Role of endothelin-1 in the development of a special type of cardiomyopathy.

The role of endothelin-1 (ET-1) in certain pathological states is still unclear. We have investigated the effect of anthracyclines (maximum dose, 450 mg/m(2) of body surface) on left ventricular systolic and diastolic function and how it influences the level of plasma ET-1 in 21 patients (12 female and nine male) with Hodgkin and non-Hodgkin lymphoma. We also studied the association between plasma ET-1 concentration and echocardiographic parameters. Serum ET-1 was measured by ELISA. Left ventricular function was analysed by echocardiography: ejection fraction (EF), velocity-time integral, E- and A-waves, E:A ratio, deceleration time (DT) and Doppler index were all measured. Statistical analysis was made by the Wilcoxon rank test. EF and serum ET-1 level decreased significantly (EF, 56.29+/-5.0% to 48.57+/-5.9%, P<0.0001; ET-1, 6.45+/-4.0 pg/ml to 2.9+/-1.0 pg/ml, P<0.0001). DT increased significantly (179.8+/-47.8 ms to 215.5+/-66.7 ms, P<0.01) after anthracycline therapy. There was no difference in other echocardiographic parameters before and after therapy. The decrease in serum ET-1 concentration might be a result of anthracycline's direct cytotoxic effect and the decreasing level of ET-1 can play a role in the reduction of EF. However, more studies are needed to evaluate the presence and severity of endothelial damage.

Factor XIII of blood coagulation as a nuclear crosslinking enzyme.

Intracellular localization and distribution of Factor XIII subunit A (FXIIIA) was investigated in association with monocyte-macrophage differentiation in a long term culture of human monocytes by light- and electron microscopical as well as biochemical and immunobiochemical techniques. To allow the detection of FXIIIA in cells with well-preserved ultrustructure, immunosera against glutaraldehyde-derivatized recombinant FXIIIA were developed in rabbits, then characterized and used in this study. In the early phase of macrophage differentiation intranuclear accumulation of FXIIIA was detected as a transient phenomenon in cells of the 2nd day culture by optical sectioning with 0,7 microm steps in laser scanning confocal microscopy and immunoblotting technique. FXIIIA could be detected by immunoelectron microscopic postembedding staining over electrodense DNA-containing areas. Fluoresceinated monodansylcadaverine incorporation assay was used to demonstrate that FXIIIA is not only present in the nuclei, but also expresses its transglutaminase activity. Our finding of the nuclear accumulation of FXIIIA in differentiating human macrophages is also unique in that a blood clotting factor has, for the first time, been localized in nuclei and has been shown to be an intracellular crosslinking enzyme. The possible role of nuclear FXIIIA in association with cellular processes involving chromatin structure remodeling, such as cell death, cell differentiation or cellular proliferation requires further in-depth investigation.

Knock-out mouse for Canavan disease: a model for gene transfer to the central nervous system.

BACKGROUND: Canavan disease (CD) is an autosomal recessive leukodystrophy characterized by deficiency of aspartoacylase (ASPA) and increased levels of N-acetylaspartic acid (NAA) in brain and body fluids, severe mental retardation and early death. Gene therapy has been attempted in a number of children with CD. The lack of an animal model has been a limiting factor in developing vectors for the treatment of CD. This paper reports the successful creation of a knock-out mouse for Canavan disease that can be used for gene transfer. METHODS: Genomic library lambda knock-out shuttle (lambdaKOS) was screened and a specific pKOS/Aspa clone was isolated and used to create a plasmid with 10 base pair (bp) deletion of exon four of the murine aspa. Following linearization, the plasmid was electroporated to ES cells. Correctly targeted ES clones were identified following positive and negative selection and confirmed by Southern analysis. Chimeras were generated by injection of ES cells to blastocysts. Germ line transmission was achieved by the birth of heterozygous mice as confirmed by Southern analysis. RESULTS: Heterozygous mice born following these experiments have no overt phenotype. The homozygous mice display neurological impairment, macrocephaly, generalized white matter disease, deficient ASPA activity and high levels of NAA in urine. Magnetic resonance imaging (MRI) and spectroscopy (MRS) of the brain of the homozygous mice show white matter changes characteristic of Canavan disease and elevated NAA levels. CONCLUSION: The newly created ASPA deficient mouse establishes an important animal model of Canavan disease. This model should be useful for developing gene transfer vectors to treat Canavan disease. Vectors for the central nervous system (CNS) and modulation of NAA levels in the brain should further add to the understanding of the pathophysiology of Canavan disease. Data generated from this animal model will be useful for developing strategies for gene therapy in other neurodegenerative diseases.

Stage-related superoxide anion production of granulocytes of gynecologic cancer patients.

OBJECTIVE: To measure superoxide anion production of polymorphonuclear leukocytes (PMNLs) in 58 gynecologic cancer patients and compare to that of healthy controls. METHODS: PMNLs were separated from peripheral blood samples by Ficoll and subsequent Percoll gradient sedimentation. Baseline and phorbol-dibutyrate (100 nmol/l) stimulated superoxide anion production was measured spectrophotometrically as superoxide dismutase inhibitable reduction of ferricytochrome c (50 micromol/l) absorbance. Differences between the mean superoxide anion production of different patient groups and the control group were assessed by Student's t-test. RESULTS: The mean superoxide anion production of PMNLs of healthy controls was 1.855 nM/min/3 x 10(5) cells (SD=0.211 nM/min/3 x 10(5) cells). Superoxide anion production of gynecologic cancer patients and healthy controls varied in a wide range. PMNLs of patients had lower baseline and stimulated activity than those of healthy volunteers. The frequency of a mean superoxide production at least 2 x SD below the control showed a parallel increase with advancing stage. CONCLUSION: Granulocytes of gynecologic cancer patients have reduced capacity and inducibility of superoxide anion production already at an early stage of disease.

[Congenital laryngeal cysts]. / Angeborene Kehlkopfzysten.

Congenital cysts of the larynx occur rarely. Their incidence amounts to about 1.8/100,000 newborns. Most frequently, these cysts are located in the supraglottic region, 75% of them within the plica aryepiglottica. The leading symptoms, inspiratoric stridor, cyanosis, and jugular and epigastric retractions, are caused by blockage of the airway. Therefore, laryngeal cysts often lead to severe respiratory obstruction immediately post partum, giving rise to an emergency intervention. The case report demonstrates a newborn with a laryngeal cyst located in the region of the right plica aryepiglottica. After emergency intubation, a primary punction and, some days later, a secondary endoscopic cyst removal were performed.

Single-cell measurement of superoxide anion and hydrogen peroxide production by human neutrophils with digital imaging fluorescence microscopy.

Besides flow cytometry, fluorescence microscopy combined with computerized image analysis offers an alternative tool for assessing phagocyte oxidant generation at the single-cell level. This technique provides an opportunity for the direct visualization of cells and simultaneous measurement of cellular fluorescence intensity. Thus, we developed a simple method for the quantitative evaluation of intracellular superoxide anion and hydrogen peroxide production with image cytometry by using hydroethidine and dihydrorhodamine 123 dyes, respectively. Human neutrophils stimulated with phorbol dibutyrate and labeled by these fluorogenic substrates showed intense, well recognizable red or green fluorescence. The intensity of signals from individual granulocytes of cytospin preparations were quantitatively measured in digitized images. There was a great heterogeneity in response to the stimulus within the granulocyte population as shown by the integrated fluorescence intensity values. In agreement with the results of parallel flow cytometric experiments, this simple image analysis performed on cells of cytospin preparations was able to detect the defects in the oxidative metabolism of neutrophils from patients with cervix carcinoma. We demonstrated that even minor alterations in superoxide anion/hydrogen peroxide generation can be detected by image cytometry as efficiently as by flow cytometry. This result validates imaging microscopy as an alternative to flow cytometry in such experiments. In addition, the image cytometric technique allows the observation of the kinetics of free radical production in individual cell under adherent conditions. Therefore, we carried out image analysis of the oxidative burst of neutrophils adherent to uncoated glass and fibronectin- and type IV collagen-coated surfaces in response to stimulation with phorbol dibutyrate or N-formyl-methionyl-leucyl-phenylalanine. We elaborated a calibration technique for the quantitative measurement of the ethidium bromide generation mediated by superoxide anion within individual adherent granulocytes. The ethidium bromide production varied between 0.48 and 1.17 amol/cell/min.

Comparative FISH analysis of numerical chromosome 7 abnormalities in 5-micron and 15-micron paraffin-embedded tissue sections from prostatic carcinoma.

Interphase fluorescence in situ hybridization (FISH) was performed on 15-micron-thick paraffin sections from prostatic carcinomas using a chromosome 7-specific alpha-satellite DNA probe. A confocal laser scanning microscope (CLSM) was used for optical sectioning of the thick sections and reconstruction of 3D images. The number of FISH signals was determined by a gallery of optical sections evaluating only complete nuclei. To investiate the influence of section thickness and truncation and nuclei on scoring results, we compared the FISH data from 15-micron sections with signal counts obtained from 5-micron sections. The latter were evaluated by conventional fluorescence microscopy in the same tumor regions previously defined and marked on the slides. After statistical analysis of spot frequencies in tumor and non-tumorous cells (chi 2 test), we transferred the signal frequencies into a cytogenetic classification (-7, +7, polysomy 7). Based on this classification, most cases showed more than one chromosome 7 aberration type. Trisomy 7 (+7) became apparent in 15-micron thick sections in all 19 tumors, polysomy 7 (> 3 spots) in 18/19 cases, and monosomy 7 (-7) in 13/19 cases. In 5-micron sections, however, trisomy 7 and polysomy 7 were found in only 7/19 and 13/19 cases, respectively, and monosomy 7 in 7/19 cases. When comparing the classification results of tumor cells of the same tumor regions originating either from 5-micron or 15-micron sections, the following discrepancies were noted: in 15-micron sections exclusively, in 12/19 tumors, trisomy 7 was found; in 6/19 cases, polysomy 7; in 8/19 cases, monosomy 7. The high proportion of cases with tumor nuclei expressing only one hybridization signal of chromosome 7 in 15-micron sections could be confirmed as monosomy 7 in five selected cases by double-hybridization using centromere-specific probes for chromosomes 7 and 12. These results demonstrate that numerical chromosome 7 aberrations are more frequently observed in thick (15-micron) paraffin-embedded tissue sections by evaluating only complete nuclei. The use of routine sections (5-micron) for interphase cytogenetic analyses is compromised by a remarkable underestimation of the real chromosome copy numbers.

Combined cytogenetic and molecular genetic analyses of fifty-nine untreated human prostate carcinomas.

G-banding analyses and molecular genetic investigations (fluorescence in situ hybridization (FISH) and loss of heterozygosity (LOH) studies) were performed in 59 tumor and nontumorous samples of human prostate carcinoma. Clonal chromosome aberrations were detected in 16 tumors of which nine were poorly differentiated (G3) and 11 in an advanced stage (pT3). Six cases showed numerical chromosome aberrations. The most common numerical aberrations were trisomy 7 and loss of the Y chromosome each present in three tumors. Clonal structural aberrations were detected in 12 tumors. Deletions could be observed in two cases affecting chromosome 6q23 and in two cases affecting chromosomal region 16q. A structural variant of the pericentromeric heterochromatin of chromosome 9 became apparent in six cases. The Y chromosome was involved in clonal translocations in two cases, additionally an inversion occurred on chromosome 19 in one case. All clonal chromosomal changes were found exclusively in the tumor sample. For an analysis of the pericentromeric heterochromatin of chromosome 9, FISH using a chromosome 9-specific sat III DNA probe was carried out on metaphase preparations of tumor and nontumorous tissues of two cases showing var(9)(qh). The FISH data suggest a deletion in the pericentromeric heterochromatin. Loss of heterozygosity studies on chromosomal regions 10q and 16q were carried out because both chromosomes were frequently affected by nonclonal structural aberrations. Loss of heterozygosity could be verified in 11 cases.

Changes in superoxide anion production and phagocytosis by circulating neutrophils during tumor progression in a rat model.

The functional state of circulating neutrophils was monitored in a rat model of mesoblastic nephroma during tumor progression. Superoxide anion (O2.-) production in response to PMA and phagocytosis of yeast particles (Saccharomyces cerevisiae) were measured every second day after tumor cell implantation. Both phagocytosis and PMA-induced 02.- generation were found to be enhanced in the first period (on Days 6, 8, and 10), while they became significantly reduced in the advanced stage of cancer (on Days 12, 14, 16, and 18). The suppression of PMNL functions was accompanied with tumor progression and an increased number of neutrophils in the peripheral blood. Studies were also carried out on PMNLs isolated from normal rats and the cells were treated with plasma samples obtained from tumor-bearing animals at different stages of nephroma. Incubation of the normal cells with plasmas separated on the 2nd and 8th days of tumor growth influenced neither the 02.- generation nor the phagocytosis. In contrast, plasma preparations obtained on the 14th day significantly inhibited both 02.- production and phagocytosis by normal neutrophils. The alterations in 02'- generation and phagocytosis by PMNLs were observed in close association with tumor growth, thus they could be considered as indicators of tumor progression. However, further studies are required to see whether the granulocyte dysfunctions observed in our animal model could provide additional prognostic information in the case of human malignancies as well as to clarify the origin of inhibitory factor(s) present in the blood of tumorous animals.

Two-phase short-term culture method for cytogenetic investigations from human prostate carcinoma.

An improved technique for primary short-term culture of prostate carcinoma cells in two phases, with and without serum, for subsequent cytogenetic analysis is reported and compared with four other methods. After mechanical disaggregation and a brief collagenase treatment of tumor specimens, cell clusters were seeded in RPMI 1640 and 15% fetal calf serum (FCS) without any other supplement in the first phase. The culture medium was changed to a serum-free medium supplemented with bovine pituitary extract (BPE) and epidermal growth factor (EGF) when the first outgrowth became apparent. During this second phase, fibroblast growth could be virtually abolished within 48 hr. The epithelial and prostatic origin of the cultured cells was confirmed by immunocytochemical methods in each culture. Metaphase analysis revealed chromosome aberrations in over 80% of cases (both clonal and nonclonal alterations) indicating the presence of neoplastic cells. Clonal numerical chromosome aberrations, found by conventional cytogenetic analysis, were used to provide the reliability of the culture system in interphase nuclei of corresponding uncultured tumor tissue by fluorescence in situ hybridization (FISH). The main points of the described method are: 1) combined mechanical/enzymatic disaggregation, 2) seeding of the disaggregated cell clumps rather than of single cells, 3) initialization of the cultures in RPMI 1640 medium with 18% FCS without any other supplements, and (4) stimulating of selective epithelial proliferation by changing the culture conditions through serum-free medium.

Numerical abnormalities of chromosome 7 in human prostate cancer detected by fluorescence in situ hybridization (FISH) on paraffin-embedded tissue sections with centromere-specific DNA probes.

Fluorescence in situ hybridization (FISH) using chromosome-specific alpha-satellite DNA probes for chromosomes 7, 8, and 12 was performed on paraffin-embedded tissue sections and touch imprint preparations of 53 cases of human prostate cancer. Subsequent haematoxylin and eosin (H & E) staining of the hybridized tissue sections allowed unambiguous assignment of hybridization signals either to tumour or to non-tumorous parenchyma. Fifty-three cases of human prostate cancer were evaluated for numerical aberrations of chromosome 7. Scoring 200 cells of tumour and non-tumorous parenchyma in each case revealed abnormalities exclusively in tumour parenchyma in 41 cases (77 per cent). Ten of 41 cases (24 per cent) showed trisomy 7, and 15 cases (37 per cent) monosomy 7 or trisomy 7 in combination with monosomy 7, respectively. Sixteen cases (39 per cent) exhibited polysomy 7 in cells of the tumour parenchyma. In the tumour tissue in one case, different polyploid clones (triploid, tetraploid) and polysomy 7 could be identified by double hybridization with chromosome-specific DNA probes for chromosome 7, plus 8 or 12. The indicated numerical aberrations of chromosome 7 were correlated with 78 per cent of advanced pathological stages or poorly differentiated tumours (pT3/4 or G3) of prostate carcinomas. A statistical analysis of the data revealed significant relationships of particular numerical abnormalities of chromosome 7 to different pathological categories (pT, G, pN) of tumour classification. For the T-classification, the frequency of cells carrying polysomy 7 and polysomy 7/+7 increases significantly from pT1 to pT3/4 (P = 0.022).(ABSTRACT TRUNCATED AT 250 WORDS)

The separation of the granulocytes from different rat strains. A comparative study.

Different Percoll density gradients were used to purify granulocytes from Long-Evans, Fischer, Sprague-Dawley, and Fischer/Long-Evans hybrid rats. Three different discontinuous gradient types were developed which permitted the separation of polymorphonuclear leukocytes (PMNLs) from the peripheral blood of the different rat strains. Our purification techniques were compared to each other in terms of purity and yield. Purities of 97.7 +/- 0.6%, 97.0 +/- 1.1%, 96.4 +/- 1.2% and 96.7 +/- 1.1% were achieved for the granulocyte fractions of LE, F344, SD and FL/F1 rats, respectively. The superoxide production of the isolated cells was also investigated and it was established that the granulocytes could be activated by phorbol-12-myristate-13-acetate (PMA).

Age-dependent changes in transmembrane signalling: identification of G proteins in human lymphocytes and polymorphonuclear leukocytes.

In human neutrophils (PMNLs) we found that in the elderly IP3 formation was significantly decreased compared to that of young subjects. For FMLP receptor binding affinity and number no measurable differences occurred upon ageing, studying both the low or the high affinity receptors. The amount of ADP-ribosylated G proteins, catalysed by pertussis toxin (PT) or cholera toxin (CT), was significantly increased in PMNLs of the elderly. In lymphocytes, the PT-catalysed ADP ribosylation of G proteins was also increased with ageing, while the CT-catalysed ribosylation was decreased. The autoradiogram of [32P]ADP-ribosylated proteins by CT in lymphocytes of young individuals showed a major polypeptide of 40,000 M(r). In contrast, in lymphocytes of the elderly, the major polypeptide was 45,000 M(r). In PMNLs, CT labelled quite strongly the 45,000 M(r) band, mainly in the elderly. When PT was used, no age-related pattern changes could be demonstrated, while differences could be observed between the two types of cells. The use of antiserum P680 (G alpha common) showed no age-related pattern changes, while the intensity of the labelled proteins varies with age and cell type. The antiserum U46 (Go alpha) could identify in lymphocytes of young subjects two polypeptides 68,000 and 41,000 M(r). The prominent polypeptide in lymphocytes of the elderly was the 70,000 M(r) and no other polypeptides could be recognized. In PMNLs of young subjects the U46 and serum identified a range of species. In PMNLs of the elderly all these bands were weakly labelled. The present data indicate changes in the pattern and the quantity of G proteins in lymphocytes and PMNLs of elderly subjects.

[Cytogenetic investigations in primary prostate carcinomas]. / Zytogenetische Untersuchungen in primären Prostatacarcinomen.

Primary cultures of 48 cases of prostate carcinoma were established. The expression of cytokeratin and prostate specific antigen were determined in the cultures routinely. G-banding analysis revealed some clonal numerical and structural aberrations: Trisomy 7 occurred in 4 cases, in one case in combination with a t(Y;22). Loss of the Y chromosome was found in 3 cases, in one case in combination with gain of chromosome #5. The most frequent clonal structural aberration var9(qh) occurred in 6 cases. A deletion of 6q(23) was found in two cases. None of these aberrations were found in normal tissue of the same patients. Numerical changes were demonstrated by fluorescence in situ hybridization (FISH) on native tissue, confirming the conventional cytogenetic findings.

Clonal aberrations of chromosomes X, Y, 7 and 10 in normal kidney tissue of patients with renal cell tumors.

By means of G-banding techniques, chromosome aberrations were studied in short-term cultures of normal renal parenchymal cells from 45 patients with renal cell carcinoma. Clonal chromosomal aberrations were detected in 29 patients; loss of the Y chromosome as well as trisomy X, 5, 7, 9, 10, 12, and 18 was found. Chromosomes 7 and 10 were involved preferentially. Results of fluorescence in situ hybridization with chromosome 7- and 10-specific DNA probes on non-cultured normal kidney cells suggested that the aberrations developed in vivo.

Transmembrane signaling changes with aging.

Altered immune response and transmembrane signaling with aging has previously been demonstrated. The aim of the present study was to characterize PMNLs and lymphocyte G proteins and to determine whether their relative amounts are altered with aging. First we studied the effects of FMLP on PMNLs IP3 formation. It was found that in any group of elderly the PMNLs IP3 formation was significantly decreased compared to that of young subjects. In FMLP receptor binding affinity no measurable difference exists in either low- or high-affinity FMLP receptors. The autoradiogram of 32P-ADP-ribosylated proteins by CT in lymphocytes of young individuals showed a major polypeptide of 40 kDa, and two much less prevalent components of 52 and 45 kDa. In contrast, in lymphocytes of elderly subjects the major polypeptide was 45 kDa, and the two others were very weakly labeled. In PMNLs, CT labeled the 45-kDa band quite strongly, mainly in the elderly, and the 52- and 40-kDa bands were very weakly labeled, mainly in young subjects. When PT was used, no age-related pattern changes could be demonstrated, while differences could be observed between the two types of cells.

Glomerulocystic kidney. A case report.

This paper presents a rare type of renal cystic disease involving the Bowman's capsule: the glomerulocystic disease associated with multiple malformation. Etiology or pathogenesis of glomerulocystic kidney remained unclear.

Focal-segmental glomerulosclerosis associated with herpes simplex viral encephalitis.

A 5-month-old boy presented nephrotic syndrome associated with severe viral encephalitis. From his various body fluids herpes simplex virus (HSV) type 1 and anti-HSV antibodies could be isolated. Renal biopsy showed focal-segmental glomerulosclerosis and cytomegaly within the mesangial region in a single glomerulus. Immunohistology demonstrated mesangial and segmental depositions of IgM and C3. HSV antigen could be detected in two of the glomeruli. Osmiophilic mesangial deposits, segmental sclerosis and foot process fusion of podocytes could be seen by electron microscopy. In the nucleoplasm of a mesangial cell of a single glomerulus typical viral particles were observed. To the best of the author's knowledge this is the first reported case in the literature on human herpes glomerulopathy confirmed by histological examination.