Calcium-binding protein and some neuropeptides in the retina of Octopus vulgaris: A morpho-histochemical study.

The existence of both calcium-binding proteins (CBPs) and neuropeptides in the retina and brain of various species of vertebrates and invertebrates is well documented. Octopus retina is particularly interesting because it represents a case of convergent evolution. The aim of this study was to characterize the distribution of two CBPs, calretinin and calbindin, in Octopus retina using morphology, in situ hybridization, immunocytochemistry and Western blot. Calretinin-like immunoreactivity was found in the photoreceptor cells, but unexpectedly also in the supporting cells. In situ hybridization and Western blot analysis confirmed these results. No immunoreactivity was found for calbindin. Two neuropeptides, Substance P and calcitonin gene-related peptide (CGRP), as well as neurofilament protein and glial fibrillary acidic protein were also localized in the Octopus retina by immunocytochemistry. Our work provides new insights about calcium-binding proteins and neuropeptide distribution in Octopus retina and suggests a functional role for calretinin, a highly conserved protein, in visual signal transduction of cephalopods.

Calretinin Immunoreactivity in the Human Testis Throughout Fetal Life.

The main functions of the testis are sex hormone and sperm cell production. Steroidogenesis occurs in the Leydig interstitial cells and spermatogenesis in the seminiferous tubules. Male gonad morphogenesis is a finely orchestrated process, mainly coordinated by hormones, whose actions can significantly affect post-pubertal testicular function. Calcium is a key intracellular messenger, which regulates many signal transduction pathways, and is also implicated in steroidogenesis. Calcium homeostasis and signaling rely on many calcium-binding proteins including calretinin, of the "EF-hand" protein family. Calretinin is a highly conserved protein mainly expressed in the nervous system but also detected in rat and human adult and fetal testis as well as in pathological conditions. Calretinin expression in the fetal testis, however, has not been thoroughly analyzed probably owing to limited availability and paucity of tissues. Here, we examined by immunocytochemistry the expression of calretinin in human fetal testis specimens, obtained from natural and therapeutic abortions, at various developmental ages. We found that calretinin-immunoreactive Leydig cells were visible throughout the timeframe studied (14th-27th week). Immunoreactivity was also observed in Sertoli cells and in the germ cells of the immature seminiferous tubules. Overall our data indicate that calretinin expression parallels the decline in Leydig cell number, suggesting that its presence is indeed correlated to their steroidogenic activity. They also suggest that the intratubular positivity of calretinin could be linked to the ability of Sertoli cells to produce locally acting hormones contributing to the histodifferentiation of the male genital tract. J. Cell. Physiol. 232: 1872-1878, 2017. © 2016 Wiley Periodicals, Inc.

Distribution of the c-MYC gene product in colorectal neoplasia.

AIMS: Recent attempts to study MYC distribution in human samples have been confounded by a lack of agreement in immunohistochemical staining between antibodies targeting the N-terminus and those targeting the C-terminus of the MYC protein. The aim of this study was to use a novel in-situ hybridization (ISH) approach to detect MYC mRNA in clinically relevant samples, and thereby determine the reliability of MYC-targeting antibodies. METHODS AND RESULTS: We performed immunohistochemistry on human formalin-fixed paraffin embedded normal colon (n = 15), hyperplastic polyp (n = 4) and neoplastic colon samples (n = 55), using the N-terminally directed antibody Y69, and the C-terminally directed antibody 9E10. The MYC protein distributions were then compared with the location of MYC mRNA, determined by ISH. We found that the localization of MYC mRNA correlated well with the protein distribution determined with the N-terminally directed antibody Y69, and was also associated with expression of the proliferation marker Ki67. The protein distribution determined with the C-terminally directed antibody 9E10 was not always associated with MYC mRNA, Y69, or Ki67, and indeed often showed a reciprocal pattern of expression, with staining being strongest in non-proliferating cells. CONCLUSIONS: The observed discrepancy between the staining patterns suggests that the significance of 9E10 in immunohistochemical staining is currently uncertain, and therefore should be interpreted with caution.

Differential expression of E-cadherin and catenins in ovarian sex cord stromal tumours.

AIMS: Sex cord stromal tumours (SCSTs) of the ovary encompass several histological tumour subtypes that are defined by characteristic histological features. Some can show morphological overlap with other subtypes of SCSTs, as well as with non-SCSTs. The E-cadherin/catenin complex constitutes the adherens junction, which is well developed in epithelial tissue, but the constituent molecules are also expressed in several non-epithelial tumours. The aim of this study was to determine whether the expression patterns of E-cadherin and catenins in ovarian SCSTs can be of diagnostic utility. METHODS AND RESULTS: We studied the expression of E-cadherin, α-, ß- and γ-catenin in 55 tumours using immunohistochemistry. We found that all tumour subtypes showed nuclear expression of E-cadherin, while only microcystic stromal tumours (MCSTs) displayed a distinct profile, with nuclear localization of all three catenins in almost all cases. CONCLUSIONS: We conclude that the E-cadherin expression profile in SCSTs can assist in distinguishing between SCSTs and non-SCSTs in which there is no nuclear expression of E-cadherin. The nuclear localization of catenins may be of potential use in distinguishing MCST from other subtypes of SCST.

Calcium/Calmodulin-Dependent Kinases in the Hypothalamus, Pituitary, and Pineal Gland: An Overview.

We review the literature on the little-known roles of specific CaMKs in regulating endocrine functions of the pineal gland, the pituitary gland, and the hypothalamus. Melatonin activates hippocampal CaMKII, which then influences dendritogenesis. In the pituitary gland, the signal pathways activated by the CaMK in lower vertebrates, such as fishes, differ from those of mammals. In the teleost anterior pituitary, the activation of CaMKII induces the expression of somatolactin by glucagon b. In rats and humans, CaMKIVs have been associated with gonadotropes and thyrotropes and CaMKII with several types of human tumor cells and with a specific signaling pathway. Neuropeptides such as vasopressin and endothelin are also involved in the CaMKII signaling chain, as is the CaMKIIδ isoform which participates in generating the circadian rhythms of the suprachiasmatic nucleus. What arises from this review is that most of the hypothalamic CaMKs are involved in activities of the endocrine brain. Furthermore, among the CaMKs, type II occurs with the highest frequency followed by CaMKIV and CaMKI.

Correlation of CXCL12 expression and FoxP3+ cell infiltration with human papillomavirus infection and clinicopathological progression of cervical cancer.

Human cervical cancer is an immunogenic tumor with a defined pattern of histopathological and clinical progression. Tumor-infiltrating T cells contribute to immune control of this tumor; however, cervical cancer dysregulates this immune response both through its association with human papillomavirus (HPV) infection and by producing cytokines and chemokines. Animal tumor models have revealed associations between overproduction of the chemokine stromal cell-derived factor-1 (SDF-1 or CXCL12) and dysregulation of tumor-specific immunity. We therefore proposed that CXCL12 expression by cervical precancerous and cancerous lesions correlates with histopathological progression, loss of immune control of the tumor, and HPV infection. We found a significant association between cancer stage and CXCL12 expression for squamous and glandular lesions as well as with the HPV16+ (high-risk) status of the neoplastic lesions. Cancer progression was correlated with increasing levels of FoxP3 T-cell infiltration in the tumor. FoxP3 and CXCL12 expression significantly correlated for squamous and glandular neoplastic lesions. These observations were supported by enzyme-linked immunosorbent assay and Western blotting. In addition, we demonstrated CXCL12 expression by dyskaryotic cells in ThinPrep cervical smears. This study robustly links increased CXCL12 expression and FoxP3(+)-cell infiltration to HPV infection and progression of cervical cancer. It supports the detection of CXCL12 in cervical smears and biopsies as an additional biomarker for this disease.

Copper/Zinc Superoxide Dismutase in Human Skin: Current Knowledge.

Superoxide dismutase is widespread in the human body, including skin and its appendages. Here, we focus on human skin copper/zinc superoxide dismutase, the enzyme that protects skin and its appendages against reactive oxygen species. Human skin copper/zinc superoxide dismutase resides in the cytoplasm of keratinocytes, where up to 90% of cellular reactive oxygen species is produced. Factors other than cell type, such as gender, age and diseased state influence its location in skin tissues. We review current knowledge of skin copper/zinc superoxide dismutase including recent studies in an attempt to contribute to solving the question of its remaining unexplained functions. The research described here may be applicable to pathologies associated with oxidative stress. However, recent studies on copper/zinc superoxide dismutase in yeast reveal that its predominant function may be in signaling pathways rather than in scavenging superoxide ions. If confirmed in the skin, novel approaches might be developed to unravel the enzyme's remaining mysteries.

Novel zinc-based fixative for high quality DNA, RNA and protein analysis.

We have developed a reliable, cost-effective and non-toxic fixative to meet the needs of contemporary molecular pathobiology research, particularly in respect of RNA and DNA integrity. The effects of 25 different fixative recipes on the fixed quality of tissues from C57BL/6 mice were investigated. Results from IHC, PCR, RT-PCR, RNA Agilent Bioanalyser and Real-Time PCR showed that a novel zinc-based fixative (Z7) containing zinc trifluoroacetate, zinc chloride and calcium acetate was significantly better than the standard zinc-based fixative (Z2) and neutral buffered formalin (NBF) for DNA, RNA and protein preservation. DNA sequences up to 2.4 kb in length and RNA fragments up to 361 bp in length were successfully amplified from Z7 fixed tissues, as demonstrated by PCR, RT-PCR and Real-Time PCR. Total protein analysis was achieved using 2-D gel electrophoresis. In addition, nucleic acids and proteins were very stable over a 6-14-month period. This improved, non-toxic and economical tissue fixative could be applied for routine use in pathology laboratories to permit subsequent genomic/proteomic studies.

Copper/Zinc-Superoxide Dismutase in Human Epidermis: An Immunochemical Study.

The localization of copper and zinc-superoxide dismutase in normal and neoplastic human skin was examined with immunochemical techniques. Skin samples were taken from males and females of different ages, UV exposed and non-exposed areas and basal-/spino-cellular carcinomas. The enzyme was localized diffusely in the cytoplasm and was also found in the nuclei of epidermal cells, endothelial cells and other dermis cell types. The dismutase content in the epidermis was higher in males than females, UV-exposed than non-exposed and young than old people. In the tumors, the enzyme content of the superficial epidermal layers was higher than in the deep tumoral epithelial cells. These data suggest that the localization of Cu, Zn-SOD in skin tissues reflects the gender and age of the subject, the cell types and their normal or diseased state. Further studies based on the investigation of systemic changes of this enzyme in physiological and pathological epidermis could provide additional information on tumor cell generation.

Sporadic and endemic Burkitt lymphoma have frequent FOXO1 mutations but distinct hotspots in the AKT recognition motif.

FOXO1 has an oncogenic role in adult germinal center-derived lymphomas, in which mutations, predominately within the AKT recognition motif, cause nuclear retention of FOXO1, resulting in increased cell proliferation. To determine the prevalence and distribution of FOXO1 mutations in pediatric Burkitt lymphoma (BL), we sequenced a large number of sporadic and endemic BL patient samples. We report a high frequency of FOXO1 mutations in both sporadic and endemic BL at diagnosis, occurring in 23/78 (29%) and 48/89 (54%) samples, respectively, as well as 8/16 (50%) cases at relapse. Mutations of T24 were the most common in sporadic BL but were rare in endemic cases, in which mutations of residue S22, also within the AKT recognition motif, were the most frequent. FOXO1 mutations were almost always present in the major tumor cell clone but were not associated with outcome. Analysis of other recurrent mutations reported in BL revealed that FOXO1 mutations were associated with mutations of DDX3X and ARID1A, but not MYC, TCF3/ID3, or members of the phosphatidylinositol 3-kinase signaling pathway. We further show common nuclear retention of the FOXO1 protein, irrespective of mutation status, suggesting alternative unknown mechanisms for maintaining FOXO1 transcriptional activity in BL. CRISPR/Cas9 knockout of FOXO1 in an endemic cell line produced a significant decrease in cell proliferation, supporting an oncogenic role for FOXO1 in endemic BL. Thus, FOXO1 is frequently mutated in both sporadic and endemic BL and may offer a potential therapeutic target for pediatric BL patients worldwide.

The status of epidermal growth factor receptor in borderline ovarian tumours.

The majority of borderline ovarian tumours (BOTs) behave in a benign fashion, but some may show aggressive behavior. The reason behind this has not been elucidated. The epidermal growth factor receptor (EGFR) is known to contribute to cell survival signals as well as metastatic potential of some tumours. EGFR expression and gene status have not been thoroughly investigated in BOTs as it has in ovarian carcinomas. In this study we explore protein expression as well as gene mutations and amplifications of EGFR in BOTs in comparison to a subset of other epithelial ovarian tumours. We studied 85 tumours, including 61 BOTs, 10 low grade serous carcinomas (LGSCs), 9 high grade serous carcinomas (HGSCs) and 5 benign epithelial tumours. EGFR protein expression was studied using immunohistochemistry. Mutations were investigated by Sanger sequencing exons 18-21 of the tyrosine kinase domain of EGFR. Cases with comparatively higher protein expression were examined for gene amplification by chromogenic in situ hybridization. We also studied the tumours for KRAS and BRAF mutations. Immunohistochemistry results revealed both cytoplasmic and nuclear EGFR expression with variable degrees between tumours. The level of nuclear localization was relatively higher in BOTs and LGSCs as compared to HGSCs or benign tumours. The degree of nuclear expression of BOTs showed no significant difference from that in LGSCs (mean ranks 36.48, 33.05, respectively, p=0.625), but was significantly higher than in HGSCs (mean ranks: 38.88, 12.61 respectively, p< 0.001) and benign tumours (mean ranks: 35.18, 13.00 respectively, p= 0.010). Cytoplasmic expression level was higher in LGSCs. No EGFR gene mutations or amplification were identified, yet different polymorphisms were detected. Five different types of point mutations in the KRAS gene and the V600E BRAF mutation were detected exclusively in BOTs and LGSCs. Our study reports for the first time nuclear localization of EGFR in BOTs. The nuclear localization similarities between BOTs and LGSCs and not HGSCs support the hypothesis suggesting evolution of LGSCs from BOTs. We also confirm that EGFR mutations and amplifications are not molecular events in the pathogenesis of BOTs.

Centrocytoid plasma cells of the germinal center.

Germinal centers within the lymph node follicles are T-cell-dependent, antigen-driven B-cell proliferations that develop from the rapid clonal expansion of a few founder cells. The end results of this B-cell expansion are memory B cells or plasma cells. Two morphologic forms of plasma cell can be recognized in the germinal center: classic plasma cells, characterized morphologically by peripherally clumped arrangement of nuclear chromatin, and cells with a nuclear morphology more resembling that of the centrocytes, which the authors have termed "centrocytoid plasma cells." In this study the authors examined the distribution and immunohistochemical characteristics of these two populations of germinal center plasma cells. The centrocytoid plasma cells were arranged in a band stretching from the junction of the dark and light zone to the periphery of the germinal centers, while the classic plasma cells were mainly present at the germinal center periphery. Both marked with CD38, CD138, and VS38c, recognized markers for plasma cells; however, EMA and CD31 were present only in the classic form of plasma cell. The proliferation marker Ki67 was present in less than 1% of the cells labeling with CD138. Others have demonstrated Ki67 in 50% of the cells labeled with Blimp-1, which is consistent with Blimp-1 appearing earlier than CD138 in ontogeny. CD10 is co-expressed with CD138 in about 10% of cells and CD45 with CD138 in about 5% of cells. Their topographic features, together with the progressive acquisition of plasma cell markers, suggest that the centrocytoid plasma cells may be the precursors of the classic plasma cells. Of note, both the forms of plasma cell were absent in follicles of follicular lymphoma, which supports the concept that in this disease, lymphocytes fail to differentiate and mature beyond the centrocyte stage.

Advances in immunocytochemistry.

Recent advances in immunohisto/cytochemical methods have been directed towards new fluorescent labels, and to increasing sensitivity and improving methods for multiple immunostaining. The newest fluorescent dyes come in many colours, are much more stable than fluorescein isothiocyanate and give brighter fluorescence. The greatest impact on sensitivity comes from heat-induced antigen retrieval on paraffin sections. Biotinylated tyramide can also increase enormously the amount of label on a preparation. The immunogold method with silver enhancement has been improved by nanogold, which is smaller than colloidal gold, attracts more silver and increases the sensitivity even further. The difficulties of multiple labelling with two or more primary antibodies raised in the same species due to cross-binding between reagents have largely been overcome. There are new methods of blocking spare reactive sites on the first reaction by strong heat, e.g. from microwaving in buffer. Alternatively, one of the antibodies can be directly biotinylated very easily with a new commercially available kit.

Evaluation of NMP179 for the detection of squamous intraepithelial neoplasia in ThinPrep cervical slides using a combination of double immunostaining and morphometric methods of analysis.

This study investigates the potential value of the nuclear matrix protein NMP179 as a marker of abnormal squamous cells in ThinPrep slides. Forty-six cervical scrapes were collected as cell suspensions and ThinPrep slides were prepared. They were double-immunostained for NMP179 and Cytokeratin 18 (CK18), an endocervical cell marker. The method of analysis adopted for the study was designed to distinguish the abnormal squamous cells from benign epithelial cell so that the percentages of abnormal squamous cells that expressed the marker could accurately be determined. Initially, an attempt was made to identify benign and abnormal cells in the ThinPrep slides on the basis of their morphology and immunostaining patterns. Discrimination between the various types of epithelial cells was incomplete using this approach and a more precise method of discrimination between the different epithelial cell types was carried out using a combination of double immunostaining (NMP179 and CK18) and morphometry using nuclear area and nuclear cytoplasmic ratios. Once the different epithelial cell types had been identified, the specificity and sensitivity of NMP179 were determined. The optimal sensitivity (89.9%) was achieved at the N/C ratio 0.36; however, the specificity of NMP179 was very low for all N/C ratios and ranged from 38.8% to 42.2%.

The frequency and significance of WT-1 expression in serous endometrial carcinoma.

Serous endometrial carcinoma is an aggressive type of endometrial carcinoma. Wilms tumor gene 1 (WT-1) is commonly expressed in ovarian serous carcinomas and considered a diagnostic marker of these tumors. However, it is generally believed that WT-1 is rarely expressed by endometrial serous carcinoma. The aim of this study was to evaluate the frequency and significance of WT-1 expression in endometrial serous carcinoma. We studied the expression of WT-1 in formalin-fixed, paraffin-embedded tumor sections from 77 cases of endometrial serous carcinoma. Thirty-four tumors showed positive expression for WT-1 (44%). There was a statistically significant association between the presence of WT-1 expression and disease-free survival (DFS), where patients with tumors expressing WT-1 had a shorter DFS compared with those with no WT-1 expression (P = .031; median DFS, 15 and 38 months, respectively). By multivariate Cox regression analysis, DFS was independent from other clinicopathological data (tumor stage, presence of lymphovascular space invasion, cervical involvement, and extrauterine spread), indicating that WT-1 expression is independently associated with DFS. Our study shows that WT-1 is expressed in a considerable percentage of endometrial serous carcinomas, suggesting a role for WT-1 in the pathology of these tumors. This has therapeutic significance, as WT-1 is an emerging target for immunotherapy. Moreover, our results show that WT-1 has prognostic value, being predictive of DFS. As a potential prognostic marker and therapeutic target, we recommend that WT-1 expression should be included in histopathologic reports of endometrial serous carcinoma.

The expression of interleukin (IL)-6, IL-8, and their receptors in fallopian tubes with ectopic tubal gestation.

OBJECTIVE: To investigate the level of expression of the cytokines interleukin (IL) -8 and IL-6 and their receptors in fallopian tubes with tubal ectopic gestation. DESIGN: Immunohistochemistry (IHC) was used to study the expression of IL-8, CXCR1, CXCR2, IL-6, and IL-6 receptor alpha in fallopian tubes with tubal ectopic gestation in comparison with normal fallopian tubes. SETTING: Hospital. PATIENT(S): We studied fallopian tubes from 50 patients with tubal ectopic gestation and normal fallopian tubes from 25 patients who had hysterectomy and bilateral salpingo-oopherectomy for uterine or ovarian lesions. INTERVENTION(S): The expression of the chemokines/cytokines and their receptors was evaluated by IHC. MAIN OUTCOME MEASURE(S): Level of expression and tissue distribution of the different antigens. RESULT(S): IHC results showed that the expression levels of IL-6 and IL-8, as well as CXCR1, were significantly up-regulated, particularly near the implantation site in fallopian tubes with tubal gestation, while the expression levels of CXCR2 and IL-6Rα were not changed in comparison with normal fallopian tubes. CONCLUSION(S): The results suggest a role for IL-6, IL-8, and CXCR1 in the pathophysiology of tubal ectopic gestation.

Burkitt's lymphoma: maximising the use of fine needle aspirates by long-term preservation for diagnosis and research.

Fine needle aspirates from Burkitt's lymphoma and other tumours transferred directly into ThinPrep® PreservCyt® (Cytyc UK Ltd, Crawley, UK) buffered alcohol fixative retain their cellular and viral antigens and nucleic acids for many months at ambient temperatures. Despite the presence of blood and debris, cells dried onto slides from droplets and post-fixed in formalin, or sections of paraffin-embedded cell blocks from formalin post-fixed pellets, prove adequate for morphology, immunocytochemistry, in-situ hybridization and molecular biological analyses. Where there is lack of expertise in making thin smears or hospitals lack pathology laboratories and services, PreservCyt® provides an excellent medium for transport elsewhere for diagnosis and research.

Nitric oxide regulates the release of somatostatin from cultured gastric rabbit primary D-cells.

BACKGROUND & AIMS: Neuronal nitric oxide synthase (nNOS) is present in gastric D-cells. Mucosal somatostatin is diminished in H. pylori gastritis, where production of nitric oxide (NO) is increased. Therefore, we investigated the role of NO in D-cell function and the effects of prolonged exposure of D-cells to NO. METHODS: Rabbit gastric D-cells were cultured. Somatostatin-14 was measured after 2 hours to examine the effects of arginine, nitric oxide sythase (NOS) inhibitors, and NO donors. Some cells were preincubated with a slow releasing NO donor for 12 hours. Results are expressed as percentage of total cell content. Nitrate content was measured by chemiluminescent assay. RESULTS: L-arginine increased somatostatin-14 release in the presence of CCK8 from 4.4% +/- 0.5% to 6.4% +/- 0.4% (P < 0.02), and this was accompanied by NO release from 27 +/- 7 micromol/L to 86 +/- 12 micromol/L (P = 0.001). D-arginine and L-lysine had no effect. NOS inhibitors LNNA, SMT, and 7NI significantly attenuated the stimulatory response to L-arginine. NO donors sodium nitroprusside (SNP), 1 mmol/L, and S-nitroso-N-acetyl-D-L-penicillamine, 0.1 mmol/L, significantly increased basal and cholecystokinin-8 (CCK8) stimulated somatostatin release. Oxyhemoglobin attenuated the effect of SNP but not of L-arginine. Neither cyclic guanosine monophosphate nor guanylate cyclase were involved in the response to NO. However, inhibition of adenosine diphosphate (ADP) ribosyltransferase significantly decreased the response to L-arginine. Preincubation for 12 hours with 150 micromol/L (Z)-1-[(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate; IP3, inositol triphosphate decreased the 2-hour cellular response to CCK8 and SNP. CONCLUSIONS: NO regulates rabbit D-cells. Acute exposure stimulates somatostatin mediated by ADP ribosylation, whereas long-term exposure reduces cellular responses to stimuli. The latter pathway may be responsible for the suppression of somatostatin in H. pylori gastritis.

Expression of lymphotoxin-beta (LT-beta) in chronic inflammatory conditions.

Functional studies in gene-knockout and transgenic mice systems have shown that lymphotoxin-alpha and lymphotoxin-beta (LT-alpha and LT-beta) are of fundamental importance in peripheral lymphoid organ development, but it remains unclear what role these cytokines have to play in the adult immune response and in the pathogenesis of disease. In this study, a polyclonal anti-serum to human LT-beta was used to investigate the distribution of LT-beta by immunohistochemistry in normal and diseased tissues. In the gut, lymph nodes, spleen, and tonsil, there was some LT-beta present on a variety of lymphoid cell types. In contrast, strong staining for LT-beta was observed on plasma cells and a subpopulation of CD4+ T cells in tissues affected by chronic inflammatory disease or infection, for example in inflammatory bowel disease, and in lymph nodes obtained from patients with sarcoidosis and tuberculosis. In tuberculous and sarcoid lymph nodes, LT-beta expression also occurred on some but not all epithelioid histiocytes within granulomas and on multi-nucleated giant cells. These findings support a role for LT-beta in human disease and suggest that it might represent a therapeutic target in a variety of common infective or inflammatory disorders.