Petra Reinecke

Genomic instability and myelodysplasia with monosomy 7 consequent to EVI1 activation after gene therapy for chronic granulomatous disease

Gene-modified autologous hematopoietic stem cells (HSC) can provide ample clinical benefits to subjects suffering from X-linked chronic granulomatous disease (X-CGD), a rare inherited immunodeficiency characterized by recurrent, often life-threatening bacterial and fungal infections. Here we report on the molecular and cellular events observed in two young adults with X-CGD treated by gene therapy in 2004. After the initial resolution of bacterial and fungal infections, both subjects showed silencing of transgene expression due to methylation of the viral promoter, and myelodysplasia with monosomy 7 as a result of insertional activation of ecotropic viral integration site 1 (EVI1). One subject died from overwhelming sepsis 27 months after gene therapy, whereas a second subject underwent an allogeneic HSC transplantation. Our data show that forced overexpression of EVI1 in human cells disrupts normal centrosome duplication, linking EVI1 activation to the development of genomic instability, monosomy 7 and clonal progression toward myelodysplasia.

JAK2V617F mutation status identifies subtypes of refractory anemia with ringed sideroblasts associated with marked thrombocytosis

Refractory anemia with ringed sideroblasts and marked thrombocytosis is a condition with both myelodysplastic and myeloproliferative features. This study indicates that a considerable proportion of patients with this condition carry the unique V617F mutation of JAK2, and that the mutant allele burden increases over time. See related perspective on page 4. Background Refractory anemia with ringed sideroblasts and marked thrombocytosis (RARS-T) was recently shown to be a JAK2-V617F mutation-related disorder. To determine the frequency and the prognostic significance of this mutation, we retrospectively evaluated 23 patients with platelet counts more than 600 × 109/L, 15% ringed sideroblasts or more, and at least erythroid marrow dysplasia. Design and Methods An allele-specific polymerase chain reaction for JAK2-V617F was used to determine the allelic ratio of the mutated JAK2 allele in DNA samples extracted from bone marrow biopsies. Hematologic and survival data of the JAK2-V617F positive vs. the JAK2-V617F negative patients were statistically analyzed. Allele-specific polymerase chain reaction was also used to screen for MPL-W515 mutations. Results The JAK2-V617F mutation was present in 11 patients (48%) and was associated with significantly higher erythrocyte and white blood cell counts (p=0.009 and 0.011, respectively). In 6/11 RARS-T patients the allelic ratio of JAK2-V617F was above 50%, indicating the presence of cells homozygous for the mutation. In two of these patients a transition from JAK2-V617F heterozygosity to homozygosity was documented and was accompanied by rising platelet counts in sequential samples. The MPL-W515L mutation was detected in one JAK2-V617F negative patient. The relative risk of death was found to be lower in the mutation-positive group than in the mutation-negative group. Conclusions RARS-T patients with JAK2-V617F have a more favorable prognosis than those without the JAK2 mutation. The prevalence of homozygous JAK2-V617F mutation in RARS-T suggests that this entity is biologically distinct from essential thrombocythemia.

DLK-1 as a marker to distinguish unrestricted somatic stem cells and mesenchymal stromal cells in cord blood

In addition to hematopoietic stem cells, cord blood (CB) also contains different nonhematopoietic CD45-, CD34- adherent cell populations: cord blood mesenchymal stromal cells (CB MSC) that behave almost like MSC from bone marrow (BM MSC) and unrestricted somatic stem cells (USSC) that differentiate into cells of all 3 germ layers. Distinguishing between these populations is difficult due to overlapping features such as the immunophenotype or the osteogenic and chondrogenic differentiation pathway. Functional differences in the differentiation potential suggest different developmental stages or different cell populations. Here we demonstrate that the expression of genes and the differentiation toward the adipogenic lineage can discriminate between these 2 populations. USSC, including clonal-derived cells lacking adipogenic differentiation, strongly expressed δ-like 1/preadipocyte factor 1 (DLK-1/PREF1) correlating with high proliferative potential, while CB MSC were characterized by a strong differentiation toward adipocytes correlating with a weak or negative DLK-1/PREF1 expression. Constitutive overexpression of DLK-1/PREF1 in CB MSC resulted in a reduced adipogenic differentiation, whereas silencing of DLK-1 in USSC resulted in adipogenic differentiation.

Functional Intactness of Stimulatory and Inhibitory Autocrine Loops in Human Renal Carcinoma Cell Lines of the Clear Cell Type

PURPOSE The aim of the present study was to analyze the contribution of different stimulatory and inhibitory growth factors to the deregulated proliferation of human RCCs. MATERIALS AND METHODS The expression of different growth factors and their corresponding receptors were analyzed by Northern blot, FACS, ELISA and immunocytochemistry in 13 permanent human RCC cell lines of the clear cell type. Moreover, the functional intactness of growth factor-related signal transduction pathways was investigated. RESULTS All RCC cell lines expressed EGF-receptor mRNA and protein and 10 cell lines secreted TGF-alpha. Exogeneously added TGF-alpha resulted in a significant (p < 0.05) stimulation of growth in 6 RCC cell lines and a significant (p < 0.05) inhibition of proliferation in 3 cell lines. PDGF B and the corresponding type beta receptor were expressed in a single cell line. mRNA expression of PDGF A and PDGF-alpha-receptor as well as IGF-1 and its receptor could not be detected in any cell line. Eleven RCC cell lines expressed TGF-beta 1 mRNA and in all cell lines TGF-beta 1 secretion into the supernatant could be demonstrated. Whereas all cell lines exhibited TGF-beta type II-receptor mRNA, type I-receptor mRNA could be detected only in 3 cell lines. TGF-beta type III-receptor was observed in 1 cell line. Exogeneously added TGF-beta1 resulted in a significant (p < 0.05) inhibition of proliferation in 7 RCC cell lines. CONCLUSION Clear cell RCCs exhibit a complex and heterogeneous expression pattern for various growth factors and their receptors. Growth factor secretion and intact signal transduction pathways in most clear cell RCCs facilitate an intricate modulation of RCC growth by autocrine and paracrine interactions between tumor cells and host tissue.

Fabry disease in patients with hypertrophic cardiomyopathy (HCM)

Morbus Fabry ist eine x-chromosomale rezessiv vererbte lysosomale Speicherkrankheit mit variablem Phänotyp. Aufgrund eines Mangels von alpha-Galaktosidase A kommt es zu einer Akkumulation von Glykosphingolipiden im Gewebe.    Die kardiale Variante des Morbus Fabry ist, im Gegensatz zur klassischen Variante mit multipler Organbeteiligung, eine auf das Herz beschränkte myokardiale Speicherkrankheit.    Klinisch imponiert eine myokardiale Hypertrophie, so dass die kardiale Variante des Morbus Fabry das Bild einer hypertrophischen obstruktiven und vor allem nicht obstruktiven (HNCM) Kardiomyopathie imitiert.    Bei Patienten mit unklarer linksventrikulärer Hypertrophie kann die Diagnose eines Morbus Fabry durch licht- und elektronenmikroskopische Untersuchung von endomyokardialen Katheterbiopsien und/oder laborchemische Untersuchungen (verminderte Aktivität der alpha-Galaktosidase A im Plasma bzw. in Leukozyten) gestellt werden. Wie in mehreren Studien gezeigt werden konnte, weisen zwischen 4% und 8% von nicht selektierten Patienten mit dem klinischen Bild einer HNCM eine kardiale Variante des Morbus Fabry auf.    Deshalb sollte bei jedem Patienten mit myokardialer Hypertrophie unklarer Ätiologie eine verborgene myokardiale Speicherkrankheit, insbesondere ein kardialer Morbus Fabry, ausgeschlossen oder diagnostiziert werden. Der endomyokardialen Katheterbiopsie kommt hierbei eine entscheidende Rolle zu.    Dies ist von besonderer Bedeutung durch die kürzlich publizierte alpha-Galaktosidase A-Enzymersatztherapie bei Morbus Fabry, die eine kausale Therapie dieser Erkrankung eröffnet.    Jedoch fehlen bisher randomisierte Studien, die belegen, dass die Enzymersatztherapie zu einer signifikanten Verbesserung des klinischen Bildes der kardialen Variante des Morbus Fabry führt und die Krankheitsprogredienz in bisher klinisch asymptomatischen Patienten aufhält. Fabry disease is an X-linked recessive lysosomal storage disorder with variable phenotype characterized by the accumulation of glycosphingolipid in various tissues. Unlike patients with the classical systemic Fabry disease entity, who present with multiple organ involvement, patients with a cardiac variant of Fabry disease are characterized mainly by myocardial hypertrophy. Therefore, the cardiac variant of Fabry disease may be defined as a cardiomyocytic storage disorder, thus, mimicking the clinical features of hypertrophic obstructive and especially non-obstructive cardiomyopathy. In patients with unexplained left ventricular hypertrophy the diagnosis of a cardiac variant of Fabry disease is performed by light- and electron microscopic evaluation of endomyocardial catheter biopsy specimens and/or serologic investigations (decreased activity of alpha-galactosidase A in plasma or leucocytes).    Several studies show that between 4% and 8% of unselected patients with the clinical features of hypertrophic non-obstructive cardiomyopathy have a cardiac variant of Fabry disease. In each patient with unexplained myocardial hypertrophy concealed myocardial storage disease, especially cardiac Fabry disease has to be considered and should be ruled out or confirmed by endomyocardial catheter biopsy. This is important because of the recently reported alpha-galactosidase A enzyme replacement therapy in Fabry disease. Randomized, multicenter studies are mandatory to test the hypothesis that enzyme replacement therapy leads to a beneficial clinical effect in the cardiac variant form of Fabry disease and may prevent the progression of the disease in asymptomatic patients.

Secretion of GM-CSF and M-CSF by human renal cell carcinomas of different histologic types

OBJECTIVES To analyze the secretion of hematopoietic growth factors and the expression of their corresponding receptors in 40 newly established renal cell carcinoma (RCC) cell lines of different histologic types. Little is known about the secretion and function of hematopoietic growth factors by human RCCs. METHODS The expression of the hematopoietic growth factors (ie, erythropoietin, interleukin [IL]-3, IL-5, granulocyte colony-stimulating factor [G-CSF], granulocyte-macrophage colony-stimulating factor [GM-CSF], and macrophage colony-stimulating factor [M-CSF]) was determined by enzyme-linked immunosorbent assay analysis under different culture conditions, including suspension culture and monolayer cultures (plastic and Matrigel-coated culture flasks). The expression of their corresponding receptors was defined by fluorescence activated cell scanner analysis and by reverse-transcriptase polymerase chain reaction. The response of the RCC cell lines to exogenous hematopoietic growth factors was analyzed by MTT assay. RESULTS In almost all of the cell lines, significant amounts of GM-CSF and M-CSF were secreted, and in four cell lines, a secretion of G-CSF was detected. Fourteen RCC cell lines showed secretion of IL-3, and production of IL-5 and erythropoietin was not observed in any cell line. Secretion of GM-CSF and M-CSF was affected by the substratum offered for cell attachment in the adherent cultures. GM-CSF secretion was more pronounced under culture conditions with a reduced frequency of cell-to-cell contacts. Two cell lines were shown to express receptors for M-CSF, but receptors for G-CSF and GM-CSF could not be detected in any cell line. Exposure to exogenous G-CSF, GM-CSF, and M-CSF did not affect the proliferation of our RCC cell lines. CONCLUSIONS The results of our study clearly demonstrate that human RCC cells can secrete significant amounts of G-CSF, GM-CSF, M-CSF, and IL-3, and are thereby theoretically able to modulate the hosts tumor-directed immune response.

Differential response to transforming growth factor (TGF)-α and fibroblast growth factor (FGF) in human renal cell carcinomas of the clear cell and papillary types

The clear cell and the papillary types of human renal cell carcinoma (RCC) are distinct tumour entities with marked differences in their biological properties. Because growth factors are considered to affect profoundly the biological behaviour of malignant tumours, we compared the expression and function of transforming growth factor (TGF)-alpha and fibroblast growth factor (FGF) in both types of RCCs. Both in vivo and in vitro expression of TGF-alpha, epidermal growth factor-receptor (EGF-R), FGF-2 and FGF type 3- and 4-receptors was found in RCCs of both types. However, marked differences between clear cell and papillary RCCs became evident for TGF-alpha secretion, which could be demonstrated in 20 out of 24 (83%) clear cell RCCs but in only two out of four (50%) papillary tumours. Moreover, the mean TGF-alpha secretion rate in clear cell RCCs significantly (P<0. 05) exceeded that of papillary RCCs. Because the expression of growth factor receptors could not prove the corresponding signalling cascades were functional, tumour cell proliferation was tested after exposure to exogenous TGF-alpha or FGF-1. These experiments demonstrated that papillary RCCs did not respond significantly to exogenous TGF-alpha or FGF-1, whereas eight (33%) (TGF-alpha) and 11 (46%) (FGF-1) out of 24 clear cell RCCs responded with significant (P<0.05) growth stimulation. In conclusion, our investigation presents data indicating that TGF-alpha and FGF are functionally involved in the progression of clear cell RCCs, directly stimulating proliferation by autocrine and/or paracrine actions. In contrast, TGF-alpha and FGF did not directly stimulate the proliferation of our papillary RCCs, thereby suggesting functional defects or a blockade in the corresponding signalling cascades. This differential functionality might contribute to the more aggressive behaviour of clear cell RCCs.

Antiproliferative effects of paclitaxel (Taxol®) on human renal clear cell carcinomas in vitro

The aim of this study was to analyse the direct antiproliferative effects of paclitaxel on 20 different renal clear cell carcinoma (RCCC) cell lines comparing the effects of paclitaxel dissolved in either DMSO or Cremophor EL/ethanol (Taxol). The MTT assay was used to determine the growth inhibition of the cell lines by paclitaxel. In addition, micronuclei and microtubule alterations were examined by light and immunofluorescence microscopy. A significant (P < 0.05) dose-dependent inhibition of proliferation was evident in 19 out of 20 cell lines after exposure to paclitaxel dissolved in DMSO and in all cell lines after exposure to paclitaxel in Cremophor EL/ethanol. The extent of response markedly varied between the different cell lines ranging from modest effects to reduction of cell viability down to 1-2% of the control. The effects of paclitaxel in Cremophor EL/ethanol proved to be more pronounced than the effects of paclitaxel dissolved in DMSO. This observation could be explained by additional growth inhibitory effects of Cremophor EL alone. Light microscopy revealed extensive micronucleus formation after treatment with paclitaxel. However, the failure to demonstrate differences of micronucleus formation in paclitaxel-responsive and non-responsive RCCC cell lines argued against a causal relationship between micronucleus formation and growth inhibition. Immunofluorescence microscopy revealed no differences in the formation of abnormal microtubules in cell lines responsive or non-responsive to the growth inhibitory effects of paclitaxel. Further investigations, therefore, are needed to understand the mechanisms determining the response of RCCCs to paclitaxel treatment.

Solitary submucous neurofibroma of the mandible: review of the literature and report of a rare case

Solitary neurofibroma is a rare benign non-odontogenic tumor. Particularly in the oral cavity, neurogenic tumors are rare, especially if they are malignant. Neurofibromas may present either as solitary lesions or as part of the generalised syndrome of neurofibromatosis or von Recklinghausens disease of the skin. Clinically, oral neurofibromas usually appear as pediculated or sessile nodules, with slow growth and mostly without pain. The diagnosis can be confirmed by histological examination. Neurofibromas are immunopositive for the S-100 protein, indicating its neural origin. Treatment is surgical and the prognosis is excellent. For illustration a rare case of a solitary neurofibroma in the mandible is presented.

Myocardial T2 Mapping Increases Noninvasive Diagnostic Accuracy for Biopsy-Proven Myocarditis

Detection of myocardial inflammation in patients with clinically suspected acute myocarditis (sAMC) is of prognostic importance but remains a challenge in routine clinical practice [(1)][1]. Compared with endomyocardial biopsy (EMB), the diagnostic gold standard, cardiovascular magnetic resonance (