vitro as well as ex vivo, and to yield signal intensity increases in the order of 150-400% of the average signal, in the geometric center of a brachytherapy seed and a needle, respectively. The geometric accuracy of the resultant images is confirmed by computed tomography.

Towards inherently distortion-free MR images for image-guided radiotherapy on an MRI accelerator.

In MR-guided interventions, it is mandatory to establish a solid relationship between the imaging coordinate system and world coordinates. This is particularly important in image-guided radiotherapy (IGRT) on an MRI accelerator, as the interaction of matter with γ-radiation cannot be visualized. In conventional acquisitions, off-resonance effects cause discrepancies between coordinate systems. We propose to mitigate this by using only phase encoding and to reduce the longer acquisitions by under-sampling and regularized reconstruction. To illustrate the performance of this acquisition in the presence of off-resonance phenomena, phantom and in vivo images are acquired using spin-echo (SE) and purely phase-encoded sequences. Data are retrospectively under-sampled and reconstructed iteratively. We observe accurate geometries in purely phase-encoded images for all cases, whereas SE images of the same phantoms display image distortions. Regularized reconstruction yields accurate phantom images under high acceleration factors. In vivo images were reconstructed faithfully while using acceleration factors up to 4. With the proposed technique, inherently undistorted images with one-to-one correspondence to world coordinates can be obtained. It is a valuable tool in geometry quality assurance, treatment planning and online image guidance. Under-sampled acquisition combined with regularized reconstruction can be used to accelerate the acquisition while retaining geometrical accuracy.

Effects of medetomidine and ketamine on the regional cerebral blood flow in cats: a SPECT study.

Brain perfusion can be investigated using single photon emission computed tomography (SPECT) and the intravenous injection of (99m)technetium ethyl cysteinate dimer ((99m)Tc-ECD). However, sedation using medetomidine, an α(2)-agonist, or anaesthesia using medetomidine and ketamine, an N-methyl-d-aspartate-(NMDA)-antagonist, may be required for SPECT studies in cats but can affect the regional cerebral blood flow (rCBF). The effects of medetomidine, with or without ketamine, on regional brain perfusion were therefore investigated in six cats under three conditions. Injection of tracer occurred before sedation or anaesthesia (condition A), following intramuscular (IM) sedation with medetomidine (condition M) or after IM anaesthesia with medetomidine and ketamine (condition MK). Medetomidine and medetomidine with ketamine caused a significantly higher total tracer uptake in all brain regions. Semi-quantification of brain perfusion gave lower perfusion indices in several sub-cortical regions in conditions M and MK, compared to A. Left-right differences were observed in the temporal cortex (A), the temporal, parietal cortex and the thalamus (M) and the frontal cortex (MK). A significantly higher perfusion index in the sub-cortical regions, compared to the whole cortex, was only present in condition A. This study showed that caution is needed when quantifying brain perfusion indices when using sedative or anaesthetic agents that may affect rCBF.

Correction of gradient echo images for first and second order macroscopic signal dephasing using phase derivative mapping.

Gradient echo techniques are often hampered by signal dephasing due to macroscopic phase perturbations because of system imperfections (shimming) or object induced perturbations of the magnetic field (hemorrhagic lesions, calcified tissue, air-tissue interfaces). Many techniques have been proposed to reduce the effects of macroscopic phase variations. Among these techniques are tuned pulse sequences, fitting techniques and reconstruction algorithms. These methods, however, suffer from one or more of the following drawbacks: they require longer acquisition times, require additional acquisitions, compensate only locally, can only be applied to multi-gradient echo data or may result in inaccurate results. In this work a generally applicable post-processing technique is presented to evaluate and compensate signal alterations invoked by first and second order macroscopic phase incoherences. In this technique, the derivatives of the signal phase are determined by applying the Fourier derivative theorem on the complex data. As a result, the phase derivatives are obtained without phase unwrapping and without compromising the resolution. The method is validated for single and multi-echo acquisitions by experiments on a co-axial cylinder phantom with known macroscopic field disturbances. The potential of the method is demonstrated on a multi-gradient echo acquisition on the head of a human volunteer. In general a first order correction is shown to be sufficient, however higher order correction is found to be beneficial near sharp transitions of the magnetic field.

Prevalence of squamous abnormalities in women with a recent smear without endocervical cells is lower as compared to women with smears with endocervical cells.

The purpose of this study was to determine the prevalence rate ratio of squamous lesions in women with recent smears without endocervical component (ECC-) versus women having a smear with ECC+ and to estimate the true prevalence of these lesions in women with ECC- smears by addition of short-term follow-up results of negative ECC- smears. Results of initial smears in a 3-year period, as well as follow-up results of negative ECC- smears in the same period were retrieved. Women were categorized into two groups: having ECC- and ECC+ smears. The data were analysed for three outcome parameters, ASCUS or higher (ASCUS+), LSIL or higher (LSIL+) and HSIL or higher (HSIL+). Squamous abnormalities occurred far less frequently in women with initial ECC- than with ECC+ smears. Prevalence rate ratio (PRR) was 0.27 for ASCUS+, 0.39 for LSIL+ and 0.36 for HSIL+. Addition of follow-up results of negative ECC- smears, as a correction for false-negative ECC- smears, results in PRRs which are still significantly lower than 1, and most marked in subset HSIL+ (PRR = 0.60). We conclude that the true prevalence of squamous lesions in women with recent ECC- smears is significantly lower as compared with ECC+ smears. These findings lent support to the decision to abolish the repeat of ECC- smears in the Dutch population screening programme.

The effect of Replens on vaginal cytology in the treatment of postmenopausal atrophy: cytomorphology versus computerised cytometry.

BACKGROUND: After the menopause decreased concentrations of oestrogen may result in insufficient maturation of the vaginal epithelium, which can lead to a range of vaginal discomforts. This state of vaginal atrophy may be treated with oestrogen replacement treatment. Replens, a non-hormonal alternative to oestrogen replacement treatment has been shown to be effective in relieving symptoms related to vaginal atrophy in previous studies. AIMS: To study the effect of Replens on the maturation of the vaginal epithelium and morphology of the vaginal cells and to compare the results of a recently developed cytomorphometric method with manual assessment of the degree of maturation in vaginal smears. METHODS: Vaginal smears from 38 postmenopausal women suffering from symptoms related to vaginal atrophy were analysed manually and by cytomorphometry. The maturation value (MV) and the percentages of (para)basal, intermediate, and superficial cells (maturation index; MI) were measured by both methods before and after treatment with Replens. Cytomorphometry also measured mean cellular area, mean nuclear area, and mean area ratio. RESULTS: A correlation was shown between the two methods in the assessment of percentages of (para) basal and intermediate cells and MV. Cytomorphometric data showed a significant increase in mean cellular area, indicating a positive effect of Replens on the maturation of the vaginal epithelium. Changes in nuclear area and ratio between nuclear and cellular areas were not significant. Treatment with Replens did not influence MI or MV, as assessed by the two methods. CONCLUSIONS: Replens did have an effect on vaginal morphology. The automated procedure may be useful for the assessment of maturation in vaginal smears and is more sensitive to small (subvisual) changes.

Small GTP-binding protein Ral modulates regulated exocytosis of von Willebrand factor by endothelial cells.

Weibel-Palade bodies are endothelial cell-specific organelles, which contain von Willebrand factor (vWF), P-selectin, and several other proteins. Recently, we found that the small GTP-binding protein Ral is present in a subcellular fraction containing Weibel-Palade bodies. In the present study, we investigated whether Ral is involved in the regulated exocytosis of Weibel-Palade bodies. Activation of endothelial cells by thrombin resulted in transient cycling of Ral from its inactive GDP-bound to its active GTP-bound state, which coincided with release of vWF. Ral activation and exocytosis of Weibel-Palade bodies were inhibited by incubation with trifluoperazine, an inhibitor of calmodulin, before thrombin stimulation. Functional involvement of Ral in exocytosis was further investigated by the expression of constitutively active and dominant-negative Ral variants in primary endothelial cells. Introduction of active Ral G23V resulted in the disappearance of Weibel-Palade bodies from endothelial cells. In contrast, the expression of the dominant-negative Ral S28N did not affect the amount of Weibel-Palade bodies in transfected cells. These results indicate that Ral is involved in regulated exocytosis of Weibel-Palade bodies by endothelial cells.

Alternative splicing of the human Rab6A gene generates two close but functionally different isoforms.

Analysis of the human Rab6A gene structure reveals the presence of a duplicated exon, and incorporation of either of the two exons by alternative splicing is shown to generate two Rab6 isoforms named Rab6A and Rab6A', which differ in only three amino acid residues located in regions flanking the PM3 GTP-binding domain of the proteins. These isoforms are ubiquitously expressed at similar levels, exhibit the same GTP-binding properties, and are localized to the Golgi apparatus. Overexpression of the GTP-bound mutants of Rab6A (Rab6A Q72L) or Rab6A' (Rab6A' Q72L) inhibits secretion in HeLa cells, but overexpression of Rab6A' Q72L does not induce the redistribution of Golgi proteins into the endoplasmic reticulum. This suggests that Rab6A' is not able to stimulate Golgi-to-endoplasmic reticulum retrograde transport, as described previously for Rab6A. In addition, Rab6A' interacts with two Rab6A partners, GAPCenA and "clone 1," but not with the kinesin-like protein Rabkinesin-6, a Golgi-associated Rab6A effector. Interestingly, we found that the functional differences between Rab6A and Rab6A' are contingent on one amino acid (T or A at position 87). Therefore, limited amino acid substitutions within a Rab protein introduced by alternative splicing could represent a mechanism to generate functionally different isoforms that interact with distinct sets of effectors.

Assembly of multimeric von Willebrand factor directs sorting of P-selectin.

We designed a model system to study the role of von Willebrand factor (vWF) in the sorting of P-selectin and the biogenesis of Weibel-Palade body (WPB)-like organelles. For that purpose, a human epithelial cell line (T24) that synthesizes P-selectin mRNA, but which is devoid of vWF mRNA synthesis and storage organelles, was transfected with full-length vWF cDNA or a deletion mutant thereof. Stable transfectants of T24 with full-length vWF cDNA revealed the generation of WPB-like organelles as demonstrated by colocalization of vWF and P-selectin with double-labeling immunofluorescence. In contrast, T24 cells transfected with vWF delD'D3 cDNA, encoding a mutant that is unable to form vWF multimers, displayed only perinuclear vWF staining, whereas no indication was found for the presence of WPB-like organelles. The contents of the organelles in full-length vWF cDNA-transfected T24 cells were released on activation of the protein kinase C pathway, similar to the situation with genuine endothelial cells. The expression of vWF did not affect the biosynthesis of P-selectin, as deduced from the observation that untransfected and vWF cDNA-transfected T24 cells contained the same amount of P-selectin mRNA. We propose that the biosynthesis of multimeric vWF directs the generation of WPB-like organelles, as evidenced by the sequestering and anchoring of P-selectin into these storage granules.

Small GTP-binding protein RalA associates with Weibel-Palade bodies in endothelial cells.

In endothelial cells von Willebrand factor (vWF) and P-selectin are stored in dense granules. so-called Weibel-Palade bodies. Upon stimulation of endothelial cells with a variety of agents including thrombin, these organelles fuse with the plasma membrane and release their content. Small GTP-binding proteins have been shown to control release from intracellular storage pools in a number of cells. In this study we have investigated whether small GTP-binding proteins are associated with Weibel-Palade bodies. We isolated Weibel-Palade bodies by centrifugation on two consecutive density gradients of Percoll. The dense fraction in which these subcellular organelles were highly enriched, was analysed by SDS-PAGE followed by GTP overlay. A distinct band with an apparent molecular weight of 28,000 was observed. Two-dimensional gel electrophoresis followed by GTP overlay revealed the presence of a single small GTP-binding protein with an isoelectric point of 7.1. A monoclonal antibody directed against RalA showed reactivity with the small GTP-binding protein present in subcellular fractions that contain Weibel-Palade bodies. The small GTPase RalA was previously identified on dense granules of platelets and on synaptic vesicles in nerve terminals. Our observations suggest that RalA serves a role in regulated exocytosis of Weibel-Palade bodies in endothelial cells.

Small GTP-binding proteins in human endothelial cells.

Small GTP-binding proteins of the Ras superfamily control an extensive number of intracellular events by alternating between GDP- and GTP-bound conformation. The presence of members of this protein family was examined in human umbilical vein endothelial cells employing RT-PCR. Sequence analysis of 215 cDNA clones revealed the presence of a total of 28 different partial cDNAs encoding small GTP-binding proteins. Two sequences corresponded to novel isoforms of Rab2 and Rab9. In addition, human analogues of Rab4b, Rab7, Rab9, Rab14 and Rab15 were identified. Besides Rab proteins, members of other subfamilies were detected as well. As a first step towards elucidation of the function of the different small GTP-binding proteins identified we have isolated full length cDNA corresponding to Rab30 from a human endothelial cell cDNA library. In order to assess the subcellular localization of Rab30, we expressed epitope-tagged Rab30 cDNA in monkey kidney COS-1 cells. Immunoelectron-microscopy of transfected COS-1 cells indicated that Rab30 is associated with Golgi stacks.

Persistent gestational trophoblastic disease: DNA image cytometry and interphase cytogenetics have limited predictive value.

DNA flow cytometry has shown a wider spectrum of DNA content in the complete hydatidiform mole (CM) than the originally reported diploidy. Conflicting results have been published about the relationship of DNA content and the occurrence of persistent gestational trophoblastic disease (PGTD). In the present study, 71 cases of CM and 4 cases of partial mole accompanied by PGTD and 100 cases of CM without PGTD were evaluated with DNA image cytometry for differences in DNA-ploidy pattern, expressed as the 2.5c and 5c exceeding rates. A pilot study of 20 cases of each group was performed using interphase cytogenetics to detect differences in the frequency of numerical chromosomal aberrations and in sex chromosome composition. For this purpose, DNA probes specific for the pericentromeric regions of chromosomes 1 and X and for the long arm of chromosome Y were incubated on 6-micron paraffin tissue sections. The results showed no differences between CMs with or without PGTD; DNA polyploidy occurred in 99% and 98% of cases, respectively; the 2.5c exceeding rate and 5c exceeding rate were 62.6 and 62.4, and 6.5 and 6.0, respectively. The frequency of numerical chromosomal aberrations as detected by interphase cytogenetics was 23.4 and 22.8%. An XY pattern was found in 3 of 20 cases of CM with PGTD and in 4 of 20 cases of CM without PGTD. The four cases of partial mole showed a DNA-ploidy pattern identical to that of a CM. For this reason, they would be better reclassified as CMs, despite the presence of nucleated red blood cells or amnion. Although nuclear atypia and corresponding increased DNA content is pronounced but variable in CMs, the occurrence of PGTD is not related to variations in quantitative DNA content nor to gross heterology or homology in sex chromosomes.

Quantitative immunohistological analysis of the microvasculature in untreated human glioblastoma multiforme. Computer-assisted image analysis of whole-tumor sections.

Because histologically prominent microvascular proliferation is frequently present in glioblastoma multiforme, it has been hypothesized that this neoplasm is particularly dependent on neovascularization for its continued growth and that antiangiogenic therapy might be especially useful. To quantify the histological aspects of microvascular proliferation in glioma, a feasible and reproducible method was developed for computer-assisted image analysis of the visualized microvasculature in glial tissue. This method was used to compare several vascular parameters in histological whole-tumor sections of untreated human glioblastoma multiforme with those in histologically normal cerebral cortex and white matter. There was a significant increase in mean number, area, and perimeter of blood vessels per microscopic field in glioblastoma multiforme compared to normal cerebral white matter. In a substantial number of tumor fields, however, the vascular density was in the same range as that of normal cerebral white matter. The striking heterogeneity of the microvasculature within glioblastoma multiforme was illustrated by the significantly higher standard deviation for the vascular parameters in tumor tissue. The results of this study suggest that many regions of glioblastomas multiforme are not overtly angiogenesis dependent and may be difficult to treat by antiangiogenic therapy alone.

Localization of the cellular retinoic acid binding protein (CRABP) gene relative to the acute promyelocytic leukemia-associated breakpoint on human chromosome 15.

A human genomic fragment comprising the cellular retinoic acid binding protein (CRABP) gene was isolated. By using a panel of somatic cell hybrids, this gene could be assigned to human chromosome 15. Subsequently, a possible involvement of the CRABP gene in translocation (15;17) (q22;q11) positive acute promyelocytic leukemia (APL) was investigated. Although transposition of the CRABP gene could be demonstrated, we did not observe any gross CRABP rearrangement in a series of primary APL patients, nor in the acute myeloblastic leukemia cell line HL-60. Thus, the observed lack of CRABP expression in these leukemic cells may not be caused by disruption of its gene. CRABP maps to the region 15q22-qter.

Cytophotometric analysis of corresponding cytological and histological cervical intraepithelial neoplasia grade III specimens.

Cytophotometric analysis of cervical intraepithelial neoplasia grade III (CIN III) was performed in 22 cytological smears (CS) and in 22 corresponding cytospin specimens retrieved from selected areas of paraffin-embedded tissues (PEC). The average time interval between cytological and histological diagnosis was 6 weeks. CIN III nuclei in CS and PEC specimen were Thionin-Feulgen stained and digitized. Beside the visual classification of DNA ploidy patterns, the 2.5c and 5c exceeding rates and the specimen mean and standard deviation values of 21 photometric features were also analyzed. It was shown that, although there was a significant correlation between DNA ploidy patterns in corresponding PEC and CS specimen, the DNA patterns were dissimilar in eight of 22 cases. The DNA index, as represented by 2.5c and 5c exceeding rates, was significantly higher in the CS specimen. High-resolution cytophotometric analysis of cell nuclei in CS and PEC specimens showed significant differences for a large number of nuclear photometric features. These findings can possibly be explained by differences in selection of CIN III cells from CS and PEC specimens and by differences between fixation procedures as used for the two techniques. It was concluded that cytophotometric data of CS and PEC specimens representing CIN III lesions should not be regarded as interchangeable.

Conformational analysis of m4(2)C-m4(2)C-m6(2)A: a chemically modified 3'-acceptor end of tRNA, studied by NMR and CD methods.

A study on the conformation of the title compound, C-C-A, and on its constituent dinucleotides is presented. 1H-NMR spectra at 360 and 500 MHz were completely assigned by decoupling experiments. Computer simulation of the spectra yielded precise proton-proton and proton-phosphorus coupling constant values. The coupling constants are analyzed in terms of torsion angles and of N- and S-type sugar pucker. 31P-NMR spectra gave some information about P-O backbone torsion angles alpha and zeta. CD spectroscopy was used to obtain insight in the base-base interaction. The C(1) and C(2) unit in C-C-A show normal preference for N-type conformation of the sugar ring, whereas the A(3) residue appears rather biased towards the S-conformation. The zeta and alpha backbone torsion angles in the C-C phosphodiester linkage in C-C-A appear to assume normal g-, g- conformation, the zeta, alpha combination in the C-A linkage is proposed to have a g+, t conformation. In the C-C fragment in C-C-A a regular stack is indicated; it is suggested that the C-A part adopts an unusual antiparallel base stack.

Influence of the 2'-hydroxyl group and of 6-N-methylation on the conformation of adenine dinucleoside monophosphates in solution. A nuclear magnetic resonance and circular dichroism study.

Proton NMR studies at 360 MHz are reported on the adenine dinucleoside monophosphates N6-dimethyladenyly(3'-5')-N6-dimethyladenosine (m(6)(2)Apm(6)(2)A), ApA, rApdA, dAprA and on the methyl phosphate esters of the monomers m(6)(2)Ap, pm(6)(2)A, Ap and pA. Complete 1H-NMR spectral assignments are given. The dimers were also investigated by means of circular dichroism to obtain accurate thermodynamic parameters of the stacking equilibrium. With the aid of the thermodynamic data NMR coupling constants are extrapolated to values appropriate to the stacked conformers. A modernized version of pseudorotation analysis is used to delineate the conformational behaviour of the ribose and 2'-deoxyribose rings. It is shown that the unmethylated dimers can be arranged in two groups (dApdA/dAprA vs ApA/rApdA) according to their melting temperatures. ApA and the fully N6-methylated dimer m(6)(2)Apm(6)(2)A prefer to adopt the classical right-handed N-N stacked conformation. Both dimers with a 2'-deoxyribose ring at the 5'-OH end (dApdA and dAprA) behave similarly and occur in solution as a 75:25 mixture of S-S and S-N stacked states. The fully stacked hybrid dimer rApdA displays an unexpectedly large amount of S conformers (greater than 40%) in both sugar rings. This finding is rationalized by the postulation of a right-handed helical S-S stacked state on the basis of NMR and circular dichroic data.