Layer Of Flattened Cells Research Articles

Ultrastructural and morphometric characterization of buffalo ( Bubalus bubalis) ovarian preantral follicles

The main objective of the present study was to characterize buffalo preantral ovarian follicles. Parts of ovarian cortex, collected from postpubertal buffalo females that were having estrous cycles at regular intervals, were selected under stereomicroscopy and processed for optic and transmission electron microscopy. Primordial follicles were characterized as an oocyte encircled by one layer of flattened cells. The buffalo primordial follicle has a mean diameter of 35 μm and the oocyte diameter is 24.9 μm. The oocyte nucleus is relatively large and eccentric; and in the cytoplasm a large amount of mitochondria, vesicles and endoplasmic reticulum cistern, mainly of the smooth type is observed. The primordial follicles cells are rich in plasma membrane invaginations, which are observed within the cell and between the cell and the oocyte. The primary follicles (mean diameter of 41.8 μm) consist of an oocyte, with a medium diameter of 26.9 μm, surrounded by one layer of cubical granulosa cells. At this follicular stage, the beginning of zona pellucida deposition can also be seen in areas between the oocyte and follicular cells. The secondary follicles, which are surrounded by more than one layer of cubical cells, have a diameter of 53.3 μm, and the oocyte has a mean diameter of 29.4 μm. The ultrastructural analysis showed a large amount of coalescent vesicles, more evident in the oocyte periphery. The zona pellucida (ZP) is thicker at this stage and contains a large quantity of glycoproteins. In general, the ultrastructure of buffalo preantral follicles was similar to that of other mammalian species, but some differences were observed, which indicate species specific characteristics. The main differences observed were cytoplasmic vesicles quantity, mitochondria shape and inner content, ZP deposition and granulosa cell–oocyte junctions. In conclusion, the morphological differences described in this paper, could be responsible for some functional differences observed in Bubalus bubalis in vitro embryo production and follicular dynamics, when compared with Bos taurus or Bos indicus species.

Testicular Cell Conditioned Medium Supports Differentiation of Embryonic Stem Cells into Ovarian Structures Containing Oocytes

Previous reports and the current study have found that germ cell precursor cells appear in embryoid bodies (EBs) formed from mouse embryonic stem cells as identified by positive expression of specific germ cell markers such as Oct-3/4, Mvh, c-kit, Stella, and DAZL. We hypothesized that if exposed to appropriate growth factors, the germ cell precursor cells within the EBs would differentiate into gametes. The source for growth factors used in the present study is conditioned medium collected from testicular cell cultures prepared from the testes of newborn males. Testes at this stage of development contain most growth factors required for the transformation of germ stem cells into differentiated gametes. When EBs were cultured in the conditioned medium, they developed into ovarian structures, which contained putative oocytes. The oocytes were surrounded by one to two layers of flattened cells and did not have a visible zona pellucida. However, oocyte-specific markers such as Fig-alpha and ZP3 were found expressed by the ovarian structures. The production of oocytes using this method is repeatable and reliable and may be applicable to other mammalian species, including the human.

Cell dynamics in the Growth and Differentiation of Dental Epithelium during Tooth Development: Stratum Intermedium Cells Originated From Inner Enamel Epithelium

The stratum intermedium develops as flattened cell layer on the proximal side of the ameloblast layer during the bell stage of tooth development. Stratum intermedium cells strongly express alkaline phosphatase (ALP) activity and have been considered to play a complementary role in the enamel mineralization, however the origin and/or the role of these cells have not been elucidated. In the present study, we focused on the lineage of stratum intermedium cells in continuously growing rodent incisors and analyzed it by using DiI tracers experiment and using the incisors organ culture. The results indicated that some stratum intermedium cells were originated from the inner enamel epithelium. Immunohistochemical and in situ hybridization studies showed that the stratum intermedium cells expressed the Notch-1, Notch-2, and Hes1, while the inner enamel epithelium and ameloblasts expressed their ligands Jagged-1. Furthermore, we examined th role of Notch signaling in the development of the stratum intermedium cells by use of the dental epithelial cell line, HAT-7. Recombinant Jagged1 protein enhanced the appearance of the stratum intermedium cells in HAT-7.On the other hand, anti-sense Notch1 decreased the number of stratum intermedium cells.Taken together, we propose a hypothesis that the lineage of the stratum intermedium differentiates from the ameloblasts lineage through Notch signaling.

A Case Report of Multiple Pigmented Perineal Hidrocystomas

Apocrine hidrocystomas occur most commonly as solitary nodules on the face and neck. Case reports of patients with multiple lesions on the face, particularly in the periorbital region, have been described. They present as cystic, translucent, blue or brown papules or nodules. The color is attributed to the Tyndall effect, as pigment such as melanin or hemosiderin has rarely been demonstrated within the lesions. We present a 43‐year‐old Caucasian female with over thirty blue‐gray macules on her perineum and vulva. The patient was unaware of the lesions though she noted perineal and inguinal pruritus after exercise. Punch biopsy showed a cluster of cysts within a loose stroma, located in the deep dermis. The cysts were lined by a single layer of flattened cells with eosinophilic cytoplasm and focal areas of papillation and decapitation secretion. A peripheral layer of myoepithelial cells was present. The epithelium contained brown pigment confirmed to be melanin by Fontana‐Masson stain. Iron stain was negative. GCDFP‐15 (gross cystic disease fluid protein) labelled the epithelial cells. S‐100 and Mart‐1 immunohistochemical stains were negative. Unusual features of this case include the anatomic site, number of lesions, and demonstration of melanin as the cause of the clinical pigmentation.

Morphometric and ultrastructural characterization of Bos indicus preantral follicles

The aim of the present study was to characterize the ultrastructure of zebu cow preantral follicles (PAFs). Ovarian cortex samples were processed for light and transmission electron microscopy. Primordial follicles consisted of an oocyte surrounded by one layer of flattened or flattened-cuboidal granulosa cells. The oocyte contained a large and usually eccentric nucleus. Most organelles were located at the perinuclear ooplasm. Round shaped mitochondria, which contained electron-dense granules, smooth and rough endoplasma reticulum and a Golgi apparatus were also observed. Vesicles and coated pits were often observed in the cortical ooplasm. In primary follicles, the oocyte was surrounded by one layer of cuboidal granulosa cells. Short microvilli were observed on the oolema. Secondary follicles consisted of an oocyte surrounded by a variable number of layers of cuboidal granulosa cells. Small secondary follicles had an ultrastructure very similar to that observed in primary follicles. At this follicular stage, the zona pellucida was beginning to form around the oocyte. In large secondary follicles, the zona pellucida was totally developed around the oocyte. Several granulosa cell projections could be detected that were encroaching into the zona pellucida and protruding towards the oocyte, where gap junctions were observed between oocyte and granulosa cell membranes. Organelles within the oocyte were located at the periphery of the ooplasm, and clusters of cortical granules were observed. Round mitochondria were abundant in all developmental stages. In conclusion, this study described the ultrastructure of zebu cow PAFs, and some unique characteristics could be observed as compared with what has been reported for follicles of Bos taurus cattle.

Ovarian hemangioma presenting with hyperandrogenism and endometrial cancer: a case report

Background. Hemangiomas are very rare tumors of the ovary. Here, we report a case of a mixed capillary and cavernous ovarian hemangioma and endometrial carcinoma presenting with postmenopausal bleeding, male pattern receding frontal hairline, and high serum androgen and estradiol levels. Case. A 70-year-old White female underwent laparotomy for endometrial carcinoma. Intraoperative frozen-section examination of the uterus revealed a 3.5 × 3 cm, grade 1 endometrioid adenocarcinoma of the endometrium with more than 50% myometrial invasion. The left ovary contained a 1.5 × 1 × 1 cm, well-circumscribed hemorrhagic nodule on the cut surface. Final histopathological examination of the small nodule demonstrated multiple, enlarged, blood-filled vascular channels lined by a single layer of flattened regular endothelial cells with no atypical features. Vascular spaces within the tumor were of different sizes, ranging from small to large, and were separated by connective tissue. The surrounding ovarian stroma was hyperplastic and contained clusters of luteinized stromal cells. Microscopy of the right ovary showed minimal stromal proliferation and no luteinization of the ovarian stroma. Conclusion. This is the first case of an ovarian hemangioma synchronous with a well-differentiated endometrial carcinoma. Absence of estrogen and progesterone receptors in the endothelial cells of the hemangioma suggests that ovarian hemangiomas may occur independent of stimulation by estrogen and progesterone.

Androgen receptor mRNA expression in the bovine ovary

Previous studies have shown that androgen receptor (AR) is expressed in granulosa cells of healthy, growing ovarian follicles in rats and primates. However, AR expression in the bovine ovary has not been examined. Therefore, a 346-base pair segment of the bovine AR was cloned and sequenced. Using a ribonuclease protection assay, AR expression was detected in total RNA from bovine ovarian cortex. Expression (absence or presence) of AR mRNA was detected by in situ hybridization in bovine ovarian cortex. Follicles ( n=32) were classified as follows: type 1 (1 layer of flattened granulosa cells), type 2 (1–1.5 layers of cuboidal granulosa cells), type 3 (2–3 layers of granulosa cells), type 4 (4–6 layers of cuboidal granulosa cells and formation of thecal layer), and type 5 (>6 layers of cuboidal granulosa cells, defined theca layer, and antrum formation). Frequency of AR mRNA expression increased ( P<0.001) as follicles entered the growing pool. Expression of AR mRNA was absent in type 1 follicles ( n=8), but present in the granulosa cells of 41% of type 2 follicles ( n=12). In types 3–5 follicles, AR mRNA expression was present in granulosa cells of 100% of follicles examined ( n=4, 4, and 4, respectively) and was greater than type 1 follicles ( P=0.002). These data provide evidence of AR mRNA expression in bovine follicles and suggest that AR mRNA increases during early follicle development.

Induction of differentiation of CD34+ cells in vitro and application in endothelialization of blood vessel prostheses

To induce differentiation of CD(34)+ cells from varying sources into epithelial cells for potential application of blood vessel prostheses. CD(34)+ cells were isolated from canine peripheral blood and bone marrow or human umbilical cord blood by an immune magnetic cell sorting system. The isolated CD(34)+ cells were induced to differentiate into endothelial cells in a liquid culture with VEGF. Endothelial cells were evaluated by immunocytochemistry and transmission electron microscopy. CD(34)+ cells were seeded on PTFE prostheses, which were harvested and observed by electron microscopy. The average isolated CD(34)+ cells from peripheral blood, bone marrow and umbilical cord blood were (26.30+/-2.42)%,(41.84 +/-3.65)%and (74.62+/-4.46)%, respectively. The number of CD(34)+ cells increased with the culture duration and reached to the highest level at the 14th d. Immunostaining showed positive signal for CD31 in endothelial cells and positive for factor VIII in cell culture. Transmission electron microscopy found Weibel-Palade bodies in the cytoplasm. Scanning electron microscopy demonstrated a single- layer of flat cells in the innermost layer of PTFE prostheses, and the cells were not of tropism. CD(34)+ cells isolated from peripheral blood, bone marrow and umbilical cord blood can be induced into endothelial cells; bone marrow and umbilical cord blood can be used as the sources of seeding cells for endothelialization of prostheses.

Human Corneal Organotypic Cultures

Purpose. In order to test irritancy levels of commercial products, the Draize animal test has been the universally accepted choice. However, since this test uses live animals, many researchers have been trying to develop an alternative organotypic culture. These organotypic cultures, however, utilize animal cells, and it is our belief that a model using human cells would be more predictive for determining human irritancy levels. Methods. Primary human corneal epithelial cells and fibroblasts were separately isolated from a limbal rim and grown in culture. SV40-transformed mouse corneal endothelial cells were seeded onto a membrane and grown to confluence. A homogenous fibroblast/collagen mixture was then added and allowed to gel. After about a week, the epithelial cells were seeded on top of the gel and the constructs were kept submerged in culture for 3-7 days. The constructs were then airlifted to allow for the stratification of the epithelial cells. Following this, the constructs were either fixed and processed for methacrylate sectioning to study morphology, or they were frozen and sectioned for indirect immunofluorescence. Indirect immunofluorescence was performed with ZO-1, a marker of tight junctions; keratins 3 and 12, markers for differentiation; and laminin, a marker of basement membrane components. Initially, a SV40-transformed human endothelial cell line was to be used; however, it did not grow well in this culture system. Results. In these experiments, we tested two methods of isolating epithelial cells. One method, the explant technique-a method we previously used to isolate rabbit epithelial cells-could not be maintained beyond two passages, and when added to the construct only stratified to 2-3 layers of flattened cells. However, with the second method, the dispase technique, the epithelial cells obtained grew rapidly and could be maintained beyond two passages. These cells, when added to the construct, stratified to 5-7 layers and exhibited a more epithelium-like morphology. Staining with ZO-1 indicated that tight junctions in the superficial cells were formed. Staining with keratins 3 and 12 indicated that the epithelial cells were differentiating, and staining with laminin indicated that basement membrane components were being synthesized. Conclusion. Constructing an organotypic culture with human corneal cells is possible, and the morphology of the construct appears to be equivalent to a construct already created with bovine cells that has been used for irritancy studies.

Urothelial Mesh - A New Technique of Cell Culture on Biomaterials

Urogenital malformations, trauma or tumours may demand surgical reconstruction in children. Cell culture is an important technology in biomaterial research and tissue engineering. Tissue-engineering of urothelial organs is of interest in children, because the number of complications and re-operations may be reduced. Actually, monolayer cultures of urothelium are used for tissue engineering and biocompatibility testing. A culture system that more closely mimics the physiologic environment of the urothelium would be of interest. The aim of this study was to determine the biological and mechanical characteristics of urothelial mesh cultured in vitro. Meshes containing urothelium, lamina propria, and submucosal tissue were generated using a skin mesh graft cutter. Meshes were cultured in 6-well plates, on collagen I/III, polydioxanone/polylactic acid and silicone matrices. Cell morphology was examined by inversion microscopy, histology, and scanning electron microscopy. It was compared to urothelium cultured by methods reported in the literature. To define the basic mechanical properties, meshes were extended longitudinally by a servohydraulic testing machine and strain diagrams generated. Urothelium was reproducibly cultured from meshes. Cell growth could be induced onto fibrillary collagen, polydioxanone-polylactic acid matrices and shaped polyurethane surfaces. Cells formed confluent layers of flat cells, resembling native urothelium. The meshes have unique mechanical properties, allowing for stable fixation, surgical handling and mechanical stimulation. Meshes can be used for cell culture on biomaterials. They maintain epithelial-stromal integrity and mechanic stability. The small size of tissue bridges allows in vitro culture for long periods with many potential advantages for tissue engineering and biologic research. Applications are possible both in vitro and in vivo.

The microstructure of almond ( Prunus dulcis (Mill.) D.A.Webb cv. ‘Nonpareil’) cotyledon

Microstructure of almond ( Prunus dulcis (Mill.) D.A. Webb cv. ‘Nonpareil’) cotyledon was observed with light, scanning and transmission electron microscopy. The objective of this study was to characterise almond cotyledon surfaces as well as to describe internal and subcellular organisation. The testa has an outer epidermis, which consists of relatively large thin-walled cells, which range from 100 to 300 μm in width. The major portion of the testa consists of approximately 14–20 layers of flattened parenchymal cells with the total thickness of the layers ranging from 80 to 120 μm. The remainder of the testa was comprised of a small amount of vascular tissue. The embryo consisted primarily of parenchymal tissue with relatively thin cell walls (1–3 μm in thickness) and a small amount of provascular tissue. Protein bodies up to 12 μm in width and spaces once occupied by lipid bodies up to 3 μm in width were present in all cells of the embryo.

Gonadotropin and steroid hormones stimulate proliferation of the rat ovarian surface epithelium.

The ovarian surface epithelium (OSE) is a single layer of flattened or cuboidal cells covering the ovary. Ninety percent of all human ovarian malignancies arise from this layer of cells. Incessant ovulation, hyperovulation induced by infertility treatment, and hormone replacement therapy have been suggested as risk factors for ovarian cancer. In this study, two groups of rats, with and without surgically induced injury to the ovary, were treated with 17beta-estradiol, pregnant mare's serum gonadotropin (PMSG), human chorionic gonadotropin (hCG), or the combination PMSG/hCG, and the proliferative response of the OSE cells was measured using bromodeoxyuridne (BrdU) and (3)H-thymidine. All hormones, alone or in combination with ovarian surgery, were found to increase significantly the rate of proliferation of the rat OSE. These data demonstrate that hormones associated with infertility treatments and hormone replacement therapy, as well as injury- or ovulation-induced rupture of the ovarian surface, stimulate the rat OSE, and hence could have a role in the development of ovarian cancer via proliferation-associated mutagenesis, or alternatively, by promoting the rapid selection of OSE cells with accumulated mutations.

Development of the glandular epithelium of the bovine parotid gland during ontogenesis.

The development of the parotid gland was examined in 36 bovine embryos and foetuses with a crown-rump-length (CRL) from 28 up to 1000 mm by light, transmission electron microscopical and actin-immunohistochemical methods. The anlage of the parotid gland in an embryo with 28 mm CRL can be found at the lateral angle of the primitive oral cavity as a local thickening of the epithelium. During the second month, the differentiation of primary ducts and endbuds starts and a lumen develops in the primary ducts. At the end of the second month a lumen appears in the terminal endbuds. In the immature endpiece cells first secretory granules can be seen from a CRL of 240 mm. In the third month differentiation between intra- and inter-lobular ducts is possible. Immature myoepithelial cells present as a basal layer of flattened cells between the epithelial cells and the basement membrane at the end of the second month. During further development they increase in number, become more flattened and form long cellular processes. At the end of the fourth month isolated actin filament bundles are formed, which were also detected by an antibody against smooth muscle actin. The actin filaments condense continuously until they fill the cell processes completely at the end of foetal development.

Growth differentiation factor 9 and bone morphogenetic protein 15 are essential for ovarian follicular development in sheep.

The aim of this study was to test the hypothesis that both growth differential factor 9 (GDF9) and bone morphogenetic protein (BMP15; also known as GDF9B) are essential for normal ovarian follicular development in mammals with a low ovulation rate phenotype. Sheep (9-10 per group) were immunized with keyhole limpet hemocyanin (KLH; control), a GDF9-specific peptide conjugated to KLH (GDF9 peptide), a BMP15-specific peptide conjugated to KLH (BMP15 peptide), or the mature region of oBMP15 conjugated to KLH (oBMP15 mature protein) for a period of 7 mo and the effects of these treatments on various ovarian parameters such as ovarian follicular development, ovulation rate, and plasma progesterone concentrations evaluated. Also in the present study, we examined, by immunohistochemistry, the cellular localizations of GDF9 and BMP15 proteins in the ovaries of lambs. Both GDF9 and BMP15 proteins were localized specifically within ovarian follicles to the oocyte, thereby establishing for the sheep that the oocyte is the only intraovarian source of these growth factors. Immunization with either GDF9 peptide or BMP15 peptide caused anovulation in 7 of 10 and 9 of 10 ewes, respectively, when assessed at ovarian collection. Most ewes (7 of 10) immunized with oBMP15 mature protein had a least one observable estrus during the experimental period, and ovulation rate at this estrus was higher in these ewes compared with those immunized with KLH alone. In both the KLH-GDF9 peptide- and KLH-BMP15 peptide-treated ewes, histological examination of the ovaries at recovery (i.e., approximately 7 mo after the primary immunization) showed that most animals had few, if any, normal follicles beyond the primary (i.e., type 2) stage of development. In addition, abnormalities such as enlarged oocytes surrounded by a single layer of flattened and/or cuboidal granulosa cells or oocyte-free nodules of granulosa cells were often observed, especially in the anovulatory ewes. Passive immunization of ewes, each given 100 ml of a pool of plasma from the GDF9 peptide- or BMP15 peptide-immunized ewes at 4 days before induction of luteal regression also disrupted ovarian function. The ewes given the plasma against the GDF9 peptide formed 1-2 corpora lutea but 3 of 5 animals did not display normal luteal phase patterns of progesterone concentrations. The effect of plasma against the BMP15 peptide was more dramatic, with 4 of 5 animals failing to ovulate and 3 of 5 ewes lacking surface-visible antral follicles at laparoscopy. By contrast, administration of plasma against KLH did not affect ovulation rate or luteal function in any animal. In conclusion, these findings support the hypothesis that, in mammals with a low ovulation rate phenotype, both oocyte-derived GDF9 and BMP15 proteins are essential for normal follicular development, including both the early and later stages of growth.

Localized malignant mesothelioma of the epithelial type occurring as a primary hepatic neoplasm: A case report with review of the literature

We report a localized malignant mesothelioma of the epithelial type, occurring as a primary hepatic neoplasm in a 64‐year‐old male. He was found to have a mass located in the right lobe of the liver. Surgery was carried out with resection of the mass from the right hepatic lobe, with partial resection of the diaphragm. Grossly, an ill‐defined tumor was present in the hepatic parenchyma. Histologically, the tumor displayed a predominant tubular pattern of growth with a desmoplastic stroma. The tubules were lined by a single layer of cuboidal or flattened cells with pleomorphic vesicular nuclei. A hyaluronidase‐digestible, mucin‐like substance was demonstrated in the lumen and tumor cytoplasm. The tumor cells were immunohistochemically positive for calretinin, HBME‐1, cytokeratin, i.e. AE1/AE3 and CAM 5.2, but negative for carcinoembryonic antigen, CD 34 and Leu M1. Moreover, the tumor cells showed nuclear accumulation of the p53 oncopotein and reacted frequently with Ki‐67 antibody. These findings support the concept that malignant mesothelioma of the epithelial type may occur at extrapleural sites. To the best of our knowledge, this is the first reported case of localized malignant primary mesothelioma arising in the liver.

Caveolin expression during chondrogenesis in the avian limb

Caveolin is the principal component and critical structural and functional element of caveolae, omega-shaped plasmalemmal invaginations, which have been implicated in a wide range of cellular processes in several different tissues. In the present study, we have investigated both the spatial and temporal expression of caveolin proteins during chondrogenesis in the avian tibiotarsus at days 10-20 of embryonic development. By using semiquantitative Western blotting, we found that caveolin-1 was clearly expressed in developing avian cartilage. The positive expression of caveolin-1 in cartilage showed an upward trend of accumulation temporally, with the highest levels of expression at 20 days of development. By using immunocytochemistry, we detected all three caveolin proteins in the cells of the outer fibrous articular surface, although caveolin-1 demonstrated the strongest and most consistent reactivity. In all cases, however, immunoreactivity appeared to be concentrated in cells facing the articular cavity. In the epiphyseal cartilage, immunocytochemistry revealed that caveolin-1 was present in the majority of chondrocytes within all layers of the cartilage and at all stages examined. A discrete, intense band of caveolin-1 immunoreactivity was apparent within the layer of flattened cells immediately underlying the proliferating rounded chondrocytes and suggests that caveolin-1 might be involved in regulating the progression of cells through these gradually maturing cell layers. In contrast to the results for caveolin-1, in the case of caveolin-2 and -3, chondrocytes were devoid of immunoreactivity in all regions of the epiphyseal cartilage. Overall, this study demonstrates that caveolin-1, -2, and -3 are expressed during chondrogenesis in the developing avian limb, although the patterns of expression are restricted both spatially and temporally throughout the differentiating cell layers of the cartilage. The results suggest that caveolin proteins might play a differentiation-dependent role during avian chondrogenesis.

Gene transfer to the pleural mesothelium as a strategy to deliver proteins to the lung parenchyma.

The pleura covers the lung parenchyma, chest wall, and diaphragm with a single layer of flat cells that are easy to genetically modify with adenovirus (Ad) vectors. Although intrapleural gene therapy has been used to treat intrapleural disorders, we hypothesized that it may also be used to deliver extracellular gene products to the lung parenchyma. In this context, this study is based on the concept that administration of adenovirus gene transfer vectors into the pleural cavity will mediate expression of gene products in mesothelial cells, and that the extracellular products produced by these genetically modified cells will reach the lung parenchyma. To assess this concept, Ad(beta)gal (expressing beta-galactosidase [beta-Gal]) or AdLuc (expressing luciferase) was administered into the right pleural cavity of BALB/c mice, as compared with intravenous injection via the jugular vein or the intratracheal route. Histologic assessment of lungs and pleural surface after intrapleural administration of Ad(beta)gal demonstrated beta-Gal expression limited to the pleural mesothelium and cells adjacent to the pleural surface. Right intrapleural administration of AdLuc showed higher level of luciferase in both the right and left lung (right vs. left, p > 0.8), compared with the intratracheal (p < 0.05) or intravenous routes (p < 0.02), that is, unilateral intrapleural administration is sufficient to transfer genes bilaterally to the pleura. To assess the ability of intrapleural gene transfer to modify lung parenchymal processes, CT26.CL25 tumor cells (3 x 10(5)) were injected via the jugular vein to generate tumor metastases in the lung parenchyma followed 24 hr later by administration to the right pleura of 5 x 10(8) PFU of Adsflt (an Ad "antiangiogenesis" vector expressing a soluble, secreted, extracellular portion of the Flt-1 receptor for vascular endothelial growth factor). Compared with phosphate-buffered saline, or the control vector AdNull (no transgene), mice receiving Adsflt by the intrapleural route had a marked suppression of tumor growth in the parenchyma of both lungs as assessed 12 days after tumor administration (p < 0.005). Treatment of preestablished lung metastases with Adsflt administered by the intrapleural route significantly improved long-term survival as compared with control animals (p < 0.0001). Thus, although intrapleural administration of an Ad vector encoding an intracellular protein mediates gene expression only in mesothelial cells and the local tissues, intrapleural delivery of a vector expressing a secreted protein can be used to modify processes throughout the lung parenchyma. In the context that intravascular gene transfer is an ineffective strategy to deliver gene products to the lung parenchyma, and that intratracheal administration is associated with alveolar inflammation secondary to host defenses against Ad vectors, these findings demonstrate that intrapleural administration represents a strategy that can be used to effectively deliver extracellular gene products to the lung parenchyma via a site that is readily accessible, and where inflammation against the vector will not have significant pathophysiologic consequences.

A clinicopathological study of venous loops and reduplications in diabetic retinopathy

To study possible causes of the gradual venous occlusion that precedes the formation of venous loops and reduplications in diabetic retinopathy using histopathological techniques. Casts of the retinal vascular system from six eyes of five diabetic patients were used to identify venous loops and reduplications. Subsequently the lesions were studied by immunohistochemistry using antibodies against cellular and non-cellular components previously shown to be disturbed in the diabetic retina. In all eyes the venous abnormalities identified on the casts were accompanied by abnormal wall partitions dividing the venous lumen. These partitions consisted of a double layer of flat cells displaying immunoreactivity to von Willebrand factor and actin (endothelial cells), but not to glial fibrillary acid protein or S-100 protein (glial cells), CD3, CD20, CD68, or neutrophil elastase (leucocytes). Neutrophile granulocytes adhering to the walls of larger retinal venules were unrelated to the venous partition and to capillary occlusion in the adjacent retinal areas. Venous loops and reduplications are associated with partitions of the larger retinal venules consisting of a double layer of endothelial cells anchored to a thin basement membrane. An elucidation of the factors distinguishing this endothelial cell proliferation from preretinal new vessel formation may be important for understanding the pathophysiology of proliferative diabetic retinopathy.

Effects of induced aestivation in Protopterus annectens: a histomorphological study.

The aim of this research was to induce, at will in the laboratory, aestivation of the Dipnoan Protopterus annectens, in order to compare the structure of organs in lungfish adapted to aquatic or aestivating conditions. The animals were placed in a glass tank containing warm water, and the bottom of the tank was filled with clay and sand. To start aestivation the water was allowed to slowly evaporate; as soon as the fish was in a dry environment, it began to excavate a hole in the mud and to burrow into it. Scanning electron microscopy and histological techniques compared the morphology of skin, gills, and lungs in aestivating and free-swimming animals. In the aestivating animals, the secondary lamellae of the gills became thick and were covered by mucus that pasted the lamellae together. The epidermis of the skin was thin and composed of layers of flattened cells. In contrast, in free-swimming animals, the secondary lamellae of the gills were widely separated and the epidermis of the skin was thick and contained numerous mucus-laden cells. The lungs, thin bloodless threads in the aquatic conditions, were, in the air-breathing animals, rich in blood and showed thick walls with ridges and pillars that protruded into the lung cavity, producing small alveolar protrusions. The features of the skin and lungs were similar to that of amphibians, testifying to the convergence of some tissue morphology in aquatic animals utilizing land as a cohabitat.

The mouse zona pellucida: folliculogenesis, fertility and pre-implantation development

Perinatally, oocytes within the mouse ovary become surrounded by a layer of flattened granulosa cells and form primordial follicles. The subsequent accretion of the zona pellucida between the oocytes and granulosa cells provides a biochemical marker of folliculogenesis. In mice, the zona matrix is composed of three proteins (ZP1, ZP2, ZP3). Mouse lines lacking either ZP1 or ZP3 have been established and have abnormal folliculogenesis. Without ZP1, structurally defective zonae are formed resulting in decreased fecundity due to early embryonic loss. More strikingly, without ZP3, the zona matrix is absent, no 2-cell embryos are formed and females are infertile. The structural integrity of the zona matrix can be restored by substituting human homologues for the missing mouse protein and these ‘humanized’ zona matrices should prove useful in investigating the molecular basis of fertilization.