Cerebral Ketones Detected by 3T MR Spectroscopy in Patients with High-Grade Glioma on an Atkins-Based Diet.

BACKGROUND AND PURPOSE: Ketogenic diets are being explored as a possible treatment for several neurological diseases, but the physiologic impact on the brain is unknown. The objective of this study was to evaluate the feasibility of 3T MR spectroscopy to monitor brain ketone levels in patients with high-grade gliomas who were on a ketogenic diet (a modified Atkins diet) for 8 weeks. MATERIALS AND METHODS: Paired pre- and post-ketogenic diet MR spectroscopy data from both the lesion and contralateral hemisphere were analyzed using LCModel software in 10 patients. RESULTS: At baseline, the ketone bodies acetone and ß-hydroxybutyrate were nearly undetectable, but by week 8, they increased in the lesion for both acetone (0.06 ± 0.03 ≥ 0.27 ± 0.06 IU, P = .005) and ß-hydroxybutyrate (0.07 ± 0.07 ≥ 0.79 ± 0.32 IU, P = .046). In the contralateral brain, acetone was also significantly increased (0.041 ± 0.01 ≥ 0.16 ± 0.04 IU, P = .004), but not ß-hydroxybutyrate. Acetone was detected in 9/10 patients at week 8, and ß-hydroxybutyrate, in 5/10. Acetone concentrations in the contralateral brain correlated strongly with higher urine ketones (r = 0.87, P = .001) and lower fasting glucose (r = -0.67, P = .03). Acetoacetate was largely undetectable. Small-but-statistically significant decreases in NAA were also observed in the contralateral hemisphere at 8 weeks. CONCLUSIONS: This study suggests that 3T MR spectroscopy is feasible for detecting small cerebral metabolic changes associated with a ketogenic diet, provided that appropriate methodology is used.

Cerebral Reorganization after Hemispherectomy: A DTI Study.

BACKGROUND AND PURPOSE: Hemispherectomy is a neurosurgical procedure to treat children with intractable seizures. Postsurgical improvement of cognitive and behavioral functions is observed in children after hemispherectomy suggesting plastic reorganization of the brain. Our aim was to characterize changes in DTI scalars in WM tracts of the remaining hemisphere in children after hemispherectomy, assess the associations between WM DTI scalars and age at the operation and time since the operation, and evaluate the changes in GM fractional anisotropy values in patients compared with controls. MATERIALS AND METHODS: Patients with congenital or acquired neurologic diseases who required hemispherectomy and had high-quality postsurgical DTI data available were included in this study. Atlas- and voxel-based analyses of DTI raw data of the remaining hemisphere were performed. Fractional anisotropy and mean, axial, and radial diffusivity values were calculated for WM and GM regions. A linear regression model was used for correlation between DTI scalars and age at and time since the operation. RESULTS: Nineteen patients after hemispherectomy and 21 controls were included. In patients, a decrease in fractional anisotropy and axial diffusivity values and an increase in mean diffusivity and radial diffusivity values of WM regions were observed compared with controls (P < .05, corrected for multiple comparisons). In patients with acquired pathologies, time since the operation had a significant positive correlation with white matter fractional anisotropy values. In all patients, an increase in cortical GM fractional anisotropy values was found compared with controls (P < .05). CONCLUSIONS: Changes in DTI metrics likely reflect Wallerian and/or transneuronal degeneration of the WM tracts within the remaining hemisphere. In patients with acquired pathologies, postsurgical fractional anisotropy values correlated positively with elapsed time since the operation, suggesting a higher ability to recover compared with patients with congenital pathologies leading to hemispherectomy.

Local physeal widening on MR imaging: an incidental finding suggesting prior metaphyseal insult.

OBJECTIVE: To offer a descriptive review which characterizes and evaluates the significance of local physeal widening, (cartilaginous signal extending from the physis into the adjacent metaphysis), identified on magnetic resonance (MR) imaging. MATERIALS AND METHODS: MR images (recollected from exams performed between 1988 and 1995) of 31 metaphyses in 22 children where we recognized local physeal widening were examined retrospectively. These areas of physeal widening were evaluated for morphology, depth, location, signal intensity, and the coexistence of epiphyseal alterations. The characteristics of the signal abnormalities were correlated with the duration and type of any identifiable insult to the adjacent metaphysis, and with the development of growth disturbance. RESULTS: Twenty-six metaphyses had identifiable insults (19 single event and 7 sustained or repetitive). The widened physes were of focal tongue (n = 15), broad band (n = 10), or mixed (n = 6) morphology. Most (n = 27) areas of widening were isointense with the physeal cartilage on all sequences. Subsequent growth disturbance was more likely when the metaphyseal insult was a single event rather than sustained or repetitive (P = 0.023), with focal tongues (P = 0.029), and with centrally located lesions (P = 0.030). In five cases, the adjacent epiphysis showed signal abnormalities; all developed growth disturbance. Histologic examinations available in two limbs confirmed that the MR findings represented extensions of hypertrophic physeal chondrocytes into the metaphysis. CONCLUSION: Incidentally observed local physeal widening in a growing bone may represent the imprint of a previous or ongoing interference with endochondral ossification from a prior metaphyseal insult, rather than a primary metaphyseal disorder. Single event insults, physeal widening of focal tongue morphology, central distribution in the metaphysis, and concomitant epiphyseal signal abnormalities on MR imaging are significant predictors of subsequent growth disturbance.

Binding preferences of the POU domain protein Brain-4: implications for autoregulation.

The POU domain-containing transcription factor Brain-4 (Brn-4, RHS-2) was examined for its sites of expression and DNA binding preferences. In the rat, Brn-4 is expressed in 76 and 65% of vasopressin neurons in the paraventricular and supraoptic nuclei, respectively; but in only 10% of corticotropin-releasing factor neurons in the paraventricular nucleus of the hypothalamus. From these data we speculate that genes expressed within vasopressinergic neurons are more likely to be regulated by Brn-4 than those in corticotropin-releasing factor neurons. Random oligonucleotide site selection indicates Brn-4 prefers binding the DNA element CAATATGCTAAT and is inflexible in its spacing requirement between putative CAATAT and TAAT half sites, preferring 2 nucleotides between these elements. Electrophoretic mobility shift and DNase I footprinting analyses show five regions between nucleotides -457 and +22 of the Brn-4 promoter that are bound by Brn-4. Furthermore, Brn-4 can transactivate from this region of the Brn-4 promoter, suggesting that Brn-4 expression may be autoregulated.

Diffusion of small solutes in cartilage as measured by nuclear magnetic resonance (NMR) spectroscopy and imaging.

The ability of water and solutes to move through the cartilage matrix is important to the normal function of cartilage and is presumed to be altered in degenerative diseases of cartilage such as osteoarthritis and rheumatoid arthritis. Nuclear magnetic resonance (NMR) spectroscopy and magnetic resonance imaging (MRI) techniques were used to measure a self diffusion coefficient (D) for small solutes in samples of explanted cartilage for diffusion times ranging from 13 ms to 2 s. With a diffusion time of 13 ms, the intratissue diffusivity of several small solutes (water, Na+, Li+, and CF3CO2-) was found consistently to be about 60% of the diffusivity of the same species in free solution. Equilibration of the samples at low pH (which titrates the charge groups so that the net matrix charge of -300 mM at pH 8 becomes approximately -50 mM at pH 2) did not affect the diffusivity of water or Na+. These data, and the similarity between the D in cartilage relative to free solution for water, anions, and cations, are consistent with the view that charge is not an important determinant of the intratissue diffusivity of small solutes in cartilage. With 35% compression, the diffusivity of water and Li+ dropped by 19 and 39%, respectively. In contrast, the diffusivity of water increased by 20% after treatment with trypsin (to remove the proteoglycans and noncollagenous proteins). These data and the lack of an effect of charge on diffusivity are consistent with D being dependent on the composition and density of the solid tissue matrix. A series of diffusion-weighted proton images demonstrated that D could be measured on a localized basis and that changes in D associated with an enzymatically depleted matrix could be clearly observed. Finally, evidence of restriction to diffusion within the tissue was found with studies in which D was measured as a function of diffusion time. The measured D for water in cartilage decreased with diffusion times ranging from 25 ms to 2 s, at which point the measured D was roughly 40% of the diffusivity in free solution. Although changes in matrix density by compression or digestion with trypsin led to a decrease or increase, respectively, in the measured D, the functional change in measured diffusivity with diffusion time remained essentially unchanged. In a different type of study, in which bulk transport could be observed over long periods of time, cartilage was submerged in 99% D2O and MRI studies were performed to demonstrate the bulk movement of water out of the cartilage matrix.(ABSTRACT TRUNCATED AT 400 WORDS)

The merit and significance of clinical nurse specialists.

The authors studied the merit and significance of the role of the clinical nurse specialist (CNS) by collecting quantitative and qualitative data documenting the activities performed in each of the four role components: practitioner, educator, scholar, and consultant. This follow-up study continues to substantiate the impact of the CNS role on quality patient outcomes and the major contributions of the CNS to the nursing organization and the hospital.

Conserved tissue-restricted expression of HUC 1-1 actin phenotype among eumetazoan organisms.

Actin has evolved from a single protein into a family of more than six distinct isoforms in mammals. Based on amino acid sequence data, actins segregate into two major classes, the "cytoplasmic" or nonmuscle actins, present in all animals, and the "a-" or muscle actins, a group restricted to vertebrate muscle. We have recently identified two unique actin isoforms in rat intestinal brush border which combine features of these two classes. The amino terminal regions of these actins indicate that they are of a cytoplasmic type and yet the carboxy terminal regions contain an epitope (defined by Mab HUC 1-1) which, among mammalian actins, is restricted to the muscle isoforms. We report here that in addition to the rat, all species thus far examined which have an intestinal "brush border" express actins containing the HUC 1-1 epitope in this region. Furthermore, we show that the actins present in the muscle tissue of nonvertebrate eumetazoans, which are all of the cytoplasmic type, also contain this epitope. Thus these findings suggest that the HUC 1-1 epitope appeared early on a subset of cytoplasmic-type actins and was retained among actins expressed in muscle tissue throughout the evolutionary divergence of these cytoplasmic-type actins to the "a-" muscle actins.

Exercise-induced ovarian dysfunction in the rat.

The effect of treadmill running on estrous cycles was studied in the rat. Additional effects of cortisol acetate treatment and adrenalectomy were studied in both exercising and sedentary rats. Sedentary rats given the vehicle or cortisol acetate, or which had been adrenalectomized, all exhibited estrous cycles with diestrous phases that were uniformly less than 4 days. However exercising rats had extended estrous cycles; 50-62% of cycles were incomplete within 11 days and 78% of rats had cycles with diestrous phases that were more than 4 days long. There were no difference in duration of estrous cycles of running rats that received vehicle, received cortisol acetate, or had been adrenalectomized. We conclude that the running regimen resulted in a delay of the normal ovulatory period in rats, and that this effect of running was not affected by the presence or absence of glucocorticoids.

Expression of actin isoforms in developing rat intestinal epithelium.

A minimum of six very similar but distinct actin isoforms are encoded by the mammalian genome. Developmental regulation of these genes results in a tissue-specific distribution of the isoforms in the adult. Using a panel of actin specific monoclonal antibodies (MAb), we recently reported the expression of two unique actin isoforms in adult rat intestinal brush border. In this report, we examine the developmental expression of these and other actin isoforms in rat intestinal epithelial cells. Isoforms containing the HUC 1-1 and/or C4 epitopes are present by day 15 of gestation and are continuously expressed throughout adult life. Unexpectedly, the gamma-enteric smooth muscle isoactin, defined by the B4 epitope, is transiently expressed in these non-muscle cells late in gestation. The alpha-vascular smooth muscle isoform, however, is not expressed in intestinal epithelial cells during development and, as previously reported, both smooth muscle isoforms are absent in epithelial cells of adult intestine. In addition, we demonstrate that although multiple isoforms are expressed simultaneously in these cells, they are not uniformly distributed at the subcellular level, suggesting that the cell recognizes the actin isoforms as functionally distinct entities.

Nuclear morphology of follicular center cell-associated T-cells: an immunoultrastructural study of follicular hyperplasia and follicular center cell lymphomas.

Although the immunophenotypic nature and distribution of T-cells in normal follicular centers and in follicular center cell lymphomas have been studied in great detail, the morphology of these cells has been largely ignored. Immunoultrastructural studies using UCHT-1, an antibody to the T-cell receptor-associated CD3 antigen, were therefore performed on four tonsils with reactive follicular hyperplasia and on four follicular center cell lymphomas. T-cells present in normal follicular centers had a mean maximum nuclear diameter of 4.9 +/- 0.93 microns and were not significantly different from those associated with neoplastic follicular center cells which had a maximum nuclear diameter of 4.9 +/- 1.2 microns. In contrast, the T-cells in normal interfollicular T-zones had a mean maximum nuclear diameter of only 4.4 +/- 0.81 microns which was significantly smaller than either of the previous two groups (P = 0.002 and P = 0.004, respectively). Similarly, T-cells in a relatively uninvolved T-zone in a follicular center cell lymphoma had a mean maximum nuclear diameter of 4.5 +/- 0.78 microns. The nuclear configuration of the follicular center cell-associated and interfollicular T-cells ranged from round to clefted with the greatest number of clefted T-cells being associated with the follicular center cell lymphomas. These data demonstrate that T-cells associated with either normal or neoplastic follicular center cells tend to be larger than interfollicular T-cells. Because of the very variable nuclear configurations of these T-cells, not all cells with clefted nuclei in follicular centers or follicular center cell lymphomas can be assumed to be of B-cell origin.

Intrathymic ontogeny of the T cell receptor associated CD3 (T3) antigen.

Intrathymic T cell maturation has been studied in great detail, yet much of this process remains controversial. One of the critical steps in thymocyte maturation is the surface expression of the T cell antigen receptor associated CD3 (T3) antigen. The expression and ultrastructural localization of this antigen was therefore studied in tissue sections of five human thymuses using the monoclonal antibody UCHT-1. Thymocytes were also independently categorized morphologically as small nonblastic, large blastic, or intermediate. CD3 positivity was identified in 89% of cortical thymocytes and 92% of medullary thymocytes. Of the CD3+ cells, staining was present in the perinuclear region in 48% of cortical thymocytes and in only 11% of medullary thymocytes. Surface membrane staining was present in 87% of CD3+ cortical thymocytes and 99% of CD3+ medullary thymocytes. Of the CD3+ cortical large blastic thymocytes, 97% had perinuclear staining, but only 31% had surface positivity. Conversely, 98% of CD3+ medullary large blastic thymocytes had surface staining, but only 17% had perinuclear positivity. Almost all cortical and medullary small nonblastic thymocytes had surface membrane CD3 positivity, whereas perinuclear positivity was rare in these cells. These data suggest that the most immature CD3+ thymocytes (the large blasts with perinuclear CD3 positivity only) are present almost exclusively in the cortex. Maturation defined by the loss of perinuclear CD3 positivity and the acquisition of surface CD3 also occurs in the cortex. The vast majority of medullary thymocytes have surface CD3 positivity only.

C3 dependent, C5 independent immune complex glomerulopathy in the mouse.

This study examines the role of complement in a murine model of accelerated nonproliferative immune complex glomerulopathy. Two C5 deficient strains (DBA/2J and B10.D2oSnJ) as well as normocomplementemic mice consistently develop heavy proteinuria and glomeruli show loss of normal visceral epithelial cell architecture within 4 days of intravenous antigen administration. In contrast, animals depleted of C3 with cobra venom factor fail to develop proteinuria and retain discrete foot processes. Semiquantitative evaluation of antigen and antibody in glomeruli shows equivalent deposition in mice from all groups. The localization of these deposits, however, is different in C3-depleted mice. There is extensive accumulation of deposits along the subepithelial aspect of the glomerular basement membrane of normocomplementemic and C5 deficient mice while deposits in glomeruli of C3-depleted animals accumulate in the subendothelial region and do not cross the glomerular basement membrane. These data demonstrate that in this model, glomerular injury is dependent on complement components generated up thru C3 but not C5 or latter components. In addition, our data suggest that C3 is important in the movement of immune complexes across the glomerular basement membrane. Although the mechanism by which complement is mediating injury in this model is not known, it does not appear to involve an inflammatory cell infiltrate or the terminal complement components.

Unique isoactins in the brush border of rat intestinal epithelial cells.

The mammalian genome contains 20-30 genes encoding a family of actins. To date, however, only six proteins (four muscle and two nonmuscle isoforms) encoded by this multigene complex have been identified. We have isolated two actins from the brush border of rat intestinal epithelial cells that have isoelectric points and N-terminal peptides characteristic of the cytoplasmic beta- and gamma-actins. However, using a panel of actin-specific monoclonal antibodies, we show that these actins contain a set of epitopes that distinguishes them from any of the known cytoplasmic or muscle isoforms. These unique actins share features of both the nonmuscle and muscle isoforms, suggesting that they represent an intermediate in the evolution of the specialized muscle actins.

T-cells in follicular hyperplasia and follicular center-cell lymphomas: an immunoultrastructural study of the T-cell receptor-associated CD3 antigen.

T-cells are an integral part of normal follicular centers and many follicular center-cell (FCC) lymphomas. Because the functional role of these cells remains imprecisely determined and because ultrastructural localization of the T-cell antigen receptor-associated CD3 antigen has not been previously reported, an immunoultrastructural study of four tonsils and four FCC lymphomas was performed using an anti-CD3 antibody (UCHT-1). Normal interfollicular CD3-positive T-cells always demonstrated surface membrane positivity, as did 94% of normal and 88% of neoplastic FCC-associated CD3-positive cells. Conversely, whereas only 5% of normal interfollicular CD3-positive cells showed perinuclear positivity, 58% of normal and 38% of neoplastic FCC-associated CD3-positive cells did (p less than 0.001). Definite endoplasmic reticulum (ER) staining for CD3 was identified in 4% and 8% of cells associated with normal or neoplastic FCC, respectively, but in none of the T-zone lymphocytes. Because the perinuclear space is reported to be a site of protein synthesis in cells with little ER, and because of the occasional ER staining observed in cells with perinuclear staining, perinuclear CD3 positivity probably represents CD3 synthesis in mature tonsillar T-cells. The frequency of perinuclear positivity in the FCC-associated T-cells, together with the loss of surface positivity in some cells at this site, suggests that this could represent in vivo T-cell "activation" in follicular centers, with modulation and resynthesis of the CD3 antigen. Furthermore, the results demonstrate a similar phenomenon in the T-cells associated with neoplastic follicular center cells.

Studies on the structure and carbohydrate binding properties of lobster agglutinin 1 (LAg1), a sialic acid-binding lectin.

Lobster agglutinin 1 (LAg 1) was isolated from the hemolymph of the American lobster (Homarus americanus) by a sequential combination of ammonium sulfate precipitation, preparative starch block electrophoresis, gel filtration and affinity chromatography on Sepharose-Fetuin and Sepharose-Colominic acid columns. Two types of protomeric structures with molecular weights of 700 and 500 Kilodaltons respectively were isolated. These molecules are composed of noncovalently held subunits with a molecular weight of 70 Kilodaltons. Analysis of preparations by double immunodiffusion, polyacrylamide gel electrophoresis and isoelectrofocusing indicates that the LAg 1 obtained was a single molecular species. Hemagglutination inhibition experiments indicated that the best inhibitors were bovine mucin, glycophorin, fetuin and human IgM in that order. The desialylated forms of some of these proteins still bound lectin, although to a lesser degree than their intact sialylated counterparts. Affinity chromatography experiments indicated that LAg 1 binds to N-acetylneuraminic acid, N-acetylglucosamine and N-acetylgalactosamine. LAg 1 does not contain sialic acid nor neuraminidase activity: oligosaccharides associated with it appear to be either of the oligomannosyl or biantennary type. The sialic acid binding specificity of this lectin was used to separate immature mouse thymocytes (low sialic acid content) from mature thymocytes (high sialic acid content).

Stimulation of B cells, but not T cells or thymocytes, by a sialic acid-specific lectin.

Haemolymph of the American lobster, Homarus americanus, contains several lectins. One of these, lobster agglutinin 1 (LAg1) is specific for N-acetylneuraminic acid and agglutinates mouse and human erythrocytes. In addition, this lectin agglutinates peripheral T cells and cortisone-resistant thymocytes, but agglutinates whole thymocytes poorly. Because this material is being used to prepare purified populations of cortical thymocytes, and then to study their maturation, it was important to determine if it is mitogenic for thymocytes and T cells. Thus, the studies described here were conducted to find if LAg1 is a mitogen for mouse lymphocytes, and if so for what cell populations. The data show that purified LAg1, but not purified lobster agglutinin 2 (LAg2) is a mitogen for mouse spleen cells, and that LAg1 is a polyclonal activator. Furthermore, LAg1 is a B-cell mitogen since it stimulates nude spleen cells, nude spleen cells depleted of pre-T cells, and normal spleen cells which have been treated with rabbit anti-mouse brain antiserum and complement. Moreover, LAg1 does not stimulate division by thymocytes or T cells, that is, spleen cells which do not adhere to nylon wool columns. Mitogenic activity of LAg1 but not of LPS is inhibited by N-acetylmannosamine, demonstrating that the mitogenic effects of LAg1 are unlikely to be due to contaminants.

Distribution of kinetochore (centromere) antigen in mammalian cell nuclei.

Antigens associated with mammalian centromeres were localized at the high and electron microscopic levels using the peroxidase-labeled antibody method. The antibody used was of a type naturally occurring in the sera of patients with scleroderma. At the light microscopic level, it reacts specifically with the centromere regions of chromosomes in a variety of mammalian species and strains in discrete foci in interphase nuclei. We find that the number of foci approximates the number of chromosomes present in the various cell types. At the ultrastructural level, the antigenic foci are confirmed to lie in the kinetochore regions of each chromosome. In interphase nuclei, the antigenic foci were usually associated either with the inner surfaces of the nuclear envelope or with the nucleoli. These observations indicate that the centromere regions of the chromosomes in interphase are not randomly distributed within the nucleus but are usually fixed either to the inner surface of the nuclear envelope or to nucleoli.

Intracellular localization of antigens with backscatter mode of SEM using peroxidase-labeled antibodies.

The peroxidase-labeled antibody method was employed for immunohistochemical localization of intracellular and extracellular antigens with SEM. With this method, the antigenic sites may be localized with high sensitivity on tissue sections mounted on carbon-coated glass slides by detecting 0s04-DAB complexes using the backscatter mode. The sections may be fresh frozen fixed on slide, frozen sections of fixed tissue, or sections of immunostained tissue embedded in epon. The method introduces another utilization of SEM and the immunoenzyme histochemistry.

Immunocytochemical localization of intracellular antigens with SEM.

A method for the localization of intracellular antigens with a scanning electron microscope using peroxidase-labelling antibodies is described. A search for a hydrogen donor which may be deposited at the sites of antigen by enzymatic action and emit secondary electrons or generate backscatter electrons was made. It was found that when 4-chloro-1-naphthol was used, the peroxidase deposited reaction product which resulted in a strong secondary electron emission at the site of antigen. With this method, the presence of luteinizing hormone in secretion granules and other cytoplasmic structures of gonadotropic cells was demonstrated. The level of detection of intracellular antigens with this method is not as high as that detectable with light microscopical examination of the same specimens, that is, more reaction product at the site of antigen is required to be detectable with scanning electron microscopy than with light microscopy. In spite of the lack of high sensitivity, the intracellular antigens may be localized with the method described.