P16 as a marker of carcinoma in effusions and peritoneal washing.

BACKGROUND: Considering the potential of p16 as a marker for diagnosis, prognosis and therapeutic response, the aim of this study was to assess its presence, via immunocytochemistry, in metastatic carcinoma of different primary sites and histological types obtained from effusions and peritoneal washings. A total of 118 samples including 85 of metastatic carcinoma and 33 samples of benign effusion/peritoneal washing were prepared by the plasma/thromboplastin method. Immunocytochemistry reactions were performed on cell block sections using antibodies against p16, claudin-4, MOC-31, calretinin, HBME and CD68. RESULTS: P16 overexpression was observed in 88.23% of all carcinoma samples. All cervix adenocarcinoma samples showed p16 overexpression. Overexpression in adenocarcinomas of ovary, lung and breast was observed in 93.75, 93.10 and 75% of the samples, respectively. Overexpression was observed in all different histological types analyzed: small cell carcinoma (lung), squamous cell carcinoma (cervical) and urothelial carcinoma (bladder). The specificity of p16 for carcinoma detection was of 96.96%. CONCLUSION: Overexpression of p16 was observed in most metastatic carcinoma, from different primary sites and histological types, obtained from effusions and peritoneal washings. Due to its high frequency of overexpression in metastatic carcinoma, p16 may play a possible role in tumor progression and it may be considered as a complementary diagnostic marker depending on histological type and primary site of carcinoma.

Diagnostic Accuracy of a Limited Immuno-panel of Calretinin and Ber-EP4 for Diagnosis of Malignant Effusions.

OBJECTIVE: To differentiate between adenocarcinoma cells and reactive mesothelial cells (RMC) in serous effusions using a limited immuno-panel of Ber-EP4 and Calretinin. STUDY DESIGN: Descriptive study. PLACE AND DURATION OF STUDY: Department of Pathology, Allama Iqbal Medical College, Lahore, in collaboration with the Departments of Surgery, Pulmonology and Oncology, Jinnah Hospital, Lahore, from March 2015 to March 2016. METHODOLOGY: Ninety-seven clinically and radiologically proven cases of peritoneal and pleural effusion and peritoneal wash of patients with suspicion of malignancy were included in the present study. Diagnostic accuracy of a limited immuno-panel of Calretinin and Ber-EP4 for diagnosis of malignant effusions was calculated using histopathology as gold standard. RESULTS: The sensitivity of Ber-EP4 for malignant cases was 98.6%, specificity 100%, positive predictive value (PPV) 100%, negative predictive value (NPV) 96%, and diagnostic accuracy 98.9%. Sensitivity of Calretinin as positive staining for RMC was 79.2%, specificity and positive predictive value (PPV) 100%, negative predictive value (NPV) 93.6%, and diagnostic accuracy 94.8%. CONCLUSION: Limited immuno-panel of Calretinin and Ber-EP4 had a high positive and negative predictive value and is cost-effective in resource limited set-up for identification of adenocarcinoma cells and reactive mesothelial cells in challenging cases of serous effusions.

Ecto-Calreticulin is essential for an efficient immunogenic cell death stimulation in mouse melanoma.

Skin melanoma remains one of the most aggressive and difficult to treat human malignancy, with an increasing incidence every year. Although surgical resection represents the best therapeutic approach, this is only feasible in cases of early diagnosis. Furthermore, the established malignancy is resistant to all therapeutic strategies employed so far, resulting in an unacceptable patient survival rate. Although the immune-mediated therapeutic approaches, based on anti-PD1 or anti-CTLA4, are very promising and under clinical trial experimentation, they could conceal not yet fully emerged pitfalls such as the development of autoimmune diseases. Therefore, alternative therapeutic approaches are still under investigation, such as the immunogenic cell death (ICD) process. Here we show that the lack of calreticulin translocation onto mouse melanoma cell membrane prevents the stimulation of an effective ICD response in vivo.

Glypican-1 immunohistochemistry is a novel marker to differentiate epithelioid mesothelioma from lung adenocarcinoma.

Histological morphology alone is not sufficient for the pathological diagnosis of malignant mesothelioma. Positive and negative immunohistochemical markers are necessary to differentiate it from lung adenocarcinoma. As calretinin and D2-40, the recognized positive markers of mesothelioma, are expressed in lung adenocarcinoma to some extent, novel markers with high specificity are desirable. In this study, we investigated the applicability of glypican-1 immunohistochemistry to differentiate epithelioid mesothelioma from lung adenocarcinoma. We investigated 82 cases of epithelioid mesothelioma and 97 cases of lung adenocarcinoma for glypican-1 expression by immunohistochemistry using a commercially available antibody. All 82 cases of epithelioid mesothelioma showed glypican-1 expression, most with diffuse and strong reactivity. In contrast, only three cases of lung adenocarcinoma showed focal glypican-1 expression. Glypican-1 expression showed 100 sensitivity, 97% specificity, and a 98% accuracy rate to differentiate epithelioid mesothelioma from lung adenocarcinoma. The sensitivity of glypican -1 immunohistochemistry is as high as that of calretinin and D2-40, and its specificity is far better than that of calretinin and D2-40. Therefore, we recommend including glypican -1 immunohistochemistry as a positive marker of epithelioid mesothelioma.

Immunoreactivities of calbindin­D28k, calretinin and parvalbumin in the somatosensory cortex of rodents during normal aging.

Calbindin­D28k (CB), calretinin (CR) and parvalbumin (PV), which regulate cytosolic free Ca2+ concentrations in neurons, are chemically expressed in γ­aminobutyric acid (GABA)ergic neurons that regulate the degree of glutamatergic excitation and output of projection neurons. The present study investigated age­associated differences in CB, CR and PV immunoreactivities in the somatosensory cortex in three species (mice, rats and gerbils) of young (1 month), adult (6 months) and aged (24 months) rodents, using immunohistochemistry and western blotting. Abundant CB­immunoreactive neurons were distributed in layers II and III, and age­associated alterations in their number were different according to the species. CR­immunoreactive neurons were not abundant in all layers; however, the number of CR­immunoreactive neurons was the highest in all adult species. Many PV­immunoreactive neurons were identified in all layers, particularly in layers II and III, and they increased in all layers with age in all species. The present study demonstrated that the distribution pattern of CB­, CR­ and PV­containing neurons in the somatosensory cortex were apparently altered in number with normal aging, and that CB and CR exhibited a tendency to decrease in aged rodents, whereas PV tended to increase with age. These results indicate that CB, CR and PV are markedly altered in the somatosensory cortex, and this change may be associated with normal aging. These findings may aid the elucidation of the mechanisms of aging and geriatric disease.

Identification of Aâ ¡ amacrine, displaced amacrine, and bistratified ganglion cell types in human retina with antibodies against calretinin.

Antibodies against calretinin are markers for one type of rod pathway interneuron (AⅡ amacrine cell) in the retina of some but not all mammalian species. The AⅡ cells play a crucial role in night-time (scotopic) vision and have been proposed as a target for optogenetic restoration of vision in retinal disease. In the present study we aimed to characterize the AⅡ cells in human retina. Postmortem human donor eyes were obtained with ethical approval and processed for calretinin immunofluorescence. Calretinin-positive somas in the inner nuclear and the ganglion cell layer were filled with the lipophilic dye DiI. The large majority (over 80%) of calretinin-immunoreactive cells is located in the inner nuclear layer, is immunopositive for glycine transporter 1, and shows the typical morphology of AⅡ amacrine cells. In addition, a small proportion of calretinin-positive cells in the inner nuclear layer and in the ganglion cell layer is glutamic acid decarboxylase-positive and shows the morphology of widefield amacrine cells (stellate, semilunar, and thorny amacrine cells). About half of the calretinin cells in the ganglion cell layer are bistratified ganglion cells resembling the small bistratified (presumed blue-ON/yellow-OFF) and the G17 ganglion cell previously described in primates. We conclude that in human retina, antibodies against calretinin can be used to identify AⅡ amacrine cells in the inner nuclear layer as well as widefield amacrine and small bistratified ganglion cells in the ganglion cell layer.

Regulation of calreticulin-major histocompatibility complex (MHC) class I interactions by ATP.

The MHC class I peptide loading complex (PLC) facilitates the assembly of MHC class I molecules with peptides, but factors that regulate the stability and dynamics of the assembly complex are largely uncharacterized. Based on initial findings that ATP, in addition to MHC class I-specific peptide, is able to induce MHC class I dissociation from the PLC, we investigated the interaction of ATP with the chaperone calreticulin, an endoplasmic reticulum (ER) luminal, calcium-binding component of the PLC that is known to bind ATP. We combined computational and experimental measurements to identify residues within the globular domain of calreticulin, in proximity to the high-affinity calcium-binding site, that are important for high-affinity ATP binding and for ATPase activity. High-affinity calcium binding by calreticulin is required for optimal nucleotide binding, but both ATP and ADP destabilize enthalpy-driven high-affinity calcium binding to calreticulin. ATP also selectively destabilizes the interaction of calreticulin with cellular substrates, including MHC class I molecules. Calreticulin mutants that affect ATP or high-affinity calcium binding display prolonged associations with monoglucosylated forms of cellular MHC class I, delaying MHC class I dissociation from the PLC and their transit through the secretory pathway. These studies reveal central roles for ATP and calcium binding as regulators of calreticulin-substrate interactions and as key determinants of PLC dynamics.

Intrinsic neuroendocrine cells in the outer wall of the human pyriform recess.

The extrinsic neural supply of the hypopharynx is well established. However, little is known about the intrinsic neurons and neuroendocrine cells (NECs) of the human hypopharynx that are under the influence of the extrinsic nerves. We aimed to identify and characterize such cell populations within the outer wall of the pyriform recess. We applied antibodies for neuron-specific enolase (NSE), calretinin (CR) and neurofilaments (NF) to autopsy samples from four donor cadavers. Within the lamina propria and the muscle layer of the pyriform recess outer wall, usually in perivascular areas, we found NSE-, CR- and NF-positive cells, mostly apolar, that were considered on a histological and immunohistochemical basis to be NECs. Although these cells have not, to our knowledge, been described previously in this anatomical location, their presence within the hypopharynx wall may explain the appearance of rare forms of local primary neuroendocrine carcinomas.

Developmental markers of ganglion cells in the enteric nervous system and their application for evaluation of Hirschsprung disease.

Hirschsprung disease (HSCR) is a congenital disease resulting from failure of neural crest-derived ganglion cells to colonize the colon. Conventional diagnostic methods are insufficient for evaluating the 'functional' prognosis of HSCR. In order to elucidate the maturation of ganglion cells, 17 immunohistochemical markers were examined. We examined the digestive tracts of 2 human early delivery patients, 2 miniature swine fetuses, 4 little infants, 3 infants, 3 children, 6 adults, and 3 aged individuals. With increasing age, the labeling index (LI) for both calretinin and tyrosine hydroxylase (TH) increased, whereas that for SOX10 decreased. We then examined the 'transitional zone' of HSCR in 21 affected patients and 18 controls for these three markers. The LI of calretinin and TH were significantly lower than in the controls (median: 3.7 in HSCR and 8.2 in controls, P < 0.001, median: 27.9 in HSCR and 44.4 in controls, P < 0.001, respectively). In contrast, the LI for SOX10 showed no significant difference (median: 33.7 in HSCR and 29.2 in controls, P = 0.666) however, hierarchical cluster analysis was able to divide HSCR patients into two groups. These results suggest that immature ganglion cells are present in the transitional zone of HSCR, and that HSCR may have two different pathophysiological processes.

What neurons hide behind calretinin immunoreactivity in the human gut?

Calretinin (CALR) is often used as an immunohistochemical marker for the histopathological diagnosis of human intestinal neuropathies. However, little is known about its distribution pattern with respect to specific human enteric neuron types. Prior studies revealed CALR in both myenteric and submucosal neurons, most of which colabel with choline acetyl transferase (ChAT). Here, we specified the chemical code of CALR-positive neurons in small and large intestinal wholemounts in a series of 28 patients. Besides other markers, we evaluated the labeling pattern of CALR in combination with vasoactive intestinal peptide (VIP). In colonic submucosa, CALR and VIP were almost completely colocalized in about three-quarters of all submucosal neurons. In the small intestinal submucosa, both the colocalization rate of CALR and VIP as well as the proportion of these neurons were lower (about one-third). In the myenteric plexus of both small intestine and colon, CALR amounted to 11 and 10 %, respectively, whereas VIP to 5 and 4 % of the whole neuron population, respectively. Colocalization of both markers was found in only 2 and 3 % of myenteric neurons, respectively. In section specimens, nerve fibers coreactive for CALR and VIP were found in the mucosa but not in the muscle coat. Summarizing the present and earlier results, CALR was found in at least one submucosal and two myenteric neuron populations. Submucosal CALR+/VIP+/ChAT± neurons innervate mucosal structures. Furthermore, CALR immunoreactivity in the myenteric plexus was observed in morphological type II (supposed primary afferent) and spiny type I (supposed inter- or motor-) neurons.

The changes of calretinin immunoreactivity in paraquat-induced nephrotoxic rats.

Calcium-binding proteins are present in the kidneys: calbindin D-28k in the distal tubules and calretinin in the proximal tubules. Since paraquat causes degeneration in the brush border-bearing proximal tubule cells in rat kidneys, we investigated the changes of calretinin immunoreactivity in the proximal tubule cells of paraquat-induced nephrotoxicity in experimental male Sprague-Dawley rats following chitosan oligosaccharide pretreatment to investigate its protective properties. Paraquat (60 mg/kg) was administered intraperitoneally with or without chitosan oligosaccharide (500 mg/kg, p.o.) pretreatment. The changes on calretinin were compared with those of calbindin D-28k by immunohistochemistry and Western Blot analysis. Calretinin was immunolocalized on the apical surface of proximal tubule cells in the deeper cortex of normal kidney, and disappeared after paraquat administration with minor changes of calbindin D-28k immunoreactivity in the distal tubules and collecting ducts. Chitosan oligosaccharide pretreatment caused increased expression of calretinin and calbindin D-28k before paraquat injection and helped preserve proximal tubules after paraquat treatment. However, Western blot analysis on calretinin and calbindin D-28k could not explain the degeneration of the proximal tubule cells in paraquat-induced nephrotoxicity. These findings suggested that calretinin is a possible and more useful histopathological marker for proximal tubule cells in paraquat-induced nephrotoxic rats.