Calcium/calmodulin-dependent kinases can regulate the TSH expression in the rat pituitary.

PURPOSE: The endocrine secretion of TSH is a finely orchestrated process controlled by the thyrotropin-releasing hormone (TRH). Its homeostasis and signaling rely on many calcium-binding proteins belonging to the "EF-hand" protein family. The Ca2+/calmodulin (CaM) complex is associated with Ca2+/CaM-dependent kinases (Ca2+/CaMK). We have investigated Ca2+/CaMK expression and regulation in the rat pituitary. METHODS: The expression of CaMKII and CaMKIV in rat anterior pituitary cells was shown by immunohistochemistry. Cultured anterior pituitary cells were stimulated by TRH in the presence and absence of KN93, the pharmacological inhibitor of CaMKII and CaMKIV. Western blotting was then used to measure the expression of these kinases and of the cAMP response element-binding protein (CREB). TSH production was measured by RIA after time-dependent stimulation with TRH. Cells were infected with a lentiviral construct coding for CaMKIV followed by measurement of CREB phosphorylation and TSH. RESULTS: Our study shows that two CaM kinases, CaMKII and CaMKII, are expressed in rat pituitary cells and their phosphorylation in response to TRH occurs at different time points, with CaMKIV being activated earlier than CaMKII. TRH induces CREB phosphorylation through the activity of both CaMKII and CaMKIV. The activation of CREB increases TSH gene expression. CaMKIV induces CREB phosphorylation while its dominant negative and KN93 exert the opposite effects. CONCLUSION: Our data indicate that the expression of Ca2+/CaMK in rat anterior pituitary are correlated to the role of CREB in the genetic regulation of TSH, and that TRH stimulation activates CaMKIV, which in turn phosphorylates CREB. This phosphorylation is linked to the production of thyrotropin.

Differential expression of IL-8 and IL-8 receptors in benign, borderline and malignant ovarian epithelial tumours.

INTRODUCTION: Ovarian Cancer is the leading cause of death from gynecological malignancy. The poor prognosis is mainly due to presentation at a late stage and poor response to therapy. Much research is needed to identify diagnostic and prognostic biomarkers as well as therapeutic targets for ovarian cancer. Interleukin-8 is expressed by many tumour types and is known to have mitogenic, motogenic and angiogenic effects on tumour cells. AIMS: The aim of this study was to investigate the expression of IL-8 and IL-8 receptors (IL-8RA and IL-8RB) in different histological subtypes of ovarian tumours, as potential prognostic biomarkers in ovarian tumours. MATERIALS AND METHODS: Immunohitochemistry was used to study the expression of IL-8 and IL-8 receptors in 115 ovarian tumours including 21 benign tumours, 25 borderline tumours and 69 carcinomas of serous, clear cell, endometrioid and mucinous types. The correlation of expression profile, tumour type, stage, and progression free survival and overall survival was statistically analysed. RESULTS: IL-8 and IL-8 receptors were expressed in all types of tumours with variable intensity and subcellular distribution. There was a statistically significant correlation between levels of expression and tumour stage and tumour type, being mostly significant in serous tumours. No correlation with patient progression free survival or overall survival was found. CONCLUSION: This is the first study investigating the expression of IL-8 and IL-8 receptors using immunohistochemistry in different types of ovarian tumours, including benign and borderline tumours. IL-8 and IL-8RA are potential prognostic biomarkers and therapeutic targets in ovarian cancer, particularly in ovarian serous carcinoma.

Purified malignant mammary epithelial cells maintain hormone responsiveness in culture.

Currently, the therapy for breast cancer is determined by immunohistochemical staining of the primary tumour for oestrogen receptor alpha (ERalpha). However, a proportion of ERalpha-positive patients fail to respond to tamoxifen and a proportion of ERalpha-negative patients show response. Here, we describe a novel procedure for the purification of malignant breast epithelial cells in an attempt to identify these patients at an early stage. Using this procedure, we are able to purify malignant cells to >90% purity as determined by immunohistochemical staining, cytology and fluorescent in situ hybridisation (FISH). While the malignant cells can be maintained in culture they do not proliferate in contrast to purified breast epithelial cells from reduction mammoplasties. Moreover, ERalpha and progesterone receptor (PR) expression is maintained in malignant cells, whereas normal epithelial cells rapidly lose ERalpha and PR. Functional studies were performed on the separated malignant cells in terms of their response to oestradiol and tamoxifen. Four out of the seven ERalpha-positive tumours showed a significant reduction in cell numbers after tamoxifen treatment compared to oestradiol, ERalpha negative tumours failed to show a response. We conclude that (a) it is possible to purify and maintain breast cancer cells for a sufficient period to permit functional studies and (b) ERalpha is retained in culture facilitating the use of these cells in studies of the mechanism of endocrine response and resistance in vitro.

Rapid flow carbon dioxide laparoscopy disperses cancer cells into the peritoneal cavity but not the port sites in a new rat model.

BACKGROUND: The role of carbon dioxide (CO2) in the pathogenesis of tumor recurrence after laparoscopy remains controversial. Using a new rat model, we studied the effect of different CO2 flow rates on the dispersal of free cancer cells. METHODS: A novel model of desufflation without trocar was developed, and 55 Fischer rats were randomized into three flow groups: group A (rapid, 0.67 l/min; n = 20), group B (slow, 0.44 l/min; n = 20), and group C (gasless, n = 15). We vented CO2 via a portless surgical valve that filtered cells. After the abdominal wall had been suspended to create space, half of the animals in each group (nonrecovery) received 7.5 x 10(6) immunolabeled rat colon cancer cells (RCC2) intraperitoneally, whereas the other half (recovery) received 7.5 x l0(6) viable RCC2 before insufflation or gasless laparoscopy. Nonrecovery animals were killed after 20 l of insufflation. Parietal peritoneal and port-site specimens were examined for RCC2 by fluorescence microscopy (FM) and flow cytometry (FC). The recovery animals were killed at 4 weeks for evidence of wound recurrence. RESULTS: Nine of 10 nonrecovery animals in A had RCC2 on FM or FC, as compared with 2 animals in each of the nonrecovery groups B and C (p = 0.018, Fisher's exact test). Two of the nine animals in group A also had RCC2 in their portless valves. Two recovery (A) animals developed wound recurrence as compared with none in the other groups (p = 0.315). CONCLUSION: In this model, rapid CO2 flow dispersed free cancer cells into the peritoneal cavity, but not into the port sites, thus supporting a role for CO2 in the intraperitoneal dispersal of free cancer cells, but not in wound recurrence.

Estrogen receptor beta in breast cancer.

Estrogen is essential for normal growth and differentiation in the mammary gland. It also supports growth of approximately 50% of primary breast cancers. For this reason, removal of estrogen or blocking of its action with the anti-estrogen, tamoxifen, is the main treatment for estrogen receptor alpha (ERalpha)-positive tumors. In 1996, when oncologists became aware of a second ER, ERbeta, there was some doubt as to whether this receptor would be of importance in breast cancer because the clinical consensus was that responsiveness to tamoxifen is related to the presence of ERalpha in breast cancer. Today we know that ERalpha and ERbeta have distinct cellular distributions, regulate separate sets of genes and can oppose each other's actions on some genes. We also know that ERbeta is widely expressed in both the normal and malignant breast and that there are proliferating cells in the breast which express ERbeta. In this review we summarize what is known about ERbeta in breast cancer and examine the possibility that ERbeta-selective ligands may well represent a useful class of pharmacological tools with a novel target, namely proliferating cells expressing ERbeta.

Estrogen receptors and proliferation markers in primary and recurrent breast cancer.

To elucidate the clinical importance of estrogen receptor (ER) beta in breast cancer, 29 archival primary breast cancer specimens, six locally recurrent cancers, and five benign mammary tumors were examined histochemically for ERalpha, ERbeta and the proliferation markers Ki67 and cyclin A. In benign tumors, most epithelial cells contained ERbeta, but ERalpha was rare. In primary cancers, both ERalpha and ERbeta occurred in epithelial cells, the presence of ERbeta being associated with elevated expression of Ki67 and cyclin A, and ERalpha with decreased levels. Thus, the highest content of proliferation markers was seen in primary cancers that were ERalpha(-) ERbeta(+). Most Ki67-containing cells coexpressed ERbeta, but few showed ERalpha. In locally recurring cancers, ERalpha, ERbeta, and Ki67 were more highly expressed than in the corresponding primary tumors, and many cells containing ERbeta, but few with ERalpha, expressed Ki67. Surprisingly, ERbeta, but not ERalpha, was seen in the stromal cells of both primary and recurrent cancers. Because the response of breast cancers to tamoxifen therapy is correlated with the presence of ERalpha, cancer cells that lack ERalpha but contain ERbeta and proliferation markers represent a novel population of apparently proliferating cells that probably are not targeted by the current antiestrogens. Thus, appropriate ERbeta-specific ligands, perhaps in combination with tamoxifen, may be useful in improving the treatment of breast cancers.

Enhanced susceptibility to superantigen-associated streptococcal sepsis in human leukocyte antigen-DQ transgenic mice.

Bacterial superantigens are believed to cause septic shock, although, because of the lack of superantigen-sensitive infection models, proof that superantigenicity underlies shock pathogenesis is lacking. This work demonstrates a clear superantigen effect in septic shock resulting from bacterial infection. Transgenic expression of human leukocyte antigen (HLA)-DQ, but not HLA-DR, specifically augments lymphocyte responses to streptococcal pyrogenic exotoxin A (SPEA). HLA-DQ transgenic mice had increased mortality after administration of SPEA or infection with Streptococcus pyogenes. Immune activation during infection was HLA-DQ transgene-dependent and was manifested by Vbeta-specific T cell repertoire changes and widespread lymphoblastic tissue infiltration. Unlike earlier models, which used toxin-induced shock, these T cell superantigen responses and lymphoblastoid changes were observed during invasive streptococcal sepsis. Lymphoid activation was undetectable in HLA-DQ mice infected with an isogenic SPEA(-) strain, which proves that a single superantigen can play a role in sepsis pathogenesis.

Failure of ultrasonic vibration as a means of antigen retrieval in routine diagnostic immunocytochemistry.

It was recently reported that certain antigens can be retrieved from paraffin sections of formalin-fixed tissues by ultrasonication using either an expensive and sophisticated ultrasonic cell disrupter probe (cytokeratins 13 and 16) or an inexpensive and generally available ultrasonic cleaning bath (bcl-1). We wished to investigate the routine use of the latter method and therefore tried to retrieve from various tissues 11 antigens that usually require heat-mediated retrieval in citrate buffer. We applied ultrasonic vibration for periods of 30 seconds to 1.5 minutes in a cleaning bath containing citrate buffer or water, with and without the addition of heat, or for 1 minute in hot citrate buffer after microwaving for 10 minutes in the same buffer. Although a slight effect of ultrasound was noted for a few antigens, in no case did the immunostaining reach the level achieved after standard microwave heating in citrate buffer. We conclude that, under the conditions we used, ultrasonic antigen retrieval cannot be used for immunocytochemistry in a routine histopathology laboratory.

High expression of CD23 in the proliferation centers of chronic lymphocytic leukemia in lymph nodes and spleen.

Chronic lymphocytic leukemia (CLL) is a B-cell neoplasm composed of a heterogeneous mixture of cells, including small lymphocytes, prolymphocytes, and large transformed cells; these last cells appear to represent the proliferating compartment. CLL cells express, in addition to B cell markers, the transmembrane receptor CD23. CD23 functions as the receptor for IgE and also appears to play a role in controlling the growth and proliferation of lymphocytes. Its level of expression among the different cells in CLL has not been examined. In this study, we show that CD23 expression is much higher in the large transformed CLL cells than in the small lymphoid population. This may provide an explanation for the observed correlation between a circulating CD23 cleavage product (soluble CD23) and prognosis in CLL. In addition, we have shown that proliferation in splenic CLL occurs preferentially in the white pulp zones, even in cases in which both the white and red pulp are extensively infiltrated.

E-cadherin/catenin complex in benign and malignant melanocytic lesions.

E-cadherin is a calcium-dependent cell-cell adhesion molecule expressed by melanocytes and responsible for their adhesion to keratinocytes in vitro. In this study, the expression of E-cadherin and its associated cytoplasmic proteins alpha-, beta-, and gamma-catenin was evaluated in melanocytic lesions by immunohistochemistry. E-cadherin expression was evaluated in 70 malignant melanomas and the catenins in 35 of these specimens. Twenty benign melanocytic naevi were also evaluated for E-cadherin and catenin expression. In normal epidermis, E-cadherin/catenin immunostaining was localized at the intercellular borders. In melanomas, a differential loss of E-cadherin expression was observed. Membranous E-cadherin staining was absent in dermal nests of melanomas in their radial growth phase and in Clark level II and III lesions, whereas it was present in a high proportion of melanomas in their vertical growth phase, in Clark level IV and V lesions and in metastasizing melanomas. In contrast, superficial compartments of naevi showed membranous E-cadherin immunoreactivity and junctional naevus cell nests displayed heterogeneous or diffuse cytoplasmic staining. Cytoplasmic alpha- and beta-catenin, but not gamma-catenin staining were detected in all benign and malignant lesions. These findings indicate that qualitative changes in the expression and cellular localization of E-cadherin and of alpha-, beta-, and gamma-catenin occur in melanocytic lesions and may reflect different stages in their progression.

The interaction of CGRP and adrenomedullin with a receptor expressed in the rat pulmonary vascular endothelium.

An abundant, seven trans-membrane domain receptor related to the calcitonin receptor has been studied by a number of groups without identification of its ligand. A recent report claimed that the receptor was a type 1 CGRP receptor (Aiyar et al J. Biol. Chem. 271 11325-11329 (1996)). We have studied the equivalent rat sequence in transfected cells. When expressed in 293 cells the receptor interacts with CGRP and adrenomedullin with KD values of 1.2 nM for CGRP and 11 nM for adrenomedullin. Both ligands cause an elevation of intracellular cAMP with EC50 values of 4 nM and 20 nM respectively and these effects are inhibited by the antagonist CGRP8-37. The receptor is expressed at high levels in the pulmonary vascular endothelium. Both the pharmacological data and the localisation are consistent with the conclusion that the orphan receptor is a type J CGRP receptor. However, when expressed in COS-7 cells, no receptor activity could be demonstrated suggesting that 293 cells contain a factor necessary for functional receptor expression.

The intestine and endocrine pancreas of the African elephant: a histological immunocytochemical and immunofluorescence study.

Histological, immunocytochemical and immunofluorescence methods were employed to study the intestine and endocrine pancreas of the elephant. The histological findings were in line with those in monogastric mammals. In the mucosa of intestine, endocrine cells were immunoreactive to somatostatin, gastrin, CCK, GIP, secretin, motilin, glucagon and NPY. Nerve cells immunoreactive to somatostatin, substance P, VIP, PHI, NPY, bombesin and CGRP were detected. No immunoreactivity to neurotensin was observed, islets of the pancreas had insulin cells in their cores and glucagon and somatostatin cells in their mantles. The antisera employed failed to demonstrate PP cells in the pancreas, but NPY-immunoreactive cells were present.

Acetyl cholinesterase is expressed in the follicular dendritic cells of germinal centres: differences between normal and neoplastic follicles.

Acetyl cholinesterase (AcChE) was demonstrated by histochemistry in the follicular dendritic cells (FDCs) of the germinal centres of lymph nodes, tonsils, and bowel lymphoid tissue. Its presence in the FDCs was confirmed by double immunostaining for CD21 or DRC-1. AcChE-positive FDCs are concentrated in the inner portion of the light zone of the germinal centre, being absent from the dark zone. In the lymphoid tissue surrounding the germinal centres are AcChE-positive blood vessels; double staining shows that the AcChE is present in the pericytes surrounding the endothelium of the blood vessels. In contrast to the reactive follicle, the AcChE reactivity in FDCs of follicle centre lymphoma is absent or minimally expressed, although the dense FDC mesh is well stained with CD21 or DRC-1. This suggests that the AcChE is not constitutively expressed in FDCs but that its expression is influenced by the state and activity of the lymphoid cells in the germinal centre. The reduced level of AcChE staining can be profitably employed in the diagnosis of follicle centre lymphoma.

Morphological interactions of human first trimester placental villi co-cultured with decidual explants.

Abnormalities of pregnancy such as pre-eclampsia and intrauterine growth retardation are characterized by shallow trophoblastic invasion of the placental bed, the precise molecular pathophysiology of which remains to be fully elucidated. An in-vitro model involving a co-culture of first trimester placental villi and decidua parietalis explants (of 8-12 weeks gestation) was developed and used to characterize the migration and local invasion of trophoblast cells. Trophoblast proliferation (confirmed by Ki-67 immunostaining), differentiation and loose attachment of placental villi to the underlying decidual epithelium or stroma occurred within the first 24 h of co-culture. This was followed by erosion of the syncytial layer of the placental villi and commencement of a progressive cytotrophoblast invasion after 48 h of co-culture, which continued until 120 h, when the experiments were terminated. E-cadherin was expressed at the interfaces between trophoblast cells within the villi, but expression of this adhesion molecule seemed to be down-regulated in the invasive trophoblast cells. Our results suggest that the model could be useful in investigating the factors that control early human placentation and the feto-maternal interface.

Expression of the multifunctional extracellular matrix protein thrombospondin in crescentic glomerulonephritis.

Thrombospondin is a multifunctional 450 kD glycoprotein which may be secreted into the extracellular matrix by a wide variety of cells. Occasional foci of immunoreactive thrombospondin have previously been demonstrated within normal human glomeruli. A specific polyclonal antibody directed against thrombospondin 1 was used to examine the distribution of this regulatory glycoprotein in renal biopsies from patients with a variety of renal diseases, including rapidly progressive glomerulonephritis associated with circulating antibodies to neutrophils, active or quiescent systemic lupus erythematosus, and membranous nephropathy, together with normal renal tissue. The results demonstrated the marked up-regulation of thrombospondin expression in acutely inflamed renal tissue with strongly positive, predominantly extracellular staining of glomerular crescents, although cytoplasmic staining of epithelial cells was also seen, indicating that these cells may contribute to thrombospondin accumulation at these sites. Occasional segmental mesangial staining was seen in cases of active lupus and rapidly progressive glomerulonephritis, while some focal interstitial staining around peritubular capillaries was seen in all renal tissue examined. These results suggest that thrombospondin may play an important role in the regulation of cellular recruitment, proliferation, and function in crescentic glomerulonephritis.

Expression of a B-cell marker, CD24, on nasopharyngeal carcinoma cells.

Random sequencing of clones from a lambda gt10 cDNA library, made from mRNA expressed in an Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC) has revealed the gene transcript of human CD24. The CD24 antigen, a glycosylphosphatidylinositol-anchored cell surface molecule, has been identified as a B-cell marker that is lost during cell maturation. We show here that it is expressed on 3 NPC xenografts, previously defined as consisting of poorly differentiated epithelial cells, and on an NPC biopsy. In the case of the former, the level of expression of CD24 corresponds to the EBV load. A B-lymphoblastoid cell line carrying the same EBV genome as one of the tumours, C15, and an EBV-negative Burkitt's lymphoma cell line do not display the antigen, but epithelial-like cells of a laryngeal tumour cell line (Hep2) do express it. Our data suggest that CD24 may be a marker of cell differentiation not only for B cells but also for epithelial cells and may have an indirect association with EBV gene expression.

Virulence of Aspergillus fumigatus double mutants lacking restriction and an alkaline protease in a low-dose model of invasive pulmonary aspergillosis.

To investigate the pathogenicity of Aspergillus fumigatus mutants lacking putative virulence factors, we have developed a new murine model of invasive pulmonary aspergillosis based on neutropenia, the major factor predisposing patients to this infection. Mice were treated with cyclophosphamide and inoculated by the intranasal route with 5 x 10(3) conidia, a significant reduction from inoculum levels used in previous models. Evidence for the production of the extracellular alkaline protease (Alp) in lung tissue was obtained by using a fungal transformant harboring an alp::lacZ reporter gene fusion. The pathogenicities of single mutant strains lacking either Alp or the ribotoxin restrictocin and of a double mutant strain lacking both proteins were assessed in this infection model. There were no significant differences between the mutant and the wild-type strains in terms of mortality or histological-features. Inoculations with mixtures of conidia showed that the double mutant strain is slightly less virulent than the wild-type strain. We conclude that Alp and restrictocin are not important virulence determinants in pulmonary infection.

False positive immunoreaction in products of conception.

False positive immunostaining for cytomegalovirus in products of conception was revealed using an avidinbiotinylated peroxidase complex. The cause was shown to be endogenous biotin. The use of a non-avidin-biotin method avoided the problem.

The oesophagus and stomach of the African elephant: a histological, immunocytochemical and immunofluorescence study.

Histological, immunocytochemical and immunofluorescence methods were employed to study the oesophagus and stomach of the elephant. The histological findings were in line with the situation in monogastric species like swine and man. In the mucosa of the stomach, endocrine cells were immunoreactive to gastrin, somatostatin, chromogranin A and serotonin. Nerve cells immunoreactive to somatostatin, bombesin, VIP, PHI and CGRP were detected in the submucosal and myenteric plexus of the stomach. In the stomach, the absence of glucagon cells and the presence of endocrine cells immunoreactive to PYY, are in contrast to the situation in mammals and need further investigation. Small gastric ulcers were observed in some of the specimens.

The cell blot assay in analysis of rat anterior pituitary cell secretion.

We have investigated the efficacy of the cell blot assay in analysis of the secretion of hormones and peptides from rat anterior pituitary cells. The dissociated cells are cultured on pieces of translucent polyvinylidene difluoride membrane, on which their secretory products are adsorbed and subsequently immunostained. The area and integrated optical density of the stained 'halo' surrounding individual cells is measured by microscopical image processing and the values for basal secretion of a particular hormone or peptide are compared with those after application of secretagogues or inhibitors. Our experiments tested established responses of dissociated rat anterior pituitary cells; in general, the results were as expected. Double immunoenzymatic staining could be used to show secretion of two products from the same or different cells in one preparation, and immunofluorescence with fluorescein- and/or rhodamine-labelled antibodies could be used instead of enzyme-linked immunolabelling. Optimal dilutions of immunoreagents were much higher than those used for immunocytochemistry on tissue sections. Although the cell blot assay does not provide absolute quantification, since some of the secreted product escapes into the medium, it is a relatively easy and economical way for morphologists to compare secretion from individual cells under varying conditions.