A morphologic and immunofluorescent analysis of primary guinea pig glomerular cell types grown in chemically defined media. Evidence for clonal growth and cell differentiation

Primary kidney guinea pig glomerular cells were successfully grown in chemically defined media containing insulin, transferrin, and fibronectin or glycylhistidyllysine and fibronectin. Morphologic analysis of glomerular cells grown in either of these chemically defined media provided identical results with respect to cell growth properties and cell types involved. Electron microscopic studies of glomeruli early after they had been placed in culture showed definite evidence of "dedifferentiation" of some glomerular cells. Most glomerular cells in later cultures were undifferentiated. However, since electron microscopic analyses of glomeruli in confluent cultures demonstrated that the majority of cells in culture grow from the epithelial side of the glomerular basement membrane, we suggest that these cells were some form of epithelial cell. This conclusion was further strengthened by the fact that cells resembling well differentiated glomerular epithelial cells were seen in cultures of glomeruli grown in chemically defined media; these cells have never been observed in glomeruli grown in calf serum. Fluorescent microscopy of cell stained with the mitochondrial stain rhodamine 123 allowed identification of several glomerular cell types according to distribution, number, and morphology of mitochondria. Similarly, indirect immunofluorescent microscopy studies using antibodies to fibronectin or laminin provided evidence that glomerular cells separated into cell types according to mitochondrial staining properties were unique biochemically. Using these histochemical criteria it was possible to demonstrate that certain of the glomerular cell types could be selectively grown by addition of the enzyme galactose oxidase to the media. Analysis of our morphologic and histochemical results suggests the possibility that clonal growth and differentiation of glomerular epithelial cells occurs when glomeruli are placed in chemically defined media, and our results are compatible with the hypothesis that either "stem cells" or "dedifferentiated" cells are the primary cells dividing in culture.     

Tetrahedral recording of 3-D BAEPs: evidence for the centered dipole model

Three-dimensional brain-stem auditory evoked potentials (3-D BAEPs) were recorded from 12 normal subjects using a new tetrahedral montage, as well as two other bipolar montages previously described for 3-channel Lissajous' trajectories (3-CLTs). Mean responses, as well as between-subject and within-subject variability were described. A mathematical transformation was applied to the recorded trajectories to render them in a common canonical form to test the assumption that the BAEP conforms to a centrally generated dipolar field. Apex, segment, and plane orientations were measured for each trajectory, and discrepancies between montages were evaluated to judge the adequacy of the centered dipole model. For the vector means of apices, segments, and planes, median angles of discrepancy between montages ranged from 10 to 23°. These results support the validity of a centered dipole model for the BAEP and affirm the rationale for employing the 3-channel recording technique. Among the montages studied, the tetrahedron provided maximum economy by using fewer electrodes, avoided certain problematic recording sites, and produced less variable data.     

Confocal fluorescence microscopy of plant cells

Summary The confocal laser scanning microscope (CLSM) has become a vital instrument for the examination of subcellular structure, especially in fluorescently stained cells. Because of its ability to markedly reduce out-of-focus flare, when compared to the conventional wide-field fluorescence microscope, the CLSM provides a substantial improvement in resolution along the z axis and permits optical sectioning of cells. These developments have been particularly helpful for the investigation of plant cells and tissues, which because of their shape, size, and optical properties have been difficult to analyze at high resolution by conventional means. We review the contribution that the CLSM has made to the study of plant cells. We first consider the principle of operation of the CLSM, including a discussion of image processing, and of lasers and appropriate fluorescent dyes. We then summarize several studies of both fixed and live plant cells in which the instrument has provided new or much clearer information about cellular substructure than has been possible heretofore. Attention is given to the visualization of different components, including especially the cytoskeleton, endomembranes, nuclear components, and relevant ions, and their changes in relationship to physiological and developmental processes. We conclude with an effort to anticipate advances in technology that will improve and extend the performance of the CLSM. In addition to the usual bibliography, we provide internet addresses for information about the CLSM.     

Effect of 50-Hz Powerline Exposed Magnetized Water on Rat Kidney

Double distilled water samples were exposed for 48 hr. to 50 Hz-powerline electromagnetic field (EMF) strength of 51.2 μT (36.2 RMS). This EMF exposed water was made available to experimental adult Charles-Foster male rats for drinking ad libitum for 30 days. On the 31st day the rats were anaesthetised with ether and then fixed by perfusion with 10% neutral formalin. The Kidneys were dissected out and further fixed in the same fixative. The corresponding control rats provided with unexposed triple distilled water were similarly treated. On gross examination, no anomaly was observed in the kidney of the exposed group. On histological examination, marked spongiform changes leading to degeneration and compensatory proliferation of the glomerular tufts and degeneration of the lining epithelia of the tubules was observed. This study adds a link in demonstrating that powerline exposure induces stable changes in water structures and effects biomechanisms of tissue fluid.     

CXC Chemokine Receptor 7 (CXCR7) Regulates CXCR4 Protein Expression and Capillary Tuft Development in Mouse Kidney

Background

The CXCL12/CXCR4 axis is involved in kidney development by regulating formation of the glomerular tuft. Recently, a second CXCL12 receptor was identified and designated CXCR7. Although it is established that CXCR7 regulates heart and brain development in conjunction with CXCL12 and CXCR4, little is known about the influence of CXCR7 on CXCL12 dependent kidney development.

Methodology/Principal Findings

We provided analysis of CXCR7 expression and function in the developing mouse kidney. Using in situ hybridization, we identified CXCR7 mRNA in epithelial cells including podocytes at all nephron stages up to the mature glomerulus. CXCL12 mRNA showed a striking overlap with CXCR7 mRNA in epithelial structures. In addition, CXCL12 was detected in stromal cells and the glomerular tuft. Expression of CXCR4 was complementary to that of CXCR7 as it occurred in mesenchymal cells, outgrowing ureteric buds and glomerular endothelial cells but not in podocytes. Kidney examination in CXCR7 null mice revealed ballooning of glomerular capillaries as described earlier for CXCR4 null mice. Moreover, we detected a severe reduction of CXCR4 protein but not CXCR4 mRNA within the glomerular tuft and in the condensed mesenchyme. Malformation of the glomerular tuft in CXCR7 null mice was associated with mesangial cell clumping.

Conclusions/Significance

We established that there is a similar glomerular pathology in CXCR7 and CXCR4 null embryos. Based on the phenotype and the anatomical organization of the CXCL12/CXCR4/CXCR7 system in the forming glomerulus, we propose that CXCR7 fine-tunes CXCL12/CXCR4 mediated signalling between podocytes and glomerular capillaries.     

Ambulatory cassette EEG in epilepsy diagnosis

The electroencephalographic evaluation of patients with possible or proven epilepsy is no longer limited to routine laboratory EEGs or intensive inpatient monitoring. Expanded temporal sampling of the EEG, which increases the probability of documenting, characterizing, and quantitating the electrographic manifestations of these illnesses, is now available on a portable, outpatient, and less cumbersome inpatient basis by means of ambulatory cassette recordings. The technological advances which have made this technique feasible include small multi-channel tape recorders, miniature preamplifiers, and rapid video/audio playback units. New designs in montages and analysis techniques have made the procedure practical. Clinical series and controlled trials have confirmed the usefulness of cassette EEG monitoring in the evaluation of epilepsy and a wide range of other paroxysmal neurologic disorders. Ambulatory EEG diagnostic yields have been shown to be superior to routine laboratory studies and nearly as good as inpatient telemetry evaluations. The role of cassette recordings in clinical electroencephalography continues to be defined as new applications are established.     

Flow cytometric analysis of pollen grains collected from individual bees provides information about pollen load composition and foraging behaviour

Background and Aims

Understanding the species composition of pollen on pollinators has applications in agriculture, conservation and evolutionary biology. Current identification methods, including morphological analysis, cannot always discriminate taxa at the species level. Recent advances in flow cytometry techniques for pollen grains allow rapid testing of large numbers of pollen grains for DNA content, potentially providing improved species resolution.

Methods

A test was made as to whether pollen loads from single bees (honey-bees and bumble-bees) could be classified into types based on DNA content, and whether good estimates of proportions of different types could be made. An examination was also made of how readily DNA content can be used to identify specific pollen species.

Key Results

The method allowed DNA contents to be quickly found for between 250 and 9391 pollen grains (750–28 173 nuclei) from individual honey-bees and between 81 and 11 512 pollen grains (243–34 537 nuclei) for bumble-bees. It was possible to identify a minimum number of pollen species on each bee and to assign proportions of each pollen type (based on DNA content) present.

Conclusions

The information provided by this technique is promising but is affected by the complexity of the pollination environment (i.e. number of flowering species present and extent of overlap in DNA content). Nevertheless, it provides a new tool for examining pollinator behaviour and between-species or cytotype pollen transfer, particularly when used in combination with other morphological, chemical or genetic techniques.     

Measurement of single kidney glomerular filtration rate in dogs using dynamic contrast-enhanced magnetic resonance imaging and the Rutland-Patlak plot technique

Background

Nephropathies are among the most common diseases in dogs. Regular examination of the kidney function plays an important role for an adequate treatment scheme. The determination of the glomerular filtration rate (GFR) is seen as the gold standard in assessing the kidney status. Most of the tests have the disadvantage that only the complete glomerular filtration rate of both kidneys can be assessed and not the single kidney glomerular filtration rate. Imaging examination techniques like dynamic contrast-enhanced magnetic resonance imaging have the potential to evaluate the single kidney GFR. There are studies in human medicine describing the determination of the single kidney GFR using this technique. To our knowledge there are no such studies for dogs.

Results

An exponential fit was found to describe the functional interrelation between signal intensity and contrast medium concentrations. The changes of contrast medium concentrations during the contrast medium bolus propagation were calculated. The extreme values of contrast medium concentrations in the kidneys were reached at nearly the same time in every individual dog (1st maximum aorta 8.5 s, 1st maximum in both kidneys after about 14.5 s; maximum concentration values varied between 17 and 125 µmol/mL in the aorta and between 4 and 15 µmol/mL in the kidneys). The glomerular filtration rate was calculated from the concentration changes of the contrast medium using a modified Rutland-Patlak plot technique. The GFR was 12.7?±?2.9 mL/min m² BS for the left kidney and 12.0?±?2.2 mL/min/m² BS for the right kidney. The mean values of the coefficient of determination of the regression lines were averagely 0.91?±?0.08.

Conclusions

The propagation of contrast medium bolus could be depicted well. The contrast medium proceeded in a similar manner for every individual dog. Additionally, the evaluation of the single kidney function of the individual dogs is possible with this method. A standardized examination procedure would be recommended in order to minimize influencing parameters.

     

Demonstration of the in vitro phagocytosis of Treponema pallidum by rabbit peritoneal macrophages.

Evidence has been provided for the in vitro phagocytosis of virulent Treponema pallidum by stimulant-induced peritoneal macrophages. After the 4-hr incubation of macrophages with T. pallidum, treponemal antigens associated with the macrophages are specifically stained using indirect immunofluorescent techniques. Phagocytized treponemes appear within the cytoplasm of macrophages as round, brightly fluorescent "bodies" observable in increasing numbers as the duration of the treponeme-phagocyte interaction increases. Their presence is significantly reduced in the cytoplasm of macrophages that have been treated with cytochalasin B, a known inhibitor of phagocytosis, and in nonphagocytic fibroblasts. Additionally, supportive evidence for T. pallidum phagocytosis in vitro has been provided by electron microscopic examination in which treponemes have been demonstrated within typical phagocytic vacuoles. This study also provides evidence that immune serum factor(s) significantly promote the phagocytosis of T. pallidum, although a contribution by heat-labile serum factors has not been demonstrated. The possible mechanisms of immune serum contribution and the implications of the demonstration of T. pallidum phagocytosis are discussed.     

An overview of the assessment of aquatic ecosystem health using benthic invertebrates

Community structure or species composition of benthic invertebrates has frequently been used in environmental monitoring and assessment of aquatic systems. Three general approaches have been taken: the saprobic approach, which requires detailed knowledge of taxonomy and is most effective in measuring impacts from sewage effluents; diversity indices, which do not require detailed knowledge of species requirements but ignore information provided by important species and tend to lose information; and biotic indices, which combine both approaches. In the past few years considerable advances have been made by applying multivariate statistical techniques to large data matrices and relating benthic community structure to key environmental variables. Using these techniques it is possible to establish reference communities for a set of environmental conditions, to predict the benthic community that should occur at new sites and thus measure deviation from an expected community type. This suggests that environmental criteria and objectives can be established based on biological variables as opposed to the more traditional chemical approach.Measurement of ecosystem health using functional attributes of benthic invertebrates is generally in the development stage. In the future, functional measures of ecosystem health, such as chronic measures of toxicity or stress, should be incorporated into any assessment process.     

Diagnostic value of urine erythrocyte morphology in the detection of glomerular disease in SurePath™ liquid‐based cytology compared with fresh urine sediment examination

H. Ohsaki, T. Hirouchi, N. Hayashi, E. Okanoue, M. Ohara, N. Kuroda, E. Hirakawa and Y. Norimatsu
Diagnostic value of urine erythrocyte morphology in the detection of glomerular disease in SurePath? liquid‐based cytology compared with fresh urine sediment examination Objective: To assess whether the morphology of urine erythrocytes can be an effective tool for distinguishing glomerular disease from lower urinary tract disease in SurePath^? liquid‐based cytology (SP‐LBC). Methods: We examined four morphological parameters of erythrocytes: (1) irregular erythrocytes (of all types including fragmented forms) comprising greater than or equal to 20% of erythrocytes; (2) uniform erythrocytes (>80%); (3) doughnut or target‐like shaped (D/T) erythrocytes (≥1%); and (4) acanthocytes (≥1%) in glomerular disease (n = 32) and lower urinary tract disease (n = 20) with SP‐LBC slides in cases that had also been assessed by fresh urine sediment examination. Results: Sensitivity of D/T erythrocytes and acanthocytes (dysmorphic erythrocytes) for glomerular disease were 100% and 87.5%, respectively, with urine sediment examination, and 81.3% and 46.9%, respectively, in SP‐LBC slides. Specificity was 100% for D/T erythrocytes and acanthocytes using either procedure. While irregular erythrocytes were specific for glomerular disease using urine sediment examination, they were seen in 70% of those with lower urinary tract disease using SP‐LBC slides as a result of the deformation of erythrocytes by the fixative. Conclusions: Although the sensitivity of D/T erythrocytes and acanthocytes for glomerular disease was lower in SP‐LBC slides than fresh urine sediment examination, their specificity was equally high. Therefore, urine erythrocyte morphology is useful in the detection of glomerular disease with the SP‐LBC slides. However, morphological features apart from D/T erythrocytes and acanthocytes are not useful in SP‐LBC slides.     

Computed tomography in the evaluation of tumor regression after neoadjuvant therapy: X-ray surgery and X-ray morphological comparisons

An analysis was made of chest computed tomograms of 38 small-cell lung cancer patients subjected to radical surgical treatment after neoadjuvant therapy. CT data were compared with the findings of macro- and microscopic examination of surgical specimens. In 24 (63.2%) patients, computed tomograms made before surgery showed complete tumor response confirmed by gross examination in 22 (96.1%) of them. However, microscopic examination found cancer cells in 8 (33.3%) patients. In 2 (8.3%) patients, small residual tumors could be detected by sight, which was confirmed by pathological examination. In 14 (36.8%) patients with partial response, radiological and gross examination findings fully coincided. Nevertheless, in 2 (14.3%) cases the "residual tumor" appeared to be a segment of fibrocicatrical tissue under pathological examination. On the basis of the CT findings the values of diagnostic sensitivity, accuracy and specificity in tumor response evaluation were calculated which made up 87.5%, 68.4% and 54.5% respectively. With sufficiently high sensitivity, CT specificity is low. CT makes it possible to objectively define the response of small-cell lung cancer to neoadjuvant therapy. However, the conclusion about complete response to the treatment can be made only on the basis of a comprehensive evaluation of the results of all available investigation methods and pathological examination.     

Global expression profiling applied to plant development

Plant development is controlled by both endogenous genetic programs and responses to exogenous signals. Microarray experiments are being used to identify the genes involved in these developmental processes. Most of the analyses conducted to date have been conducted on whole organs. Although these studies have provided valuable information, they are limited by the composite nature of plant organs that consist of multiple cell types. Technical advances that have made it possible to study global patterns of gene expression in individual cell types promise to increase greatly the information revealed by microarray experiments.     

Biophysical basis of glomerular permselectivity

Summary The mammalian glomerular capillary wall normally restricts the transmural passage of plasma proteins while offering little resistance to the filtration of water and small solutes. The basis for this selectivity has been explored extensively in recent years, through clearance measurements of endogenous (mainly albumin, transferrin, and immunoglobulins) and exogenous (horseradish peroxidase) proteins, and a variety of nonprotein polymers such as dextrans and polyvinylpyrrolidone. In conjunction with efforts to localize particulate and soluble tracers by high resolution ultrastructural techniques, such measurements have now made it possible to define the determinants of the glomerular filtration of macromolecules in terms of discrete structural barriers as well as such biophysical influences as hemodynamics and the molecular size- and charge-selective characteristics of the capillary wall.These experimental approaches have been aided greatly by the development of theoretical models that enable investigators to describe macromolecular filtration in terms of hydrodynamic principles applied to isoporous membranes. Although the initial models failed to consider the important role of membrane fixed negative-charge characteristics in influencing protein filtration, this shortcoming has led to the recent introduction of a theoretical model that also takes this factor into consideration. The aim of this brief review is to summarize these various theoretical approaches to the understanding of glomerular permselectivity and, wherever possible, to cite specific tests of these theories based on experimental studies in humans and animals.     

The role of cell-extracellular matrix interactions in glomerular injury

Glomerulosclerosis is characterized by excessive deposition of extracellular matrix within the glomeruli of the kidney, glomerular cell death, and subsequent loss of functional glomeruli. While in physiological situations the levels of extracellular matrix components are kept constant by a tight balance between formation and degradation, in the case of injury that results in fibrosis there is increased matrix deposition relative to its breakdown. Multiple factors control matrix synthesis and degradation, thus contributing to the development of glomerulosclerosis. This review focuses primarily on the role of cell-matrix interactions, which play a critical role in governing glomerular cell cues in both healthy and diseased kidneys. Cell-extracellular matrix interactions are made possible by various cellular receptors including integrins, discoidin domain receptors, and dystroglycan. Upon binding to a selective extracellular matrix protein, these receptors activate intracellular signaling pathways that can either downregulate or upregulate matrix synthesis and deposition. This, together with the observation that changes in the expression levels of matrix receptors have been documented in glomerular disease, clearly emphasizes the contribution of cell-matrix interactions in glomerular injury. Understanding the molecular mechanisms whereby extracellular matrix receptors regulate matrix homeostasis in the course of glomerular injury is therefore critical for devising more effective therapies to treat and ideally prevent glomerulosclerosis.     

Characteristics of the galvanic skin response and electrocardiogram in active and passive subjects under test conditions

Simultaneous examination of two subjects using a method of signal detection in a homogeneous series made it possible to detect some regular changes in the electrocardiogram and galvanic skin response in both passive and active subjects. A passive subject concealed from an active participant information kept in mind, whereas the latter subject aimed at detecting the information significant for the partner. This detection was mainly based on voluntary analysis of unconscious changes in the voice quality, variability in the latent periods of verbal responses, and enhancement of the general motor activity of the partner. At the same time, the phenomenon of unconscious decision-making by an active partner was observed, which possibly took the form of an insight into the information concealed by the passive subject. Some other mechanisms of this phenomenon are also possible.     

Genetic control of cardiac tube formation in Drosophila

In Drosophila, the heart is composed of a simple linear tube constituted of 52 pairs of myoendothelial cells which differentiate during embryogenesis to build up a functional mature organ. The cardiac tube is a contractile organ with autonomous muscular activity which functions as a hemolymph pump in an open circulatory circuit. The cardiac tube is organized in metamers which contain six pairs of cardioblasts per segment. Within each metamer the cardioblasts express a combination of genetic markers underlying their functional diversity. For example, the two most posterior cardiac cells in segments A5 to A7 differentiate into ostiae which allow the inflow of hemolymph in the tube. An additional axial information along the anteroposterior axis orchestrates the subdivision of the cardiac tube into an "aorta" in the anterior region and a "heart" in the posterior region which behave as distinct functional entities. The major pacemaker activity is located in the most caudal part of the heart. This analysis has being made possible by the identification and the utilization of specific morphological and genetic markers and an in vivo observation of cardiac function in the embryo. Functional organogenesis of the cardiac tube is accurately controlled by genetic programs that have been in part identified. Hox genes are responsible for the axial subdivision of the tube into functional modules. They activate, in their specific domains of expression, target genes effectors of the terminal differentiation. On the other hand, part of the information required for segmental information is provided by Hedgehog, a morphogen secreted by dorsal ectoderm, whose activity triggers the ostiae formation in the heart domain.     

Normal glomerular organization of the antennal lobes is not necessary for odor-modulated flight in female moths

A prominent hypothesis for the function of the glomerular structures in the primary olfactory neuropil of many groups of vertebrate and invertebrate animals is that they enable the processing and coding of information about the chemical compounds that compose complex odors. Previous studies have indicated that various degrees of glomerulus formation in the antennal lobes of the brain of the moth Manduca sexta can be effected by reducing the number of olfactory sensory axons that grow from the antenna into the antennal lobe during metamorphosis. To test the hypothesis that the presence of glomerular structure is necessary to process and identify odors, we substantially reduced, by surgery, the number of antennal segments in developing moths and upon metamorphosis we observed and quantified behavioral responses known to be elicited by odors. Intact and lesioned adult female moths were challenged to fly upwind to the source of an attractive host-plant odor in a wind tunnel. Some of the moths that had developed with reduced olfactory input flew upwind to the odor source. The flight behavior of these individuals was similar to the odor-mediated flight typically observed in moths that had developed normally. Histological analysis of the moths antennal lobes revealed that the lobes of more than half of the respondents that had been lesioned during development lacked normal glomerular organization. The neuropil of these abnormally developed antennal lobes was mostly aglomerular, but with a few isolated, clearly abnormal glomerulus-like structures. This suggests either that even a few abnormal glomeruli are sufficient to mediate this specific behavior or that canonical glomerular organization per se is not necessary for this odor-mediated behavior.