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PubMed2005

Targeted calretinin expression in granule cells of calretinin-null mice restores normal cerebellar functions.

Ca2 binding proteins such as calretinin, characterized by the presence of EF-hand motifs that bind Ca2+ ions, are involved in the shaping of intraneuronal Ca2+ fluxes. In the cerebellar cortex, information processing tightly relies on variations in intracellular Ca2+ concentration in Purkinje and granule cells. Calretinin-deficient (Cr-/-) mice present motor discoordination, suggesting cellular and network cerebellar dysfunctions. To determine the cell specificity of these alterations, we constructed transgenic Cr-/- mice exhibiting a selective reexpression of calretinin in granule cells through the promoter function of the GABAA receptor alpha6 subunit gene. Normal granule cell excitability and wild-type Purkinje cell firing behavior in awake mice were restored while the emergence of high-frequency oscillations was abolished. Behavioral analysis of these calretinin-rescue mice revealed that normal motor coordination was restored as compared with Cr-/- mice. These results demonstrate that calretinin is required specifically in granule cells for correct computation in the cerebellar cortex and indicate that the finetuning of granule cell excitability through regulation of Ca2+ homeostasis plays a crucial role for information coding and storage in the cerebellum.

Animals↗
PubMed2006

Dietary choline deficiency alters global and gene-specific DNA methylation in the developing hippocampus of mouse fetal brains.

The availability of choline during critical periods of fetal development alters hippocampal development and affects memory function throughout life. Choline deficiency during fetal development reduces proliferation and migration of neuronal precursor cells in the mouse fetal hippocampus and these changes are associated with modifications in the protein levels of some cell cycle regulators and early differentiation markers. We fed C57 BL/6 mouse dams diets deficient or normal in choline content from days 12 to 17 of pregnancy, and then collected fetal brains on embryonic day 17. Using laser-capture micro-dissection we harvested cells from the ventricular and subventricular zones of Ammon's horn and from the prime germinal zone of the dentate gyrus (hippocampus). In the ventricular and subventricular zones from the choline-deficient group, we observed increased protein levels for kinase-associated phosphatase (Kap) and for p15(INK4b) (two cell cycle inhibitors). In the dentate gyrus, we observed increased levels of calretinin (an early marker of neuronal differentiation). In fetal brain from mothers fed a choline-deficient diet, DNA global methylation was decreased in the ventricular and subventricular zones of Ammon's horn. We also observed decreased gene-specific DNA methylation of the gene (Cdkn3) that encodes for Kap, correlating with increased expression of this protein. This was not the case for p15(INK4b) or calretinin (Cdkn2b and Calb2, respectively). These data suggest that choline deficiency-induced changes in gene methylation could mediate the expression of a cell cycle regulator and thereby alter brain development.

Animals↗
PubMed1996

Calretinin: a novel immunocytochemical marker for mesothelioma.

Immunohistochemistry is a powerful diagnostic adjunct in the differential diagnosis between malignant mesothelioma (especially of the epithelial type) and adenocarcinoma metastatic to the serous membranes. Most of the immunological probes commonly used, however, recognize antigens expressed by the epithelial malignancies and absent from mesothelial cells and mesotheliomas. Probes suitable for the positive identification of mesotheliomas are comparatively scarce and much less commonly used because of their reduced sensitivity and specificity, their unsuitability for staining routinely fixed and embedded tissues, or their lack of commercial availability. We now document that two different polyclonal antisera to calretinin consistently immunostain mesothelial cells and malignant mesotheliomas both in routinely fixed and embedded tissue sections and in cytological preparations of serous effusions. The diagnostic sensitivity of this novel immunocytochemical approach reached 100%, allowing immunostaining of all 44 mesotheliomas investigated, which included five biphasic and three sarcomatoid types. The specificity of calretinin immunoreactivity was checked against 294 adenocarcinomas of different origin (19 serosal metastases and 275 primary tumors potentially able to metastatize to serosal membranes) relevant for the discussion of the differential diagnosis with malignant mesothelioma: only 28 cases showed focal immunoreactivity for calretinin. We conclude that calretinin is a most useful marker for the positive identification of malignant mesotheliomas.

Adenocarcinoma↗
PubMed1997

The immunohistochemical diagnostic panel for epithelial mesothelioma: a reevaluation after heat-induced epitope retrieval.

The immunohistochemical diagnosis between epithelial mesothelioma and adenocarcinoma is currently based on the use of a panel of antibodies to adenocarcinoma-associated antigens and a few antibodies to mesothelial-associated antigens. Since the introduction of epitope retrieval methods, the sensitivity of many antibodies has been enhanced. Thus, a reevaluation of the mesothelioma/adenocarcinoma diagnostic panel becomes necessary. We studied 268 paraffin-embedded formalin-fixed tumor samples that included 57 epithelial mesotheliomas and 211 adenocarcinomas of various origins, comparing an extensive antibody panel with and without heat-induced epitope retrieval (HIER). Marked increase in the sensitivity of several antibodies, with no loss of specificity, was found when HIER was used. After statistical analysis, the antibodies to the epithelial glycoproteins carcinoembryonic antigen, BerEp4, and Bg8 emerged as the best discriminators between adenocarcinoma and epithelial mesothelioma within the entire panel. The mesothelium-associated antibodies, HBME-1, calretinin, and thrombomodulin were less sensitive and less specific than the former, although they were found to be useful on certain cases. Antibodies to cytokeratins and vimentin, although of minor diagnostic value in this context, may be helpful to evaluate the quality of antigen preservation. This study confirms the value of immunohistochemistry to accurately distinguish mesothelioma from adenocarcinoma when an antibody panel approach is used. The addition of heat-induced epitope retrieval methods increases the effectiveness of the procedure and is recommended for most of the antibody panel members.

Adenocarcinoma↗
PubMed1999

Selective distribution of calretinin in adenocarcinomas of the human colon and adjacent tissues.

The expression of calretinin, a calcium-binding protein, has been studied in a series of 82 human colorectal adenocarcinomas. In 22.5% of the cases, part of the tumor cells were calretinin-positive, whereas the cells of the normal and paratumoral mucosa were always negative. Two types of cells from the tumoral mass reacted positively and selectively with calretinin-antisera: the tumor cells and giant fibroblasts. The neurons of enteric ganglia and reactive mesothelial cells also reacted positively to the same antibody. The results obtained by immunochemistry have been confirmed by Western blot analysis and in situ hybridization for calretinin mRNA. There is a correlation between the expression of calretinin and the degree of differentiation of the tumor. Well-differentiated tumors express calretinin in only 5% of the cases, whereas this percentage is 20% for moderately differentiated tumors and 66.6% for poorly differentiated or undifferentiated tumors. We conclude that calretinin is expressed by most undifferentiated colorectal adenocarcinomas, but only by a limited number of cells in well-differentiated tumors. The degree of its expression coincides also with additional signs of malignancy, such as an increase in the number of metastases in the regional lymph nodes and in other organs.

Adenocarcinoma↗
PubMed2001

Immunohistochemical panels for differentiating epithelial malignant mesothelioma from lung adenocarcinoma: a study with logistic regression analysis.

Immunohistochemistry provides an important indicator for differential diagnosis between pleural malignant mesothelioma and lung adenocarcinoma, which have complex therapeutic and medicolegal implications. To pinpoint a reliable, restricted panel of markers, the authors evaluated the efficacy of select commercial antibodies in a series of patients with confirmed clinicopathologic diagnosis of mesothelioma or lung adenocarcinoma with the aid of multiple logistic classification tables. Specimens of 46 mesotheliomas and 20 lung adenocarcinomas were examined with calretinin, thrombomodulin, cytokeratins (CKs) 5/6, and high-molecular weight CKs (indicators of mesothelioma), alongside MOC 31, Ber-EP4, and carcinoembryonic antigen (CEA; indicators of lung adenocarcinoma). Of the mesotheliomas, 40 of 46 (87%) were positive with calretinin, 29 of 46 (63%) with thrombomodulin, 40 of 46 (87%) with CKs 5/6, and 41 of 46 (89%) with high-weight CKs; five of 46 mesotheliomas (11%) were focally reactive with MOC 31, four of 46 (9%) with Ber-EP4, and two of 46 (4%) with CEA. Of the lung adenocarcinomas, 18 of 20 (90%) were positive with MOC 31, 20 of 20 (100%) with Ber-EP4, and 17 of 20 (85%) with CEA; and two of 20 (10%) were focally reactive with calretinin, one of 20 (5%) with thrombomodulin, none of 20 (0%) with CKs 5/6, and five of 20 (25%) with high-weight CKs. Multiple logistic modeling indicated two batteries of three antibodies permitting more than 98% overall accuracy: Ber-EP4 plus CKs 5/6 plus calretinin, and Ber-EP4 plus CKs 5/6 plus CEA.

Adenocarcinoma↗
PubMed2001

Calretinin and other mesothelioma markers in synovial sarcoma: analysis of antigenic similarities and differences with malignant mesothelioma.

Synovial sarcoma (SS) is a mesenchymal neoplasm that typically shows epithelial differentiation. SS commonly metastasizes to lung and pleura, and has also been reported as the primary in these locations. The histologic distinction of SS from mesothelioma may be difficult because of the combination of epithelioid and spindle cells, potentially shared locations, and antigenic expression. In this study the authors examined 103 well-documented SSs including 41 biphasic, 44 monophasic, and 18 poorly differentiated SSs in comparison with 23 epithelioid and seven sarcomatous mesotheliomas. Most biphasic SSs (29 of 41, 71%) had fields or foci of calretinin-positive tumor cells. The spindle cell components were more often positive (55%), whereas 14% of tumors had positive epithelial cells. The monophasic and poorly differentiated SSs commonly had foci of calretinin-positive cells (in 52% and 56% of cases respectively). In comparison, all 23 epithelioid mesotheliomas (EM) were extensively calretinin positive and seven sarcomatoid mesotheliomas were variably calretinin positive. HBME-1 positivity was similarly detected in biphasic SS and EM (100% and 87% respectively). Among the other sarcomas, two of 15 malignant peripheral nerve sheath tumors were focally calretinin positive, whereas 16 epithelioid sarcomas, 20 leiomyosarcomas, 20 gastrointestinal stromal tumors, and 20 angiosarcomas were negative. Biphasic SSs differed from mesotheliomas by their more common BerEp4 positivity (90%) whereas EMs showed focal reactivity in 13% cases. Marked CD15 reactivity was rare in both. Wilms tumor protein-1 (WT1) was not detected in SS, but was present in 12 of 17 EMs. CD141 was rare in SS, limited to spindle cell components, whereas EMs typically showed prominent membrane staining in epithelial cells. Simple epithelial keratins were present in all epithelial cells of biphasic SS and mesothelioma (keratin 7[K7], K19), but were only focal in monophasic and poorly differentiated SS. Biphasic SSs were extensively K14 positive (89% of cases), whereas epithelial and sarcomatoid mesotheliomas typically showed only scattered positive cells. The potentially shared calretinin patterns in SS and mesothelioma require the use of other markers. The discriminating features include extensive BerEp4 positivity, rarity of CD141, and lack of WT1 in SS. Global expression of K7 and K19 in mesotheliomas versus focal expression in monophasic and poorly differentiated SSs, and differential patterns of K14 expression may also be helpful.

Adolescent↗
PubMed2003

Expression of calretinin, thrombomodulin, keratin 5, and mesothelin in lung carcinomas of different types: an immunohistochemical analysis of 596 tumors in comparison with epithelioid mesotheliomas of the pleura.

Several immunohistochemical markers, among them calretinin, thrombomodulin (CD141), keratin 5, and mesothelin, have been documented or suggested as useful markers for positive identification of mesothelioma and to differentiate it from pulmonary adenocarcinoma; numerous studies have documented their variable specificity. However, expression of these markers in other types of lung carcinomas has not been systematically explored, although these tumors can enter in the differential diagnosis of mesothelioma. In this study we immunohistochemically evaluated 596 lung carcinomas of different types for the four above-mentioned mesothelioma markers, all of which reacted with a great majority of epithelioid mesotheliomas studied for comparison. Calretinin expression was common in giant cell carcinomas (67%), small cell carcinomas (49%), and large cell carcinomas (38%), whereas it was rare in usual adenocarcinomas but slightly more common in those with neuroendocrine differentiation (11% and 17%, respectively). Thrombomodulin was present in all keratinizing squamous carcinomas and the great majority (87%) of nonkeratinizing tumors in a membrane-staining pattern. It was moderately common in small cell (27%) and large cell carcinomas (25%) but relatively rare in adenocarcinomas (13%). Keratin 5 was expressed in all keratinizing and the great majority (87%) of nonkeratinizing squamous carcinomas, and a majority of large cell carcinomas (56%) and some small cell carcinomas (27%). It was rare in acinar adenocarcinomas (12%) and absent in those with neuroendocrine differentiation. Mesothelin was present in more than half (53%) of adenocarcinomas and a minority (13%) of large cell carcinomas but was absent in small cell carcinomas. In squamous carcinomas it was more often seen in nonkeratinizing versus keratinizing tumors (31% vs 16%). These results show that each of these "mesothelioma" markers reacts with different subsets of pulmonary carcinomas with a variable frequency; this should be considered when using these markers in the differential diagnosis of thoracic tumors.

Biomarkers, Tumor↗
PubMed1994

Calretinin labels a specific neuronal subpopulation in primate globus pallidus.

All pallidal neurones are believed to share the same chemical and morphological phenotype in primates. At variance with this idea is the present finding that calretinin (CR), a calcium binding protein, occurs only in a subset of pallidal neurones in squirrel monkeys. This chemospecific subpopulation comprises both large and small bipolar and multipolar neurones distributed according to a rostrocaudal decreasing gradient. The large neurones outnumber small neurones throughout the pallidum; they abound principally in the external pallidal segment, whereas the small neurones prevail in the internal segment. Some pallidal CR-positive neurones display dorsoventrally elongated dendrites, while others show dendrites radiating in all directions. These findings reveal that pallidal neurones form a chemically and morphologically heterogeneous population in primates.

Animals↗
PubMed1995

Calretinin-positive Cajal-Retzius cells persist in the adult human neocortex.

The occurrence of Cajal-Retzius (CjRe) cells was studied in four cortical areas of five normal adult humans using antibodies against calretinin. Calretinin immunofluorescence and autofluorescence of lipofusin granules in CjRe cells were visualized by dual channel confocal laser scanning microscopy. Three types of CjRe cells existed in the adult human cortex: horizontal, triangular and multipolar, and their number did not decrease with ageing. Horizontal CjRe cells were found in all cortical areas; they contained no lipofusin or less than other cells. Triangular CjRe cells with descending dendrites were less numerous. Multipolar CjRe cells were rare and contained more lipofuscin. We conclude that calretinin-immunoreactivity can be used to study CjRe cell morphology in normal and diseased adult human brain.

Adult↗
PubMed1999

Cellular changes in rat brain areas associated with neonatal hippocampal damage.

The neonatal destruction of the ventral hippocampus was introduced as a model to recreate features of schizophrenia in the rat. While behavioral consequences of this intervention have been studied in detail, less is known about the cellular processes underlying the deviant behavior. We studied in rats (neonatally or adult lesioned, controls) brain areas receiving or not receiving hippocampal projections. The number of neurons and the expression of the cell markers L-ornithine decarboxylase, nitric oxide synthase/NADPH diaphorase, calretinin and GFAP were estimated. Reduced numbers of neurons and increased immunostaining for ornithine decarboxylase and nitric oxide synthase in the prefrontal, perirhinal and entorhinal cortex of neonatally but not adult lesioned rats or controls demonstrate persistent cellular changes after ventral hippocampus damage.

Aging↗
PubMed1999

Calretinin-immunoreactive neurons in rostral migratory stream: neuronal differentiation.

The purpose of the present study was to examine immunohistochemically the expression of nestin and polysialylated neural cell adhesion molecule in calretinin-immunoreactive neurons of the rostral migratory stream, the restricted pathway through which neuronal precursor cells migrate towards the olfactory bulb. Using mirror sectioning, calretinin-immunoreactive neurons of the rostral migratory stream were shown to co-express nestin (20.8%) and polysialylated neural cell adhesion molecule (61.8%). The results show that calretinin-immunoreactive neurons in the rostral migratory stream still express immature neural and neuronal phenotypes.

Animals↗
PubMed2000

Sulcogyral variation in NMDA receptor 2A/B subunit immunoreactivity in human brain.

NMDA receptors (NR) are important in many neurological processes. Using a large series of human brain tissue, we show that the distribution of NR2A/B immunoreactivity varies according to position along a gyrus. For pyramidal neurons in laminae II and III, immunoreactivity is most marked at gyral crown and gyral lips, diminishes along sulcal wall, and is barely detectable in sulcal floor cortex. In contrast, in some cases, immunoreactivity in laminae V and VI pyramidal neurons shows the reverse pattern. Neurofilament and calretinin immunoreactivity do not show this phenomenon. The findings suggest novel functional regionalization at the sulcogyral level in normal human brain.

Adolescent↗
PubMed2002

Weightlessness affects cytoskeleton of rat utricular hair cells during maturation in vitro.

The aim of this study was to investigate whether an altered gravitational environment affected the phenotype of vestibular hair cells during maturation. We developed, using an automated incubator, a 3D culture of utricles from newborn rats. These cultures were subjected to weightlessness for 1 or 3 days, and then compared with control cultures developed in natural and induced 1G gravity. Immunocytochemistry for alpha-tubulin and calretinin revealed disorganisation of the microtubules and a loss of hair cell shape in cells subjected to weightlessness during maturation. We conclude that the lack of gravitational strain affected cytoskeletal dynamics, resulting in loss of the specific morphological phenotype of the cells.

Animals↗
PubMed2005

Social behavior modulates songbird interpeduncular nucleus function.

Male zebra finches produce the same song while alone and during courtship of a female. However, singing-related activity in the anterior forebrain nuclei lateral magnocellular anterior nidopallium and Area X markedly depends on the social context. Thus, the anterior forebrain should receive a signal of social context from outside the song system. Here we investigated a possible source of such modulation, the midbrain interpeduncular nucleus, by monitoring immediate early genes and synaptic activity. The level of immunoreactivity for egr1 was high and calretinin was low following courtship directed singing, but the opposite pattern was seen after solo undirected singing. Further, pairs of stimulation caused depression of synaptic responses after directed singing, but facilitation after undirected singing.

Animals↗
PubMed1999

Sclerosing stromal tumors, thecomas, and fibromas of the ovary: an immunohistochemical profile.

The immunohistochemistry of 11 sclerosing stromal tumors (SSTs), 11 fibromas, and 5 thecomas was studied to determine criteria for the assessment of 5 densely sclerotic, calcified ovarian tumors of uncertain diagnosis occurring in young women. The results indicate that the staining pattern for alpha glutathione S-transferase can be used to distinguish SSTs, fibromas, and thecomas. CD34, by highlighting the vascular pattern and density, can be used to distinguish between SSTs and other tumors in the thecoma-fibroma group. Alpha-inhibin and calretinin mirrored the alpha glutathione S-transferase staining. Vimentin, smooth muscle actin, and muscle specific actin were generally positive, but desmin was negative in all but one tumor. These results suggest that at least four of the five tumors of uncertain diagnosis were SSTs that had undergone end-stage sclerosis and calcification.

Actins↗
PubMed2001

Expression of calretinin in human ovary, testis, and ovarian sex cord-stromal tumors.

Calretinin, a calcium-binding protein, is primarily expressed in certain subtypes of neurons. It has also been found to be present in mesothelial cells and mesotheliomas but not in many types of carcinomas. Using a polyclonal anti-calretinin antibody, we investigated the expression of calretinin immunohistochemically in nonneoplastic human ovaries and testes and ovarian sex cord-stromal tumors (SCSTs). In ovaries, calretinin was expressed in theca interna cells, hilus cells, and scattered individual stromal cells. Oocytes, granulosa cells, theca externa cells, rete ovarii, and most stromal cells were negative. Expression of calretinin was also seen in the ovarian surface epithelium and in collapsed and flat epithelial inclusion glands (EIGs), but not in round, columnar, and ciliated EIGs. In some glands, a transition from calretinin-positive to calretinin-negative epithelium was observed. In postpubertal testes, calretinin was expressed in Leydig cells, but not in germ cells or most rete testes and Sertoli cells. In ovarian SCSTs, strong calretinin staining was seen in all hilus cell tumors (4/4) and the Leydig cell component of Sertoli-Leydig cell tumors (10/10). The Sertoli cell component showed focal weak positivity in 5/10. Fibrothecomas were completely negative (0/8). In granulosa cell tumors, the tumor cells were either completely negative (8/14) or weakly positive at the periphery of the tumor (6/14) while scattered stromal cell staining was seen in 9/14 cases. The expression of calretinin in normal Leydig cells, theca interna cells, the Leydig cell component of Sertoli-Leydig cell tumors, and hilus cell tumors suggests its functional relationship with androgen production. Its pattern of expression in ovarian SCSTs is useful in the differential diagnosis of these tumors. The presence of a transition from calretinin-positive, flat, nonciliated epithelium to calretinin-negative, columnar, ciliated epithelium in the same glands provides strong evidence for mullerian metaplasia.

Calbindin 2↗
PubMed2002

Adenomatoid tumors of the uterus: an analysis of 60 cases.

Sixty cases of uterine adenomatoid tumors (ATs) are reported. All except four were incidental findings in hysterectomy specimens, three of these being discovered preoperatively as large multicystic tumors. ATs were classified into two distinctive macroscopic patterns: small, solid tumors and large, cystic ones. The 56 small, solid ATs ranged from 0.2 to 3.5 cm, (average 2.1 cm); 48 were nodular and 8 diffuse. The four large, cystic tumors ranged from 7 to 10 cm. Inflammation occurred in 65% of the tumors, and a smooth muscle reaction, identified by an increased Ki-67 index, was present in most cases. Both types were histologically similar except for the presence of short papillae in cystic tumors, which also showed serosal involvement. Both were immunoreactive for cytokeratins, calretinin, HMBE-1, and vimentin. Estrogen and progesterone nuclear receptors and EMA were negative. These tumors represent a spectrum ranging from small and solid to large and cystic ATs in the female genital tract, whereas outside the genital tract they are morphologically similar to multicystic mesothelioma. Although a reactive origin for ATs often seems plausible, especially when inflammation is present, their neoplastic nature should not be ignored.

Adenomatoid Tumor↗