Endothelin-1 and endothelin converting enzyme-1 in human atherosclerosis--novel targets for pharmacotherapy in atherosclerosis.

The role of chronic inflammation in the pathogenesis of the acute coronary syndromes has received increasing attention since active plaques rich in macrophages (Mphi's) are more prone to rupture whereas plaques rich in myofibroblasts are considered to be stable. Functionally, active plaques show a locally enhanced vasoreactivity. Endothelin-1 (ET-1) a potent vasoconstrictor acts in a paracrine fashion to regulate vascular tone. ET-1 is also produced by inflammatory cells suggesting a role for ET-1 in inflammation. Additionally, ET-1 is a mitogen. Endothelin converting enzyme-1 (ECE-1) activates ET-1 and may thus contribute to the regulation of vascular tone and cell growth during atherosclerosis. We evaluated the presence of ECE-1 and big ET-1/ET-1 and the activity of ECE-1 in different plaque types. Together with ET-1, ECE-1 is present in endothelial cells (ECs), vascular smooth muscle cells (VSMCs) and Mphi's. ECE-1 activity and ET-1-immunoreactivity (IR) both are upregulated during the progression of atherosclerosis from a non-inflammatory to an inflammatory stage. Thus, enhanced production of active ET-1 may contribute to cell growth and regulation of vascular tone in advanced plaques and also in very early stages of atherosclerosis. Furthermore, we examined the presence of ET-1 in coronary plaque tissue obtained by directional coronary atherectomy. ET-1 IR localized to plaque components indicative of chronic inflammation. Semiquantitative analysis of ET-1 IR revealed significantly higher staining grades in active coronary lesions compared with nonactive lesions. The increased ET-1 content in active coronary lesions may be beneficial to the stabilization of the vessel wall after plaque rupture and disadvantageous because it may lead to vasospasm and to the progression of atherosclerosis.

Formation of thermal vacancies on the Si sublattice of the intermetallic compound MoSi2.

For a detailed understanding of high-temperature processes in complex solids the identification of the sublattice on which thermal defects are formed is of basic interest. Theoretical studies in intermetallic compounds favor a particular sublattice for thermal vacancy formation. In the present study we detect in ordered MoSi2 thermal vacancies with a low formation enthalpy of H(F)(V)=(1.6+/-0.1) eV, and we succeed in showing by experimental and theoretical efforts that they are preferentially formed on the Si sublattice. By these data self-diffusion in MoSi2 can be understood.

Study of an order-disorder phase transition on an atomic scale: the example of decagonal Al-Ni-Co quasicrystals.

In the present paper we succeeded in studying structural phase transitions from an atomistic point of view by positron annihilation Doppler broadening. This differs and is complementary to conventionally used diffraction experiments with large coherence lengths. In the exemplary case of the 1140 K order-disorder transition in decagonal Al71.5Ni14Co14.5 quasicrystals the importance of this atomistic approach and its wide scope of application is demonstrated.

Evidence of clonality in chronic neutrophilic leukaemia.

BACKGROUND: Chronic neutrophilic leukaemia (CNL) is a rare myeloproliferative disorder of elderly patients characterised by sustained neutrophilia and splenomegaly. The diagnosis of CNL requires the exclusion of BCR/ABL positive chronic myelogenous leukaemia (CML) and of leukaemoid reactions (LRs). The differentiation between CNL and LR is problematic because both conditions share similar morphological features; it is also important because patients with CNL generally have a poor prognosis. AIMS: To determine whether CNL and LR could be distinguished on the basis of different clonality patterns. METHODS: Blood samples from 52 women were studied using the human androgen receptor gene assay (HUMARA). RESULTS: Monoclonality was found in the neutrophils in all 17 patients with different myeloproliferative syndromes (MPSs), including those with CNL. In four of the patients with CNL, autologous T cells were also monoclonal, suggesting that they belonged to the neoplastic clone. This finding was in contrast to other MPSs in which T cells were almost always polyclonal. Of nine patients with clinically suspected LR, the neutrophils of five were polyclonal, whereas three patients had monoclonal neutrophils, suggesting that they might be in the process of developing an MPS. Among 26 healthy blood donors, 20 had polyclonal neutrophils and five showed skewed clonality patterns. One case of LR and one normal blood donor were scored "not informative" at the HUMARA locus. CONCLUSIONS: Clonality studies of blood neutrophils using HUMARA aid in distinguishing female patients with monoclonal CNL from those with LR. For the diagnosis of CNL, monoclonality of the neutrophils should be demonstrated whenever possible.

[Clonality as a criterion in the differential diagnosis of chronic neutrophilic leukaemia]. / Klonalität als differenzialdiagnostisches Kriterium bei chronischer Neutrophilenleukämie.

Chronic neutrophilic leukaemia (CNL) is a rare BCR/ABL negative myeloproliferative disorder of elderly patients, showing sustained neutrophilia and splenomegaly. Differentiation between CNL and leukaemoid reactions (LR) is problematic since both conditions share similar morphological features but is essential because CNL patients generally have a poor prognosis. We studied blood samples from 10 female patients with CNL or LR using the HUMARA assay to determine clonality patterns in neutrophils. T-lymphocytes of the patients were investigated as an internal control cell population. In all five CNL patients the neutrophils, and in four of them also T-lymphocytes were monoclonal, indicating that the latter may also originate from the neoplastic clone. In LR patients the neutrophils and T-lymphocytes were generally polyclonal except in one patient showing monoclonal neutrophils suggesting that this patient might be in the process of developing a myeloproliferative disorder. In females clonality studies of blood neutrophils using HUMARA aid in distinguishing patients with monoclonal CNL from polyclonal LR.

[Coincidence of chronic idiopathic myelofibrosis and chronic lymphocytic leukaemia. A rare phenomenon?]. / Koinzidenz von chronischer idiopathischer Myelofibrose und chronischer lymphatischer Leukämie. Ein seltenes Phänomen?

In 1986 we diagnosed chronic idiopathic myelofibrosis (CIMF) in a 45-year-old asymptomatic patient with hepatosplenomegaly. In 1996 splenectomy was performed because of hypersplenism, and chemotherapy with hydroxyurea was initiated. In 1999 generalised lymphadenopathy of chronic lymphocytic leukaemia (B-CLL) developed. A trephine biopsy showed leukaemic bone marrow infiltration. On heteroduplex analysis we found a clonal rearrangement of IgH in the leukaemic lymphocytes. The coincidence of chronic myeloproliferative and lymphoproliferative diseases in the same patient is a rare phenomenon. According to the relevant literature, seven cases with a combination of CIMF/CLL have been reported. Possible pathomechanisms for the development of such coincidences are: 1) a bilineage manifestation of a pluripotent stem cell proliferation, 2) independent proliferations of two distinct cell lines under a common leukaemogenic stimulus or 3) an accidental association. These coincidence cases often showed a mild clinical course and also in our case, the patient is still alive and in a stable disease condition 16 years after the initial diagnosis.

Chronic neutrophilic leukaemia: 14 new cases of an uncommon myeloproliferative disease.

BACKGROUND: Chronic neutrophilic leukaemia (CNL) is a distinct BCR/ABL negative myeloproliferative disorder of elderly patients characterised by sustained neutrophilia and splenomegaly. The bone marrow shows expansion of neutrophilic granulopoiesis, without excess of myeloblasts. To date, only 129 cases of CNL have been reported in the literature. AIMS: To report the findings from a large group of 14 new cases of CNL, consisting of eight women and six men (mean age, 64.7 years). METHODS: A review of the 14 new cases of CNL and the investigation of BCR/ABL translocations in these patients. RESULTS: Three quarters of the patients died within two years after diagnosis, mostly as a result of severe cerebral haemorrhage. Two younger patients were successfully treated with allogeneic bone marrow transplantation or interferon, which resulted in haematological remission for years. CONCLUSION: CNL is a rare myeloproliferative disease mostly taking a fatal clinical course, despite the presence of mature neutrophils as leukaemic cells in the blood. Thus, it is important to recognise CNL to develop appropriate therapeutic strategies for affected patients.

Identification of lattice vacancies on the two sublattices of SiC.

The identification of atomic defects in solids is of pivotal interest for understanding atomistic processes and solid state properties. Here we report on the exemplary identification of vacancies on each of the two sublattices of SiC by making use of (i) electron irradiation, (ii) measurements of the positron lifetimes, (iii) coincident Doppler broadening studies of the positron-electron annihilation radiation, and (iv) a comparison of the experimental data with theoretical studies. After 0.3 MeV electron irradiation, carbon vacancies V(C) are identified, where, after 0.5 MeV electron irradiation, predomi-nantly silicon vacancies V(Si) are observed. After 2.5 MeV irradiation, divacancies V(Si)-V(Si) are detected. The present results are expected to be of general importance for reliable identification of defects and atomic processes in complex solids.

Coexpression of endothelin-converting enzyme-1 and endothelin-1 in different stages of human atherosclerosis.

BACKGROUND: Endothelin-converting enzyme (ECE)-1 activates endothelin-1 (ET-1) and may thus contribute to the regulation of vascular tone and cell growth during atherosclerosis. METHODS AND RESULTS: To evaluate ECE-1 immunoreactivity concerning big ET-1/ET-1, we performed qualitative and quantitative immunohistochemistry in normal internal mammary arteries (n=10), in coronary arteries with adaptive intimal fibrosis (n=10), in aortic fatty streaks (n=10), and in distinct regions of advanced carotid plaques (n=15). Furthermore, we determined ECE-1 activity in the control specimens and in the inflammatory intimal regions of carotid plaques. Double immunolabeling showed that ECE-1 was present in endothelial cells, vascular smooth muscle cells, and macrophages. All ET-1(+) cells were simultaneously ECE-1(+). Most importantly, there were significantly more ET-1(+) cells in the intima and media when atherosclerosis was in an inflammatory stage than when it was in a noninflammatory stage. Moreover, ECE-1 activity was upregulated in the intima of carotid plaques, although immunohistochemically, there were no significant differences between the number of ECE(+) cells in the different compartments of the arterial wall. CONCLUSION: Together with ET-1, ECE-1 is abundantly present in human arteries and at different stages of atherosclerotic plaque evolution. The upregulation of the ECE-1/ET-1 system is closely linked to the presence of chronic inflammation and is present in very early stages of plaque evolution. Therefore, enhanced production of active ET-1 may substantially contribute to cell growth and the regulation of vascular tone in advanced atherosclerotic lesions and in the very early stages of plaque evolution, when a plaque is still imperceptible clinically.

Bone marrow features improve prognostic efficiency in multivariate risk classification of chronic-phase Ph(1+) chronic myelogenous leukemia: a multicenter trial.

PURPOSE: Multivariate risk classifications for chronic (stable)-phase Ph(1+) chronic myelogenous leukemia (CML) are generally focused on hematologic variables, and the putative prognostic property of bone morphology has been neglected or even contested so far. PATIENTS AND METHODS: A total of 510 consecutively recruited patients in first chronic phase Ph(1+) CML and pretreatment bone marrow biopsy specimens were entered onto this multicenter observational trial to evaluate the effect of bone marrow histopathology. According to generally accepted criteria, patients with any signs of accelerated disease were excluded. Treatment modalities included administration of interferon alfa-2b (IFN) and chemotherapy with hydroxyurea (HU) or busulfan. Immunohistochemical and morphometric techniques were applied to identify marrow cells and to quantify fiber density. Patients were separated into learning and validation samples, and classification and regression tree (CART) analysis was performed to establish a prognostic decision tree. RESULTS: CART analysis of the validation sample (123 patients with HU therapy) revealed the amount of erythroid precursors in the bone marrow, myelofibrosis, and splenomegaly as the most important prognostic features. Three risk profiles with significantly different survival patterns were established, with median survival times ranging from 33 to 108 months (two-sided log-rank test, P =.0001). The new score was confirmed by application to the learning sample with IFN therapy (two-sided log-rank test, P =.0002). Furthermore, risk status defined by the new score was significantly correlated with the occurrence of blast transformation. CONCLUSION: Our data strongly implicate that prognostic classification of chronic-phase Ph(1+) CML can be significantly improved by the inclusion of morphologic parameters. The variables of the presented scoring system may be easily assessed by routinely processed aspirates and bone marrow trephines.

Prognostic impact of bone marrow erythropoietic precursor cells and myelofibrosis at diagnosis of Ph1+ chronic myelogenous leukaemia--a multicentre study on 495 patients.

A multicentre clinicopathological study was performed on 495 patients with chronic-phase Ph1+ chronic myelogenous leukaemia (CML) to determine bone marrow characteristics that exert a significant impact on survival under standard treatment regimens. Immunohistochemical and morphometric techniques were applied to identify nucleated erythroid precursor cells in the bone marrow and to quantify argyrophilic fibre density. Application of the Sokal index and another recently proposed CML score failed to distinguish three clearly defined risk groups. A borderline increase in fibre content (i.e. doubling of the normal density) and a relevant reduction of medullary erythropoiesis proved to be important predictors for survival, even in low-risk classified patients, according to both clinical scores. With regard to optimal treatment strategies, patients with manifest myelofibrosis showed no significant difference in survival rates under interferon or hydroxyurea treatment. Multivariate analysis confirmed the prognostic value of histological features. A risk model based on three variables (fibre density, erythropoietic precursors and spleen size) was constructed that enabled a distinct discrimination of risk profiles. In conclusion, the presented data provide compelling evidence that bone marrow features at diagnosis exert a significant impact on prognosis in CML. In this context, the generally clinical-based multivariate risk classification can be improved by consideration of morphological variables that are acting independently of treatment modalities.

Therapy-related changes of CD34+ progenitor cells in chronic myeloid leukemia: a morphometric study on sequential trephine biopsies.

In chronic myeloid leukemia (CML), it has been assumed that the number of CD34(+) progenitor cells (PGCs) provides useful diagnostic and prognostic information regarding the evolution of accelerated phase and blastic crisis. However, until now no information is available about changes of this peculiar precursor cell population during therapy or possible associations with the other bone marrow constituents. For this reason, a retrospective clinicopathological study was performed on 83 patients with CML including 209 sequential bone marrow biopsies (intervals ranging between 6 and 143 months) and immunohistological staining of CD34(+) cells (QBEND10), megakaryocyte precursors (CD61), and erythropoiesis (Ret 40f). According to treatment modalities, three different groups of patients could be distinguished that received either monotherapy by interferon-alpha2b (IFN-alpha2b) or hydroxyurea (HU) and a combination of both. In comparison with a control group, morphometry revealed a significant increase in the quantity of CD34(+) PGCs per hematopoiesis (cellularity) in the CML bone marrow before treatment. Independently of treatment modalities and presentation of clinical findings nonresponding patients were generally characterized by a higher amount of progenitors in the initial biopsy specimens. Furthermore, calculation of the CD34(+) cell growth index showed a significant and rapid progression in nonresponding patients and in those developing an accelerated or blastic phase during therapy. This feature was prominently expressed following IFN treatment and related to a failing regeneration of nucleated erythroid precursors. In patients with a myelofibrotic bone marrow at onset no differences in the number of CD34(+) PGCs were recognizable in the pretreatment biopsies. This finding contrasted a significant and gradual change in progenitor cell frequency under treatment and evolving myelofibrosis. Opposed to HU therapy, the latter feature was explicitly detectable in the IFN group. In conclusion, the incidence of CD34(+) PGCs in the CML bone marrow reflects therapeutic efficacy. By demonstrating a significant relationship between fiber content and quantity of CD34(+) cells during treatment, experimental findings concerning the complex functional interactions between the fibrous stroma compartment and progenitor cell differentiation and proliferation are elucidated.

Age-dependent leiomuscular atrophy in vertebral arteries of individuals under low fat diet.

The volumetric density of vascular smooth muscle cells (VSMC) in the proximal vertebral artery was investigated. In order to identify VSMC, paraffin-embedded sections of the proximal vertebral artery, obtained from autopsy specimens, were immunostained for smooth muscle alpha-actin by a modified ABC-technique. The 63 autopsy specimens, including 35 males and 28 females, covered the entire range from 2 months to 85 years. The volumetric density of alpha-actin positive VSMC in the tunica media was morphometrically assayed by the point-counting method. It is important to note that the morphometrical evaluation was performed on arteries obtained from autopsy specimens of the years 1953/54, a post-war time characterized in Germany by low fat diet as compared to the present-day nutrition of most industrial nations. Probably due to their origin, the vertebral arteries showed almost no atheroma. As the main purpose of this study was to find out about the atherosclerosis-independent process of aging, these arteries seemed particularly suitable. The evaluation showed a strictly age-dependent leiomuscular atrophy which became morphometrically evident in early adulthood. The average degree of regression was measured at 0.62% per year. These results may justify the conclusion that the leiomuscular atrophy of the media represents a primary age-related process and does not in any way result as a secondary event from an atheromatous transformation of the intima.

Changing patterns of histological subgroups during therapy of Ph1+ chronic myelogenous leukaemia.

AIMS: Bone marrow histopathology reveals a striking heterogeneity at diagnosis of Philadelphia chromosome positive (Ph1+) chronic myelogenous leukaemia (CML). Based on semiquantitative evaluations of the number of megakaryocytes and the content of fibres, various histological subtypes have been postulated. However, little information exists on whether these groups represent stable categories of the different classification systems and whether therapeutic regimes exert any influence on the putative shift of histological patterns. METHODS AND RESULTS: A retrospective clinicopathological study was performed on 396 bone marrow biopsies derived from 173 patients. There were at least two representative trephines taken at diagnosis and at median intervals of 16 months. Processing of the specimens involved immunostaining with CD61 (megakaryopoiesis) and Ret40f (erythropoiesis) and Gomori's silver impregnation technique. Based on morphometric analysis and in accordance with the general appearance of bone marrow histology three different histological subtypes were distinguished. These consisted of a granulocytic (51 patients), a predominantly megakaryocytic (73 patients) and a myelofibrotic pattern (49 patients). Follow-up biopsies revealed that a significant transition of histological groups occurred and that, independently of treatment modalities, the myelofibrotic category was associated with an unfavourable prognosis. Of the 124 patients without myelofibrosis at onset, 42% later transformed into the myelofibrotic subtype. However, these patients showed no prevalence of either a pre-existing granulocytic or megakaryocytic growth. Myelofibrotic changes were significantly associated with interferon (IFN) and busulfan (BU) therapy. On the other hand, a transition of a myelofibrotic into a nonfibrotic subtype was detectable in 17 of the 49 patients under study and related to hydroxyurea (HU) treatment. CONCLUSIONS: Histological classification systems of bone marrow features in CML do not represent stable patterns, but may be significantly altered by therapy, in particular IFN and HU.

Therapy-related changes of CD20+ and CD45RO+ lymphocyte subsets in chronic myeloid leukemia (CML): an immunohistochemical and morphometric study on sequential trephine biopsies of the bone marrow.

Little information exists about the amount of CD45RO+-T- and CD20+-B-lymphocytes in the bone marrow of patients with Philadelphia chromosome-positive chronic myelogenous leukemia (Ph1+-CML) at presentation or regarding corresponding changes during therapy. On the other hand, quantification of this cell compartment seems to be imperative for two reasons: first, the presumed association of immunocompetent lymphocyte subsets in the expansion of the leukemic cell clone; and second, a speculated relationship with the complex generation of myelofibrosis. Therefore, an immunohistological and morphometric study was performed on 219 representative trephine biopsies of the bone marrow derived from 70 patients with repeated examinations during the course of Ph1+-CML. For the identification of the different lymphocyte populations, the monoclonal antibodies UCHL-1 (CD45RO) and L26 (CD20) were applied on formaldehyde-fixed and decalcified specimens. In comparison to a control group and calculated per hematopoietic cells, the CML bone marrow showed about a 50% decrease in the total amount of lymphocytes. Determination of CD45RO+ and CD20+ subsets revealed a significant enhancement during treatment. Because of the different intervals (range, 10 to 25 mo) between first and last biopsy in the various therapeutic groups, results had to be modified by considering dynamic features. This calculation included changes of the lymphocyte subpopulations related to time. Contrasting the CD45RO+ lymphocytes, a relevant increase in the CD20+ subset could be observed after interferon-a treatment or corresponding combination regimens. No significant correlations were found between fiber density at onset (first biopsy) or development of fibrosis and lymphocyte proliferations in the course of CML. Our results are in keeping with the finding that a proper immune response consistent with an increased lymphocyte growth seems to be associated with a regression of the clonally-transformed cell population. Opposed to a repeatedly discussed pathomechanism, we failed to demonstrate any quantitative relationships between the extent of lymphocyte proliferations and occurrence or progression of myelofibrosis.

Effects of chemotherapy (busulfan-hydroxyurea) and interferon-alfa on bone marrow morphologic features in chronic myelogenous leukemia. Histochemical and morphometric study on sequential trephine biopsy specimens with special emphasis on dynamic features.

We performed a retrospective clinicopathologic study on sequential biopsy specimens from 90 patients with Philadelphia chromosome-positive chronic myelogenous leukemia to study therapy-specific effects of busulfan (28 patients), hydroxyurea (32 patients), and interferon-alfa (IFN-alfa; 30 patients). Bone marrow specimens were evaluated by morphometry after silver impregnation and staining with monoclonal antibodies to identify reticulin fibers, nucleated erythroid precursors, megakaryocytes, and macrophages. To compute dynamics of histopathology implicating corresponding changes in time, relevant indices were calculated. Quantification of megakaryocytopoiesis and its precursor cell population showed a significant increase in the IFN-alfa and busulfan groups compared with the hydroxyurea group. These changes were associated with a development of myelofibrosis during therapy. Although a significant increase in fiber density was detectable in the busulfan group, the progression index proved to be twice as high after IFN-alfa therapy. In contrast, a considerable number of patients displayed a regression of myelofibrosis after hydroxyurea treatment. The general association of the megakaryocyte lineage with myelofibrosis was in line with experimental findings. The mature macrophage population and its activated subfraction revealed a marked proliferation (IFN-alfa group) during treatment. Growth and activation of macrophages may be compatible with their putative function during erythrocytopoietic regeneration and with stimulation of their phagocytic properties.