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PubMed2004

Use of immunohistochemical marker calretinin in the diagnosis of a diffuse malignant metastatic mesothelioma in an equine.

Mesotheliomas are rarely reported in animal species. In this report, the occurrence of a diffuse, metastatic mesothelioma in a 6-year-old gray Arabian mare is described. The mare was presented on clinical examination with ascites, bilateral pleural effusion, and pleural roughening. Necropsy revealed abundant fluid in the abdominal and thoracic cavities. The surface of all organs was thick and fibrosed with multiple raised nodules and hemorrhages. Histology was characteristic of a generalized, biphasic mesothelioma with vascular and lymph nodes metastases. It is believed that the primary tumor developed in the pericardium and spread through lymphatics. In this report, calretinin was used as an immunohistochemical marker in the diagnosis of mesothelioma in an equine species for the first time.

Animals↗
PubMed2006

Leydig cell tumor of the testis with histological and immunohistochemical features of malignancy in a 1-year-old boy with isosexual pseudoprecocity.

The article reports the clinical, histopathological, and immunohistochemical findings of a 1-year-old boy presenting with isosexual pseudoprecocity attributable to a functioning Leydig cell tumor of the testis. The case appears to represent the youngest patient ever recognized with this well-known syndrome. Malignancy features were also for the first time initially assessed using criteria, retrospectively developed from the literature, for metastasizing Leydig cell tumor. All the following were found: infiltrative borders, cellular pleomorphism, high mitotic index (12-14/high-power field), high MIB-1 index (40%), P53 positivity in 50% of the cells, and bcl-2 positivity in 15% of the cells. Immunohistochemistry proved the cells of the tumor to be positive for inhibin, Melan-A, synaptophysin, cytokeratin, and calretinin and negative for S-100 and chromogranin A. Notably, lipochrome and crystals of Reinke were not found in the tumor cells. Although the neoplasm fulfilled the criteria for a potentially metastasizing Leydig cell tumor, there was no evidence of that event having occurred, perhaps as a result of early treatment or as indication that criteria developed for Leydig cell tumor of adults may not apply to children.

Antigens, Neoplasm↗
PubMed2004

Newborn horizontal cells migrate bi-directionally across the neuroepithelium during retinal development.

Cell migration plays an important role during the development of the retina. In this work we have studied the migration of newborn horizontal cells in avian embryonic retina. Using the pattern of the early expressed transcription factors Lim1 and Prox1 we have shown that horizontal cells migrate bi-directionally from their site of birth, close to the ventricular side, to the adjacent (vitreal) side of the neuroepithelium, where they align just next to the prospective ganglion cell layer before migrating back again to their final laminar position in the external part of the inner nuclear layer. The migration occurs between Hamburger and Hamilton stages 24 and 33, which is equivalent to embryonic day 4.5 and 8. Between stages 26 and 30 the horizontal cells reside close to the ganglion cell layer and intra ocular injections of a cytochalasin D, an actin polymerisation blocker that inhibit migration, at stage 29 interfered with the migration of the horizontal cells to their final destination. Furthermore, using biolistic gene transfer with a green fluorescence protein expression vector of retinal slices we were able to record ventricle-directed migration by time-lapse microscopy. Combining biolistics with immunohistochemistry we showed that transfected cells, which have also been translocated in a ventricular direction were positive for the horizontal cell markers Lim1 and Prox1. The alternative path of migration that is described in this work differs from the generally accepted one for horizontal cells and this knowledge will influence the view of how the molecular determination of horizontal cells is specified.

Animals↗
PubMed2004

Coordinated regulation of gene expression by Brn3a in developing sensory ganglia.

Mice lacking the POU-domain transcription factor Brn3a exhibit marked defects in sensory axon growth and abnormal sensory apoptosis. We have determined the regulatory targets of Brn3a in the developing trigeminal ganglion using microarray analysis of Brn3a mutant mice. These results show that Brn3 mediates the coordinated expression of neurotransmitter systems, ion channels, structural components of axons and inter- and intracellular signaling systems. Loss of Brn3a also results in the ectopic expression of transcription factors normally detected in earlier developmental stages and in other areas of the nervous system. Target gene expression is normal in heterozygous mice, consistent with prior work showing that autoregulation by Brn3a results in gene dosage compensation. Detailed examination of the expression of several of these downstream genes reveals that the regulatory role of Brn3a in the trigeminal ganglion appears to be conserved in more posterior sensory ganglia but not in the CNS neurons that express this factor.

Animals↗
PubMed1996

Embryonic taste buds develop in the absence of innervation.

It has been hypothesized that taste buds are induced by contact with developing cranial nerve fibers late in embryonic development, since descriptive studies indicate that during embryonic development taste cell differentiation occurs concomitantly with or slightly following the advent of innervation. However, experimental evidence delineating the role of innervation in taste bud development is sparse and equivocal. Using two complementary experimental approaches, we demonstrate that taste cells differentiate fully in the complete absence of innervation. When the presumptive oropharyngeal region was taken from a donor axolotl embryo, prior to its innervation and development of taste buds, and grafted ectopically on to the trunk of a host embryo, the graft developed well-differentiated taste buds. Although grafts were invaded by branches of local spinal nerves, these neurites were rarely found near ectopic taste cells. When the oropharyngeal region was raised in culture, numerous taste buds were generated in the complete absence of neural elements. Taste buds in grafts and in explants were identical to those found in situ both in terms of their morphology and their expression of calretinin and serotonin immunoreactivity. Our findings indicate that innervation is not necessary for complete differentiation of taste receptor cells. We propose that taste buds are either induced in response to signals from other tissues, such as the neural crest, or arise independently through intrinsic patterning of the local epithelium.

Ambystoma↗
PubMed1999

Calretinin immunoreactive nerve endings in the trachea and bronchi of the rat.

Nerve endings showing calretinin immunoreactivity were examined in the lower respiratory tract of the adult rat. Tree-like nerve endings were immunostained in the tracheal and bronchial smooth muscle layer. The endings that arose from thick nerve fibers and formed corpuscles composed of many arborized nerve terminals. A few of the nerve endings were also observed in the lamina propria of the tracheal mucosa, close to the epithelial layer. Immunoelectron microscopy revealed that the immunoreactive terminals were filled with mitochondria and scattered among the intermuscular collagen fibrils. Schwann cell sheath and collagen fibrils were intercalated between the smooth muscle cells and nerve endings. The calretinin immunoreactive nerve endings observed in the present study seem to be slowly adapting stretch receptors.

Animals↗
PubMed2004

Differential expression of histochemical characteristics in the developing olfactory receptor cells in a flatfish, barfin flounder (Verasper moseri).

Differentiation of the histochemical characteristics of the olfactory receptor cells (ORC) was examined by immunohistochemistry for protein gene product 9.5 (PGP 9.5) and calretinin (CR) and lectin histochemistry for Phaseolus vulgaris agglutinin-L (PHA-L) in the developing olfactory epithelium (OE) of the barfin flounder. PGP 9.5 immunoreactivity was diffuse and CR immunoreactivity was restricted at day 7, but these immunoreactivities became intense in the OE toward day 91. Crypt cells were first identified at day 56. PHA-L staining was faint at day 28, but became intense toward day 91. These findings suggest that PGP 9.5-immunopositive cells, CR-immunopositive cells, crypt cells and PHA-L-reactive cells differentiate independently in the developing OE and constitute subsets of the ORC in the OE.

Animals↗
PubMed2006

Age-related changes in calretinin-immunoreactive periglomerular cells in the rat main olfactory bulb.

The changes of calretinin (CR)-immunoreactive periglomerular cells in the glomerular layer of the main olfactory bulb (MOB) were investigated in rats differing ages from postnatal month 1 (PM 1) to PM 24. The number of cresyl violet-positive periglomerular cells was similar between PM 1 and PM 12, but they decreased slightly in the PM 24 group. The size of CR-immunoreactive periglomerular cells in the glomerular layer increased with age, while their numbers did not change significantly in the PM 6-PM 24 groups. In the PM 24 group, numbers of CR-positive periglomerular cell bodies and their processes decreased, while the size of CR-positive cell bodies in the glomeruli was larger than that of the previous groups. These results suggest that CR-immunoreactive periglomerular cells in the rat MOB are well-developed in the PM 6 group, and that periglomerular cells in the PM 24 group show poor CR-immunoreactivity compared to those in the PM 6 group.

Aging↗
PubMed2000

Lymphohistiocytoid mesothelioma. An often misdiagnosed variant of sarcomatoid malignant mesothelioma.

Three cases of lympho-histiocytoid mesothelioma, a rare variant of pleural sarcomatoid malignant mesothelioma, are described. Histologically, the neoplasms were characterized by a diffuse discohesive proliferation of atypical histiocytoid cells intermixed with a marked lymphocytic and lesser plasmacytic infiltrate. One case initially was misdiagnosed as a ganglioneuroma, a second case was misinterpreted as malignant lymphoma, and a third case was sent in consultation with the differential diagnosis of inflammatory pseudotumor vs mesothelioma. Immunohistochemical studies showed strong and generalized expression of cytokeratins and vimentin by the neoplastic histiocytoid cells in all 3 cases. Two cases were positive for calretinin, one of which also was positive for HBME-1, thrombomodulin, and LeuM1. None of the cases stained with the epithelial glycoprotein markers carcinoembryonic antigen, B72.3, and Ber-EP4, or the blood group antigen, BG-8. The immunophenotype of the lymphoplasmacytic infiltrate revealed predominantly reactive, mature T cells, with fewer polytypic plasma cells, histiocytes, and B cells. In lymphohistiocytoid mesothelioma, as in the usual examples of sarcomatoid mesothelioma, the demonstration of cytokeratin expression by the neoplastic cells is the most useful diagnostic finding that allows exclusion of other neoplasms with which this entity may be confused.

Aged↗
PubMed2003

Expression of calretinin and the alpha-subunit of inhibin in granular cell tumors.

Granular cell tumors (GCTs) typically express S-100 protein, which has been used as a marker in differential diagnosis. Calretinin, a calcium-binding protein related structurally to S-100, and inhibin, a polypeptide hormone secreted primarily by ovarian granulosa cells and testicular Sertoli cells and functioning as an inhibitor for pituitary follicle-stimulating hormone secretion, are potentially useful but not well-evaluated markers for GCTs. We studied 43 cases of GCT with antibodies to calretinin, the inhibin alpha-subunit, and S-100 protein. All tumors were positive for inhibin alpha-subunit and S-100 protein, with 50% or more cells showing moderate to strong staining. Forty tumors (93%) were positive for calretinin, ranging from focal weak to diffuse strong staining. Enhanced staining for calretinin in the tumor cells adjacent to hyperplastic squamous epithelium was observed in 9 of 13 cases showing pseudoepitheliomatous hyperplasia. Calretinin and the inhibin alpha-subunit are useful markers for GCTs. The expression of calretinin, a primarily neuronal protein, in GCTs further supports its neural differentiation or derivation. The elevated calretinin expression in the tumor cells adjacent to the hyperplastic squamous epithelium suggests a role for calretinin in the tumor cells-squamous epithelium interaction.

Biomarkers, Tumor↗
PubMed2001

Thrombomodulin, calretinin and c-kit (CD117) expression in cardiac myxoma.

The immunohistochemical profile of cardiac myxoma has been debated. The tumor is thought to be derived from multipotential undifferentiated mesenchymal cells. A consistent marker for this tumor has not been found. In this article an immunohistochemical study of 23 cardiac myxomas was accomplished. This study comprised the immunoreactivity of the tumors for thrombomodulin, calretinin and and c-kit (CD117). To the best of our knowledge, thrombomodulin and c-kit have not been tested in cardiac myxoma. Calretinin expression has been recently demonstrated in cardiac myxoma, although this finding has not been yet validated. Surface lining cells, tumor vascular endothelium, cells around the vascular slits and stromal cells embedded in the myxoid matrix were assessed independently. All tumors showed reactivity for thrombomodulin in the surface cells and in the endothelium of neoplastic vessels. 82.6% of cardiac myxomas expressed thrombomodulin in the stromal cells and 69.6% of the tumors were reactive in the perivascular cells. 73.9% of cardiac myxomas expressed calretinin in the stromal cells and in the perivascular cells. All myxomas were negative for c-kit. Thrombomodulin and calretinin may be important diagnostic aids for cardiac myxoma. Cardiac myxoma cells do not express embryonic/fetal endothelial antigens.

Adult↗
PubMed2004

Differential expression of calretinin in the developing and regenerating zebrafish visual system.

Calretinin is a calcium-binding protein which participates in a variety of functions including calcium buffering and neuronal protection. It also serves as a developmental marker of retinal ganglion cells (RGCs). In order to study the role of calretinin in the development and regeneration of RGCs, we have studied its pattern of expression in the retina at different developmental stages, as well as during optic nerve regeneration by means of immunohistochemistry. During development, calretinin is found for the first time in RGCs when they connect with the optic tectum. Optic nerves from adult zebrafish were crushed and after different survival times, calretinin expression in the retina, optic nerve tract and optic tectum was studied. From the day of crushing to 10 days later, calretinin expression was found to be downregulated within RGCs and their axons, as was also observed during the early developmental stages of RGCs, when they are not committed to a definite cell phenotype. Moreover, 13 days after lesion, when the regenerating axons arrived at the optic tectum, a recovery of calretinin immunoreactivity within the RGCs was observed. These results indicate that calretinin may play an important role during optic nerve regeneration, Thus, the down-regulation of Calretinin during the growth of the RGC axons towards the target during development as well as during their regeneration after injury, indicates that an increase the availability of cytosolic calcium is integral to axon outgrowth thus recapitulating the pattern observed during development.

Animals↗
PubMed2005

Metabotropic glutamate receptor 8-expressing nerve terminals target subsets of GABAergic neurons in the hippocampus.

Presynaptic metabotropic glutamate receptors (mGluRs) show a highly selective expression and subcellular location in nerve terminals modulating neurotransmitter release. We have demonstrated that alternatively spliced variants of mGluR8, mGluR8a and mGluR8b, have an overlapping distribution in the hippocampus, and besides perforant path terminals, they are expressed in the presynaptic active zone of boutons making synapses selectively with several types of GABAergic interneurons, primarily in the stratum oriens. Boutons labeled for mGluR8 formed either type I or type II synapses, and the latter were GABAergic. Some mGluR8-positive boutons also expressed mGluR7 or vasoactive intestinal polypeptide. Interneurons strongly immunopositive for the muscarinic M2 or the mGlu1 receptors were the primary targets of mGluR8-containing terminals in the stratum oriens, but only neurochemically distinct subsets were innervated by mGluR8-enriched terminals. The majority of M2-positive neurons were mGluR8 innervated, but a minority, which expresses somatostatin, was not. Rare neurons coexpressing calretinin and M2 were consistently targeted by mGluR8-positive boutons. In vivo recording and labeling of an mGluR8-decorated and strongly M2-positive interneuron revealed a trilaminar cell with complex spike bursts during theta oscillations and strong discharge during sharp wave/ripple events. The trilaminar cell had a large projection from the CA1 area to the subiculum and a preferential innervation of interneurons in the CA1 area in addition to pyramidal cell somata and dendrites. The postsynaptic interneuron type-specific expression of the high-efficacy presynaptic mGluR8 in both putative glutamatergic and in identified GABAergic terminals predicts a role in adjusting the activity of interneurons depending on the level of network activity.

Animals↗
PubMed2006

Neurogenesis in the caudate nucleus of the adult rabbit.

Stem cells with the potential to give rise to new neurons reside in different regions of the adult rodents CNS, but in vivo only the hippocampal dentate gyrus and the subventricular zone-olfactory bulb system are neurogenic under physiological condition. Comparative analyses have shown that vast species differences exist in the way the mammalian brain is organized and in its neurogenic capacity. Accordingly, we have demonstrated recently that, in the adult rabbit brain, striking structural plasticity persists in several cortical and subcortical areas. Here, by using markers for immature and mature neuronal and glial cell types, endogenous and exogenously administered cell-proliferation markers, intraventricular cell tracer injections coupled to confocal analysis, three-dimensional reconstructions, and in vitro tissue cultures, we demonstrate the existence of newly formed neurons in the caudate nucleus of normal, untreated, adult rabbit. Our results suggest that neurogenesis in the caudate nucleus is a phenomenon independent from that occurring in the adjacent subventricular zone, mostly attributable to the activity of clusters of proliferating cells located within the parenchyma of this nucleus. These clusters originate chains of neuroblasts that ultimately differentiate into mature neurons, which represent only a small percentage of the total neuronal precursors. These results indicate that striatum of rabbit represents a favorable environment for genesis rather than survival of newly formed neurons.

Animals↗
PubMed2004

Origins of cortical interneuron subtypes.

Cerebral cortical functions are conducted by two general classes of neurons: glutamatergic projection neurons and GABAergic interneurons. Distinct interneuron subtypes serve distinct roles in modulating cortical activity and can be differentially affected in cortical diseases, but little is known about the mechanisms for generating their diversity. Recent evidence suggests that many cortical interneurons originate within the subcortical telencephalon and then migrate tangentially into the overlying cortex. To test the hypothesis that distinct interneuron subtypes are derived from distinct telencephalic subdivisions, we have used an in vitro assay to assess the developmental potential of subregions of the telencephalic proliferative zone (PZ) to give rise to neurochemically defined interneuron subgroups. PZ cells from GFP+ donor mouse embryos were transplanted onto neonatal cortical feeder cells and assessed for their ability to generate specific interneuron subtypes. Our results suggest that the parvalbumin- and the somatostatin-expressing interneuron subgroups originate primarily within the medial ganglionic eminence (MGE) of the subcortical telencephalon, whereas the calretinin-expressing interneurons appear to derive mainly from the caudal ganglionic eminence (CGE). These results are supported by findings from primary cultures of cortex from Nkx2.1 mutants, in which normal MGE fails to form but in which the CGE is less affected. In these cultures, parvalbumin- and somatostatin-expressing cells are absent, although calretinin-expressing interneurons are present. Interestingly, calretinin-expressing bipolar interneurons were nearly absent from cortical cultures of Dlx1/2 mutants. By establishing spatial differences in the origins of interneuron subtypes, these studies lay the groundwork for elucidating the molecular bases for their distinct differentiation pathways.

Animals↗
PubMed2002

Calretinin-containing interneurons innervate both principal cells and interneurons in the CA1 region of the human hippocampus.

Hippocampal interneurons consist of functionally diverse cell types, most of them target the dendrites or perisomatic region of pyramidal cells with a few exceptions, like the calretinin-containing cells in the rat: they selectively innervate other interneurons. However, no electron microscopic data are available about the synaptic connections of calretinin-immunoreactive neurons in the human hippocampus. We aimed to provide these data to establish whether interneuron-selective interneurons indeed represent an essential feature of hippocampal circuits across distant species. Two types of calretinin-immunostained terminals were found in the CA1 region: one of them presumably derived from the thalamic reuniens nucleus, and established asymmetric synapses on dendrites and spines. The other type originating from local interneurons formed symmetric synapses on both pyramidal and interneuron dendrites. Distribution of postsynaptic targets showed that 26.8% of the targets were CR-positive interneuron dendrites, and 25.2% proved to be proximal pyramidal dendrites. CR-negative interneuron dendrites were also contacted (12.4%). Small caliber postsynaptic dendrites were not classified (28%). Somata were rarely contacted (7.6%). The present data suggest that calretinin-positive boutons do show a preference for other interneurons, but a considerable proportion of the targets are pyramidal cells. We propose that interneuron-selective inhibitory cells exist in the human Ammon's horn, and boutons innervating pyramidal cells derive from another cell type that might not exist in rodents.

Adult↗
PubMed2005

Ber-EP4 and anti-calretinin antibodies: a useful combination for differential diagnosis of various histological types of ovarian cancer cells and mesothelial cells.

The differential diagnosis between reactive mesothelial cells and ovarian carcinoma cells is often difficult in cytologic specimens. Immunocytochemical procedures have been utilized in assisting this differential diagnosis, with limitations. Furthermore, previous studies examined only serous type but not other histological types of ovarian carcinoma cases. Therefore, we evaluated the practical value of various epithelial and mesothelial markers in differential diagnosis of these two types of cells. Various types of ovarian carcinoma (serous, n = 22; mucinous, n = 10; endometrioid, n = 7; clear cell, n = 10) and benign mesothelial tissues (n = 15) were studied by immunohistochemistry. We then studied effective panels of antibodies by immunohistochemistry in 43 cytologic specimens of ascites or peritoneal lavage fluid consisting of 20 reactive mesothelium and 23 adenocarcinomas of the ovary. In the tissue specimens, Ber-EP4, a monoclonal antibody of epithelial antigen, and a polyclonal antibody against calretinin, which is expressed in mesothelium, are used in differentiating reactive mesothelial cells from ovarian carcinoma. In cytologic specimens, the sensitivity and specificity of Ber-EP4 were 100% and 90%, respectively. The sensitivity and specificity of the anti-calretinin antibody were 90% and 91%, respectively. Using multiple regression analysis, the correlation coefficient between epithelial antigen and calretinin reactivity was r = 0.938, with a significance level of p < 0.0001. In conclusion, the combined immunostaining of cytologic specimens for Ber-EP4 and the anti-calretinin antibody is helpful for the differential diagnosis between mesothelial cells and not only serous type, but also mucinous, endometrioid and clear cell types of ovarian cancer cells.

Antibodies↗
PubMed1999

Postnatal expression of calretinin-immunoreactivity in periodontal Ruffini endings in the rat incisor: a comparison with protein gene product 9.5 (PGP 9.5)-immunoreactivity.

The postnatal expression of immunoreactivity for calretinin, one of the calcium binding proteins, and for protein gene product 9.5 (PGP 9.5), a general neuronal marker, was investigated in mechanoreceptive Ruffini endings in the periodontal ligament of the rat incisor. Age-related changes in the expression of these two proteins in periodontal nerves were further quantified with a computerized image analysis. At 1 day after birth, a few PGP 9.5-immunoreactive nerve fibers and a still smaller number of calretinin-positive fibers were found in the periodontal ligament: they were thin and beaded in appearance and no specialized nerve terminals were recognized. Tree-like terminals, reminiscent of immature Ruffini endings, were recognizable in 4-day-old rats by PGP 9.5-immunohistochemistry, while calretinin-immunostaining failed to reveal these specialized endings. At postnatal 7-11 days when PGP 9.5-immunostaining could demonstrate typical Ruffini endings, calretinin-immunopositive nerve fibers merely tapered off without forming the Ruffini type endings. A small number of Ruffini endings showing calretinin-immunoreactivity began to occur in the periodontal ligament at 24-26 days after birth when the occlusion of the first molars had been established. At the functional occlusion stage (60-80 days after birth), the Ruffini endings showing calretinin-immunoreactivity drastically increased in number and density, but less so than those positive for PGP 9.5-immunoreaction. The delayed expression of calretinin suggests that the function of the periodontal Ruffini endings is established after the completion of terminal formation because Ca2+, which binds to calcium binding proteins including calretinin with high affinity, plays an important role in mechano-electric transduction.

Age Factors↗