Histology and histopathology Vol.21, nº10 (2006)
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- PublicationOpen AccessComparison of the established standard complement-dependent cytotoxicity and flow cytometric crossmatch assays with a novel ELISA-based HLA crossmatch procedure(Murcia : F. Hernández, 2006) Altermann, W.W.; Seliger, B.; Sel, S.; Wendt, D.The detection of donor-specific anti-HLA antibodies by standard procedures such as complementdependent cytotoxicity assay (CDC) or flow cytometric (FACS) analysis is limited by its low sensitivity and the quality of the donor cells. Therefore, an ELISA-based technique was employed using solid phase-immobilized monoclonal antibodies to capture HLA class I or class II molecules of the donor, respectively. In this HLA class I and class II antibody monitoring system (AMS) the donor-specific anti-HLA antibodies from the sera of recipients bind to the HLA molecules of the donor which have been immobilized by monoclonal antibodies (mAb) recognizing non-polymorphic epitopes. Upon binding of donor-specific anti-HLA antibodies they are recognized by secondary enzyme-conjugated anti-human immunoglobulin (Ig) antibodies. A newly established modification of the standard protocol allows the differentiation between bound antibodies of the IgG and IgM isotype. Furthermore, this assay was adapted for investigating small amounts of solid tissue of donors from whom no other cells (e.g. from blood) were available. We here provide an overview of the classical crossmatch methods with their advantages and limits. In addition, the design of the novel AMS-ELISA is described in terms of quality and sensitivity of the approach using exemplary cases of different application. The selected cases show that the AMS-ELISA represents a valuable tool for the post-transplantation monitoring of donor-specific anti-HLA antibodies during reaction crisis, after transfusion reactions and in particular cases of tissue transplantations lacking single cells.
- PublicationOpen AccessAntiangiogenic and radiotherapy for cancer treatment(Murcia : F. Hernández, 2006) Kobayashi, H.; Lin, P.C.Tumor growth and progression depends on tumor angiogenesis, the growth of tumor blood vessels, therefore, targeting tumor angiogenesis is a very promising approach for controlling tumor growth and/or causing regression. Tumor blood vessels have been recognized as a critical component of radiation response to the point of being independent of tumor oxygenation during radiation. An anti-angiogenic approach has been considered less likely to develop drug resistance. But recent findings suggest that anti-angiogenesis causes hypoxia that selects tumor cells (due to genetic instability) that are less dependent on blood supply and leads to drug resistance. The approach of combination of anti-angiogenesis with ionizing radiation by targeting both endothelial and tumor cells should minimize this possibility. The combination may produce a synergistic anti-tumor effect.
- PublicationOpen AccessSpinal intradural müllerianosis, a case report(Murcia : F. Hernández, 2006) Barresi, Valeria; Cerasoli, S.; Vitarelli, E.; Donati, R.Müllerianosis is a term used to indicate lesions composed of an admixture of two or three types of müllerian-derivation glands in heterotopic sites. In this report we describe a case of spinal cord müllerianosis which occurred in a 42-year-old woman. The patient had suffered from catamenial lumbago and sciatica of three years duration before undergoing laminectomy of L2-L3 with excision of a polypoid mass that compressed nerve trunks. At histological examination, the lesion was composed of endocervical, endometrial and tubal glands within a smooth muscle nodule. These features were consistent with a diagnosis of müllerianosis. This is a very uncommon form of presentation of müllerianosis that must be correctly identified since patients can benefit from hormonal therapy.
- PublicationOpen AccessGamma-aminobutyric acid GABA and cell proliferation, focus on cancer cells(Murcia : F. Hernández, 2006) Watanabe, M.; Maemura, K.; Oki, K.; Shiraishi, N.; Shibayama, Y.; Katsu, K.In addition to its role in the adult mammalian nervous system as an inhibitory neurotransmitter, g-aminobutyric acid (GABA) is involved in the proliferation, differentiation, and migration of several kinds of cells including cancer cells. GABA is synthesized predominantly from glutamate by glutamate decarboxylase and exerts its effects via ionotropic GABAA receptors and/or metabotropic GABAB receptors. In this review, the current state of knowledge regarding the role of the GABAergic system in peripheral nonneuronal cell proliferation is described, and recent advances in elucidation of the mechanisms leading to cell proliferation are discussed.
- PublicationOpen AccessHistology and ultrastructure of the pineal organ in the domestic goose(Murcia : F. Hernández, 2006) Prusik, M.; Lewczuk, B.; Nowicki, M.; Przybylska-Gornowicz, B.The pineal organs of 14-week-old domestic geese were investigated with light and electron microscopy. The pineals consisted of a wide distal part and a narrow middle-proximal one. The glands were attached to the intercommissural region via the choroid plexus. The pineal parenchyma was formed by round or elongated follicles. The follicular wall was composed predominantly by cells immunoreactive with antibodies against hydroxyindolo-O-methyltransferase (HIOMT) or glial fibrillary acid protein (GFAP). They formed two or more layers. HIOMT-positive elements were represented by elongated cells bordering the follicular lumen and oval cells located in the external layer of the follicular wall. These cells were identified in ultrastructural studies as rudimentary-receptor pinealocytes and secretory pinealocytes, respectively. Among rudimentary-receptor pinealocytes two types of cells, designed as A and B, were distinguished due to structural differences. Type A cells extended through the whole follicular wall and showed regular stratified distribution of organelles in well-recognizable zones with rough endoplasmic reticulum, the Golgi apparatus and mitochondria. Type B cells, like type A pinealocytes, contacted the pineal lumen and showed polarity of their internal structure. However, they were markedly shorter than the cells of type A and lacked stratified distribution of organelles. Secretory pinealocytes contained irregularly dispersed organelles. A prominent feature of all types of goose pinealocytes was the presence of numerous dense core vesicles. The population of GFAP-positive cells consisted of ependymal-like supporting cells and astrocyte-like cells.
- PublicationOpen AccessExpression of gonadotrophin-releasing hormone binding sites in somatic tissues of the gilthead seabream (Sparus aurata): a quantitative autoradiographic study(Murcia : F. Hernández, 2006) González-Martínez, D.; Sarasquete, C.; Pascual, E.; Muñoz-Cueto, J.A.In this study, we have analysed the expression of gonadotrophin-releasing hormone (GnRH) binding sites in somatic tissues (intestine, liver, gill, skeletal muscle, ovary, heart, stomach, kidney and spleen) of the gilthead seabream, Sparus aurata using 3- [125I]iodototyrosyl5-mammalian GnRH and autoradiographic techniques. The qualitative and quantitative analysis showed the existence of a basal expression of specific GnRH binding sites in intestine, skeletal muscle, ovary, stomach and spleen. Furthermore, our data suggest that the level of expression of GnRH binding sites can be significantly enhanced by GnRH treatment in intestine, gill, heart, stomach, kidney and spleen. This study shows that GnRH can exert direct effects in both reproductive and non-reproductive somatic tissues of the gilthead seabream.
- PublicationOpen AccessEarly administration of methylprednisolone decreases apoptotic cell death after spinal cord injury(Murcia : F. Hernández, 2006) Vaquero, J.; Zurita, M.; Oya, S.; Aguayo, C.; Bonilla, C.The purpose of this study is to evaluate, in an experimental model of spinal cord injury (SCI), the presence of apoptotic cell death after trauma and if early administration of a single bolus of methylprednisolone (MP) influences apoptosis in the zone of trauma and in adjacent spinal cord segments. For this study, a total of 96 adult female Wistar rats were subjected to spinal contusion at the T6-T8 level, producing immediate paraplegia. Forty-eight animals (treated group) received a single intraperitoneal injection of MP, at a dose of 30 mg/kg body weight, 10 minutes later. Cells undergoing apoptosis were detected by means of immunohistochemical labeling with the monoclonal antibody Apostain (anti-ssDNA MAb F7-26), in the injured spinal cord tissue, both in the zone of the lesion and in the adjacent spinal segments (rostral and caudal zones), 1, 4, 8, 24 and 72 hours and 1 week after injury. Apoptosis was detected in neurons and glial cells in the zone of the lesion 1 hour after trauma, with a pattern that showed no changes 4 hours later. Between 4 and 8 hours postinjury, the number of apoptotic cells increased, after which it decreased over the following days. In the adjacent spinal segments, apoptotic cells were detected 4 hours after trauma, and increased progressively over the remainder of the study, the number of apoptotic cells being similar in the lesion zone and in rostral and caudal zones one week after injury. When the group of MPtreated animals was considered, significant decreases in the number of apoptotic cells were detected in the lesion zone 24 hours after injury, and in the rostral and caudal zones, at 72 hours and at 1 week after trauma. These findings show that early administration of a single bolus of MP decreases apoptotic cell death after SCI, supporting the utility of MP in reducing secondary damage in injured spinal cord tissue.
- PublicationOpen AccessRecruiting of somatotroph cells after combined somatostatin, GHRH and growth hormone (GH) secretagogue stimulation in a study of pituitary GH reserve in prepuberal female rats(Murcia : F. Hernández, 2006) Jiménez Reina, L.; García-Martínez, E.; Rojas, J.P.; Cañete, M.D.; Bernal, G.; Cañete, R.Diagnostic confirmation of growth hormone (GH) deficiency in children and adults is based on stimulation tests designed to assess the pituitary reserve by measuring the amount of GH released into the bloodstream; however, the results obtained by this means cannot provide any direct indication of the amount of GH actually produced by pituitary somatotroph cells. The present paper sought to test the hypothesis that release of GH following administration of specific stimuli does not accurately reflect the somatotroph cell response, and that the amount of GH released into the bloodstream may often be greater or smaller than the amount synthesized. GH release and changes in the proportion of somatotroph cells were charted in prepuberal female Wistar rats, following administration of several different GH stimuli: GHRH (1 µg/kg), GHRP-6 (1 µg/kg), GHRELIN (1 µg/kg) and combined GHRH-based treatments, with or without SRIH pretreatment (1 µg/kg) 90 minutes earlier. Peak serum GH values were recorded 15 minutes after administration of GHRH+GHRELIN and GHRH+GHRP-6; maximum stimulation in terms of an increased proportion of somatotroph cells occurred 15 minutes after combined adminstration of GHRH + GHRELIN. SRIH pretreatment (- 90 min) inhibited GH release, with a subsequent "escape" and lack of response to stimulation which lasted at least 30 minutes except following administration of GHRH. However, combined administration of GHRH+GHRELIN maintained stimulation of the somatotroph cell population. In conclusion, the results suggest that the enhanced GH release prompted by stimulation tests used to diagnose GH deficiency in prepuberal female rats does not fully reflect somatroph cell dynamics, and that not all the GH produced and stored by somatotroph cells is released into the bloodstream.
- PublicationOpen AccessThe histopathology of Candida albicans invasion in neonatal rat tissues and in the human blood-brain barrier in culture revealed by light, scanning, transmission and immunoelectron microscopy scanning(Murcia : F. Hernández, 2006) Lossinsky, A.S.; Jong, A.; Fiala, M.; Mukhtar, M.; Buttle, K.F.; Ingram, M.The present studies examined the effects of Candida albicans yeast and hyphal morphologies on tissue pathologies and transmigration properties of the fungus in two experimental models: 1) an in vivo, neonatal rat model, and 2) a cell culture model of human brain microvascular endothelial cells (ECs) (BMVEC). We inoculated a hyphae-producing strain (CAI4-URA3) and a non-hyphae-producing strain (CAI4) of C. albicans into 4-10 day old rats and BMVEC cultures. Animals were inoculated by intraperitonal (i.p.), intranasal (i.n.), oral (p.o.) and intracerebral (i.c.) routes and several tissues were examined after 24-48 hrs. Rats inoculated i.p. with the hyphae-producing strain showed pathology in the kidneys, liver, spleen, and other tissues associated with inoculation tracks of the nose, and muscle and connective tissues of the abdominal wall. Few animals inoculated i.p., however, presented evidence of meningitis. The non-hyphae phase yeast produced neither tissue pathology nor meningitis. Animals inoculated i.c. with the hyphae strain after 1 and 3 hrs expressed minimal meningitis, with an increasing neutrophillic meningitis between 4 and 18 hrs after inoculation. At 18 hrs after i.c. inoculation, however, the inflammatory foci and brain pathology were extensive and demonstrated mycelia within the lateral ventricles associated with necrosis of adjacent brain tissue. Neutrophillic meningitis at this time period was pronounced. BMVEC co-cultured 1-2 hrs with both C. albicans strains showed EC phagocytosis of hyphae and blastospores into intercellular adhesion molecule-1 (ICAM-1)-labeled caveolae suggesting a transcellular role for ICAM-1 in the internalization process of C. albicans.
- PublicationOpen AccessImmunolocalizations of VEGF, its receptors flt-1, KDR and TGF-ß's in epithelial ovarian tumors(Murcia : F. Hernández, 2006) Inan, S.; Vatansever, S.; Celik-Ozenci, C.; Sanci, M.; Dicle, N.; Demir, R.Objective: Angiogenesis is an essential factor for growth, differentiation, invasion and metastasis of tumors. In this study, we aimed to evaluate the immunolocalizations of vascular endothelial growth factor (VEGF), its receptors flt-1, KDR/flk-1, and transforming growth factor-beta’s (TGF-ß) in epithelial ovarian tumors, utilizing indirect immunohistochemistry to understand the role of the angiogenic events in ovarian neoplasia. Methods: Tissue blocks from 40 patients who had ovarian pathology (borderline serous–mucinous tumor and malignant serous–mucinous adenocarcinoma of the ovary) were included in this study. All formalin-fixed, paraffin-embedded tissue sections were stained with hematoxylin-eosin or primary antibodies against VEGF, flt-1, KDR/flk-1, TGF-ß1, TGF-ß2 and TGF-ß3 using the avidin-biotin-peroxidase method. H-SCORE, a semi-quantitative grading system, was used to compare immunohistochemical staining intensities. Results: Positive VEGF immunoreactivity was concentrated in the epithelial and stromal parts of all the ovarian samples and the endothelial cells in the stroma were also stained. Increased immunoreactivity of VEGF was observed in malignant ovarian adenocarcinomas compared to the borderline tumors of the ovary. VEGF receptors, flt-1 and KDR/flk-1 immunoreactivities were detected not only in vascular endothelial cells, but also in tumor cells at malignant sites. Immunoreactivities of VEGF and its receptors were coexpressed in tumor cells of the ovarian carcinoma. While immunoreactivities of TGF-ß1 and TGF-ß2 were both overexpressed in malignant ovarian carcinomas, immunoreactivity of TGF-ß3 was still mild. Conclusion: Our results suggest that overexpression of VEGF, its receptors flt-1, KDR/flk-1 and TGF-ß interaction may play an important role in the ovarian cancer biology, with potential effects on tumor growth and angiogenesis. New therapeutic strategies using VEGF and TGF-ß antagonists could obtain an additional approach to the treatment ovarian carcinoma by inhibiting angiogenesis.
- PublicationOpen AccessHistomorphogenesis and immunohistochemical study of the bovine pineal gland (Bos taurus) during prenatal development (160 days of gestation to birth)(Murcia : F. Hernández, 2006) Regodón, S.; Pozo, D.; Roncero, V.The ontogenesis of the pineal gland of 20 bovine embryos (Bos taurus) has been analysed from 160 days of gestation to birth by means of optical microscopy and immunohistochemical techniques. For this study, the specimens were grouped into two stage in accordance with the most relevant histological characteristics: Stage 1 (160 to 200 days of prenatal development) and Stage 2 (220 days of prenatal development to birth). At 160 days of gestation some rounded structures with a central lumen, which we refer to as glandular rosettes, begin differentiation from the epithelium of the pineal recess, experiencing an extraordinary increase in number and size at 200 days of intrauterine life. In the interior of the pineal parenchyma we observed some morphologically rounded cells with oval euchromatic nuclei and a well-differentiated nucleolus that we refer to as the pinealoblasts. We also observed other cells characterised by the presence of low cytoplasm and rounded and highly stained nuclei that we refer to as the interstitial cells. The glandular stroma is formed from the capsular, trabecular, and perivascular connective tissue as well as from the reticular network that comes from the cellular processes of the interstitial cells. The blood vessels, at 240 of gestation, show wellformed walls where the endothelial cells stand out. At 160 days of gestation we witnessed some cells with small, dense, oval nuclei, positive to the glial fibrillary acidic protein (GFAP). At this age NPY positive fibres were detected, distributed around the blood vessels and among the pinealoblasts. We conclude by clarifying that the changes detected in the morphology as well as in the number and size of glandular rosettes appear to be related to the functional activity of the pineal gland during embryonic development.