Epidural extension failure in obese women is comparable to that of non-obese women.

BACKGROUND: Management of labor epidurals in obese women is difficult and extension to surgical anesthesia is not always successful. Our previous retrospective pilot study found epidural extension was more likely to fail in obese women. This study used a prospective cohort to compare the failure rate of epidural extension in obese and non-obese women and to identify risk factors for extension failure. METHODS: One hundred obese participants (Group O, body mass index ≥ 40 kg/m2 ) were prospectively identified and allocated two sequential controls (Group C, body mass index ≤ 30 kg/m2 ). All subjects utilized epidural labor analgesia and subsequently required anesthesia for cesarean section. The primary outcome measure was failure of the labor epidural to be used as the primary anesthetic technique. Risk factors for extension failure were identified using Chi-squared and logistic regression. RESULTS: The odds ratio (OR) of extension failure was 1.69 in Group O (20% vs. 13%; 95% CI: 0.88-3.21, P = 0.11). Risk factors for failure in obese women included ineffective labor analgesia requiring anesthesiologist intervention, (OR 3.94, 95% CI: 1.16-13.45, P = 0.028) and BMI > 50 kg/m2 (OR 3.42, 95% CI: 1.07-10.96, P = 0.038). CONCLUSION: The failure rate of epidural extension did not differ significantly between the groups. Further research is needed to determine the influence of body mass index > 50 kg/m2 on epidural extension for cesarean section.

Tissue culture of isolated glomeruli in experimental crescentic glomerulonephritis.

As a means of studying mechanisms of response to injury in glomerulonephritis, glomeruli from normal sheep and rabbits and from sheep and rabbits with experimental crescentic glomerulonephritis have been isolated and grown in tissue culture. The cellular outgrowths from the normal and diseased glomeruli have been compared. The outgrowth of glomeruli from normal animals contained only two cell populations whose microscopic and ultrastructural appearances were of epithelial and mesangial cells. The same cells were also observed in the outgrowths of glomeruli from animals with crescenti nephritis but in addition a third population of cells was present in large numbers. These cells were identified as macrophages by their mobility, ultrastructure, phagocytic capacity, and presence of Fc receptors. Glomerular outgrowth from sheep with crescentic glomerulonephritis contained 170 +/- 20 (SEM) macrophages and outgrowths from rabbits with crescentic nephritis contained 64 +/- 6 (SEM) macrophages per glomerulus. We have previously observed large numbers of macrophages in the outgrowth of isolated glomeruli from humans with rapidly progressive crescentic glomerulonephritis. The predominance of the macrophage in cultures of glomeruli from both human and animal crescentic glomerulonephritis suggests that this is an important cell in the inflammatory reaction occurring in crescentic glomerulonephritis and may comprise a substantial proportion of the cells forming the crescent.

A type II keratin is expressed in glial cells of the goldfish visual pathway.

The predominant intermediate filament proteins of the goldfish visual pathway consist of neuronal and non-neuronal isoelectric variants (58 kd). We have isolated a cDNA clone for the glial intermediate filament protein (ON3) from an optic nerve expression library. The predicted amino acid sequence of this clone reveals that it codes for a type II keratin representing the goldfish equivalent of mammalian keratin K8. K8 has been shown to be associated with embryogenesis and development. Unlike the mammalian visual system, the goldfish visual pathway displays a remarkable capacity for functional regeneration. The expression of K8, a protein not usually expressed in glial cells but shown to be associated with development, in the goldfish optic nerve may be involved with the processes of growth and regeneration in the goldfish visual pathway.

Plasticin, a novel type III neurofilament protein from goldfish retina: increased expression during optic nerve regeneration.

The goldfish visual pathway displays a remarkable capacity for continued development and plasticity. The intermediate filament proteins in this pathway are unexpected and atypical, suggesting these proteins provide a structure that supports growth and plasticity. Using a goldfish retina lambda gt10 library, we have isolated a full-length cDNA clone that encodes a novel type III intermediate filament protein. The mRNA for this protein is located in retinal ganglion cells, and its level dramatically increases during optic nerve regeneration. The protein is transported into the optic nerve within the slow phase of axonal transport. We have named this protein plasticin because it was isolated from a neuronal pathway well known for its plasticity.

Keratin-associated protein 5-5 controls cytoskeletal function and cancer cell vascular invasion.

Cancer cell vascular invasion is a crucial step in the malignant progression toward metastasis. Here we used a genome-wide RNA interference screen with E0771 mammary cancer cells to uncover drivers of endothelial monolayer invasion. We identified keratin-associated protein 5-5 (Krtap5-5) as a candidate. Krtap5-5 belongs to a large protein family that is implicated in crosslinking keratin intermediate filaments during hair formation, yet these Krtaps have no reported role in cancer. Depletion of Krtap5-5 from cancer cells led to cell blebbing and a loss of keratins 14 and 18, in addition to the upregulation of vimentin intermediate filaments. This intermediate filament subtype switching induced dysregulation of the actin cytoskeleton and reduced the expression of hemidesmosomal α6/ß4-integrins. We further demonstrate that knockdown of keratin 18 phenocopies the loss of Krtap5-5, suggesting that Krtap5-5 crosstalks with keratin 18 in E0771 cells. Disruption of the keratin cytoskeleton by perturbing Krtap5-5 function broadly altered the expression of cytoskeleton regulators and the localization of cell surface markers. Krtap5-5 depletion did not impact cell viability but reduced cell motility and extracellular matrix invasion, as well as extravasation of cancer cells into tissues in zebrafish and mice. We conclude that Krtap5-5 is a previously unknown regulator of cytoskeletal function in cancer cells that modulates motility and vascular invasion. Thus, in addition to its physiologic function, a Krtap can serve as a switch toward malignant progression.

Restricted expression of the homeobox gene prox 1 in developing zebrafish.

Prox 1 is a vertebrate homeobox gene which is homologous to the Drosophila transcription factor, prospero. We have isolated a prox 1 cDNA from zebrafish, which encodes a protein that has 82%, 84% and 83% amino acid identity with chicken, mouse and human Prox 1, respectively. Antibodies raised against human Prox 1 cross-react with zebrafish Prox 1 and are used here to determine the expression patterns of Prox 1 during zebrafish embryogenesis by whole-mount immunohistochemistry. In the 10-somite embryo, Prox 1 is expressed over the prospective lens placode and over a broad region of epithelium extending from the eye to the otic vesicle. As embryogenesis proceeds, Prox 1 expression in the eye lens becomes intense, and is detected in maturing muscle pioneer cells and superficial muscle cells. In the CNS, Prox 1 is expressed in a stripe along the forebrain-midbrain boundary, in a segmented pattern in the ventral hindbrain, and in selected cells of the ventral spinal cord. Additional sites of Prox 1 expression include the lateral line primordium, the trigeminal ganglia, the otic vesicle and occasional endodermal cells.

Cell growth density modulates cancer cell vascular invasion via Hippo pathway activity and CXCR2 signaling.

Metastasis of cancer cells involves multiple steps, including their dissociation from the primary tumor and invasion through the endothelial cell barrier to enter the circulation and finding their way to distant organ sites where they extravasate and establish metastatic lesions. Deficient contact inhibition is a hallmark of invasive cancer cells, yet surprisingly the vascular invasiveness of commonly studied cancer cell lines is regulated by the density at which cells are propagated in culture. Cells grown at high density were less effective at invading an endothelial monolayer than cells grown at low density. This phenotypic difference was also observed in a zebrafish model of vascular invasion of cancer cells after injection into the yolk sac and extravasation of cancer cells into tissues from the vasculature. The vascular invasive phenotypes were reversible. A kinome-wide RNA interference screen was used to identify drivers of vascular invasion by panning small hairpin RNA (shRNA) library-transduced noninvasive cancer cell populations on endothelial monolayers. The selection of invasive subpopulations showed enrichment of shRNAs targeting the large tumor suppressor 1 (LATS1) kinase that inhibits the activity of the transcriptional coactivator yes-associated protein (YAP) in the Hippo pathway. Depletion of LATS1 from noninvasive cancer cells restored the invasive phenotype. Complementary to this, inhibition or depletion of YAP inhibited invasion in vitro and in vivo. The vascular invasive phenotype was associated with a YAP-dependent upregulation of the cytokines IL6, IL8 and C-X-C motif ligand 1, 2 and 3. Antibody blockade of cytokine receptors inhibited invasion and confirmed that they are rate-limiting drivers that promote cancer cell vascular invasiveness and could provide therapeutic targets.

Single cell reverse transcription-polymerase chain reaction analysis of rat supraoptic magnocellular neurons: neuropeptide phenotypes and high voltage-gated calcium channel subtypes.

Magnocellular neurosecretory cells (MNCs) in the hypothalamo-neurohypophysial system that express and secrete the nonapeptides oxytocin (OT) and vasopressin (VP) were evaluated for the expression of multiple genes in single magnocellular neurons from the rat supraoptic nucleus using a single cell RT-PCR protocol. We found that all cells representing the two major phenotypes, the OT and VP MNCs, express a small, but significant, amount of the other nonapeptide's messenger RNA (mRNA). In situ hybridization histochemical analyses confirmed this observation. A third phenotype, containing equivalent amounts of OT and VP mRNA, was detected in about 19% of the MNCs from lactating female supraoptic nuclei. Analyses of these phenotypes for other coexisting peptide mRNAs (e.g. CRH, cholecystokinin, galanin, dynorphin, and the calcium-binding protein, calbindin) generally confirmed expectations from the literature, but revealed cell to cell variation in their coexpression. Our results also show that the high voltage-activated calcium channel subunit genes, alpha1A-D, alpha2, and beta1-4 are expressed in virtually all MNCs. However, the alpha1E subunit gene is not expressed at detectable levels in these cells. The expression of all of the beta-subunit genes in each MNC may account for the variations in physiological and pharmacological properties of the high voltage-activated channels found in these neurons. (Endocrinology 140: 5391-5401, 1999)

Plasticin, a newly identified neurofilament protein, is preferentially expressed in young retinal ganglion cells of adult goldfish.

The adult goldfish retina and optic nerve display continuous growth, plasticity, and the capacity to regenerate throughout the animal's life. The intermediate filament proteins in this pathway are different from those in adult mammalian nerves, which do not continuously grow or normally regenerate. One novel intermediate filament protein of the goldfish visual pathway is plasticin, which is synthesized in ganglion cells and transported into the optic nerve. Using specific polyclonal antibodies raised against a plasticin fusion protein, we investigated the distribution of this protein in the normal retina and nerve and in the retina and nerve following optic nerve crush. In the normal pathway, plasticin was localized predominantly to the axons of very young ganglion cells; however, there was considerable immunoreactivity in older axons as they approach the chiasm. In addition, following optic nerve crush, all ganglion cell somata and their axons proximal to the crush site became equally immunoreactive. The results suggest that plasticin may contribute to axonal growth, plasticity, and regeneration.

Restricted expression of a new paired-class homeobox gene in normal and regenerating adult goldfish retina.

We describe the cloning and expression pattern of a new paired-class homeobox gene, Vsx-1, in the continuously growing retina of the goldfish. Vsx-1 belongs to a subset of paired-class homeobox genes that lack a second DNA binding domain, the paired-domain, and is closely related to the C. elegans ceh-10 gene. In the adult goldfish, Vsx-1 expression is restricted to the neural retina. In the central, mature retina, Vsx-1 mRNA is synthesized in a subset of differentiated cells in the inner nuclear layer in a pattern suggestive of bipolar cells. In immature retina, adjacent to the retinal margin, Vsx-1 is expressed in a relatively broader subset of newly postmitotic cells but is downregulated in some of these cells to form the mature expression pattern. Following retinal injury, during the early phase of regeneration, Vsx-1 mRNA synthesis appears to be upregulated in cells in the inner nuclear layer and is expressed de novo in cells outside this layer. By virtue of its identity as a transcriptional regulatory gene and its patterns of expression, we speculate that Vsx-1 may stabilize the differentiated state of a subset of cells in the inner nuclear layer and may be involved in cellular differentiation during retinal development and regeneration.

Differential expression of keratins in goldfish optic nerve during regeneration.

The goldfish visual pathway, unlike the visual pathway of higher vertebrates, retains continuous growth and development throughout life and is capable of functional regeneration. The structure and expression of proteins that support the physiological attributes of this system are of interest. Glial cells in this pathway express keratins as the predominant intermediate filament proteins rather than the expected glial fibrillary acidic protein. Previously we identified and characterized cDNA clones representing two type I keratins from the goldfish optic nerve, GK48 and GK49. The GK48 protein is the type I keratin partner to the type II keratin ON3, while the GK49 protein is expressed in a different cell type. Here, we extend our studies on the expression of mRNA for the GK48, GK49, and ON3 proteins at the early stages of optic nerve regeneration. RNase protection assays show that at 10 days post-crush, there is no overall change in levels of mRNA for these proteins as compared to uncrushed control nerves and nerves from unoperated fish. In addition, we show by in situ hybridization that the GK49 protein shows no changes in its distribution of mRNA in the optic nerve after crush. In contrast, the levels of GK48 and ON3 mRNA are greatly reduced within the crush zone. However, these two mRNAs are differentially expressed at different time points during regeneration, with GK48 mRNA appearing in the crush zone before ON3. These results indicate that the mRNA for the GK48 and ON3 proteins are differentially regulated during regeneration and that these two proteins are expressed in a different cell type from the GK49 protein.

Vsx-1 and Vsx-2: two Chx10-like homeobox genes expressed in overlapping domains in the adult goldfish retina.

The genetic linkages of the murine ocular retardation mutation with the Chx10 gene and the murine small eye mutation with the Pax-6 gene has demonstrated the importance of Paired class homeobox genes in the development of the mammalian retina. Previously, we identified a Paired-class homeobox gene, Vsx-1, whose expression in the adult goldfish retina is restricted to the inner nuclear layer (INL) and to postmitotic, differentiating progenitor cells in the growth zone at the retinal peripheral margin, where neurogenesis continues throughout life. Here, we report the molecular cloning and expression pattern of a new Paired class homeobox gene, Vsx-2, in the adult goldfish retina. Like Vsx-1, Vsx-2 expression is highly restricted to the retina in the adult goldfish and overlaps with Vsx-1 expression in the mature INL. At the peripheral margin, Vsx-2 is expressed in mitotically active neuronal progenitors and is downregulated as these cells become postmitotic and begin to differentiate. Comparison of the amino acid sequences of Vsx-2, Vsx-1, Chx10, and C. elegans ceh-10 reveal a conserved homeodomain and a unique domain termed the CVC domain. The similarities of the Vsx-2, Vsx-1, and Chx10 expression patterns suggest that genes containing the CVC domain have conserved functions during retinal development in vertebrates.

Complex expression of keratins in goldfish optic nerve.

Keratins are the predominant intermediate filament proteins in the nonneuronal cells of the goldfish optic nerve. At least three different keratin pairs are expressed in this tissue, indicating an unexpected complexity. Expression of the type II keratin ON3 in goldfish optic nerve astrocytes predicts the expression of a type I keratin partner. Here we report the cDNA sequence and predicted amino acid sequence of two type I keratins from the goldfish optic nerve, designated GK48 and GK49. The GK48 protein is the goldfish equivalent of mammalian keratin 18 (K18) and is the most likely type I keratin partner to the ON3 protein. The GK49 protein is similar to the GK50 protein, a type I keratin characterized previously from the goldfish optic nerve. The GK48 and ON3 mRNAs are expressed in a variety of goldfish tissues, whereas the expression of GK49 mRNA has a more limited expression. In addition, in situ hybridization experiments show that the expression of the GK48 and ON3 mRNAs are evenly distributed throughout the optic nerve, while the GK49 mRNA is expressed along longitudinal lines. These results show that there is a diversity of keratin expression within different cell types in the goldfish optic nerve.

Restricted expression of the neuronal intermediate filament protein plasticin during zebrafish development.

In the adult goldfish visual pathway, expression of the neuronal intermediate filament (nIF) protein plasticin is restricted to differentiating retinal ganglion cells (RGCs) at the margin of the retina. Following optic nerve injury, plasticin expression is elevated transiently in all RGCs coincident with the early stages of axon regeneration. These results suggest that plasticin may be expressed throughout the nervous system during the early stages of axonogenesis. To test this hypothesis, we analyzed plasticin expression during zebrafish (Danio rerio) neuronal development. By using immunocytochemistry and in situ hybridization, we found that plasticin is expressed in restricted subsets of early zebrafish neurons. Expression coincides with axon outgrowth in projection neurons that pioneer distinct axon tracts in the embryo. Plasticin is expressed first in trigeminal, Rohon-Beard, and posterior lateral line ganglia neurons, which are among the earliest neurons to initiate axonogenesis in zebrafish. Plasticin is expressed also in reticulospinal neurons and in caudal primary motoneurons. Together, these neurons establish the first behavioral responses in the embryo. Plasticin expression also coincides with initial RGC axonogenesis and progressively decreases after RGC axons reach the tectum. At later developmental stages, plasticin is expressed in a subset of the cranial nerves. The majority of plasticin-positive neurons are within or project axons to the peripheral nervous system. Our results suggest that plasticin subserves the changing requirements for plasticity and stability during axonal outgrowth in neurons that project long axons.

Chronic hypoosmolality induces a selective decrease in magnocellular neurone soma and nuclear size in the rat hypothalamic supraoptic nucleus.

The magnocellular neurones of the hypothalamo-neurohypophysial system (HNS) play a vital role in the maintenance of body homeostasis by regulating oxytocin (OT) and vasopressin (VP) secretion from the posterior pituitary. During hyperosmolality, OT and VP mRNA levels are known to increase by approximately two-fold, whereas during chronic hypoosmolality, OT and VP mRNA levels decrease to approximately 10-20% of basal levels. In these studies, we evaluated changes in cell size associated with these physiological conditions. Cell and nuclear sizes of neurones in the supraoptic nucleus (SON), the nucleus of the lateral olfactory tract (LOT) and the medial habenular nucleus (MHB) were measured from neurones identified by in situ hybridization histochemistry for beta(III)-tubulin mRNA, and measurements were made from OT and AVP magnocellular neurones in the SON after phenotypic identification by immunohistochemistry. Under hypoosmolar conditions, the cell and nuclear sizes of OT and VP magnocellular neurones decreased to approximately 60% of basal values, whereas cell and nuclear sizes of OT and VP neurones in hyperosmolar rats increased to approximately 170% of basal values. In contrast, neither hyperosmolality, nor hypoosmolality significantly affected cell and nuclear sizes in the LOT and MHB. These results confirm previous studies that showed that magnocellular neurones increase cell size in response to hyperosmolar conditions and, for the first time, demonstrate a marked decrease in cell size in the SON in response to chronic hypoosmolar conditions. These dramatic changes in cell and nuclear size directly parallel changes in OT and VP gene expression in the magnocellular neurones of the SON and, consequently, are consistent with the pronounced bidirectional changes in gene expression and cellular activity found during these osmotic perturbations. Our results therefore support the concept of global alterations in the synthetic activity of magnocellular OT and AVP neurones in response to extracellular osmolality.

Histochemistry of glomerular cells in animal models of crescentic glomerulonephritis.

A histochemical enzyme profile has been used to determine the origin of the cells of glomerular crescents in rabbit and sheep models of rapidly progressive crescentic glomerulonephritis. Crescentic glomeruli were examined for beta-glucuronidase, esterase and acid phosphatase, the characteristic phagolysosomal enzymes of the monocyte-macrophage series and its transformed tissue counterpart, the epithelioid cell. More than 50% of the cells of the glomerular crescents of animals with experimental crescentic glomerulonephritis contained large amounts of all the enzymes, whereas the cells of normal glomeruli contained only trace amounts of acid phosphatase and esterase and no glucuronidase. These findings support the hypothesis that the major proportion of the cells of glomerular crescents are monocytes which have accumulated in Bowman's space rather than intrinsic glomerular cells which have proliferated.

Tissue culture of isolated human glomeruli.

Glomerular cells have been grown in a reproducible manner from 5 normal human kidneys. A technique is described which combined mechanical disruption of renal cortex and microdissection, and provides large numbers of pure glomeruli within 30--45 mintues. Histological examination shows this technique produces intact glomeruli without cell disturbance. During tissue culture, glomeruli attach to the flask and the intrinsic cells migrate onto the flask and divide. Variations of culture conditions have shown that glomeruli are robust without fastidious culture requirements. Intact kidney tissue can be left at 4 degrees C for perios up to 24 hours prior to isolated of individual glomeruli without affecting subsequent cellular growth in culture. They grow in most commonly used media although the cells require 20% foetal calf serum for optimum growth. Their pH optimum is between 7.0 and 7.4 with temperature optimum of 37 degrees C. as glomeruli must attach prior to cell growth, minimum movement is critical to promote optimum growth. Under these optimum conditions a regular and predictable growth of cells of two distinct types, has been observed over 14 days; one of these types is probably epithelial.

Nephropathy in cyanotic congenital heart disease.

Three children with cyanotic congenital heart disease who developed transient proteinuria and edema are described. One died of an intercurrent illness but the other two are now well. Renal biopsy findings in all three children demonstrated a mesangial proliferative glomerulonephritis on light microscopy. An unusual ultrastructural appearance of localized electron-dense thickening of the basement membrane of the capillary loops was seen in all three and collagen fibers were present in the mesangium of two. There was slight fusion of foot processes in two specimens and marked fusion in the third. Immunofluorescence in two patients demonstrated IgM staining in both and fibrin in one. The cause of the glomerular lesions is unknown but, among the many possible factors involved, anoxia and increased venous pressure may be important.

Noninvasive discrimination of brachial plexus involvement in upper limb pain.

The ability of Elvey's "brachial plexus tension test" to identify pain referred from the cervical region was investigated in 50 patients with unilateral shoulder and upper arm pain: 25 reported symptoms commencing after open heart surgery, indicating a high probability of referred pain due to that procedure; 25 were athletes with injuries from throwing movements, a cause appearing much less likely to cause referred pain. Twenty-five asymptomatic subjects with no history of spinal or limb pain also were tested. Cervical and upper limb maneuvers of the test were assessed goniometrically. The cardiac group showed significantly greater test results than those of the other groups, suggesting that the test is able to discriminate referred and local sources of upper limb pain.

Small molecule inhibitors of ezrin inhibit the invasive phenotype of osteosarcoma cells.

Ezrin is a multifunctional protein that connects the actin cytoskeleton to the extracellular matrix through transmembrane proteins. High ezrin expression is associated with lung metastasis and poor survival in cancer. We screened small molecule libraries for compounds that directly interact with ezrin protein using surface plasmon resonance to identify lead compounds. The secondary functional assays used for lead compound selection included ezrin phosphorylation as measured by immunoprecipitation and in vitro kinase assays, actin binding, chemotaxis, invasion into an endothelial cell monolayer, zebrafish and Xenopus embryonic development, mouse lung organ culture and an in vivo lung metastasis model. Two molecules, NSC305787 and NSC668394, that directly bind to ezrin with low micromolar affinity were selected based on inhibition of ezrin function in multiple assays. They inhibited ezrin phosphorylation, ezrin-actin interaction and ezrin-mediated motility of osteosarcoma (OS) cells in culture. NSC305787 mimicked the ezrin morpholino phenotype, and NSC668394 caused a unique developmental defect consistent with reduced cell motility in zebrafish. Following tail vein injection of OS cells into mice, both molecules inhibited lung metastasis of ezrin-sensitive cells, but not ezrin-resistant cells. The small molecule inhibitors NSC305787 and NSC668394 demonstrate a novel targeted therapy that directly inhibits ezrin protein as an approach to prevent tumor metastasis.