Serosal Cells Research Articles

Soluble mesothelin-related protein in pleural effusion from patients with malignant pleural mesothelioma

Malignant pleural mesothelioma (MPM) is a highly aggressive neoplasm primarily arising from surface serosal cells of the pleura and is strongly associated with asbestos exposure. Patients with MPM often develop pleural fluid as initial presentation. However, cytological diagnosis using pleural fluid is usually difficult and has limited utility. A useful molecular marker for differential diagnosis particularly with lung cancer (LC) is urgently needed. The aim of the present study was to investigate the diagnostic value of soluble mesothelin-related protein (SMRP) in pleural fluid. Pleural fluids were collected from 23 patients with MPM, 38 with LC, 26 with benign asbestos pleurisy (BAP), 5 with tuberculosis pleurisy (TP) and 4 with chronic heart failure (CHF), and the SMRP concentration was determined. All data were analyzed by using non-parametric two-sided statistical tests. The median concentration of SMRP in MPM, LC, BAP, TP and CHF were 11.5 (range 0.90-82.80), 5.20 (0.05-36.40), 6.65 (1.45-11.25), 3.20 (1.65-6.50) and 2.03 (1.35-2.80) nmol/l, respectively. The SMRP concentration was significantly higher in MPM than in the other diseases (P=0.001). The area under the ROC curve (AUC) values of the MPM diagnosis was 0.75 for the differential diagnosis from the other groups. Based on the cut-off value of 8 nmol/l, the sensitivity and specificity for diagnosis of MPM were 70.0 and 68.4%, respectively. These results indicate that the SMRP concentration in pleural fluid is a useful marker for the diagnosis of MPM.

Rescue of calcineurin A alpha null mice reveals a role for calcineurin in salivary gland function

Calcineurin is a calcium‐dependent enzyme that functions in many cells including the immune system, neurons, musculature and kidney. The phenotype of mice lacking the αisoform of the catalytic subunit (CnAα‐/‐) is notable for failure to thrive and early lethality; a cause for which has yet to be identified. In this study we report the rescue of CnAα‐/‐ mice to adulthood by feeding of a modified diet. The success of this approach suggested a defect in salivary gland function. Accordingly, while the rate of salivary production was normal, there was a significant decline in salivary amylase and peroxidase activity and lower amounts of sialic acid, indicating a defect in exocrine vesicle formation. Consistent with this finding, salivary glands from null mice demonstrated a marked attenuation of secretory vesicles and granular content of serosal acinar cells in submandibular glands. Subcellular fractionation of wildtype salivary glands revealed endoplasmic reticulum (ER) and trans‐Golgi network expression and activity of the αisoform. Loss of CnAα leads to retention of secretory vesicles in these fractions. Moreover, calcineurin activity in fractions containing the αisoform can be upregulated by acetylcholine and blocked by xestospongin, demonstrating a requirement for the IP3R. In conclusion, we find a critical role for CnAα downstream of the IP3R that is required for post‐ ER trafficking of secretory vesicles.

Developmental and evolutionary basis for drought tolerance of the Anopheles gambiae embryo

During the evolution of the Diptera there is a dramatic modification of the embryonic ectoderm, whereby mosquitoes contain separate amnion and serosa lineages while higher flies such as Drosophila melanogaster contain a single amnioserosa. Whole-genome transcriptome assays were performed with isolated serosa from Anopheles gambiae embryos. These assays identified a large number of genes implicated in the production of the larval cuticle. In D. melanogaster, these genes are activated just once during embryogenesis, during late stages where they are used for the production of the larval cuticle. Evidence is presented that the serosal cells secrete a dedicated serosal cuticle, which protects A. gambiae embryos from desiccation. Detailed temporal microarray assays of mosquito gene expression profiles revealed that the cuticular genes display biphasic expression during A. gambiae embryogenesis, first in the serosa of early embryos and then again during late stages as seen in D. melanogaster. We discuss how evolutionary modifications in the well-defined dorsal–ventral patterning network led to the wholesale deployment of the cuticle biosynthesis pathway in early embryos of A. gambiae.

Proteomic analysis of parasitized Plutella xylostella larvae plasma

Insects use their innate immunity to defend themselves against foreign invaders, such as microorganisms, nematodes and parasites. Cotesia plutellae, an endoparasitoid wasp that parasitizes the diamondback moth Plutella xylostella, uses several strategies to attack the host immune system, such as injection of viruses, venom, and serosal membrane-derived cells denoted teratocytes. However, the proteome profiles related to these immune deficiency systems have yet to be clearly defined. In this study, we investigate differences in protein expression patterns in parasitized P. xylostella larvae, with a view to identifying parasitism-specific factors. Using 2D polyacrylamide gel electrophoresis, proteins in the host plasma were assessed every 48 h after parasitism by C. plutellae. A large number of protein spots (350 in total) were detected, and approximately 50 spots were differentially expressed in the parasitized P. xylostella larvae every 48 h. In total, 26 potential candidates, including P. xylostella Serpin 2 (pxSerpin 2), translationally controlled tumor protein, signal transduction histidine kinase, apolipophorin-III, and fatty-acid binding protein were identified through quadrupole time-of-flight tandem mass spectrometry and sequence homology analysis. These proteins were classified into the following functional groups: immunity, signaling, lipid metabolism, energy metabolism, amino acid/nucleotide metabolism, and others. The pxSerpin 2 gene was cloned, and its expression profile investigated during the course of parasitism. Real-time PCR analysis of pxSerpin 2 revealed a poor correlation between the mRNA level and protein abundance. Our results clearly suggest that parasitism-specific proteins participate in suppression of the host immune response.

Serosal mesothelium retains vasculogenic potential

Mesothelia comprise the epithelial covering of coelomic organs and line the cavities in which they are housed. Mesothelia contribute to the vasculature of the heart and the intestinal tract by developmental processes of epithelial-mesenchymal transition (EMT), migration, and differentiation into endothelial cells, vascular smooth muscle cells, and pericytes. Here, we establish a novel in vitro system to analyze the differentiative potential of mesothelia. Using explants from serosal mesothelium (the mesothelial covering of the gut), we demonstrate that much of the developmental program observed in embryonic mesothelia is retained in the adult structure. Namely, processes of epithelial spreading, EMT, and differentiation into smooth muscle cells from these cells are observed. Interestingly, we were unable to stimulate endothelial cell differentiation using serum or various signaling factors. Taken together, these data reveal that differentiated serosal cells retain vasculogenic potential and provide a generalizable model for future studies on the developmental and differentiative capacity of the mesothelial cell type.

Development of Meteorus pulchricornis and regulation of its noctuid host, Pseudaletia separata

The solitary endoparasitoid Meteorus pulchricornis can parasitize many lepidopteran host species successfully. In the case of parasitization of Pseudaletia separata, developmental duration of M. pulchricornis was 8–9 days from egg to larval emergence and 6 days from prepupa to adult emergence. Successful parasitism by M. pulchricornis decreased with host age. Following parasitization of day-0 4th host instar, the parasitoid embryo, whilst still enclosed in serosal cell membrane, hatched out of the egg chorion 2 days after oviposition. Subsequently, the 1st instar parasitoid emerged from the surrounding serosal cell membrane. Serosal cells dissociated and developed as teratocytes 3.5 days after oviposition. One embryo of M. pulchricornis gave rise to approximately 1200 teratocytes, a number that remained constant until 6 days after parasitization, but decreased drastically to 200 at 7 days post-oviposition. The teratocytes of M. pulchricornis were round- or oval-shaped and grew from 65 μm at 4 days to 200 μm in the long axis at 6 days post-parasitization. At 4 days post-parasitization, many cells or cell clusters with lipid particles were observed in the hemocoels of parasitized hosts. In addition, paraffin sections of parasitized hosts revealed that many teratocytes were attached to the host's fat body and contributed to disrupting the fat body tissue. Further, examination of the total hemocyte count (THC) during parasitization revealed that THC was maintained at low levels. Surprisingly, a temporal decrease followed by restoration of THC was observed in hosts injected with virus-like particles of M. pulchricornis (MpVLPs) plus venom, which contrasts with the constant THC suppression seen in parasitized hosts. This indicates that MpVLP function is temporal and is involved in regulation of the host during early parasitism. Therefore, teratocytes, a host regulation factor in late parasitism, could be involved in keeping THC at a low level.

Murine Mast Cells Express Mpl, the Thrombopoietin Receptor, and Thrombopoietin Is a Potent Regulator of Mast Cell Differentiation.

Mast cells are hematopoietic cells localized in extramedullary sites where they engage themselves in the process of allergic response and in the immune reaction against parasites. Mast cells derive from multilineage c-KitlowCD34lowSca-1pos progenitor cells present in the marrow. These cells give rise to Linnegc-KitposSca-1neg T1/ST2pos mast cell restricted progenitor cells (MCP) whose futher maturation in the marrow remains limited under steady state conditions. MCP migrate through the blood in extramedullary sites were they mature into tissue-retricted c-KitposFceRIpos mast cells characterized by a specific mast cell protease (MMCP) profiling (dermal, mucosal and serosal mast cells in skin, gut and peritoneal cavity, respectively). The molecular mechanism that, in normal mice, restricts the mastocytopoietic potential of progenitor cells to the extramedullary sites, as well as the factors that guide the tissue-restricted differentiation of these cells, are unknown. Thrombopoietin (TPO)-Mpl interactions play an important role in the regulation of hematopoietic stem/progenitor cell proliferation and differentiation in the marrow. Here we report that mast cells, and their precursors, express Mpl (both as mRNA and cell surface protein) (see Table). Furthermore, targeted deletion of this gene (Mplnull mutation) decrease the number of MCP (by 1-log) and increases that of mast cells in dermis (by 3-fold), peritoneal cavity (by 3-fold), bone marrow (2-log) and spleen (2-log). Furthermore, because of their higher (by 2-log) MMCP-7 expression, serosal Mplnull mast cells resemble more wild-type dermal rather than serosal mast cells. On the other hand, either treatment of mice with TPO or addition of TPO to bone marrow-derived mast cell cultures induces mast cell apoptosis (by Tunel and Annexin staining) and severely hampers mast cell differentiation (by expression profiling). These data are consistent with a regulatory mechanism for murine mastocytopoiesis according to which TPO favours the transition from multilineage progenitors to CMP but blocks differentiation of MCP to mature mast cells. We propose TPO as the growth factor that restrict mast cell differentiation to extramedullaty sites and that control the switch between serosal vs dermal mast cell differentiation.Mpl expressionmRNA 2-ΔCtProtein (AFU) Cy7-AProtein (AFU) Cy7-AMM2Wild type Marrow B cells (B220pos)b.d.120±4205±4Wild type Marrow Megakaryocytes (CD61pos/CD41pos)5.0±0.1 × 10-2178±3978±74*Wild type Marrow MCP (cKitpos/T1ST2pos)1.3±0.01 × 10-2139±161658±73*Wild-type Marrow Mast Cells (cKitpos/Fcε RIpos)1.9±0.1 × 10-2110±1868±71*Serosal Mast Cells (cKitpos/FcεRIpos)7.2±2.1 × 10-4393±11374±25*Mplnull Marrow Megakaryocytes (CD61pos/CD41pos)b.d.365±28469±50Mplnull Marrow Mast Cells (cKitpos/FcεRIpos)b.d107±1109±3AFU= arbitrary fluorescence intensity. p< 0.01 with respect to Cy7-A (irrilevant antibody)

Prostaglandin E2 and F2α receptors in the human Fallopian tube before and after mifepristone treatment

Prostaglandins are associated with several reproductive processes in addition to their effects on the vascular system and muscular contractility. The aim of this study was to gain information about the localization of the receptors for PGE(2) (EP1-EP4) and PGF(2alpha) (FP) in the human Fallopian tube and their regulation following treatment with mifepristone. Sixteen healthy fertile women received a single dose of 200 mg mifepristone or placebo immediately after ovulation (LH+2). Laparoscopic sterilization was performed on days LH+4 to LH+6. Biopsies were taken from the Fallopian tubes bilaterally. The expression of EP1, EP2, EP3, EP4 and FP was analysed using immunohistochemistry and RT-PCR. The co-localization of prostaglandin receptors and c-kit or e-nos was analysed using confocal microscopy. The effect of progesterone, mifepristone and prostaglandin on tubal contractility was studied. The presence of EP1-EP4 and FP in the Fallopian tube was detected using immunostaining. The receptors were expressed in serosal cells, luminal epithelial cells, and the muscular wall and vessels of the Fallopian tube. Co-localization studies showed that the endothelial cells stained positive for EP1-EP4 and FP and that co-localization was seen for EP4 and c-kit. Decreased contractility was seen after progesterone treatment, whereas increased contractility was seen after PGF(2alpha) and PGE(2) treatment. These data suggest that both the transport of the embryo and the communication between the embryo and the Fallopian tube involve the action of prostaglandins through EP and FP receptors in addition to the effect of prostaglandins on the vascular system and muscular contractility.

The serosal mesothelium is a major source of smooth muscle cells of the gut vasculature

Most internal organs are situated in a coelomic cavity and are covered by a mesothelium. During heart development, epicardial cells (a mesothelium) move to and over the heart, undergo epithelial-mesenchymal transition (EMT), and subsequently differentiate into endothelial and vascular smooth muscle cells. This is thought to be a unique process in blood vessel formation. Still, structural and developmental similarities between the heart and gut led us to test the hypothesis that a conserved or related mechanism may regulate blood vessel development to the gut, which, similar to the heart, is housed in a coelomic cavity. By using a combination of molecular genetics, vital dye fate mapping, organ culture and immunohistochemistry, we demonstrate that the serosal mesothelium is the major source of vasculogenic cells in developing mouse gut. Our studies show that the gut is initially devoid of a mesothelium but that serosal mesothelial cells expressing the Wilm's tumor protein (Wt1) move to and over the gut. Subsequently, a subset of these cells undergoes EMT and migrates throughout the gut. Using Wt1-Cre genetic lineage marking of serosal cells and their progeny, we demonstrate that these cells differentiate to smooth muscle of all major blood vessels in the mesenteries and gut. Our data reveal a conserved mechanism in blood vessel formation to coelomic organs, and have major implications for our understanding of vertebrate organogenesis and vascular deficiencies of the gut.

Ovicide-induced serosa degeneration and its impact on embryonic development in Manduca sexta (Insecta: Lepidoptera)

Eggs of Manduca sexta treated with the ovicide Ov. 165049 turn orange, and the embryos later die. The orange pigmentation is at first confined to the serosa, and is accompanied by pathological changes of serosal cells. Lipid vesicles aggregate and spindle-shaped electron-lucent vesicles—normally forming a single layer below the apical cell surface—greatly accumulate. The mitochondria swell considerably, and their matrices become electron-lucent. Subsequently, the serosal cells develop additional features of necrosis. They form many autophagic vacuoles which contain mostly degradating mitochondria, but also segregated rough endoplasmic reticulum (rER) and glycogen granules. The whole cytoplasm vesiculates, and the cells shrink considerably. The nuclei become less irregular in shape, the chromatin disperses rather evenly whereas the nucleoli persist. Neither chromatin condensation nor the production of apoptotic bodies was observed—further evidence, that the serosal cells die by necrosis rather than apoptosis. At some stage of development the damaged serosa ruptures, retracts from the embryo and forms a sphere beneath it. It is only after the rupture of the serosa, that the embryo also turns orange and disintegrates rapidly. This shows impressively the protective function which the serosa plays for the embryo. Our physiological tests indicate, that the orange pigmentation of the serosa induced by the ovicide results from a disturbance of the tryptophan/ommochrome pathway serving the excretion of potentially toxic metabolites of tryptophan-rich proteins. The results demonstrate first that the serosa represents an important target for ovicide pesticides and second that it plays a vital role as an excretory organ during embryogenesis.

Mucosal type mast cells express complement receptor type 2 (CD21)

Fragments of complement component C3 generated upon activation of the cascade play an important role in the induction and regulation of immune responses. Receptors interacting with various fragments of this versatile complement protein are expressed on a wide variety of cell types, including lymphocytes, macrophages, dendritic cells, follicular dendritic cells, granulocytes, erythrocytes and consequently, C3-products may influence several biological functions at different sites of the body, where complement activation occurs. Regarding the expression of various C3-receptors on mast cells, mainly rodent serosal type mastocytes have been investigated so far. It has been known for a long time that C3a triggers the release of mediators of immediate type hypersensitivity via binding to serosal-type cells. Complement receptor type 1 (CR1/CD35) and type 2 (CR2/CD21) interacting with the larger activation products, such as C3b and C3d, have so far been shown on serosal type mast cells only. In this study, the expression of CR1/2 on mucosal type mast cells is demonstrated. Using mouse CR1/2 specific single chain antibodies and the natural ligand C3d in cytofluorimetric measurements, we show that the rat mucosal mast cell line RBL-2H3 and mouse bone marrow-derived mast cells (BMMC) express CD21. RT-PCR experiments carried out with mouse CR1 and CR2 specific primers show CD21, but not CD35 specific products in BMMC. It is also demonstrated that, in contrast to serosal type mast cells, mucosal mastocytes do not express CD19. In an attempt to reveal the possible function of CR2 on mucosal type mast cells, the effect of receptor-clustering was tested regarding degranulation, Ca-response and IL-6 production, but no CR2-mediated change was detected in any of these processes.

The serosa of Manduca sexta (Insecta, Lepidoptera): ontogeny, secretory activity, structural changes, and functional considerations

In Manduca sexta, the blastoderm forms successively and becomes immediatelycellularized as the cleavage energids reach the surface of the oocyte. Presumptive serosal cells are large and contain 2 or 4 large polyploid nuclei; presumptive embryonic cells are small and mononuclear. All parts of the blastoderm participate in the uptake and digestion of yolk material. About 10h post-oviposition, the blastoderm breaks at the amnioserosal fold and the extraembryonic part closes above the germ band and constitutes the serosa (12h post-oviposition, i.e. 10% development completed). At once, the serosa starts to secrete a cuticle consisting of an epi- and a lamellated endocuticle. Detachment of the serosal cuticle, 22h post-oviposition, is reminiscent of apolysis of larval cuticle. Thereafter, the serosa deposits a membranous structure, the serosal membrane. The sercretory process lasts from 23h to 44h post-oviposition. At first a fine granular layer, then an amorphous, spongy-like, fibrillar layer is secreted via microvilli. This persisting membrane is tough, rubbery and very elastic. It may serve to bolster the serosa during katatrepsis (48h post-oviposition) and later embryonic movements. After detachment of the serosal membrane, 44h post-oviposition, a distinct subcellular reorganization of the serosa takes place. The nuclei become still larger and more irregular. Uptake of yolk granules, but not of lipid droplets, ceases, although interaction of serosa and yolk cells are intense. Serosal cells include many mitochondria, large areas of rER, besides some sER, increasing amounts of lysosomal bodies and prominent Golgi complexes. Most conspicuous is the assembly of spindle-shaped, electron-lucent vesicles below the apical surface. These vesicles may contain metabolic products which are released into the peripheral space. The studies show that the serosa assumes changing functions during embryogenesis: digestion of yolk substances, synthesis of a serosal cuticle and a serosal membrane, which may have a protective function, and excretion.

Embryogenesis of the dipluran Lepidocampa weberi Oudemans (hexapoda: diplura, campodeidae): formation of dorsal organ and related phenomena.

The developmental changes of embryonic membranes of a dipluran Lepidocampa weberi, with special reference to dorsal organ formation, are described in detail by light, scanning, and transmission electron microscopies. Newly differentiated germ band and serosa secrete the blastodermic cuticle at the entire egg surface beneath the chorion. Soon after, the serosal cells start to move dorsad. All the serosal cells finally concentrate at the dorsal side of the egg and form the dorsal organ. During their concentration, the serosal cells attenuate their cytoplasm to form filaments. The extensive area from which the serosa has receded is occupied by a second embryonic membrane, the amnion, which originates from the embryonic margin. The embryo and newly emerged amnion then secrete three fine cuticular layers, "cuticular lamellae I, II, and III," above which the filaments of the (developing) dorsal organ are situated. With the progression of definitive dorsal closure, the amnion reduces its extension, the dorsal organ is incorporated into the body cavity of the embryo, and the amnion and dorsal organ finally degenerate. The dorsal organ of diplurans is formed by the concentration of whole serosal cells, while that of collembolans is formed by the direct differentiation of a part of serosal cells. However, the dorsal organs of diplurans and collembolans closely resemble each other in major aspects, including that of ultrastructural features, and there is no doubt regarding their homology. The amnion, which has been regarded as being a characteristic of Ectognatha, also develops in the Diplura. This might suggest a closer affinity between the Diplura and Ectognatha than previously believed.

A scanning and transmission electron microscopic study of contractile trabecules in the rat spleen.

The fine structures of contractile trabecules in the splenic red pulp of the rat were examined by electron microscopy to elucidate their participation in the active contraction of the spleen. Numerous fine thready trabecules were developed in the red pulp. They were enveloped with a cytoplasmic layer of reticular cells and consisted of elongated smooth muscle cells, fascicles of collagenous fibrils and elastic fibers. Their fibrous components in the capsular ends extended in a triangular form of fanribs into the fibrous tunica of the capsule. Smooth muscle cell-like interstitial cells (SIC) were situated in the interfibrous spaces. Flattened SICs were affixed with cytoplasmic processes to the elastic lamina. The trabeculocapsular junctions were represented on the elastic lamina by grouped or isolated circular patches with concentrically arranged triangular processes and were also observed on the capsular serosa by plaques with scarce microvilli of serosal cells. Smooth muscle cells of the fine trabecules were equipped on the cell surface with anchoring structures to extracellular fibrous elements as previously described by Gabella (1981). Close associations were also seen between the smooth muscle cells and elastic fibers which were terminated to the fascicles of collagenous fibers. Cell-to-cell connections were expressed by fibrous connections between spiny processes and a small number of puntate intermediate junctions and nexuses. Unmyelinated nerve fibers with adrenergic terminals were seen in the intercellular spaces. We propose that for the rat spleen, the fine trabecules in the red pulp are muscular contractiles which are responsible for the active contraction due to sympathetic stimuli and the administration of alpha 1-adrenoceptor agonists, while the elastic lamina in the capsule plays a role in the comprehensive contraction of the subcapsular vascular bed.

Expression profile of telomerase subunits in human pleural mesothelioma.

Using the TRAP assay, telomerase activity was previously detected in over 90% of human pleural mesotheliomas (MMs), but not in mesothelial cell cultures (MCCs), suggesting that telomerase re-activation occurs during multi-step mesothelioma carcinogenesis. The present study determined the expression of the telomerase RNA template (hTERC), the telomerase-associated protein (hTEP1), and the telomerase catalytic sub-unit (hTERT), in 16 pleural MMs and 4 MM-derived cell lines, in two pleural solitary fibrous tumours and in six MCCs. Reverse transcription-polymerase chain reaction analysis revealed that hTERT mRNA expression parallels the activity status documented by the TRAP assay, whereas hTERC and hTEP1 mRNA are commonly expressed in all malignant and non-malignant serosal cells and tissues. Three alternatively spliced hTERT transcripts were detected in all telomerase-positive samples, whereas neither variant could be detected in the MCCs. Detection of the hTERT protein with a commercially available antibody was not successful. These results indicate that hTERT expression is rate-limiting for human telomerase activity and that re-activation, rather than up-regulation, of hTERT expression can play a critical role in MM carcinogenesis. While waiting suitable anti-hTERT antibodies, these results provide information for the design of hTERT mRNA-specific in situ probes to study telomerase in archived pre-malignant serosal lesions.

Expression profile of telomerase subunits in human pleural mesothelioma

Using the TRAP assay, telomerase activity was previously detected in over 90% of human pleural mesotheliomas (MMs), but not in mesothelial cell cultures (MCCs), suggesting that telomerase re-activation occurs during multi-step mesothelioma carcinogenesis. The present study determined the expression of the telomerase RNA template (hTERC), the telomerase-associated protein (hTEP1), and the telomerase catalytic sub-unit (hTERT), in 16 pleural MMs and 4 MM-derived cell lines, in two pleural solitary fibrous tumours and in six MCCs. Reverse transcription-polymerase chain reaction analysis revealed that hTERT mRNA expression parallels the activity status documented by the TRAP assay, whereas hTERC and hTEP1 mRNA are commonly expressed in all malignant and non-malignant serosal cells and tissues. Three alternatively spliced hTERT transcripts were detected in all telomerase-positive samples, whereas neither variant could be detected in the MCCs. Detection of the hTERT protein with a commercially available antibody was not successful. These results indicate that hTERT expression is rate-limiting for human telomerase activity and that re-activation, rather than up-regulation, of hTERT expression can play a critical role in MM carcinogenesis. While waiting suitable anti-hTERT antibodies, these results provide information for the design of hTERT mRNA-specific in situ probes to study telomerase in archived pre-malignant serosal lesions. Copyright © 2000 John Wiley & Sons, Ltd.

Ultrasonographically guided direct gene transfer in utero: Successful induction of β-galactosidase in a rabbit model

Objective: We sought to determine whether the transfer of enzyme-encoding genes in utero can be detected after birth. Study Design: An adenoviral vector carrying the gene for β-galactosidase was injected under ultrasonographic guidance into the livers of 4 rabbit fetuses per litter (3 litters total) at 27 days’ gestation. On delivery of the pups 2 to 3 days later, the livers were analyzed for β-galactosidase activity by using 5-bromo-4-chloro-3-indolyl-β- D -galactopyranoside (X-gal) staining. Polymerase chain reaction was also performed on liver extracts as an additional independent measure of successful vector delivery. Results: Successful targeting of the livers of fetal rabbits was demonstrated by β-galactosidase activity in the nuclei of liver serosal cells, parenchymal hepatocytes, or columnar cells of the gallbladder in 7 (58%) of 12 injected pups and by polymerase chain reaction in liver extracts from 10 (83%) of 12 injected pups. Conclusions: These results suggest that vectors that carry genes for specific enzymes can be delivered to fetal organs in utero and that expression of the enzyme can be detected after delivery. (Am J Obstet Gynecol 1999;181:848-52.)

Modulatory Effect of HCO−3 on Rat Mast Cell Exocytosis: Cross-Talks between Bicarbonate and Calcium

HCO−3 modulation of histamine release and its relationship with the Ca2+ signal were studied in serosal rat mast cells. Histamine release was induced by Ca2+ mobilizing stimuli, namely compound 48/80, thapsigargin, Ca2+ chelators, ionophore A23187, and PMA and ionophore A23187 in a HCO−3-buffered medium or a HCO−3-free medium. The presence of HCO−3 reduced histamine release by 48/80, Ca2+ chelators, A23187, and PMA/A23187, but increased histamine release induced by thapsigargin. Histamine release by PMA was significantly higher in a HCO−3-free medium than in a HCO−3-free medium, as it was the PMA potentiation of histamine release by A23187. [Ca2+]i changes induced by these drugs were measured in fura-2-loaded mast cells. In thapsigargin and EGTA or BAPTA preincubated mast cells [Ca2+]i increase was higher in a HCO−3-buffered medium than in a HCO−3-free medium in the presence of Ca2+. On the contrary, in compound 48/80 and PMA/A23187 activated mast cells the [Ca2+]i increase is the same both in the presence and in the absence of HCO−3. The effect of HCO−3 on histamine release in serosal rat mast cells depends on the stimulus, but it is not related to the presence of Cl−. In thapsigargin-stimulated mast cells the effect of HCO−3 on histamine release may be related to the Ca2+ signal, but in compound 48/80, EGTA, and PMA/A23187-activated mast cells there is no relationship between intracellular Ca2+ and the inhibitory effect of HCO−3 on histamine release. Additionally, the PKC pathway is implicated in the inhibitory effect of HCO−3 on histamine release, the higher the chelation of calcium rendering the higher the enhancement of the response after adding calcium in the absence of HCO−3.

Free serosal cells originating from the embryo of the wasp Diadromus pulchellus in the pupal body of parasitized leek-moth, Acrolepiosis assectella. Are these cells teratocyte-like?

In braconid species, teratocytes are derived from a serosal cell membrane which envelops the developing parasitoid embryo. On hatching, this membrane dissociates into individual cells, the teratocytes, which then circulate in the haemolymph of the host. We describe herein such a membrane, surrounding the embryo in eggs of the ichneumonid parasitoid wasp, Diadromus pulchellus. This membrane consisted of a single sheet of tightly packed cells with large 12±1.4 μm nuclei. These cells were released after hatching in vitro and cells of the same size were detected in vivo, in the vicinity of the D. pulchellus embryo. The number of nuclei detected suggests that the serosal membrane consists of about 450±150 cells. These cells did not grow after hatching of the parasitoid egg in the parasitized host, Acrolepiosis assectella, during the development of the parasitoid wasp larva. Southern blot experiments, using D. pulchellus satellite DNA or the ribosomal genes as probes, showed that free-living floating cells of wasp origin were present in the body of the parasitized host. This is the first time that free-floating teratocyte-like cells have been described in species of the Ichneumonidae.

Development and movements of extraembryonic cells in the Indian meal moth, Plodia interpunctella Hübner (Lepidoptera: Pyralidae)

Summary Time-lapse video microscopy, fluorescence microscopy, and electron microscopy provide complementary perspectives on the genesis and movements of the serosa, amnion, and yolk cells of the Indian meal moth, Plodia interpunctella Hübner (Lepidoptera: Pyralidae). Prospective serosal cells undergo a nuclear division, round up and detach from the adjoining cells of the germ band. They reattach only to other serosal cells, creating a free migrating edge at the boundary between the germ band and the serosa. The serosa engages in epiboly, spreads to enclose the embryo, attaches to the vitelline envelope, and secretes materials onto the vitelline envelope on the inner side of the egg shell. The amnion derives from the dorsal edge of the germ band; at this time the germ band is a cell sheet composed of columnar cells (palisade-type). Amniotic cells change briefly into spindle-shaped, motile cells that dissociate from the germ band. They cross the dorsal rim of the germ band, then flatten and form an epithelium. The amnion engages in epiboly and spreads across most of the embryo, starting approximately 1h after the serosa does. A lamina is secreted between the amnion and the embryo. By mid-embryogenesis, the amnion reaches over the dorsal region, including some yolk cells. The lamina and amnion loosen from the embryo during blastokinesis, the stage after germ band shortening. The yolk endoplasm partitions into yolk cells between 1 to 2h after other cells appear. Yolk cells are highly motile during early embryogenesis, then gradually slow and engage in cell shape changes without locomotion. They tend to flatten and ruffle during blastokinesis. By the end of blastokinesis, they form stable membrane contacts, creating a cell sheet. These large cells exhibit fountanoid and centripetal flows when they are motile.