Effects of HSD11B1 knockout and overexpression on local cortisol production and differentiation of mesenchymal stem cells.

Exogenous glucocorticoids increase the risk for osteoporosis, but the role of endogenous glucocorticoids remains elusive. Here, we describe the generation and validation of a loss- and a gain-of-function model of the cortisol producing enzyme 11ß-HSD1 (HSD11B1) to modulate the endogenous glucocorticoid conversion in SCP-1 cells - a model for human mesenchymal stem cells capable of adipogenic and osteogenic differentiation. CRISPR-Cas9 was successfully used to generate a cell line carrying a single base duplication and a 5 bp deletion in exon 5, leading to missense amino acid sequences after codon 146. These inactivating genomic alterations were validated by deep sequencing and by cloning with subsequent capillary sequencing. 11ß-HSD1 protein levels were reduced by 70% in the knockout cells and cortisol production was not detectable. Targeted chromosomal integration was used to stably overexpress HSD11B1. Compared to wildtype cells, HSD11B1 overexpression resulted in a 7.9-fold increase in HSD11B1 mRNA expression, a 5-fold increase in 11ß-HSD1 protein expression and 3.3-fold increase in extracellular cortisol levels under adipogenic differentiation. The generated cells were used to address the effects of 11ß-HSD1 expression on adipogenic and osteogenic differentiation. Compared to the wildtype, HSD11B1 overexpression led to a 3.7-fold increase in mRNA expression of lipoprotein lipase (LPL) and 2.5-fold increase in lipid production under adipogenic differentiation. Under osteogenic differentiation, HSD11B1 knockout led to enhanced alkaline phosphatase (ALP) activity and mRNA expression, and HSD11B1 overexpression resulted in a 4.6-fold and 11.7-fold increase in mRNA expression of Dickkopf-related protein 1 (DKK1) and LPL, respectively. Here we describe a HSD11B1 loss- and gain-of-function model in SCP-1 cells at genetic, molecular and functional levels. We used these models to study the effects of endogenous cortisol production on mesenchymal stem cell differentiation and demonstrate an 11ß-HSD1 dependent switch from osteogenic to adipogenic differentiation. These results might help to better understand the role of endogenous cortisol production in osteoporosis on a molecular and cellular level.

Metformin Improves the Hepatic Steatosis Index in Non-Obese Patients with Polycystic Ovary Syndrome.

Non-alcoholic fatty liver disease (NAFLD) is a common yet little recognized health problem in women with polycystic ovary syndrome (PCOS). In a retrospective setting, we investigated the effects of metformin treatment on the hepatic steatosis index (HSI) as a readily available biomarker panel for NAFLD. HSI values of >36 are considered to be highly suggestive for NAFLD. In our cohort, HSI values indicating NAFLD were found in 60/81 (74.1%) women at baseline. The mean HSI improved significantly after the metformin treatment from 43.2 ± 1.0 to 41.0 ± 1.1. Subgroup analyses of non-obese (body mass index (BMI) < 30 kg/m2), obese (BMI 30−35 kg/m2) and very obese (BMI > 35 kg/m2) women yielded mean baseline HSI values of 35.5 ± 4.5, 41.2 ± 2.7 and 51.2 ± 4.7, respectively. A significant improvement in the HSI of 1.5 ± 2.1 was observed after metformin treatment in non-obese women but not in the obese subgroups. The data suggest a new aspect of metformin treatment in non-obese PCOS patients, namely, a possible improvement in NAFLD. This study highlighted hepatic steatosis as a common comorbidity in PCOS patients that can severely affect their long-term health, and therefore, deserves more attention in the management of PCOS patients.

The Influence of Conventional Treatment on Symptoms and Complaints in Patients With Chronic Postsurgical Hypoparathyroidism.

Quality of life (QoL) is impaired in patients with chronic hypoparathyroidism (HypoPT). With a recently developed specific patient questionnaire, the 28-item Hypoparathyroid Patient Questionnaire (HPQ 28), we were able to demonstrate an effect of laboratory parameters on symptoms and complaints identified by scales and items of the HPQ 28. Here, we evaluated the effect of conventional treatment modalities on QoL using this specific questionnaire. In this cross-sectional study, we included 49 HypoPT (41 female and 8 male) patients. Laboratory values of total serum calcium, magnesium, phosphate, calcium-phosphate product (CPP), and 24-hour urine for calcium and phosphate were analyzed. Patients completed the HPQ 28 questionnaire during the corresponding visit. Mean age was 57.3 ± 10.5 years and duration of disease 12.6 ± 9.8 years. Most patients (86%, n = 42) were treated with the active vitamin D analogs calcitriol, alfacalcidol, or dihydrotachysterol (DHT). The use of calcium and magnesium supplements influenced scales on HPQ 28 in a dose-dependent manner. We detected a dose-dependent increase on the HPQ 28 scales "depression and anxiety" and "pain and cramps," and the item "numbness and tingling" related to calcitriol. This effect was independent of gender, age, underlying disease, kind of surgery, serum 25-hydroxyvitamin D3, calcium, or phosphate values. This study presents the first data on specific symptoms of HypoPT patients dependent on different treatment modalities. Our data suggest that in part the reduced QoL in these patients might be caused by conventional treatment. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

The rise in expression and activity of 11ß-HSD1 in human mesenchymal progenitor cells induces adipogenesis through increased local cortisol synthesis.

11ß-Hydroxysteroid dehydrogenase 1 (11ß-HSD1) plays an important role in pre-receptor glucocorticoid metabolism. This enzyme is expressed in bone, increases with age, and catalyzes the conversion of the inactive glucocorticoid cortisone into the active glucocorticoid cortisol and vice versa. Here we hypothesized that the physiological activity of 11ß-HSD1 to produce cortisol in human mesenchymal progenitor cells (hMSC) is principally sufficient to shift the differentiation potential in the direction of adipogenic. We thus investigated differentiating hMSCs and the mesenchymal stem cell line SCP-1 cultured under osteogenic conditions and stimulated with supra-physiological cortisone levels. The release of active cortisol into the medium was monitored and the influence on cell differentiation analyzed. We revealed an increase in 11ß-HSD1 expression followed by increased reductive activity of the enzyme, thereby inducing a more adipogenic phenotype of the cell models via cortisol with negative effects on osteogenesis. Through inhibition experiments with the specific inhibitor 10 j, we proved the enzyme specificity for cortisol synthesis and adipogenic differentiation. Increased expression of 11ß-HSD1 followed by higher cortisol levels might thus explain bone marrow adiposity followed by reduced bone quality and stability in old age or in situations of supra-physiological glucocorticoid exposure.

Quantification of Symptom Load by a Disease-Specific Questionnaire HPQ 28 and Analysis of Associated Biochemical Parameters in Patients With Postsurgical Hypoparathyroidism.

In hypoparathyroidism (HypoPT), patients suffer severely from reduced quality of life. The complexity of HypoPT demands a disease-specific control instrument to characterize symptom load. We employed a newly developed disease-specific Hypoparathyroid Patient Questionnaire (the HPQ 40/28) to investigate and quantify HypoPT patients' complaints and contributing factors. In this cross-sectional, two-center study, patients with postsurgical HypoPT (n = 49) were matched for gender and age and compared with patients having undergone thyroid surgery without HypoPT (n = 39) and patients with primary hyperparathyroidism (n = 35). The HPQ 40/28 was completed when patients visited the respective center. Clinical background information, blood tests, and current medication were documented by the physician. Serum calcium lay within the reference range in 87% of HypoPT patients, serum phosphate in 95.7%, and calcium-phosphate product (CPP) in 100%. HPQ 40/28 scores for the scales "pain and cramps" (PaC), "neurovegetative symptoms" (NVS), "numbness or tingling," and "heart palpitations" were significantly elevated in comparison with control groups. Correlations between complaints and laboratory parameters could be demonstrated in the HypoPT group, with serum calcium correlating with NVS (r = 0.309, p < 0.05) and serum phosphate with loss of vitality (r = 0.349, p < 0.05). CPP was the main contributor to symptom load with an influence on PaC (r = 0.295, p < 0.05), loss of vitality (r = 0.498, p < 0.001), numbness or tingling (r = 0.328, p < 0.05), and memory problems (r = 0.296, p < 0.05). In conclusion, the newly developed HPQ 40/28 successfully identified and quantified symptoms typical in HypoPT patients. Using the HPQ 40/28, the CPP was identified as the predominant factor in the severity of complaints in HypoPT. © 2020 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

The HPQ-Development and First Administration of a Questionnaire for Hypoparathyroid Patients.

Hypoparathyroidism patients suffer a variety of complaints often leading to reduced quality of life. Currently, no specific standard instrument exists to measure corresponding disease manifestations. We therefore aimed to develop a disease-characteristic questionnaire for hypoparathyroid patients. We used an analytical-empirical approach for questionnaire construction based on retrospective analysis of four well-established but non-disease-specific questionnaires (Symptom Checklist 90, revised [SCL-90-R]; Giessen Complaint List [GBB]; Short-Form-36 Health Survey [SF-36]; von Zerssen Symptom List [B-L Zerssen]) and two additional unpublished or local questionnaires (SHGdQ and GPQ) in a German hypoparathyroidism self-help group (n = 60). Retrospective data were compared with corresponding general population norms. The new questionnaire was administered prospectively over 1 year to patients with postoperative hypoparathyroidism and two control groups to validate specificity. Exploratory factor analysis (EFA) and reliability testing were applied to identify relevant scales and reduce overlapping items. In the self-help group, SCL-90-R revealed elevated symptom load in four complaint areas (p = 0.003 to p < 0.001). The SF-36 mental summary score (p < 0.001) and further scales were lowered. In the GBB, four of five scales (p = 0.009 to p < 0.001) were elevated. In the B-L Zerssen, 6 of 24 items revealed complaint areas. Based on these findings, the new 40-item "Hypoparathyroid Patient Questionnaire" (HPQ 40) was developed, tested prospectively, and further analyzed. EFA revealed five scales (pain and cramps, gastrointestinal symptoms, depression and anxiety, neurovegetative symptoms, loss of vitality), all with Cronbach's alpha >0.7. The questionnaire was revised accordingly and shortened to 28 questions to avoid redundancy. We present a new disease-characteristic questionnaire for hypoparathyroidism patients. Prospective testing revealed five major complaint areas and promising psychometric properties. This questionnaire can be tested for usefulness in further clinical trials. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Decreased Expression of the Human Urea Transporter SLC14A1 in Bone is Induced by Cytokines and Stimulates Adipogenesis of Mesenchymal Progenitor Cells.

The human urea transporter SLC14A1 (HUT11/UT-B) has been suggested as a marker for the adipogenic differentiation of bone cells with a relevance for bone diseases. We investigated the function of SLC14A1 in different cells models from bone environment. SLC14A1 expression and cytokine production was investigated in bone cells obtained from patients with osteoporosis. Gene and protein expression of SLC14A1 was studied during adipogenic or osteogenic differentiation of human mesenchymal progenitor cells (hMSCs) and of the single-cell-derived hMSC line (SCP-1), as well as in osteoclasts and chondrocytes. Localization was determined by histochemical methods and functionality by urea transport experiments. Expression of SLC14A1 mRNA was lower in cells from patients with osteoporosis that produced high levels of cytokines. Accordingly, when adding a combination of cytokines to SCP-1 SLC14A1 mRNA expression decreased. SLC14A1 mRNA expression decreased after both osteogenic and more pronounced adipogenic stimulation of hMSCs and SCP-1 cells. The highest SLC14A1 expression was determined in undifferentiated cells, lowest in chondrocytes and osteoclasts. Downregulation of SLC14A1 by siRNA resulted in an increased expression of interleukin-6 and interleukin-1 beta as well as adipogenic markers. Urea influx through SLC14A1 increased expression of osteogenic markers, adipogenic markers were suppressed. SLC14A1 protein was localized in the cell membrane and the cytoplasm. Summarizing, the SLC14A1 urea transporter affects early differentiation of hMSCs by diminishing osteogenesis or by favoring adipogenesis, depending on its expression level. Therefore, SLC14A1 is not unequivocally an adipogenic marker in bone. Our findings suggest an involvement of SLC14A1 in bone metabolism and inflammatory processes and disease-dependent influences on its expression.

Deficient knowledge in adult Turner syndrome care as an incentive to found Turner centers in Germany.

OBJECTIVE: Turner syndrome (TS) is characterized by the complete or partial loss of the second sex chromosome and associated with a wide range of clinical manifestations. We aimed to assess the medical care of adult patients with TS in Germany. DESIGN: Retrospective multicenter observational study. METHODS: Data were collected from medical records of 258 women with TS treated between 2001 and 2017 in five non-university endocrinologic centers in Germany. RESULTS: Mean age was 29.8 ± 11.6 years, mean height 152 ± 7.7 cm, and mean BMI 26.6 ± 6.3 kg/m2. The karyotype was known in 50% of patients. Information on cholesterol state, liver enzymes, and thyroid status was available in 81-98% of women with TS; autoimmune thyroiditis was diagnosed in 37%. Echocardiography was performed in 42% and cardiac MRI in 8.5%, resulting in a diagnosis of cardiovascular disorder in 28%. Data on growth hormone therapy were available for 40 patients (15%) and data concerning menarche in 157 patients (61%). CONCLUSION: In 258 women with TS, retrospective analysis of healthcare data indicated that medical management was focused on endocrine manifestations. Further significant clinical features including cardiovascular disease, renal malformation, liver involvement, autoimmune diseases, hearing loss, and osteoporosis were only marginally if at all considered. Based on this evaluation and in accordance with recent guidelines, we compiled a documentation form facilitating the transition from pediatric to adult care and further medical management of TS patients. The foundation of Turner Centers in March 2019 will improve the treatment of TS women in Germany.

Crohn's disease patient serum changes protein expression in a human mesenchymal stem cell model in a linear relationship to patients' disease stage and to bone mineral density.

BACKGROUND: Crohn's disease (CD) is associated with a higher prevalence of osteoporosis, a complication that is recognized as a significant cause of morbidity. Its pathogenesis is controversial, but the activity of CD is one contributing factor. METHODS: We stimulated SCP-1 cells (mesenchymal stem cell line) under osteogenic conditions with serum from adult patients with CD in the symptomatic phase (SP) and in remission (R) and with control sera. Concentrations of IL-6, IL-1 beta, and TNF alpha in the sera were measured. Patients were classified as normal or osteopenic/osteoporotic based on bone mineral density (BMD) T-score measurements. After 14 days in culture, protein expression and gene ontology (GO) annotation analysis was performed. RESULTS: Cytokine concentrations (IL-6, IL-1 beta, TNF alpha) varied within sera groups. None of the cytokines were significantly increased in the symptomatic phase compared to remission. Protein analysis revealed 17 proteins regulated by the SP versus R phase sera of disease. A linear relationship between CDAI (Crohn's disease activity index) and normalized protein expression of APOA1 and 2, TTR, CDKAL1 and TUBB6 could be determined. Eleven proteins were found to be differentially regulated comparing osteoporosis-positive and osteoporosis-negative sera. Gene annotation and further analysis identified these genes as part of heme and erythrocyte metabolism, as well as involved in hypoxia and in endocytosis. A significant linear relationship between bone mineral density and normalized protein expression could be determined for proteins FABP3 and TTR. CONCLUSION: Our explorative results confirm our hypothesis that factors in serum from patients with CD change the protein expression pattern of human immortalized osteoblast like cells. We suggest, that these short time changes indeed influence factors of bone metabolism.

IL-6, IL-1ß, and TNF-α only in combination influence the osteoporotic phenotype in Crohn's patients via bone formation and bone resorption.

BACKGROUND: Crohn´s disease (CD) is associated with a higher prevalence of osteoporosis. The pathogenesis of bone affliction remains controversial, especially if inflammatory cytokines or glucocorticoid therapy are the main contributors. In postmenopausal osteoporosis, bone resorption is induced by IL-6, IL-1ß and TNF-α. In contrast, in children with CD, IL-6 exclusively decreased bone formation without affecting bone resorption. OBJECTIVES: The objective of this study was to further clarify the pathophysiology of bone affliction in adult patients with CD with the use of an osteoblast and osteoclast cell model. MATERIAL AND METHODS: Inflammatory cytokines IL-6, IL-1ß, and TNF-α were measured in adult CD patients' serum. Mean values of these cytokines were applied with or without dexamethasone to the human cell line SCP-1 (osteoblastic cell model). Also, the effect of cytokines on primary human osteoclast differentiation and activity was determined. RESULTS: The combined cytokine application increased the receptor activator of NF-κB ligand/osteoprotegerin (RANKL/OPG) ratio 2-fold after 2 and 14 days. Additional application of dexamethasone to SCP-1 cells further increased the RANKL/OPG ratio 3-fold, but decreased IL-6 and IL-1ß expression to 10% and 50%, respectively. TNF-α expression was maximally suppressed to 16% by dexamethasone in the presence of cytokines. In osteoclasts, the combined cytokine treatment decreased expression of characteristic genes to approx. 30%, while increasing osteoclast resorption activity to 148%. In addition, a cytokine stimulated osteoblast cell culture-generated supernatant stimulated osteoclast resorption activity by 170%. CONCLUSIONS: Our results suggest that IL-6, IL-1ß, and TNF-α only in combination induced osteoclaststimulating activity represented by the RANKL/OPG ratio in osteoblasts. Dexamethasone further increased this effect in osteoblasts, while decreasing cytokine expression. The results in osteoclasts support a direct and osteoblast-mediated effect on bone resorption. Our in vitro results differentiate for the first time the effect of cytokines on bone turnover as measured in adult CD patients from the additional dexamethasone effect on osteoblasts as part of the pathophysiology of osteoporosis.

Immune cells in primary and metastatic gastrointestinal stromal tumors (GIST).

We have previously described immune cells in untreated primary gastrointestinal stromal tumors (GIST). Here we compare immune cells in metastatic and primary GIST, and describe their chemoattractants. For this purpose, tissue microarrays from 196 patients, 188 primary and 51 metastasized GIST were constructed for paraffin staining. Quantitative analysis was performed for cells of macrophage lineage (Ki-M1P, CD68), T-cells (CD3, CD56) and B-cells (CD20). Chemokine gene-expression was evaluated by real-time RT-PCR. Immuno-localisation was verified by immunofluorescence. Ki-M1P+ cells were the predominant immune cells in both primary and metastatic GIST (2 8.8% ± 7.1, vs. 26.7% ± 6.3). CD68+ macrophages were significantly fewer, with no significant difference between primary GIST (3.6% ± 2.1) and metastases (4.6% ± 1.5). CD3+ T-cells were the most dominant lymphocytes with a significant increase in metastases (7.3% ± 2.3 vs. 2.2% ± 1.8 in primary GIST, P < 0.01). The percentage of CD56+ NK-cells was 1.1% ± 0.9 in the primary, and 2.4 ± 0.7 (P < 0.05) in the metastases. The number of CD20+ B-cells was generally low with 0.6% ± 0.7 in the primary and 1.8% ± 0.3 (P < 0.05) in the metastases. Analysis of the metastases showed significantly more Ki-M1P+ cells in peritoneal metastases (31.8% ± 7.4 vs. 18.2% ± 3.7, P < 0.01), whilst CD3+ T-cells were more common in liver metastases (11.7% ± 1.8 vs. 4.4% ± 2.6, P < 0.01). The highest transcript expression was seen for monocyte chemotactic protein 1 (MCP1/CCL2), macrophage inflammatory protein 1α (MIP-1α/CCL3) and the pro-angiogenic growth-related oncoprotein 1 (Gro-α/CXCL-1). Whilst the ligands were predominantly expressed in tumor cells, their receptors were mostly present in immune cells. This locally specific microenvironment might influence neoplastic progression of GIST at the different metastatic sites.

Differential gene expression of chemokines in KRAS and BRAF mutated colorectal cell lines: role of cytokines.

AIM: To study KRAS/BRAF mutations in colorectal-cancer (CRC) that influences the efficacy of treatment. To develop strategies for overcoming combination of treatment. METHODS: Five colonic cell-lines were investigated: DLD-1 with KRAS (G13D) mutation, HT 29 and Colo 205 with BRAF (V600E) mutation as well as the wild type (Wt) cell-lines Caco2 and Colo-320. DLD-1 (KRAS), HT-29 (BRAF) and Caco2 (Wt) cell lines were treated with cytokines (TNFα 50 ng, IL-1ß 1 ng and IFNγ 50 ng) and harvested at different time points (1-24 h). KRAS inhibition was performed by the siRNA-approach. Two colorectal cancer cells DLD-1 and Caco2 were used for KRAS inhibition. About 70% confluency were confirmed before transfection with small interferring RNA (siRNA) oligonucleotides. All the synthetic siRNA sequences were designed in our laboratory. Total RNA and protein was isolated from the cells for RT-PCR and Western blotting. Densitometry of the Western blotting was analyzed with the Image J software (NIH). Results are shown as mean ± SD. RESULTS: RT-PCR analysis in non-stimulated cells showed a low basal expression of TNFα and IL-1ß in the DLD-1 KRAS-mutated cell-line, compared to Caco2 wild type. No detection was found for IL-6 and IFNγ in any of the studied cell lines. In contrast, pro-angiogenic chemokines (CXCL1, CXCL8) showed a high constitutive expression in the mutated cell-lines DLD-1 (KRAS), HT-29 and Colo205 (BRAF), compared to wild type (Caco2). The anti-angiogenic chemokine (CXCL10) showed a high basal expression in wild-type, compared to mutated cell-lines. KRAS down-regulation by siRNA showed a significant decrease in CXCL1 and CXCL10 gene expression in the DLD-1 (KRAS) cell-line in comparison to wild type (Caco2) at 72 h after KRAS silencing. In contrast, the specific KRAS inhibition resulted in an up-regulation of CXCL1 and CXCL10. The results of our study show a higher expression of pro-angiogenic chemokines at basal level in mutated cell-lines, which was further increased by cytokine treatment. CONCLUSION: To summarize, basal chemokine gene expression for pro-angiogenic chemokines was high in mutated as compared to wild type cell-lines. This reflects the likely existence of a different microenvironment in tumours consistent of wild type or mutated cells. This may help to rationalize the choice of molecular targets for suitable therapeutic investigation in clinical studies.

Long-lasting complete response of metastatic melanoma to ipilimumab with analysis of the resident immune cells.

Even though ipilimumab is a promising antibody used for stage IV melanoma therapy, the response varies and is difficult to predict. We here report on a case of successful treatment with ipilimumab in dacarbazine-resistant metastatic malignant melanoma, including a review of the literature on the long-term treatment results. A 62-year-old patient with a history of a resected lentigo-maligna melanoma 5 years earlier and parotideal metastasis 1 year before was admitted with a newly detected 3.5 cm liver metastasis. Atypical liver resection was performed (R1). Immunohistochemically, CD3+ T-lymphocytes and CD68+ macrophages were detected at the tumour margins and within the parotideal and hepatic melanoma metastases. A sub-analysis of the liver metastasis showed scattered FOX-P3+ regulatory T-lymphocytes as well as multiple CD8+ effector T-cells. Chemotherapy with dacarbazine 1,000 mg/m(2)/day was administered at 4-weeks intervals for 3 months. A follow-up positron-emission computed tomography and liver biopsy revealed melanoma metastases in the liver, lungs, and mediastinum. Compassionate use of ipilimumab was administered at 3 mg/kg every 3 weeks for a total of four doses. After an initial increase in tumour size, most lesions responded, but progressive axillary and cervical lymphadenopathy was observed before complete remission was achieved. Side effects included fatigue, dyspnoea, cough, upper abdominal pain with diarrhoea, and gingival hyperplasia. Now, 36 months after ipilimumab therapy and 8 years after the initial melanoma diagnosis, the tumour did not recur. It would be challenging to hypothesize that long intervals between diagnosis and need for treatment, clinical side effects, an initial increase in tumour size and the presence of intra-tumoural T-cells and macrophages might predict tumour response.

Rat model of fractionated (2 Gy/day) 60 Gy irradiation of the liver: long-term effects.

The liver is considered a radiosensitive organ. However, in rats, high single-dose irradiation (HDI) showed only mild effects. Consequences of fractionated irradiation (FI) in such an animal model have not been studied so far. Rats were exposed to selective liver FI (total dose 60 Gy, 2 Gy/day) or HDI (25 Gy) and were killed three months after the end of irradiation. To study acute effects, HDI-treated rats were additionally killed at several time points between 1 and 48 h. Three months after irradiation, no differences between FI and HDI treatment were found for macroscopically detectable small "scars" on the liver surface and for an increased number of neutrophil granulocytes distributed in the portal fields and through the liver parenchyma. As well, no changes in HE-stained tissues or clear signs of fibrosis were found around the portal vessels. Differences were seen for the number of bile ducts being increased in FI- but not in HDI-treated livers. Serum levels indicative of liver damage were determined for alkaline phosphatase (AP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyltransferase (γGT) and lactate dehydrogenase (LDH). A significant increase of AP was detected only after FI while HDI led to the significant increases of AST and LDH serum levels. By performing RT-PCR, we detected up-regulation of matrix metalloproteinases, MMP-2, MMP-9, MMP-14, and of their inhibitors, TIMP-1, TIMP-2 and TIMP-3, shortly after HDI, but not at 3 month after FI or HDI. Overall, we saw punctual differences after FI and HDI, and a diffuse formation of small scars at the liver surface. Lack of "provisional clot"-formation and absence of recruitment of mononuclear phagocytes could be one explanation for scar formation as incomplete repair response to irradiation.

Identification of CD68(+) neutrophil granulocytes in in vitro model of acute inflammation and inflammatory bowel disease.

CD-68 is widely regarded as a selective marker for human monocytes and macrophages and is commonly used in human pathology studies. The purpose of this study was to investigate the expression of CD-68 in human peripheral blood mononuclear cells (PBMCs), neutrophil granulocytes (NGs) and in inflamed intestinal tissue samples for comparison. PBMCs and NGs were isolated from heparinized human blood samples. Intestinal biopsies were obtained during routine endoscopic procedures from patients with inflammatory bowel disease (IBD), e.g. ulcerative colitis and Crohn's disease. Gene and protein expression was analyzed by real-time RT-PCR, Western blot and immunohistochemistry. Both PBMCs and NGs preparations contained cells that were positive for CD-68 and either neutrophil elastase (NE), or myeloperoxidase (MPO). CD-68(+)/NE(-)/MPO(-) cells were regarded as monocytes. CD-68 mRNA expression was detected in PBMCs and NGs preparations. With Western blot and by performing immunoprecipitation of cell lysate, we could clearly detect CD-68 in NGs, U-937, THP-1, Hep-G2, Jurkat cells and PBMCs. Identification of inflammatory cells in acutely inflamed colonic mucosa obtained from patients with IBD revealed a strong accumulation of CD-68(+)/MPO(+) cells compared to normal colonic mucosa. The uptake of the marker by phagocytosis was excluded by performing a double staining with CD-163/NE and CD-163/MPO in PBMCs, NGs cultures and in inflamed colonic mucosa. These results identify CD-68(+) NGs in peripheral blood and inflamed colonic mucosa. CD-68 is not only a marker for the macrophages-monocytes but also for NGs.

Characterization of the erythropoietin/erythropoietin receptor axis in a rat model of liver damage and cholangiocarcinoma development.

It has been recently shown that the biological effects of erythropoietin (EPO) are not limited to the hematopoietic compartment but, as pleiotropic glycoprotein, this hormone can exert pro-angiogenic and tissue-protective functions also in a wide range of non-hematopoietic organs. The role of EPO and the effective functionality of its receptor in solid tumors are still a controversial point of debate. In the present work we analyzed the gene expression of EPO and its cognate receptor (EpoR) in a rat model of thioacetamide-induced damage and tumor. An analysis of the EPO/EpoR axis was also performed on human cholangiocarcinoma (CC) cell lines. A progressive increase of EPO and EpoR mRNA can already be observed during the fibrotic-cirrhotic development with a peak of expression rising at tumor formation (24.7 ± 9.9-fold increase and 15.5 ± 1.1-fold increase, respectively, for the two genes). Co-localization studies by immunofluorescence revealed hepatocytes in the regenerative cirrhotic nodules (Hep Par-1(+)) and in the dysplastic bile duct cells (CK19(+)) as the major EPO producers in this specific condition. The same cell populations, together with endothelial cells, exhibited an increased expression of EpoR, although all the non-parenchymal cell populations in the liver exhibited modest basal mRNA levels. Challenging human CC cells, Mz-Cha-2, with a combination of EPO and SCF resulted in a synergistic effect on the gene expression of EPO, CyclinD1 and PCNA. This study suggests that the autocrine and paracrine release of endogenous EPO in the microenvironment may contribute to the development and maintenance of the CC possibly in cooperation with other signaling pathways.

Ferroportin-1 is a 'nuclear'-negative acute-phase protein in rat liver: a comparison with other iron-transport proteins.

Liver is the central organ of iron metabolism. During acute-phase-response (APR), serum iron concentration rapidly decreases. The current study aimed to compare expression and localization of iron transport protein ferroportin-1 (Fpn-1) and of other iron import proteins after experimental tissue damage induced by injecting turpentine oil in the hind limbs of rats and mice. Serum and spleen iron concentration decreased with an increase in total liver, cytoplasmic and nuclear iron concentration. In liver, mRNA amount of Fpn-1, Fpn-1a, Fpn-1b, HFE, hemojuvelin (HJV) and hephaestin (heph) genes showed a rapid decrease. Hepcidin, divalent metal transporter-1 (DMT-1), transferrin (Tf) and Tf-receptor-1 (TfR1), TfR-2 (TfR2) gene expression was increased. Western blot analysis of liver tissue lysate confirmed the changes observed at mRNA level. In spleen, a rapid decrease in gene expression of Fpn-1, Fpn-1a, Fpn-1b, DMT-1, Tf, TfR1 and TfR2, and an increase in hepcidin was observed. Immunohistochemistry of DMT-1 and TfR2 were mainly detected in the nucleus of rat liver and spleen, whereas TfR1 was clearly localized in the plasma membrane. Fpn-1 was mostly found in the nuclei of liver cells, whereas in spleen, the protein was mainly detected in the cell membrane. Western blot analysis of liver fractions confirmed immunohistochemical results. In livers of wild-type mice, gene expression of Fpn-1, Fpn-1a and Fpn-1b was downregulated, whereas hepcidin gene expression was increased. In contrast, these changes were less pronounced in IL-6ko-mice. Cytokine (IL-6, IL-1b and TNF-a) treatment of rat hepatocytes showed a downregulation of Fpn-1, Fpn-1a and Fpn-1b, and upregulation of hepcidin gene expression. Moreover, western blot analysis of cell lysate of IL-6-treated hepatocytes detected, as expected, an increase of a2-macroglobulin (positive acute-phase protein), whereas albumin (negative acute-phase protein) and Fpn-1 were downregulated. Our results demonstrate that liver behaves as a 'sponge' for iron under acute-phase conditions, and Fpn-1 behaves as a negative acute-phase protein in rat hepatocytes mainly, but not exclusively, because of the effect of IL-6. These changes could explain iron retention in the cytoplasm and in the nucleus of hepatocytes during APR.

Immunodetection of cyclooxygenase-2 (COX-2) is restricted to tissue macrophages in normal rat liver and to recruited mononuclear phagocytes in liver injury and cholangiocarcinoma.

It has been suggested that cyclooxygenase-2 (COX-2)-mediated prostaglandin synthesis is associated with liver inflammation and carcinogenesis. The aim of this study is to identify the cellular source of COX-2 expression in different stages, from acute liver injury through liver fibrosis to cholangiocarcinoma (CC). We induced in rats acute and "chronic" liver injury (thioacetamide (TAA) or carbon tetrachloride (CCl(4))) and CC development (TAA) and assessed COX-2 gene expression in normal and damaged liver tissue by RT-PCR of total RNA. The cellular localization of COX-2 protein in liver tissue was analyzed by immunohistochemistry as well as in isolated rat liver cells by Western blotting. The findings were compared with those obtained in human cirrhotic liver tissue. The specificity of the antibodies was tested by 2-DE Western blot and mass spectrometric identification of the positive protein spots. RT-PCR analysis of total RNA revealed an increase of hepatic COX-2 gene expression in acutely as well as "chronically" damaged liver. COX-2-protein was detected in those ED1(+)/ED2(+) cells located in the non-damaged tissue (resident tissue macrophages). In addition COX-2 positivity in inflammatory mononuclear phagocytes (ED1(+)/ED2(-)), which were also present within the tumoral tissue was detected. COX-2 protein was clearly detectable in isolated Kupffer cells as well as (at lower level) in isolated "inflammatory" macrophages. Similar results were obtained in human cirrhotic liver. COX-2 protein is constitutively detectable in liver tissue macrophages. Inflammatory mononuclear phagocytes contribute to the increase of COX-2 gene expression in acute and chronic liver damage induced by different toxins and in the CC microenvironment.

The anti-TNF-α antibody infliximab indirectly regulates PECAM-1 gene expression in two models of in vitro blood cell activation.

Chronic inflammatory bowel diseases can be successfully treated with antibodies against the acute phase mediator TNF-α. The process of activation and of extravasation of inflammatory cells from the blood into the 'stressed' tissue site is controlled by cytokines and chemokines, which attract leukocytes and by adhesion molecules, which mediate their attachment and transmigration toward the affected cell(s). The changes in the gene expression of adhesion molecules taking place in those cells before attachment have been less investigated. Changes of PECAM-1, ICAM-1 and vascular cell adhesion molecule-1 (VCAM-1) gene expression were studied in phytohaemagglutinin (PHA)- and lipolysaccharide (LPS)-treated human peripheral blood leukocytes (PBLs), granulocytes and the human monocyte cell line U-937. Cells were treated either with PHA or with LPS in the presence or absence of infliximab and incubated with TNF-α, IFN-γ and/or transforming growth factor beta (TGF-ß) and treated as above. Activation of PBLs by PHA or LPS treatment triggered a sharp upregulation of ICAM-1, VCAM-1 gene expression and a time-dependent downregulation of PECAM-1 gene expression reaching a minimum 4 h from start of the experiment. The anti-TNF-α antibody infliximab, by neutralizing TNF-α and IFN-γ production, completely reversed PECAM-1 mRNA downregulation and ICAM-1 and VCAM-1 upregulation. Immunostaining of PBLs cytospins with antibodies against PECAM-1 and ICAM-1 confirmed RT-PCR and western blot results. PBLs IFN-γ or TNF-α treatment downregulated PECAM-1 in parallel with the upregulation of ICAM-1 and VCAM-1 gene expression, whereas TGF-ß upregulated PECAM-1- and downregulated ICAM-1 and VCAM-1 gene expression counteracting the effect of TNF-α or IFN-γ. Similar results were obtained in human U937 cells and in granulocyte cultures by TNF-α or IFN-γ treatment. Taken together, these results suggest that infliximab, blocking TNF-α and IFN-γ production, exerts its anti-inflammatory effect through inhibiting downregulation of PECAM-1 gene expression and upregulation of ICAM-1 and VCAM-1 expression in leukocytes of the peripheral blood. These results also suggest that TGF-ß may thus be of therapeutic importance as an anti-inflammatory agent.