[Extrahepatic cholangiocarcinoma: current state of palliation therapy]. / Extrahepatische Gallenwegstumoren: aktueller Stand der palliativen Therapie.

INTRODUCTION: Decompression of bile ducts is the priority objective in the non-curative stage of hilar cholangiocarcinoma. Only this will prevent or slow down infectious complications and secondary biliary cirrhosis thereby sustaining the quality of life. KEY STATEMENTS: At present, photodynamic therapy combined with insertion of an endoprosthesis seems to be best documented and most appropriate therapy. METHODS: Data from a selective literature search combined with our clinical experience were evaluated. CONCLUSIONS: Therapeutic measures should match the dissemination and stage of the tumor: in locally advanced or progressing disease (stage III) a local ablating therapy, in systemically progressing disease (stage IV) systemic chemotherapy should be utilised.

Trans-complementing adenoviral vectors for oncolytic therapy of malignant melanoma.

BACKGROUND: Despite attempts to develop efficient viral-based gene transfer therapies for the treatment of malignant tumors, only limited progress has been made to improve the efficacy of this approach. As an alternative, the use of replicating oncolytic adenoviruses with and without the expression of therapeutic transgenes is an area of active investigation. METHODS: We used a human melanoma xenograft tumor nude mouse model to test the efficacy of a bivalent vector approach consisting of two trans-complementing replication-incompetent adenoviral vectors that resulted in tumor-restricted oncolysis. We combined an E1-deleted non-replicating adenoviral vector expressing the herpes simplex virus thymidine kinase gene (AV.C2.TK) and Ad5.dl1014, an E4-deleted/E4orf4-only expressing adenovirus, to allow full replication competence when tumor cells were co-infected with both vectors. RESULTS: A375 tumors showed apoptosis at the ultrastructural level after transduction with the trans-complementing vector system that was not seen with injection of either vector alone. Apoptotic DNA fragments could be co-localized to sites of infection with the adenoviral vectors. A significant survival benefit was achieved for the trans-complementing vector treated animals compared to animals treated with either vector alone. Interestingly, the administration of GCV did not further increase animal survival over treatment with the trans-complementing system of viruses alone, and long-term survival was only seen in the trans-complementing vector treatment group. Intraperitoneal administration of a pseudo-wild-type vector Ad.dl327 resulted in significant hepatotoxicity, while intraperitoneal administration of the trans-complementing vectors resulted in only mild liver abnormalities. CONCLUSIONS: The trans-complementing vector approach using a combination of E1- and E4-deleted adenoviral vectors showed similar antitumor efficacy as reported for monovalent replicating vector systems, but may offer additional safety by reducing the risk of dissemination of the replication-competent vectors by requiring the presence of both vectors in a cell to achieve replication competence.

A novel approach using transcomplementing adenoviral vectors for gene therapy of adrenocortical cancer.

Current therapies for adrenocortical carcinomas do not improve the life expectancy of patients. In this study, we tested whether a gene-transfer therapy based upon a suicide gene/prodrug system would be effective in an animal model of the disease. We employed E4- and E1A/B-depleted, herpes simplex virus-thymidine kinase-expressing adenoviral mutants that transcomplement each other within tumor cells, hereby improving transgene delivery and efficacy by viral replication in situ. Transcomplementation of vectors increased the fraction of transduced of tumor cells. This increase was accompanied by greater tumor volume reduction compared to non-transcomplementing approaches. Survival time improved with non-replicating vectors plus GCV compared to controls. However, transcomplementation/replication of vectors led to a further significant increment in anti-tumor activity and survival time (p < 0.02). In treated animals, we observed a high number of apoptotic nuclei both adjacent to and distant from injection sites and sites of viral oncolysis. Ultrastructural analyses exhibited nuclear inclusion bodies characteristic of virus production in situ, and provided further evidence that this therapy induced apoptotic cell death within tumor cells. We conclude that the efficacy of suicide gene therapy is significantly amplified by viral replication and, in combination with GCV, significantly reduces tumor burden and increases survival time.

Adrenal cortical activation in murine colitis.

BACKGROUND & AIMS: Proper adrenal glucocorticoid secretion is crucial in the course of inflammatory diseases. However, the function and structure of the adrenal glands have not been examined in inflammatory bowel diseases. METHODS: After induction of trinitrobenzene sulfonic acid (TNBS) colitis in SJL/J mice, plasma hormone and cytokine levels were measured, adrenal structure was analyzed by immunohistochemistry and electron microscopy, and adrenal cytokine/cytokine receptor expression were studied by RNase protection. RESULTS: Adrenals of colitic animals were enlarged and hypervascularized. These animals had a marked increase in plasma corticosterone levels during the course of colitis (270 +/- 34 vs. 16 +/- 11 ng/mL; P < 0.0001) but only a modest elevation of their concurrent adrenocorticotropin levels (57 +/- 13 vs. 29 +/- 9 pmol/L; NS). On electron microscopy, adrenocortical cells showed ultrastructural signs of marked stimulation, and intra-adrenal lymphocytes were frequently found in direct contact with these cells. Concurrent plasma levels of interleukin (IL)-6, the major cytokine activating the hypothalamic-pituitary-adrenal axis, were markedly increased (495 +/- 131 vs. 20 +/- 1.5 pg/mL; P < 0.0001), and this cytokine directly stimulated corticosterone secretion by adrenocortical cells in vitro. Intra-adrenal expression of IL-6 in animals with colitis was increased 80-fold, and the IL-6 receptor subunits IL-6R alpha and gp130 were present in the adrenal cells. Treatment of animals with neutralizing anti-IL-6 antibody reduced the TNBS-induced growth and activation of the adrenal cortices. CONCLUSIONS: Colitis is associated with a profound stimulation of adrenocortical cell function and glucocorticoid release. Direct immune-adrenal interactions seem to contribute to this activation of the adrenal glands during colitis.

Plasma dehydroepiandrosterone levels during experimental endotoxemia and anti-inflammatory therapy in humans.

OBJECTIVE: To measure the effect of experimental endotoxemia and anti-inflammatory therapy on plasma dehydroepiandrosterone (DHEA) levels in humans. DESIGN: Controlled, randomized, single-blind, prospective clinical study. SETTING: Monitored unit in research hospital. SUBJECTS: Twelve healthy volunteers served as their own controls and were randomized to receive intravenous endotoxin (Escherichia coli) or saline separated by 1 wk. Six were randomized to receive ibuprofen, a cyclooxygenase inhibitor, and six were given placebo. INTERVENTIONS: Measurement of vital signs and hormones during a 24-hr period. MEASUREMENTS AND MAIN RESULTS: All subjects given endotoxin had a significant increase in plasma DHEA, cortisol, and adrenocorticotropic hormone (ACTH) levels (all p = .02). DHEA levels were maximum at 2 hrs and returned to baseline values by 6 hrs. Ibuprofen administration significantly blunted the endotoxin-induced increase in DHEA secretion (p = .001), whereas the increase in cortisol and ACTH was not affected. CONCLUSIONS: Acute endotoxemia leads to a rise in plasma DHEA levels in humans. Maximum levels of DHEA but not cortisol or ACTH were blunted by ibuprofen, suggesting a different regulation of these synthetic pathways in the adrenal cortex inner zone during acute inflammation.

Cellular immune-endocrine interaction in adrenocortical tissues.

BACKGROUND: The detection of important immunocompetence-like features on endocrine steroid cells raises questions about direct intercellular communication between the adrenal and immune systems. This article summarizes our recent work and new data on immune-adrenal interactions. MATERIALS AND METHODS: RT-PCR and immunohistochemistry were performed to examine MHC class II (HLA-DR) expression in adrenocortical tumours. Coculture systems of NCI-H295 adrenocortical carcinoma cells and HLA-matched lymphocytes were used to examine effects on steroid production and survival of lymphocytes. RESULTS: HLA-DR m-RNA is found in both benign and malignant adrenals, except the NCI-H295 cell line. Under direct coculture conditions with NCI-H295 cell line, spontaneous apoptosis of immune cells was reduced. Synthesis of cortisol and especially of dehydroepiandrosterone production of tumour cells was markedly increased. Differences by separating CD4- and CD8- T cells were not detected. CONCLUSIONS: Direct cellular contact between lymphocytes and adrenocortical cells seems to be involved in the peripheral regulation of androgen synthesis in the adrenal. The molecular basis of this interaction is not known. With regard to normal adrenals, ligation of MHC class II antigens could be a potential mechanism for a peripheral regulation of androgen secretion.

Lymphocytes stimulate dehydroepiandrosterone production through direct cellular contact with adrenal zona reticularis cells: a novel mechanism of immune-endocrine interaction.

Adrenal androgen production was reduced by 80% in patients receiving T lymphocyte-suppressive medications compared to that in age-matched controls. In vitro, however, neither tacrolimus nor cyclosporin A reduced dehydroepiandrosterone (DHEA) release by adrenocortical cells. Therefore, we examined the potential role of lymphocytes in adrenal androgen production, using cocultures of human T lymphocytes and adrenocortical primary or transformed cells. Co-cultures led to a 4-fold elevation of DHEA levels (490.4 +/- 94.8% over basal), which was greater than the increase observed after the addition of maximal concentrations of ACTH (117.4 +/- 14.8%). Separation of cells by semipermeable membranes abolished this effect, and transfer of leukocyte-conditioned medium had little androgen-stimulating effect. These data suggested that the observed stimulation of androgen secretion required cell contact rather than soluble paracrine factor(s). Furthermore, we examined human adrenal glands for the presence of T lymphocytes and contact between these cells and steroid-secreting cells of the zona reticularis. Indeed, T lymphocytes expressing CD4 and CD8 antigens were present within human adrenal zona reticularis by immunohistochemical subtyping. Electron microscopic analyses demonstrated direct cell-cell contact between T lymphocytes and adrenocortical cells in situ. This study provides evidence for a novel mechanism of immune-endocrine interactions of direct T lymphocyte-adrenocortical cell contact-mediated stimulation of adrenal androgen secretion.

Tissue remodelling in the adrenal gland.

Adaptation of the adrenal gland to the demands of the organism is regulated functionally and structurally. Three common hypotheses on zonation in the adrenal gland, the migrational, zonal, and transformation field theories, try independently to reconcile the findings on structure, proliferation, and cell death. The classical theories on zonation are revisited in the light of recent data on cell death and renewal. In accordance with data on cell death as immunoreactivity against FAS(CD 95), an apoptosis-inducing receptor, in situ end labelling of fragmented DNA, and ultrastructural analyses, programmed cell death (PCD) occurs throughout the whole organ. The angiotensin II receptor subtypes described in the adrenal allow an additional regulation of tissue homeostasis by proliferative and even by the antiproliferative effects of the angiotensin II type 2 receptor. Proto-oncogenes are involved in the regulation of cell cycle and PCD, and adrenocorticotropin asserts its tissue integrating and differentiating effects by regulating proto-oncogenes such as c-jun, c-fos, jun-B and c-myc. Polypeptides involved in proliferation and DNA repair, such as proliferating cell nuclear antigen and Ki-67, have been found within zones of expected cell senescence. The expression of the class II major histocompatibility complex on normal adrenocortical cells allows cell-to-cell communication with the immune system and may trigger the Fas/Fas-ligand system to permit tissue regression and decreasing activity in both systems. In summary, new data allow us to reappraise and to reconcile the classical theories. Apoptosis is a physiological process in the adrenal gland. There is a differential regulation of apoptosis in the different zones. An investigation of this process may elucidate the basic mechanisms of adrenal zonation.

A new view on immune-adrenal interactions: role for Fas and Fas ligand?

The hypothalamic-pituitary-adrenal axis and the immune system interact in a bidirectional manner providing the basis for the regulation of the immune response due to a pathogenic stimulus. This interplay is commonly believed to be based on the action of hormones or cytokines, respectively. Since it has been detected that adrenocortical cells offer immunological properties such as expression of MHC class II antigens and/or CD95 (Fas antigen) and its ligand, the question has to be raised whether direct intercellular communication between immune cells and 'immunocompetent' endocrine cells contributes to the complexity of immunoregulation. Here we discuss the possible reciprocal relevance of physiological and pathological adrenal changes, as well as T-cell-mediated immune response for immune and/or adrenal pathology during disease.

The presence of chorionic gonadotrophin beta subunit in normal cyclic human endometrium.

The aim of the present study was to determine whether human endometrial cells are able to secrete beta-chorionic gonadotrophin (betaCG). Immunohistochemical studies and in-situ hybridization were performed in order to provide evidence for the occurrence of betaCG in the normal endometrium in 15 patients in the proliferative phase, two patients in the periovulatory phase and 13 patients in the secretory phase. Neither immunohistochemical nor hybridization reactions could be recognized during the proliferative phase. In contrast, both protein and betaCG mRNA were observed in the glandular cells of the endometrium during the secretory phase. The results were supported by Western blotting of secretory phase endometrium extracts and the assessment of the functional secretory capacity of primary endometrium cultures. In comparison with cultured and separated cell fractions, tissue extracts showed a higher betaCG, indicating a regulatory interaction. In conclusion, betaCG can be demonstrated in normal human cyclic endometrium, suggesting a paracrine role in endometrial physiology.

Utero-ovarian interaction in the regulation of reproductive function.

The physiological regulation of fertile reproductive cycle in mammals depends on interactions between hypothalamus-pituitary, ovarian and uterine stimuli. Over the past 20 years, much has been learned about the interrelation between the affluent and effluent lymph and vascular drainage in and around both ovarian and uterine tissues. An essential feature in the regulation of the fertile cycle is the functional status of the ovary, particularly the corpus luteum. During the time of implantation and the early pregnancy, an active corpus luteum is essential. As human chorionic gonadotrophin (HCG) is important in the maintenance of the corpus luteum, we investigated if it was produced by the cyclic endometrium. Immunohistochemical and in-situ hybridization reactions were performed but neither identified the presence of HCG during the proliferative phase. Positive staining and beta-human chorionic gonadotrophin (beta-HCG) mRNA were observed during the secretory phase in the glandular cells of the endometrium. The results were confirmed by Western blotting of secretory phase endometrium extracts and assessment of the functional secretory capacity of primary endometrial cultures. Polymerase chain reaction (PCR) investigations showed a positive result in the secretory phase. We postulate that, based on the very close morphological interrelation between the uterus and the ovary, the beta-HCG of the endometrium is the primary factor for the maintenance of the corpus luteum and early pregnancy.

The mercy of adrenocortical tumor cells on lymphocytes.

Immunologic escape includes the loss of Fas-receptor and the gain of Fas-ligand expression. Normal adrenal glands express the Fas-receptor and MHC class II molecules in inner cortical zones. A distinctive feature of adrenocortical tumors is the loss of MHC class II expression. Here we demonstrate loss of Fas and gain of Fas-ligand expression in the adrenocortical carcinoma cell line NCI-H295 by immunohistochemistry and RT-PCR. In a co-culture system of tumor cells and HLA-matched leukocytes, CD 8-positive or CD 4-positive lymphocytes, we examined the immunologic escape and the ability to induce apoptosis in the immune cells. The direct co-culture with either leukocytes, CD 8-positive or CD 4-positive lymphocytes reduced spontaneous apoptosis in immune cells from 49.9% to 13.0%, 8.6% and 15.3%, respectively, as determined by FACS analysis of Annexin V binding and LDH release in the medium. In co-culture, cortisol secretion increased up to 200%. Cellular communication does not induce apoptosis in immune cells, but promotes their survival. This may be due to partial HLA class I mismatches contributing to immunologic activity. The viability of the tumor cells was not affected, and these cells were stimulated to secrete cortisol. In summary, immune escape of adrenocortical carcinomas may occur because of altered Fas/Fas-L system expression and loss of MHC class H expression.

Relevance of major histocompatibility complex class II expression as a hallmark for the cellular differentiation in the human adrenal cortex.

Major histocompatibility complex (MHC) class II antigens are expressed on adrenocortical cells of the zona reticularis and have been shown to be a marker of dignity. This suggests a correlation to the zellular differentiation of the adrenal cortex. Therefore, we immunohistochemically investigated the MHC class II expression in the context of the ontogenesis of the zonal and cellular differentiation in fetal, postnatal, childhood, and adult adrenals. Cell types and cell turnover were studied using specific immune markers (including expression of CD95/ Fas), in situ end labeling of apoptosis, and electron microscopy. We show that prenatal (fetal and definitive) steroid cells, as well as postnatal adrenals, reveal no expression of MHC class II. In childhood, these antigens first appear by the fourth year, in parallel with the differentiation of reticularis cells. The expression index in childhood was 7.43% +/- 2.78 (mean +/- SEM), in adult adrenals 18.63% +/- 3.14 (third decade), and 15.15% +/- 1.26 (fourth through sixth decade). In conclusion, MHC class II expression and the development of the functional maturation of the adult adrenal cortex occur simultaneously. The expression of MHC class II on steroid cells may thus be involved in potential immune-adrenal interactions.

MHC class II expression--a new tool to assess dignity in adrenocortical tumours.

One in seventy randomly selected individuals is supposed to host an adrenal mass. The increasing number of incidentally detected adrenocortical adenomas requires the effective and reliable evaluation of dignity. So far this has been determined through a difficult multi-parametric analysis. Since MHC class II antigens are expressed in the normal adrenal cortex with a restriction to the zona reticularis, we examined 28 adrenocortical incidentalomas, 10 adenomas, 13 cortical carcinomas, 2 metastases, 10 controls as well as the adrenocortical carcinoma cell line NCI-H295 immunohistochemically for the expression of HLA class II antigens. We showed, that the majority of the adenomas still express class II antigens, whereas the expression is abrogated in all carcinomas examined. Our results indicate, that the detection of HLA class II positive tumour cells excludes malignancy. Therefore, MHC class II antigens may serve as a novel tumour marker in the evaluation of dignity in adrenocortical tumours. These findings could change the strategy for the assessment of adrenal masses.

Evaluation of apoptotic parameters in normal and neoplastic human adrenal.

The migration and proliferation of adrenocortical cells is accompanied by mechanisms of cellular knock-out. We compared the programmed cell death of normal and malignant adrenocortical tissues on the basis of apoptotic rates by the nonradioactive in situ end-labelling of DNA-fragments, immunohistochemistry against PCNA, CD95 and ultrastructural analysis. The highest labelling index (LI) was detectable in the outermost zones of the adrenal cortex of normal adrenals. Average LI in normal adrenal cortex was 20% whereas only 2% was detectable in adrenocortical neoplasms. MHC class II, which was previously shown to be involved in programmed cell death in lymphocyte populations (1), was detectable in normal and benign but not in malignant adrenocortical neoplastic cells. In conclusion, the analysis of apoptosis provides new aspects of normal adrenal zonation and allows the differentiation between normal and neoplastic adrenal cortex although the differentiation between malignant and benign neoplasms requires further markers.

Differential regulation of apoptosis in the normal human adrenal gland.

Analysis of apoptosis in the human adrenal appears to be of eminent importance in the understanding of adrenal structure, zonation, and function. In this study we investigated the programmed cell death of normal adrenal tissues on the basis of apoptotic index by the nonradioactive in situ end labeling of DNA fragments, proliferating cell nuclear antigen, (PCNA), CD95 (cluster of differentiation), major histocompatibility complex class II immunohistochemistry, and ultrastructural analysis. The highest apoptotic index was detected in the outermost zones of the adrenal cortex, mainly in the zona glomerulosa. A labeling index of 50.46 +/- 5.22% (mean +/- SEM) for zona glomerulosa, 9.36 +/- 1.68% for zona fasciculata, 3.90 +/- 0.78% for zona reticularis, and 7.37 +/- 1.62% for the zona medullaris was found. Immunohistochemistry was used to distinguish between apoptotic and S phase cells. Positive anti-PCNA staining occurred in the inner cortical zones, whereas anti-CD95 signals appeared throughout the whole cortex, albeit at a much weaker level. MHC class II expression, which is known to be associated with programmed cell death, was demonstrated in the inner cortical zone. The data showed that mechanisms of cell death other than necrosis occur in the adrenal. In conclusion, we found a differential regulation of cell death for each zone of the adrenal cortex; the old theories of adrenal zonation (migrational vs. zonal or transformation theory) may, in fact, correlate with each other.