[The new technologies for the analytical examination of the embryonic metabolome and its prospects]. / Nové technologie a perspektivy analýzymetabolomu embrya.

OBJECTIVE: To review current technologies for the analytical examination of the embryonic metabolome and its perspectives. DESIGN: Review article. SETTING: Department of Gynecology and Obstetrics, Faculty of Medicine, Masaryk University, and University Hospital, Brno, Department of Biochemistry, Faculty of Science and CEITEC, Masaryk University, Brno. METHODS AND RESULTS: Nowadays, very sensitive analytical technologies are available. They enable exact measurement of various molecules - products of embryo metabolism during first days of cultivation. The capillary electrophoresis is one of promising method. Recent studies analysed metabolic differences between embryos that result in a pregnancy and those that do not. Amino acid levels, glucose or pyruvate in the embryo culture media were analysed most frequently. However, results of these studies are ambiguous. CONCLUSIONS: The capillary electrophoresis with contactless conductivity detection may provide a useful data of the embryonic metabolome. A comprehensive analysis of the used culture medium may represent a valuable adjunct to morphological criteria for enhanced rates of implantation and delivery.

Bone demineralization, biochemical indices of bone remodeling, and estrogen replacement therapy in adults with Turner's syndrome.

The study centered on a controversy about whether long-term estrogen replacement therapy may ameliorate the osteoporosis seen in patients with Turner's syndrome. This study comprised 26 adult patients with Turner's syndrome (9 treated and 17 untreated or insufficiently treated) and 12 adult women with pure gonadal dysgenesis (8 untreated and 4 treated). A low bone density below -2 standard deviations from the age- and sex-matched predicted normal mean was documented by dual-photon absorptiometry of the lumbar spine in all the untreated and insufficiently treated patients, but only in 6 treated patients. The biochemical indices of bone resorption (urinary hydroxyproline excretion and plasma tartrate-resistant acid phosphatase activity), as well as osteoblastic function (serum osteocalcin and bone alkaline phosphatase isoenzyme), were significantly increased in untreated and insufficiently treated patients compared with treated patients. A significant negative correlation was found between biochemically documented osteoresorption and spinal bone mineral density corrected for age of the patients. Significant positive correlations were found between serum osteocalcin and bone alkaline phosphatase isoenzyme and between biochemical indices of bone resorption and formation. Although in the patients there was an evidence of a high bone remodeling rate, the rate of bone mass loss seemed to be low, comparable with that seen in oophorectomized women who had already passed their accelerated phase of bone loss. The results indicate that long-term hormonal replacement therapy is justified in gonadal dysgenesis, regardless of the karyotype of the patient, to prevent further bone mass loss.

Karyotype at diagnosis, subsequent leukemic transformation and survival in myelodysplastic syndrome. Czechoslovak MDS Cooperative Group.

240 patients with MDS studied cytogenetically at diagnosis between 1981 and 1990 were followed until death or until April 1992 to evaluate the prognostic significance of FAB classification, age and karyotype. 61 patients (25.4%) subsequently transformed into AML and 176 (73.3%) died during the follow-up period. Patients with blastic MDS types had a shorter survival and a higher probability of leukemic transformation. The younger age increased the probability of leukemic transformation, but was associated with a longer survival. The absence of analyzable mitoses was associated with a shorter survival. The complex chromosomal abnormalities at the initial evaluation identified a subgroup of patients with a high risk of a short survival and/or subsequent leukemia transformation. In refractory anemia the presence of complex chromosomal abnormalities was linked with a relative risk of 3.58 of leukemic transformation and shorter survival as compared with other cytogenetically defined groups.

Mixed myelodysplastic and myeloproliferative syndromes.

Aplastic anemia, myelodysplastic syndromes (MDS) and chronic myeloproliferative diseases (MPD) are stem cell disorders. There is no clear-cut demarcation of them. Hypoplastic MDS displays features of aplastic anemia and MDS, on the other side mixed myelodysplastic and myeloproliferative syndromes (MDS-MPS) develop. In our collection of 566 MDS patients, features of myelodysplasia as well as myeloproliferation, MDS-MPS, were present in 25 patients (4.4%). Twelve patients had at the time of diagnosis megakaryocytic proliferation and thrombocythemia beside signs of MDS, and seven had myelodysplasia with granulocytic proliferation and leukocytosis. In another six patients, MDS was the first diagnosis and the proliferative phase developed later during the course of the disease. These patients can be characterized as MDS-MPS in evolution. All subjects had a variable degree of anemia. While the level of thrombocythemia has been relatively stable, the number of leukocytes has been progressive, but rarely extended beyond 100 x 10(9)/l. Ring-sideroblasts and myelofibrosis were frequent findings. Two more homogeneous MDS-MPS groups emerged in our analysis: sideroblastic anemia with thrombocythemia and a group fulfilling the criteria of Philadelphia chromosome negative and bcr-abl negative "atypical chronic myeloid leukemia (aCML)'. One patient with thrombocythemia and three with leukocytosis (23%) transformed to acute myeloid leukemia (AML). Men prevailed (12/13) in patients with leukocytosis and MDS-MPS in evolution. Of the 46% MDS-MPS patients with chromosomal aberrations, del(20)(q) is of interest.

A case of type I Gaucher disease with cardiopulmonary amyloidosis and chitotriosidase deficiency.

Severe cardiopulmonary amyloidosis developed several months after a total splenectomy in a patient with type 1 Gaucher disease and led within a year to his death at 48 years of age. The autopsy findings were dominated by extensive pulmonary and cardiac amyloid infiltration. No Gaucher cells were found in the lungs. Aside from a glucocerebrosidase deficiency the patient was also deficient in chitotriosidase, an enzyme whose activity is usually greatly increased in the serum of Gaucher patients. Analysis of mutations in the glucocerebrosidase gene revealed heterozygosity for N370S and D409H mutations. The normal amount of glucocerebrosidase was found in the spleen by Western blotting. We suggest that amyloidosis should be considered in the differential diagnosis of severe cardiopulmonary disease in Gaucher patients.

Chromosome study of 85 patients with myelodysplastic syndrome.

We studied bone marrow chromosomes in 85 consecutive patients with myelodysplastic syndrome (MDS). Fifty-seven (67%) had a clone with an abnormal karyotype at diagnosis. Eight had secondary MDS, all with abnormal karyotypes. The frequency of abnormal karyotypes in the primary MDS was 64.9%. During subsequent follow-up, five patients acquired chromosome abnormalities; thus, at the end of the study, 72.0% of patients had an abnormal karyotype. The most frequent chromosome abnormalities were 5q-, +8, -7, -5, and -22. Forty patients (i.e., 70% of those with an abnormal karyotype and 47% of the whole group) had one of the karyotype abnormalities associated with secondary MDS or acute nonlymphocytic leukemia; in other words, 5q- or -5, or -7. Of all patients, 21.1% progressed into ANLL. Unfavorable prognostic factors associated with the risk of evolution into ANLL and with shorter overall survival were the presence of greater than 5% of bone marrow blasts, major chromosome abnormalities, and monosomy 7.

Cytogenetic study of cancer cells in effusions.

A cytogenetic study of 71 malignant effusions caused by different histopathologic types of cancer was performed by a direct technique. Abnormal metaphases were present in 50 effusions (70.4%); a detailed analysis of banded karyotypes was possible in 20 of these. Trisomies and monosomies were present in 18 of 20 cases. The most common trisomies were +19, +3, +5, +8, +9, +10, +16 and +22; the most frequent monosomies were -X, -2, -5, -7, and -21. Clonal structural abnormalities were identified in 18 of 20 cases. In most instances, they were extensive, and some chromosomes were involved in a nonrandom fashion. Chromosomes #1, #13, and #6 were the most frequently involved in these rearrangements. A common marker chromosome, t(7;9)(p11;q12), was observed in two patients with cancer of the endometrium. Double minutes (DMs) were present in two patients, and there was a homogeneously stained region (HSR) in one.

Multiple unrelated clones in myelodysplastic syndrome and in acute myeloid leukemia.

We identified cytogenetically unrelated clones in the bone marrow of 12 of 240 patients with myelodysplastic syndrome (MDS) and in 3 of 232 patients with acute myeloid leukemia (AML). In addition, unrelated single-cell abnormalities were found in six MDS and two AML patients. The most commonly encountered abnormalities present in the unrelated clones in patients with refractory anemia (RA) were del(5q), +8, and -7. In blastic types of MDS and AML trisomy 8 was found in two of eight patients while in the remaining cases the chromosome abnormalities were diverse and nonspecific. The presence of the chromosomally unrelated clones, together with recent data on the early appearance of monoclonality provided by molecular biology studies, support the interpretation that aberrations such as +8 and del(5q) are actually secondary abnormalities that develop during tumor progression in a cell with a primary submicroscopic genomic rearrangement.

Sequential cytogenetic study of patients after bone marrow transplantation.

Thirty-one patients (19 males and 12 females; mean age 23.9 years, range 4-41 years) were treated with bone marrow transplantation (BMT) after intensive chemoradiotherapy. Their diagnoses were as follows: chronic myeloid leukemia (CML) in 13, acute myeloid leukemia (AML) in seven, acute lymphocytic leukemia (ALL) in six, myelodysplastic syndrome (MDS) in two, aplastic anemia (AA) in two, and Fanconi anemia (FA) in one. Allogeneic BMT was performed in 28 cases (17 donors were of like sex, 11 were of unlike sex), one patient received syngenic transplant, and one received transplant of cells obtained from an unrelated donor through a computerized international registry in London. Autologous BMT was performed in three patients. BM cells were analyzed cytogenetically at diagnosis, before and serially after BMT (three to nine times). Follow-up ranged from 2 to 55.5 months. Cytogenetic examination was a very useful method for monitoring posttransplantation course in patients with CML or in those who received BM cells of unlike sex. Results of concomitant cytogenetic examinations are reported in detail.

Cartilage collagen in osteoarthrosis.

Pronase digestion of osteoarthrotic cartilage collagen indicated that this type of pathological collagenous structure is more susceptible to proteolytic degradation than human controls or bovine and calf articular cartilage. There were some differences between individual patients in the collagen susceptibility to pronase cleavage. Also, different zones of the same diseased femoral head exhibited varied susceptibility. It is suggested that changes in collagen stability are reflected in cartilage permeability and in this way also in alterations in the supply of nutrients.

Sister-chromatid exchanges and chromosal breakage in patients treated with cytostatics.

The frequency of structural chromosomal rearrangements and sister-chromatid exchanges (SCEs) was investigated in short-term phytohemagglutininstimulated lymphocyte cultures by means of bromodeoxyuridine substitution and fluorescence plus Giemsa (FPG) staining technique. Both these parameters were significantly increased in patients treated with comparatively low doses of cyclophosphamide, busulphan and adriamycin. The increased SCE rate was proportional to the number of chromosome breaks, the ratio of SCE to breaks being about 100:1. The increase in the SCE number was maintained for several months after the termination of cytostatic therapy, when the conventional analysis of chromosome breaks yielded normal results. Normal SCE values were obtained in two patients treated with low doses of fluorouracil.

Cytogenetic study of 130 patients with acute nonlymphocytic leukemia.

Results of cytogenetic examination of 130 patients (62 males and 68 females, mean age 48.7 +/- 18.7 years) with acute nonlymphocytic leukemia (ANLL) are presented. All patients were examined at the onset of the disease. According to the FAB classification, 35 (26.9%) patients were diagnosed as having M 1, 42 (32.3%) M 2,9 (6.9%) M 3, 29 (22.3%) M 4, 8 (6.2%) M 5 and 7 (5.4%) M 6 subtype of ANLL. Normal karyotype (NN) was found in 29 (22.3%) patients studied, normal and abnormal clones (AN) in 24 (18.5%) and abnormal chromosomes only (AA) in 77 (59.2%) patients. Thus 78% of patients showed a clonal karyotypic abnormality. Specific chromosomal abnormalities t(8;21), t(15;17), t(9;22), inv(16), del(16), del(11q) were found in 30.7% of AA and AN patients. Nonrandom chromosomal changes (+8, 5q--, --5, --7) were noted in 53.4% of them. Single chromosomal change was found in 40.5% of patients and complex changes with more chromosomal clones were presented in the test. Relationship between prognosis and chromosomal finding was evaluated.

Cytogenetic study of chronic myeloid leukemia.

A total of 122 patients with chronic myeloid leukemia (CML) were cytogenetically examined. At the first cytogenetic examination 68 of them were in chronic phase (CP) and 54 in blastic phase (BP) of the disease. The mean age of the whole group was 44.8 +/- 15.2 years. All patients included in this study were Ph-positive. In two of them constitutional chromosomal aberrations were ascertained. In two patients the standard Ph translocation was accompanied within onset of BP by a clone with complex translocation and in three patients Ph chromosome was found in mosaics with normal karyotype. Standard translocation t(9.22) (q34;q11) as the sole chromosomal abnormality was found in 17 (31%) patients in BP and in 47 (69%) of patients in CP. The most frequent additional abnormalities found were +8, i(17q), +Ph and their combination, and other nonrandom chromosomal changes. Clonal evolution was proved in 11 out of 24 serially examined patients during the progression of the disease. The analysis of relationship between prognosis of the disease and the results of cytogenetic examination revealed significant differences, and a more favorable course of the disease was observed for the group of patients in CP and with Ph chromosome as the sole chromosomal aberration.

Chromosome sensitivity to bleomycin in patients with dominantly inherited and sporadic tumors.

G2 chromosomal sensitivity to bleomycin (30 micrograms/ml) was tested in PHA-stimulated lymphocytes of healthy subjects and in patients with familial and sporadic tumors. These were multiple endocrine neoplasias (MEN) types 1, 2A and 2B, familial medullar thyroid cancer, Recklinghausen neurofibromatosis type I, sporadic and hereditary malignant tumors, and a preleukemic disorder, the myelodysplastic syndrome. Control subjects were either young (15-20), middle-aged (28-49) or old (70-83 years). Cells from old healthy subjects and from subjects with MEN 1 showed increased sensitivity to clastogenic effects of bleomycin. All the remaining investigated groups were insignificantly different from controls. Our data suggest that in contrast with recessively inherited syndromes with chromosome instability the mutagen hypersensitivity, as evaluated by the extent of chromosomal damage, is not a feature of most dominantly inherited tumor syndromes.

Cytogenetic study of malignant and benign effusions.

The direct chromosome study of mitotic cells in effusions was performed in 49 patients with malignant and 5 with benign effusions. Cytogenetic analysis correctly identified 69.3% of malignant effusions, standard cytology 79.6%. With both methods combined 95.9% of all cases were correctly diagnosed as malignant. The presence of mitoses in the effusions was found to be an unfavorable prognostic sign and was correlated with a shorter survival. Chromosome analysis of the malignant effusions revealed stemlines with extensive numerical and structural rearrangements. No consistent marker chromosome was detected in the histologically similar tumor types. Normal chromosomes. No 1 and 14 were preferentially lost from the karyotype of malignant cells. In two samples double minutes were observed.

Cytogenetic study of acute myeloid leukemia: comparison of data obtained in 1991-1996 and 1982-1988.

The results of the cytogenetic study of bone marrow cells from 110 consecutive patients with primary acute myeloid leukemia (AML) who were diagnosed and treated between 1991 and 1996 at one tertiary care institution were compared with similar data obtained between 1982 and 1988 in 130 patients. Despite improvements in cytogenetic techniques (namely FISH methods, applied in all patients with abnormal karyotypes since 1990) in recent years we have observed a significantly lower frequency of abnormal karyotypes: 52.7% versus 77.7% (p = 0.001). This was mainly due to the decreased frequency of patients with +8, -5, -7 and inv(16). The survival rate (excluding the patients who underwent a bone marrow transplantation) was only slightly increased.

Clastogenicity and sister chromatid exchange induction by ftorafur.

The main effect of Ftorafur at the chromosomal level is the induction of chromatid and chromosome breaks, which is some pronounced in neoplastic or transformed cells than in normal cells. Different cell lines used in the study exhibited both in vitro and in vivo varying sensitivity to Ftorafur. Ftorafur does not increase the frequency of SCE.

Isolation of rotavirus strains from naturally infected piglets.

Twenty rotavirus strains were isolated in 1991-92 from 60 faecal samples collected from diarrhoeic piglets in the Czech and Slovak Republics. Three isolates were adapted to the growth in the cell line MA-104 and produced cytopathic effect. Rotavirus was demonstrated by immunofluorescence test, electron microscopy, polyacrylamide gel electrophoresis, immunoperoxidase test and ELISA.

[Detection of porcine epidemic diarrhea virus using electron microscopy in the Czech Republic]. / Elektronove mikroskopický prukaz viru epidemické diarrhoey prasat v Ceské republice.

Coronavirus-induced porcine epidemic diarrhoea (PED) was diagnosed in two swine herds. The causal agent was demonstrated in intestinal contents by electron microscopy and identified by immunoelectron microscopy using specific immune serum to the reference strain PED-CV77. Experimental transmission to hysterectomy-derived, colostrum-deprived piglets with an intestinal contents filtrate was successful. The virus was demonstrable by electron microscopy in the intestinal contents between 12th hour and 4th day, and in small intestinal epithelial cells 18 hours after infection. Scanning electron microscopy revealed shortening and fusion of villi of small intestinal mucosa.