Marco R. Celio

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.

Perineuronal nets: past and present

Golgi ranked the peripheral reticulum--which adheres intimately to nerve cell surfaces--alongside the intracellular reticulum, or Golgi apparatus,which immortalized his name. At first dismissed as an artefact of capricious staining techniques, this peripheral reticulum, or perineuronal net, is now recognized as a genuine entity in neurocytology. It represents a complex of extracellular matrix molecules interposed between the meshwork of glial processes, from which they are indistinguishable, and nerve-cell surfaces. In no other branch of neuroscience has the waxing and waning of interest in any morphological entity been so pronounced as in the case of the perineuronal net. This review traces the history of this enigmatic structure from its conception to the present time, brings to light the keen observational powers of morphologists at the turn of the century and reveals how their sagacious forethought anticipated current thinking on the role of perineuronal nets.

Monoclonal antibodies directed against the calcium binding protein Calbindin D-28k

We have produced 25 clones secreting antibodies directed against chicken Calbindin D-28k. Two of them, 300 and 318, recognize determinants conserved in fish, chicken, mouse, rat, rabbit, monkey and human Calbindin D-28k. We demonstrate their use in the immunohistochemical localization of Calbindin D-28k, and in the detection of Calbindin D-28k on immunoblots.

Characterization of a polyclonal antiserum against the purified human recombinant calcium binding protein calretinin

We have purified recombinant human calretinin (CR) from Escherichia coli lysates and have produced a polyclonal antiserum against it. The antiserum recognizes determinants conserved in fish, chicken, rat, monkey and human CR. We show its use in the qualitative detection of CR by different methods of immunohistochemistry as well as in the detection of CR on immunoblots.

Prolonged contraction-relaxation cycle of fast-twitch muscles in parvalbumin knockout mice

The calcium-binding protein parvalbumin (PV) occurs at high concentrations in fast-contracting vertebrate muscle fibers. Its putative role in facilitating the rapid relaxation of mammalian fast-twitch muscle fibers by acting as a temporary buffer for Ca2+ is still controversial. We generated knockout mice for PV (PV -/-) and compared the Ca2+ transients and the dynamics of contraction of their muscles with those from heterozygous (PV +/-) and wild-type (WT) mice. In the muscles of PV-deficient mice, the decay of intracellular Ca2+ concentration ([Ca2+]i) after 20-ms stimulation was slower compared with WT mice and led to a prolongation of the time required to attain peak twitch tension and to an extension of the half-relaxation time. The integral [Ca2+]i in muscle fibers of PV -/- mice was higher and consequently the force generated during a single twitch was approximately 40% greater than in PV +/- and WT animals. Acceleration of the contraction-relaxation cycle of fast-twitch muscle fibers by PV may confer an advantage in the performance of rapid, phasic movements.The calcium-binding protein parvalbumin (PV) occurs at high concentrations in fast-contracting vertebrate muscle fibers. Its putative role in facilitating the rapid relaxation of mammalian fast-twitch muscle fibers by acting as a temporary buffer for Ca2+ is still controversial. We generated knockout mice for PV (PV -/-) and compared the Ca2+ transients and the dynamics of contraction of their muscles with those from heterozygous (PV +/-) and wild-type (WT) mice. In the muscles of PV-deficient mice, the decay of intracellular Ca2+ concentration ([Ca2+]i) after 20-ms stimulation was slower compared with WT mice and led to a prolongation of the time required to attain peak twitch tension and to an extension of the half-relaxation time. The integral [Ca2+]iin muscle fibers of PV -/- mice was higher and consequently the force generated during a single twitch was ∼40% greater than in PV +/- and WT animals. Acceleration of the contraction-relaxation cycle of fast-twitch muscle fibers by PV may confer an advantage in the performance of rapid, phasic movements.

Parvalbumin-lmmunoreactive Neurons in the Neocortex are Resistant to Degeneration in Alzheimer's Disease

Recent studies have stressed the fact that specific neuronal subtypes may display a differential sensitivity to degeneration in Alzheimers disease. For example, large pyramidal neurons have been shown to be vulnerable, whereas smaller neurons are resistant to pathology. Using a monoclonal antibody against the calcium-binding protein parvalbumin, we investigated the possible changes in a subpopulation of interneurons in two cortical areas known to be strongly damaged in Alzheimers disease. In the prefrontal cortex as well as in the inferior temporal cortex, we observed no differences in parvalbumin-immunoreactive cell counts or cell size in Alzheimers disease brains as compared to control cases. Moreover, the general cellular morphology of these neurons was preserved in the Alzheimers disease cases, in that their perikarya and dendritic arborizations were intact. These results suggest that paravalbumin-immunoreactive cells represent a neuronal subset resistant to degeneration, and further support the hypothesis that the pathological process in Alzheimers disease involves specific neuronal subtypes with particular morphological and molecular characteristics.

Calretinin-immunoreactive neocortical interneurons are unaffected in Alzheimer's disease

Recent studies have revealed that select neuronal populations may display a differential sensitivity to degeneration in Alzheimers disease. For example, large pyramidal neurons have been shown to be vulnerable, whereas small, local circuit neurons appear to be resistant to the pathologic process. More significantly, interneurons that contain the calcium-binding proteins parvalbumin and calbindin are particularly resistant to degeneration in Alzheimers disease. Using a polyclonal antibody to the calcium-binding protein calretinin, we analyzed the possible changes in the subset of interneurons containing this protein in two neocortical areas that are generally devastated in Alzheimers disease. In the prefrontal cortex as well as in the inferior temporal cortex, we observed no difference in the density of calretinin-immunoreactive neurons in Alzheimers disease brains as compared to control cases. Moreover, the cellular morphology of these neurons was well preserved in the Alzheimers disease cases. These data suggest that calretinin-immunoreactive neurons, like other calcium-binding protein-containing interneurons, are resistant to degeneration in Alzheimers disease. The results support the notion that the pathological process in Alzheimers disease involves specific cellular populations sharing particular morphological and neurochemical characteristics. In addition, it is possible that the presence of calcium-binding proteins confers a certain degree of resistance to degeneration in specific neuronal subsets.

Parvalbumin deficiency affects network properties resulting in increased susceptibility to epileptic seizures

Networks of GABAergic interneurons are of utmost importance in generating and promoting synchronous activity and are involved in producing coherent oscillations. These neurons are characterized by their fast-spiking rate and by the expression of the Ca(2+)-binding protein parvalbumin (PV). Alteration of their inhibitory activity has been proposed as a major mechanism leading to epileptic seizures and thus the role of PV in maintaining the stability of neuronal networks was assessed in knockout (PV-/-) mice. Pentylenetetrazole induced generalized tonic-clonic seizures in all genotypes, but the severity of seizures was significantly greater in PV-/- than in PV+/+ animals. Extracellular single-unit activity recorded from over 1000 neurons in vivo in the temporal cortex revealed an increase of units firing regularly and a decrease of cells firing in bursts. In the hippocampus, PV deficiency facilitated the GABA(A)ergic current reversal induced by high-frequency stimulation, a mechanism implied in the generation of epileptic activity. We postulate that PV plays a key role in the regulation of local inhibitory effects exerted by GABAergic interneurons on pyramidal neurons. Through an increase in inhibition, the absence of PV facilitates synchronous activity in the cortex and facilitates hypersynchrony through the depolarizing action of GABA in the hippocampus.

The Calcium Binding Protein Calretinin is a Selective Marker for Malignant Pleural Mesotheliomas of the Epithelial Type

In a series of 23 cases of mesothelioma of either the epithelial, sarcomatoid or the mixed type, the expression of three calcium-binding proteins (calretinin, parvalbumin and calbindin-D28k) was studied using immunohistochemical techniques on paraffin sections. The results show that calretinin is expressed in mesotheliomas of the epithelial type (papillary, adenomatous or solid) and by the epithelial component of the mixed tumours. The immunohistochemical reaction is specific and reproducible. The tissues of the pulmonary parenchyma and of the pleura are negative for calretinin except for the rare fibroblasts and some skeletal muscle fibres situated in the interstices of, or near the epithelial tumour mass. The sarcomatoid mesotheliomas and the sarcomatoid component of the mixed tumours do not express calretinin. Parvalbumin and calbindin-D28k are expressed neither in mesotheliomas nor in normal lung tissue. Primary adenocarcinomas of the lung are negative for all three calcium binding proteins cited. Thus, calretinin seems to represent a selective marker for mesotheliomas of the epithelial type and allows their differentiation from metastases of lung adenocarcinomas.

Alterations in Purkinje cell spines of calbindin D-28 k and parvalbumin knock-out mice

The second messenger Ca2+ is known to act in a broad spectrum of fundamental cell processes, including modifications of cell shape and motility, through the intermediary of intracellular calcium‐binding proteins. The possible impact of the lack of the intracellular soluble Ca2+‐binding proteins parvalbumin (PV) and calbindin D‐28 k (CB) was tested on spine morphology and topology in Purkinje cell dendrites of genetically modified mice. Three different genotypes were studied, i.e. PV or CB single knock‐out (PV–/–, CB–/–) and PV and CB double knock‐out mice (PV–/–CB–/–). Purkinje cells were microinjected with Lucifer Yellow and terminal dendrites scanned at high resolution with a confocal laser microscope followed by three‐dimensional (3‐D) reconstruction. The absence of PV had no significant effect on spine morphology, whereas the absence of CB resulted in a slight increase of various spine parameters, most notably spine length. In double knock‐out mice, the absence of both PV and CB entailed a doubling of spine length, an increase in spine volume and spine surface, a higher spine density along the dendrites, as well as a more clustered spine distribution. In all three genotypes, a reduction in the number of stubby spines was observed compared with wild‐type animals. These results suggest a morphological compensation for the lack of the soluble calcium buffers in the cytoplasm of Purkinje cell dendritic spines. The increase in various spine parameters, particularly volume, may counteract the lack of the calcium buffers, such as to adjust Ca2+‐transients at the transitional zone between spines and dendrites.