A short-term in vivo model for giant cell tumor of bone.

BACKGROUND: Because of the lack of suitable in vivo models of giant cell tumor of bone (GCT), little is known about its underlying fundamental pro-tumoral events, such as tumor growth, invasion, angiogenesis and metastasis. There is no existing cell line that contains all the cell and tissue tumor components of GCT and thus in vitro testing of anti-tumor agents on GCT is not possible. In this study we have characterized a new method of growing a GCT tumor on a chick chorio-allantoic membrane (CAM) for this purpose. METHODS: Fresh tumor tissue was obtained from 10 patients and homogenized. The suspension was grafted onto the CAM at day 10 of development. The growth process was monitored by daily observation and photo documentation using in vivo biomicroscopy. After 6 days, samples were fixed and further analyzed using standard histology (hematoxylin and eosin stains), Ki67 staining and fluorescence in situ hybridization (FISH). RESULTS: The suspension of all 10 patients formed solid tumors when grafted on the CAM. In vivo microscopy and standard histology revealed a rich vascularization of the tumors. The tumors were composed of the typical components of GCT, including (CD51+/CD68+) multinucleated giant cells which were generally less numerous and contained fewer nuclei than in the original tumors. Ki67 staining revealed a very low proliferation rate. The FISH demonstrated that the tumors were composed of human cells interspersed with chick-derived capillaries. CONCLUSIONS: A reliable protocol for grafting of human GCT onto the chick chorio-allantoic membrane is established. This is the first in vivo model for giant cell tumors of bone which opens new perspectives to study this disease and to test new therapeutical agents.

Biological importance of a polymorphic CA sequence within intron 1 of the epidermal growth factor receptor gene (EGFR) in high grade central osteosarcomas.

Expression of EGFR in high grade osteosarcomas has been observed to be correlated with an improved prognosis. Yet, the underlying mechanism remained unclear since amplifications of EGFR have rarely been described. Recently, the length of a polymorphic CA repeat located at a 5'-regulatory sequence in the intron 1 of the EGFR gene (SSR I) has been shown to be associated with its basal transcriptional activity. We therefore determined the allelic length of CA SSR-I in 219 cases of high grade osteosarcoma and correlated the results with EGFR expression in 34 cases, the presence of amplifications within the CA SSR-I repeat in 59 cases, and clinical follow-up. Our results confirm that in osteosarcoma patients short alleles are more frequent than longer ones, 16 CA repeats being the most frequent. The allele composition differed significantly from the one recently described in a healthy control population (P < 0.01). Short alleles tended to be associated with increased expression of EGFR. Amplifications of the EGFR gene were seen in 13.5% of cases. Significant correlations between allele length composition and neoadjuvant chemotherapy response or long term clinical outcome could not be established. While we were able to show that high frequency of EGFR expression in osteosarcomas is associated with predominantly short alleles of EGFR-CA SSR I, persisting shortcomings in the correspondence with clinical data point toward the existence of additional, putatively more important transcription control mechanisms for EGFR in osteosarcomas which might account for the good prognostic value of EGFR expression.

Influence of heme oxygenase-1 on microcirculation after kidney transplantation.

BACKGROUND: Cytoprotective proteins, such as heme oxygenase-1 (HO-1), play a decisive role in ischemia-reperfusion injury during kidney transplantation. The aim of this study was to investigate the impact of heme oxygenase-1 on microcirculation and on ischemia-reperfusion injury in an isogenic kidney transplantation rat model. MATERIALS AND METHODS: Seventy male Lewis rats were distributed into three groups. In Group 1(control), the kidneys were only mobilized. In Groups 2 and 3, bilateral nephrectomy was performed, and a kidney from another Lewis rat was orthotopically transplanted on the left side. The donor animals in Group 3 received preconditioning with the HO-1 inductor hemin. 24 h after reperfusion graft function and morphology were examined. Microcirculation was investigated by in vivo microscopy of the renal surface 1 h after reperfusion. RESULTS: HO-1 preconditioning led to significantly lower serum creatinine and serum urea, as well as less histological damage and inducible nitric oxide synthase expression. Microcirculation was improved by a significant enlargement of the vascular diameter and an increase of the capillary flow. CONCLUSIONS: Treatment with hemin improves microcirculation by induction of HO-1 and reduces ischemia-reperfusion injury after kidney transplantation. HO-1 induction was shown to be a promising approach in the preconditioning of donor kidneys.

Egfr amplification specific gene expression in phyllodes tumours of the breast.

BACKGROUND: Recently, we were able to show that amplifications of the epidermal growth factor receptor (egfr) gene and the overexpression of EGFR were associated with the initiation and progression of phyllodes tumours. METHODS: In order to gain further insights into regulation mechanisms associated with egfr amplifications and EGFR expression in phyllodes tumours, we performed global gene expression analysis (Affymetrix A133.2) on a series of 10 phyllodes tumours, of these three with and seven without amplifications of an important regulatory repeat in intron 1 of egfr (CA-SSR I). The results were verified and extended by means of immunohistochemistry using the tissue microarray method on an extensively characterized series of 58 phyllodes tumours with antibodies against caveolin-1, eps15, EGF, TGF-alpha, pErk, pAkt and mdm2. RESULTS: We were able to show that the presence of egfr CA-SSR I amplifications in phyllodes tumours was associated with 230 differentially expressed genes. Caveolin-1 and eps15, involved in EGFR turnover and signalling, were regulated differentially on the RNA and protein level proportionally to egfr gene dosage. Further immunohistochemical analysis revealed that the expression of caveolin-1 and eps15 were also significantly correlated with the expression of pAkt (p<0.05), pERK (p<0.05), mdm2 (p<0.01) and EGF (p<0.001 for caveolin-1). Eps15 and pERK were further associated with tumour grade (p<0.01 and p<0.001, respectively). CONCLUSION: Our results show that amplifications within regulatory sequences of egfr are associated with the expression of eps15 and caveolin-1, indicating an increased turnover of EGFR. The interplay between EGFR and caveolin-1, eps15, pAkt, mdm2 and pERK therefore seems to present a major molecular pathway in carcinogenesis and progression of breast phyllodes tumours.

Stress associated proteins metallothionein, HO-1 and HSP 70 in human zero-hour biopsies of transplanted kidneys.

Light microscopic alterations reflecting both previous and preservation-induced changes in the donor organ are usually not very distinctive. The ischemia/reperfusion-associated injury depends primarily on the conditions of donor organ preservation. The present study examined human kidney biopsies with special attention paid to the molecular mechanisms of preservation-induced injury preceding reperfusion. Stress-associated proteins hemeoxygenase-1 (HO-1), heat shock protein 70 (HSP 70), and metallothionein (MT) were studied in human zero-hour biopsies of transplanted kidneys prior to reperfusion in 29 patients. Protein expression was evaluated by semiquantitative immunohistochemistry and Western blotting for HO-1 and HSP 70. These findings were correlated with terminal deoxynucleotidyltransferase-mediated 2'-deoxyuridine 5'-triphosphate-digoxigenin nick end labeling (TUNEL) staining and follow up. Compared to controls, MT and HSP 70 expression was significantly higher at zero hour. In contrast, HO-1 and the number of TUNEL-positive cells were not elevated. MT and HO-1 immunoexpression were inversely associated with graft function, and hence, were of prognostic relevance. MT and HSP 70 were sensitive to the duration of cold ischemia. MT and HO-1 are suitable indicators for tissue injury during ischemia and may serve as new predictive markers that need to be validated in further independent studies.

Radiology-pathology conference: osteosarcoma in a cartilaginous exostosis of the femur.

Osteochondromas are common lesions in the metaphyseal segments of the long bones and are known to be able to degenerate into chondrosarcoma. We present the case of a 20-year-old patient with an osteosarcoma at the base of a cartilaginous exostosis and discuss the causal relationship between the two lesions on the basis of the radiologic and pathologic findings.

Upregulation of Id-1 via BMP-2 receptors induces reactive oxygen species in podocytes.

Bone morphogenetic proteins (BMPs) are secreted signaling molecules, which play a major role in kidney development and disease. Here, we show the existence of mRNA for BMP-2 and for the BMP receptors BMPR1A, BMPR1B, BMPRII, ACVR1A, ACVR2, and ACVR2B in differentiated mouse podocytes and the protein expression of BMPR1A in human glomerular podocytes. BMP-2 dose dependently increases the free cytosolic Ca(2+) concentration in podocytes proving the existence of a functional receptor in these cells. Recent data indicate that in a myoblastic cell line and in a breast cancer cell line, BMP-2 increases the expression of Id-1, a negative regulator of basic helix-loop-helix transcription factors, but the role of BMP-2 stimulated Id-1 expression in the kidney has not been further characterized. Here, we show that BMP-2 increases the expression of Id-1 in differentiated podocytes. To investigate a role of Id-1 for podocyte function, overexpression of Id-1 was induced in differentiated mouse podocytes. Id-1-overexpressing podocytes show an increased NADPH-dependent production of reactive oxygen species (ROS). This effect can be evoked by BMP-2 and can be antagonized by anti-Id-1 antisense oligonucleotides. The data indicate that BMP-2 may, via an increased expression of Id-1 and an increased generation of ROS, contribute to important cellular functions in podocytes. ROS supposedly play a major role in cell adhesion, cell injury, ion transport, fibrogenesis, angiogenesis and are involved in the pathogenesis of membranous nephropathy.

Myxoid chondrosarcoma of the maxilla in a pediatric patient.

Myxoid chondrosarcomas of the head and neck region are rare. We report the case of an 8-year-old boy with progressive unilateral nasal obstruction resulting from a highly differentiated myxoid chondrosarcoma of the maxilla extending to the nasal cavity and the ethmoid. Clinical presentation, histological findings and therapy are presented with a brief review of the literature. This case reaffirms the importance of considering sarcomas or other neoplastic lesions in the differential diagnosis of progressive nasal obstruction in children.

First evidence supporting a potential role for the BMP/SMAD pathway in the progression of oestrogen receptor-positive breast cancer.

Oestrogen receptor expression is generally a sign of better tumour differentiation and comparatively good clinical outcome in invasive breast cancer. However, oestrogen receptor-positive, poorly differentiated carcinomas with a poor clinical outcome exist. The underlying genetic mechanisms and the genes involved remain obscure, even though chromosome 7p gains seem to be associated with these uncommon tumours. In this study, we compared two subsets of oestrogen receptor-positive breast cancers, which differed in tumour grade, cytogenetic instability, and tumour proliferation, for their differential gene expression in order to identify proteins involved in the progression of oestrogen receptor-positive breast cancers. We were able to show by means of subtractive suppression hybridization, real-time reverse transcriptase PCR, and tissue microarray analysis that expression of the bone morphogenetic protein receptor IB (BMPR-IB) is a major hallmark of the progression and dedifferentiation of breast cancer. Strong expression of BMPR-IB was associated with high tumour grade, high tumour proliferation, cytogenetic instability, and a poor prognosis in oestrogen receptor-positive carcinomas. Western blot analysis revealed that downstream signalling of this receptor is mainly mediated via phosphorylation of SMAD 1 in oestrogen receptor-positive breast cancer. Even though BMPR-IB was expressed in oestrogen receptor-positive and -negative breast cancers, an impact on tumour grade, proliferation, and cytogenetic instability, as parameters of tumour progression, could only be demonstrated in oestrogen receptor-positive carcinomas. This pro-proliferative effect was complemented by significant anti-apoptotic activity, indicated by XIAP and IAP-2 expression in BMPR-IB-positive carcinomas. These results show that the BMP/SMAD pathway is activated in breast cancer and may contribute to breast cancer progression and dedifferentiation in oestrogen receptor-positive breast cancer. The definition of this pathway characterizes a new potential target in the molecular treatment of invasive breast cancer.

Long-term follow-up of double kidney transplantation using a score for evaluation of marginal donors*.

To face the problem of organ shortage, marginal grafts from 36 donors which had been refused for single transplantation were used for double-kidney transplantation (D-KTX). The residual kidney function was evaluated by the Muenster double kidney score. In a 5-year period kidneys from 57 marginal donors were transferred to our center. According to the Muenster double kidney score, the kidneys were distributed to single, double or refusal of transplantation. Sixteen male and 20 female donors were used for D-KTX (70+/-9.3 years, range 53-86). Thirty-six recipients (23 male, 13 female; 60.5+/-6.9 years) were double-grafted within a mean cold ischemic time of 19.3+/-3.4 h. Immunosuppression varied according to human leukocyte antigen (HLA)-mismatch. Graft and patient survival was observed up to 5 years. Initial graft function rate was 69%. Two recipients had a primary nonfunction (5.5%) and nine recipients suffered from delayed graft function (DGF; 25%). One-, 2-, 3-year creatinine values were 1.6 +/- 0.5, 1.9 +/- 0.6 and 2.2 +/- 0.7 mg/dl, respectively. One-, 2-, 3-, 4- and 5-year function rate was 93.7%, 93.5%, 81.8%, 76.4% and 55%, respectively (n = 32, 31, 22, 17 and 9). Acute rejection rate was 19%. 4 grafts were lost to chronic rejection (months 22, 25, 28, 48). Six (16%) died in long-term follow-up because of pneumonia (n = 2), carcinoma of the lung (n = 1), cardial complications (n = 2) and multiorgan failure (n = 1). D-KTX is a safe way to face the problem of organ shortage. However, a score for preoperative evaluation of marginal kidneys for single, dual or refusal of transplantation is essential.

Heme-induced heme oxygenase-1 (HO-1) in human monocytes inhibits apoptosis despite caspase-3 up-regulation.

Monocyte activation, apoptosis and differentiation are hallmarks of most inflammatory vascular disorders. We studied the effects of heme oxygenase-1 (HO-1) induced by its substrate hemin on apoptosis, caspase-3 expression and the differentiation of freshly isolated human monocytes. Hemin induced HO-1 in a dose- and time-dependent fashion as measured by semi-quantitative RT-PCR and flow cytometry. Apoptosis was markedly suppressed by hemin in cells rendered apoptotic by serum deprivation or dexamethasone as determined by flow cytometric detection of annexin V binding or transmission electron microscopy (TEM). The specific HO-1 inhibitor zinc protoporphyrin (ZnPP) reversed the effects of hemin on monocyte apoptosis and diminished cell lifespan. Surprisingly, the cytoprotective effects of hemin were positively correlated with caspase-3 up-regulation. Hemin-induced apoptosis suppression was enhanced by the caspase-3 inhibitor DEVD-CHO, indicating that caspase-3 was active in a pro-apoptotic fashion. Hemin inhibited CD95 as a putative cytoprotective mechanism. Morphological studies and detection of CD86 showed that monocytes differentiated into macrophages in response to hemin after relatively long incubation times, a phenomenon that might be provoked by caspase-3-regulated pathways. Our results confirm a similar cytoprotective effect of hemin/HO-1 for monocytes as has been shown for other cells, despite caspase-3 up-regulation. The fact that HO-1 may adversely affect monocyte survival and differentiation could be of particular significance in future therapies for occlusive vascular diseases or transplant rejection.

The effect of endogenous nitric oxide on cholinergic ciliary stimulation of human nasal mucosa.

OBJECTIVES/HYPOTHESIS: Endogenous nitric oxide (NO) production by inducible nitric oxide synthase is enhanced in the nasal ciliated respiratory tract epithelium of patients with allergic rhinitis. Recent experimental data have suggested endogenous NO to be strongly involved in the complex regulation of ciliary activity, the driving force of the mucociliary transport system. The authors investigated the effect of endogenous NO on acetylcholine-stimulated ciliary activity of human nasal mucosa. STUDY DESIGN: In vitro study. METHODS: Cultures of human nasal mucosa explants were incubated with tumor necrosis factor-alpha and bacterial lipopolysaccharides to enhance endogenous NO production. Expression of inducible NO synthase was morphologically demonstrated by immunohistochemistry. Ciliary beat frequency was determined by phase-contrast microscopy of ciliated epithelium, using a computerized photoelectric technique. Stimulation experiments were performed in vitro with acetylcholine and N(G)-nitro-l-arginine methyl ester (L-NAME), a NO synthase inhibitor. RESULTS: Upregulation of inducible NO synthase in the respiratory tract epithelium after stimulation with tumor necrosis factor-alpha and lipopolysaccharide was visualized by immunohistochemical analysis. Experimental inhibition of enhanced endogenous NO production by 10 mol/L L-NAME significantly reduced baseline ciliary beat frequency from 8.6 +/- 0.2 to 7.8 +/- 0.2 Hz (P < .05). Cholinergic ciliary stimulation above baseline by 10 mol/L acetylcholine was not significantly different before (11.5%) or after (10.8%) blocking of endogenous NO production. CONCLUSION: Taken together, the study results suggest that baseline ciliary activity depends on endogenous NO production but that the extent of cholinergic ciliary stimulation is independent of endogenous NO production. The combination of the two effects may improve nasal mucociliary clearance of inhaled allergens in patients with allergic rhinitis.

CGH and CD 44/MIB-1 immunohistochemistry are helpful to distinguish metastasized from nonmetastasized sporadic pheochromocytomas.

The natural course of pheochromocytomas (PCC) cannot be predicted for certain on the basis of primary histology, their malignant character can only be confirmed by the occurrence of metastases during follow-up. Based on the recently proposed PASS score for evaluation we examined 37 adrenal (36 sporadic and one familial) and six sporadic extra-adrenal paragangliomas (all designated as pheochromocytomas) with a 'malignant histology' to find additional predictive factors. Drawing upon the follow-up (18 months to 12 years, mean 5.8 years) metastasized (n=20) and nonmetastasized (n=23) courses could be distinguished. Metastasized PCC revealed significantly (P=0.03) more copy number changes on comparative genomic hybridization (CGH) (mean 8.3) than nonmetastasized tumors (mean: 4.3). The most frequent chromosomal alterations were losses on 1p (75.6%) and 3q (44%). Both were detected with identical frequency in metastasized and nonmetastasized PCC. A gain on 17q (P=0.025) was significantly predominant in malignant courses and suggests similarities in the genetic origin and progression of PCC and neuroblastomas. The proliferative activity (MIB-1 score) of metastasized PCC (n=20) was found to be significantly higher in metastasized tumors (mean 12.8% vs mean 3.5%). In contrast, the semiquantitatively scored membrane-bound staining of CD 44-S was stronger in tumors without metastases (mean 2.1 vs mean: 0.25) during the follow-up period (P<0.01). Although the results correspond to the established weight differences the tumor weight does not appear to be an independent prognostic factor. Our study suggests that CD 44-S and MIB-1 immunostaining as well as the CGH results might complement the PASS score in predicting a metastasized course of PCC. Regardless of tumor weight, tumors with a 'malignant histology' are highly prone to metastasize when more than 5% of MIB1-positive nuclei are present or CD44-S immunostaining is negative, or both. PCC with 10 or more copy number changes on CGH must be referred to as malignant tumors.

Neonatal hemoperitoneum caused by a congenital liver angioma.

Severe intraabdominal bleeding in neonates within 24 hours after birth are an extremely seldom event. In our neonate, the intraabdominal bleeding was diagnosed by the clinical picture and by abdominal ultrasound examination. The etiology for this bleeding was primarily a blunt abdominal trauma as described in most cases of the liver; however, histology showed a congenital cavernous angioma of the liver resulting in a different tensile strength of this part of the liver in comparison with the rest of the liver tissue. Therefore, a relatively blunt abdominal trauma caused a rupture of the liver in a histologically altered part, which histologically was found to be a relatively soft and fragile tumor of the liver.

Impact of renal transplantation on small vessel reactivity.

BACKGROUND: The function of large arteries is altered after renal transplantation. Whether transplantation also induces agonist-dependent functional changes in small arterial renal and extrarenal vessels has not yet been studied. METHODS: Chronic rejection was induced by grafting Lewis rats with kidneys from Fischer rats (FL). Rats that underwent transplantation were bilaterally nephrectomized. Rats that underwent syngeneic transplantation, uninephrectomized rats, uninephrectomized rats with denervated kidneys or with kidneys made ischemic, and native rats served as controls. All animals were treated with cyclosporine for 10 days. Eighteen weeks after surgery, the reactivity of small arteries (220-270 microm) was tested by myography. RESULTS: Weight gain, glomerular filtration rate, and arterial pressure were similar in all groups, whereas proteinuria was elevated in FL. Only kidneys from FL showed glomerular lesions, tubular atrophy, and vasculopathy. Responsiveness of coronary, mesenteric, and femoral resistance vessels to both constrictor and dilator agonists was similar in transplanted and nontransplanted animals. Resistance vessels obtained from both allogeneically and syngeneically transplanted kidneys were more sensitive to norepinephrine, phenylephrine, angiotensin II, and vasopressin than renal vessels from weight-matched controls. Vasodilation in response to acetylcholine and sodium nitroprusside was mitigated in transplanted versus nontransplanted kidneys. CONCLUSIONS: In rat renal transplantation, renal resistance vessel responsiveness to constrictor or dilator stimuli is altered. Extrarenal small vessel function is not affected. The changes in function of renal resistance vessels are not explained by reduction of nephron mass, denervation, ischemia, or chronic rejection.

Acute renal failure in IgA nephropathy: aggravation by gross hematuria due to anticoagulant treatment.

IgA nephropathy is one of the most common forms of glomerulonephritis. Macroscopic or microscopic hematuria with mild proteinuria are the main symptoms. Without complicating factors, IgA nephropathy has a favourable long-term prognosis. We report a case of reversible acute renal failure (ARF) as a complication of mild IgA nephropathy while oral anticoagulants were administered. Diagnosis was based on a renal biopsy showing marked granular mesangial IgA-deposition. In addition, numerous tubules were extended and completely obstructed by red blood cell casts. After hemodialysis treatment and parallel anti-inflammatory steroids and after stopping anticoagulation, renal function gradually improved up to complete remission. This report indicates that anticoagulatory treatment may have negative effects on the long-term prognosis of IgA nephropathy with respect to development of ARF or tubulo-interstitial inflammation.

Early detection of severe cholestatic hepatopathy in COACH syndrome.

COACH syndrome is a disorder with cerebellar vermis hypoplasia, oligophrenia, ataxia, coloboma, and hepatic fibrosis. Sixteen cases with certain COACH diagnosis have been reported so far. Neurologic abnormalities are the first symptoms in most cases. The majority of cases were diagnosed late in childhood or adolescence. Complications of the hepatopathy contribute extensively to the morbidity and lethality in the course of the disease. Major complications are portal hypertension, esophageal varices, and gastrointestinal bleeding. We report of a child with only mild neurologic symptoms, but severe hepatic fibrosis with cholangiopathy, and review the literature. This is the first description of profound cholestatic hepatopathy in a very young child with COACH syndrome. The patient was found to have cerebellar vermis hypoplasia, unilateral optical nerve coloboma, mild dysmorphic signs, and a ventricular septum defect. Routine laboratory investigations eventually revealed elevated liver enzymes. Prothrombin time was abnormal. Ultrasound scan of the liver was normal. Hepatotropic viral infections were excluded. We performed a liver biopsy at the age of 16 months, confirming an early stage of cirrhosis with septal fibrosis and pseudolobules, inflammatory infiltrates, signs of cholestasis, and reduced numbers of intrahepatic bile ducts. Early detection and differentiation of liver pathology are important in COACH syndrome. Progressive destructive cholangiopathy may contribute to hepatic fibrosis in COACH syndrome. Liver disease can be severe even in cases with mild neurologic deficits.