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Biomedical subjects

C V Howard

Publications and source records attributed to C V Howard.

53 records · Page 3Linked to original sources
PubMed1994

Immunocytochemical study of a vascular barrier antigen in the developing rat brain.

A rat-specific mouse monoclonal antibody, exclusively reacting with a blood-brain and blood-nerve barrier protein, was employed to assess the post-natal, age-related changes in morphology and intraparenchymal distribution of barrier competent microvessels in normal rat forebrain. Antibody binding first appeared in single cells over the external surfaces of the brain between days 3 and 6 post-partum and increased progressively with age. In mature rats, the cerebral grey matter showed higher vascularization by barrier competent microvessels than white matter. Microvessels in the hippocampus were immunocytochemically more avid but less "networked" than the rest of the neocortex. These features may be related to morphological, metabolic and haemodynamic changes associated with brain growth and development.

Animals↗
PubMed1994

Analysis of relative proliferation rates of Wilms' tumor components using proliferating cell nuclear antigen and MIB-1 (Ki-67 equivalent antigen) immunostaining and assessment of mitotic index.

BACKGROUND: Flow-cytometric analysis of proliferation index (PI) has potential use in predicting prognosis in malignancy. Its relevance to heterogeneous tumors has not been conclusively studied. In nephroblastoma, where the epithelial components are considered more differentiated than others, potentially different PIs may exist within a single lesion based on the inverse relation between differentiation and proliferation. Proliferating cell nuclear antigen (PCNA) and MIB-1 (Ki-67 equivalent antigen) demonstration in histologic sections by immunoperoxidase methods may allow for determination of PI in relation to tissue type. EXPERIMENTAL DESIGN: A consecutive unselected series of 8 pediatric nephroblastoma patients was used to study the relation between PI and histologic differentiation as established by flow-cytometric analysis of nuclear suspensions prepared from formalin-fixed and paraffin-embedded tissue and by PCNA/MIB1 staining of parallel histologic sections. PI by PCNA/MIB1 was established using 5-microns paraffin sections, immunoperoxidase, and quantification procedures detailed in the literature. The mitotic index (MI) of tissue components was separately assessed using 5-microns hematoxylin and eosin-stained sections and counting procedures detailed in the literature. RESULTS: The 8 lesions showed a PI of 4 to 20% as determined by flow cytometry. Using PCNA staining, the epithelium showed a mean PI of 55.5% (range 40 to 80%), that was significantly higher (p < 0.001, Wilcoxon's two-tailed rank sum test) than blastema (mean PI: 34.1%, range 17.5 to 76.5%) and stroma (mean PI of 14.9%, range 5 to 24%, p < 0.001, Wilcoxon's two-tailed rank sum test). Although, probably due to tissue antigen preservation, acceptable MIB-1 staining was not achieved in all lesions (5 of 8), the results, although generally with lower labeling indices, confirmed the PCNA findings. The relative MI of epithelial components was higher than that of stroma and blastema in keeping with the immunocytochemical findings. In 6 of 8 cases, the PI by flow-cytometric analysis was lower than the lowest value for the PI (labeling index) of an individual tissue type found by PCNA or MIB staining. CONCLUSIONS: The differences found between PI of the different tissue components in nephroblastoma are difficult to understand if the epithelial components (with the highest PI values) are considered as differentiation products from the other components of the lesion. The relation between PIs as determined by PCNA/MIB-1 analysis/mitotic index, for the three components and the PI as established by flow cytometry is not simply explained by the relative volume of the tissue components.

Cell Division↗
PubMed1993

PCNA independence of Ki-67 expression in HPV infection.

12 Infectious Wart lesions were stained using the streptavidin-biotin immunoperoxidase method for PCNA10, MIB-1 (Ki-67 equivalent antigen) and Human Papilloma Virus antigen to study the effect of HPV presence on epidermal proliferation. Using strict methods to avoid observer bias, Ki-67 antigen was found in a high proportion of nuclei in the suprabasal layers together with HPV antigens, in the absence of PCNA staining. This finding indicates that DNA synthesis related, Ki-67 antigen bearing structures can be raised in the human nucleus in the absence of induction of PCNA bearing structures, suggesting also structural independence between the antigen bearing molecules.

Antigens, Neoplasm↗
PubMed1993

Cerebral malaria in the rhesus monkey (Macaca mulatta): observations on host pathology.

Infection was induced in five rhesus monkeys (Macaca mulatta) by intravenous inoculation with a virulent strain of Plasmodium knowlesi. Approximately one week after inoculation, four of the exposed animals developed acute malaria and died or were killed when moribund after varying periods of illness. Post-mortem and light microscopical examination showed marked cerebral vascular congestion and widespread plugging of the brain capillaries and venules (microvessels) by heavily parasitized erythrocytes mixed with uninfected erythrocytes. Electronmicroscopically, the major changes seen were adherence of large numbers of parasitized erythrocytes and macrophages to swollen microvascular endothelial cells; increased numbers of fibroblasts and deposition of collagen bundles in the extracellular matrix around damaged and parasite-packed microvessels were also found in many areas. This animal model may prove useful for further investigation of the pathogenesis of cerebral malaria.

Animals↗
PubMed1992

"Medullary ray glomerular counting" as a method of assessment of human nephrogenesis.

Renal weight (left-right combined), as a parameter of renal development, is required to be less than half the normal value for age for a statistically confident diagnosis of hypoplasia. "Medullary ray glomerular counting" (MRGC), counting cortical glomerular generations, has been proposed as a simple technique of possibly greater sensitivity. Recent development of the Disector method for the unbiased stereological estimation of total glomerular number has provided a, hitherto unavailable, "golden standard" with which to determine the diagnostic potential of MRCG. Both "true" (actual number of generations seen) and "assumed" (a subjective "guess" of the total number of generations) MRGC counts were determined in 11 pairs of kidneys from spontaneously aborted, normally developed, non-malformed fetuses (gestational age: 15-40 weeks). Each kidney was randomly analysed blind and on two separate occasions by two paediatric pathologists using a written protocol. Results were compared with unbiased stereological estimates of glomerular number. Intra- and inter-observer and intra- and inter-(left-right)renal reproducibility were analysed. In conclusion, MRGC, using "real" counts, is a highly reproducible parameter of renal development from 15-36 weeks' gestation. Sensitivity for detection of both hypoplasia and maturation delay increase with gestational age and generally exceeds that of renal weight.

Histological Techniques↗
PubMed1992

Geostatistical and morphological methods applied to three-dimensional microscopy.

Three-dimensional (3-D) images of osteocyte lacunae were examined on a confocal microscope. Both geostatistical and morphological processing techniques were used to improve and to analyse them. By a geostastical approach, this study aims at improving 3-D confocal images before any further image processing. Optimized linear filters, which take account of the second-order statistics and the 3-D structure of the data, allow for the removal of imperfections such as noise and/or blur due to the axial convolution, and interpolate voxels on a face-centred cubic grid from an initial cubic grid. An application of this technique to 3-D biological images is demonstrated. In a second step, a 3-D binary image is digitized and cleaned with 3-D morphological filters. The standard 3-D measurements cannot be applied in this case, since all osteocytes cut the border of the field. For this reason a 3-D Boolean model has been adjusted, from which it is possible to derive all useful information on the repartition and the morphology of the osteocytes.

Humans↗
PubMed1992

Measuring the surface area of a cell by the method of the spatial grid with a CSLM--a demonstration.

The spatial grid is a method for estimating the surface area of particles. A stack of perfectly registered sections is the essential prerequisite for its use. The confocal scanning light microscope provides such a stack by optical sectioning. The spatial grid method is briefly described and applied to an osteocyte lacuna in dry mineralized human mandible. This type of cell was chosen because of its very complex shape. The variance of the area estimate is studied and compared with the results of a simulation.

Humans↗
PubMed1992

The effect of intrauterine growth retardation on the development of renal nephrons.

OBJECTIVE: To investigate the effect of Type II (asymmetrical) intrauterine growth retardation (IUGR) on renal development. DESIGN: A prospective descriptive study. SETTING: Department of Fetal and Infant Pathology, Liverpool Children's Hospital. SUBJECTS: Six (severely) affected IUGR stillbirths of known gestational age with a control group of stillbirths with birthweight greater than 10th centile, and eight liveborn IUGR infants who died within a year of birth with a control group of appropriately grown infants who died within a year of birth (postnatal groups). TECHNIQUES: The kidneys from all the groups studied were analysed using unbiased, reproducible and objective design-based stereological techniques. MAIN OUTCOME MEASURES: Total renal nephron (glomerular) numbers and average volumes of total nephron and cortical and medullary nephron segments. RESULTS: Nephron number estimates lay below the control group's 5% prediction limit in five out of the six growth-retarded stillbirths, and were significantly (P less than 0.005, IUGR at 65% of the control mean) reduced in the postnatal group. Estimates of nephron (segment) volume did not differ between control and IUGR groups. CONCLUSIONS: Type II intrauterine growth retardation may exert a profound effect on renal development. The reduced nephron number at birth, together with the lack of any early postnatal compensation in either nephron number or nephron size, emphasizes the need for vigorous antenatal surveillance for IUGR and consideration of elective preterm delivery of affected fetuses. A systematic review of other organs, which develop in a similarly rapid fashion during the late intrauterine period, is indicated by this work. With one exception, all birthweights in the growth-retarded groups were below the third centile, thus the precise quantitative relation between progressive IUGR and renal function requires further assessment.

Birth Weight↗
PubMed1992

Estimating neuron dendritic length in 3D from total vertical projections and from vertical slices.

The recently developed method of total vertical projections is illustrated to estimate the total dendritic length of a human Substantia Nigra neuron. Next, the length of the different orders of dendritic branches, and the mean segment length for each order--commonly regarded as important parameters in neuron physiology--are also estimated. Finally, it is shown how to estimate the mean dendritic length in a population of neurons from vertical slices of arbitrary and unknown thickness. Being unbiased and highly efficient, the proposed methods offer interesting alternatives to current procedures used for the metric analysis of neuron arborizations.

Dendritic Cells↗
PubMed1991

The application of total vertical projections for the unbiased estimation of the length of blood vessels and other structures by magnetic resonance imaging.

A new stereological method has recently been developed to estimate the total length of a bounded curve in 3D from a sample of projections about a vertical axis. Unlike other methods based on serial section reconstructions, the new method is unbiased (i.e., it has zero systematic error). A basic requirement, not difficult to fulfill in many cases, is that the masking of one structure by another is not appreciable. The application of the new method to real curvilinear structures using a clinical magnetic resonance (MR) imager is illustrated. The first structure measured was a twisted water-filled glass tube of known length. The accuracy of the method was assessed: With six vertical projections, the tube length was measured to within 2% of the true value. The second example was a living bonsai tree, and the third was a clinical application of MR angiography. The possibility of applying the method to other scientific disciplines, for example, the monitoring of plant root growth, is discussed.

Blood Vessels↗
PubMed1991

Human intrauterine renal growth expressed in absolute number of glomeruli assessed by the disector method and Cavalieri principle.

The disector method, a stereologic procedure unbiased by feature size, shape, or tissue-processing methods, for the estimation of total glomerular number was performed on pairs of human kidneys from 11 normal spontaneous second trimester abortions and stillbirths (15 to 40 weeks gestation). In addition, gestational age-dependent patterns of change in the average volume of the nephron and its cortical and medullary segments were analyzed. Mean glomerular number, plateauing at 36 weeks, increased from 15,000 at 15 weeks to 740,000 at 40 weeks. Average volume of the medullary nephron segment (Henle's loop) increased throughout pregnancy. Average volume of the cortical nephron segment (Tubuli Contorti) decreased from 15 weeks to 25 weeks, then increased after 36 weeks. Fractional volume of the renal cortex decreased from 15 weeks to 40 weeks. Three to 4 hours of microscopic analysis time were required per kidney on routinely processed 5-microns hematoxylin and eosin-stained paraffin sections. Average coefficient of error for number estimation was 8.02%. Average intra- and interobserver reproducibilities were 96.8 and 93.7%, respectively. The demonstrated temporal differences in the development of the cortical and medullary nephron components may result in a dissociation of function, which may explain the increased incidence of fetal hydrops in the second trimester of pregnancy, and which must be taken into account in the treatment of (very) premature infants. Although the number of kidneys included in this study is limited, as they reflect the whole period of antenatal development relevant to neonatal intensive care, the disector method of glomerular number estimation shows significant potential for the analysis and increased understanding of the development of renal function. The method appears to be more sensitive in detecting small and early deviations from normal renal growth and development than previously available parameters e.g., renal weight and (cortical) volume.

Embryology↗
PubMed1990

The total number of neurons in the human neocortex unbiasedly estimated using optical disectors.

An efficient method is presented for obtaining, in under 4 h, an unbiased estimate of the total number of neurons in the human neocortex, with a coefficient of error on the estimate of approximately 5%. The novel sampling scheme used in this study is unbiased and was designed so that only a small amount of neocortical grey matter had to be removed. Hence, the majority of the cerebral grey matter and all the internal grey matter was left intact for further resampling and analysis. Each cerebral hemisphere was subdivided into the four major neocortical regions, sliced coronally at 7-mm intervals and the volume of the neocortex determined using Cavalieri's principle. Uniform sampling of neocortex was performed in the hemisphere followed by regional subsampling with a varying sampling fraction being taken from each region. Neuronal density estimates were made in thick plastic sections using optical disectors. Shrinkage estimates were made in parallel with the number estimates and found to be negligible. The total number of neocortical neurons in the right hemisphere of five normal 80-year-old men was found to be 13.7 x 10(9) with an inter-individual coefficient of variation of 12%.

Aged↗
PubMed1990

An assessment of volume-weighted mean nuclear volume estimates as a prognostic index for neuroblastoma.

This study evaluates an objective, unbiased, stereologic parameter (volume-weighted mean nuclear volume vV) as a prognostic indicator for survival of neuroblastoma in comparison with three histopathologic grading systems. In this retrospective study 24 consecutive, nonselected patients from the registry of Royal Liverpool Children's Hospital, Alder Hey, England were analyzed. Only primary lesions obtained before chemotherapy were used. Follow-up time in surviving patients (n = 10) was 4.5 to 20 years. All lesions were regraded blind and twice by two pathologists. vV was estimated on routinely processed, hematoxylin and eosin-stained, 5-microns sections, requiring on average less than 20 min per patient. Results show an absolute cut-off point for survival at vV = 270 microns 3. No patient with vV less than this value is alive at present (n = 7). In addition, actuarial survival curves for nonsurvivors show a bimodal pattern of survival time, separating patients with vV greater than (n = 7) and less than (n = 7) 270 microns 3. In comparison with the same analysis for the results of regrading by means of the Hughes or Beckwith system, results of vV estimation were superior. In comparison with the Shimada system the results confirmed the strong dichotomy for survivors and nonsurvivors, although with more overlap. vV has the advantage of predicting length of survival in nonsurvivors. The combination of Shimada grading and vV measurement, on the basis of the material studied, seems to offer useful prediction of biologic behavior. vV was always estimated on the small cell population, reducing the problems of biopsy representability.

Aneuploidy↗
PubMedDate not supplied

Renal developmental delay expressed by reduced glomerular number and its association with growth retardation in victims of sudden infant death syndrome and in "normal" infants.

In victims of sudden infant death syndrome (SIDS), renal development has been reported to be significantly impaired. In the present study, we used stereological techniques to estimate volume of kidney cortex and total number of glomeruli in a group of human infants. Infants were classified according to cause of death-SIDS or non-SIDS. Cases were further subdivided according to birth weight-normal birth weight (NBW) or low birth weight (LBW) (we were unable to identify any non-SIDS LBW infants for our study). No significant differences were found between NBW and LBW infants (irrespective of cause of death) for cortical volume, glomerular density, or total glomerular number (p > 0.140). Kidney cortical volume, glomerular density, and total glomerular number were not significantly different between SIDS and non-SIDS infants (p > 0.510). Glomerular number was only significantly less in SIDS infants of LBW (p = 0. 032) than in controls according to the Wilcoxon rank sum test; using the Kruskal-Wallis for one-way analysis, no significant difference was found (p > 0.010). These results contrast with those from previous studies, as a reduction in glomerular number was not noted in SIDS NBW infants, and the mean value for the control (non-SIDS NBW) group was significantly reduced (p < 0.01) from those of previous studies. This indicates that glomerular number reduction is seen in SIDS NBW and non-SIDS NBW cases and is therefore directly associated with growth retardation rather than with SIDS.

Birth Weight↗
PubMedDate not supplied

Renal developmental arrest in sudden infant death syndrome.

Investigations linking sudden infant death syndrome (SIDS) and type II intrauterine growth retardation (IUGR) have thus far failed due in part to technical limitations. Recently developed stereological methods for the unbiased estimation of total nephron number in the human kidney are capable of detecting deviations from normal values of greater than 10%. We compared the total number of nephrons in the kidneys of 24 SIDS victims with those from 16 controls with the same age range. Mean nephron number was significantly (P < 0.001) reduced in ex-IUGR SIDS cases (birthweight under the 10th centile, n = 9, mean number 635,000, range 327,000-1,010,000) in comparison with controls (903,000, 740,000-1,060,000). A similarly significant (P < 0.01) reduction in the "normal birthweight" SIDS group (birthweight over 10th centile, n = 15, 690,000, 361,000-1,040,000) was found. This hitherto unreported renal developmental arrest may be only one manifestation of a general, somatic developmental defect, reflecting adverse intrauterine conditions; other organ systems, similarly critical to homeostasis may be comparably affected. The findings, although not proposed as direct cause of SIDS, may represent a potential explanation for the recognized association of IUGR and SIDS, and provide--we believe--the first quantitative evidence of intrauterine growth retardation in, at least a number of, children of average birthweight.

Birth Weight↗
PubMedDate not supplied

The effect of benomyl on neurite outgrowth in mouse NB2A and human SH-SY5Y neuroblastoma cells in vitro.

The commercial fungicide methyl 1-[(butylamino) carbonyl]-1H-benzimidazol-2-ylcarbamate (benomyl) is teratogenic in rats. Its mode of action is believed to be related to its ability to inhibit the polymerization of brain tubulin. In this study its effects were studied in cultured neuronal cells during differentiation and neurite outgrowth. Mouse NB2a and human SH-SY5Y neuroblastoma cells were induced to differentiate by addition of dibutyryl cyclic AMP and at the same time were exposed to various concentrations of benomyl. Benomyl significantly inhibited neurite outgrowth in both cell lines at concentrations of 10(-8) M and above with IC50 values of 5.9 x 10(-7) M and 1.0 x 10(-6) M in the NB2a and SH-SY5Y cells respectively. The results show that benomyl inhibits neuronal cell differentiation at concentrations likely to be achieved during the development of fetal abnormalities in rats in vivo.

Animals↗