[Diagnosis and therapy of non-occlusive mesenteric ischemia (NOMI)]. / Diagnostik und Therapie der nicht-okklusiven mesenterialen Ischämie (NOMI).

Pathophysiologically, the non-occlusive mesenteric ischemia (NOMI) results from reduced blood supply to the intestine, caused by "low cardiac output syndrome", or the use of certain drugs leading to intestinal vasoconstriction and stasis of the microcirculation. Regardless of the aetiopathogenesis, the patient's prognosis crucially depends on rapid diagnosis and initiation of adequate medical or surgical intervention. In a 10-year retrospective chart analysis (1989 to 1998) we identified a total of 62 patients that demonstrated classical features of NOMI. The investigation focused on patients' history, risk factors, clinical symptoms, diagnostic procedures and patient's clinical outcome. The most important associated risk factors and concomitant diseases were reduced cardiac output (caused by preexisting heart failure), renal diseases, diabetes and the use of some specific drugs (digitalis, furosemide, ergotamine). Except for leucocytosis, elevated serum lactate and an increased CK/CK-MB level, all laboratory findings were unspecific. Using abdominal ultrasound and plain abdominal x-ray, 80% of the cases showed positive signs of ileus, subileus and free intraabdominal fluid. The angiographic diagnostics (mesentericography) of non-occlusive mesenteric ischemia showed the typical signs of peripheral vasoconstriction in 90% of the cases. Fifty three patients (86%) presenting with peritoneal signs underwent operative bowel exploration. Necrotic bowel had to be resected in 37 cases (60%). The overall letality was 58%. The progress made in better understanding the pathophysiology of NOMI has led to differential treatment of the disease. Close cooperation between surgeons and radiologists, coupled with early diagnosis and prompt treatment are necessary to optimize the clinical outcome.

A novel hematopoietic multilineage clone, Myl-D-7, is stromal cell-dependent and supported by an alternative mechanism(s) independent of stem cell factor/c-kit interaction.

A strictly stroma-dependent hematopoietic clone, Myl-D-7, with lympho-myeloid potential has been isolated. A subset of cells expresses myeloid-macrophage (Mac-1 and Gr-1), erythroid (TER119), and lymphoid (Thy-1 and B220) lineage markers. Spontaneous differentiation to the myeloid-macrophage, erythroid, or lymphoid pathway can be seen by morphologic criteria, detection of beta major globin synthesis, or expression of the early lymphoid specific transcription factor, Ikaros. By sorting lineage marker (Mac-1, Gr-1, B220, and TER119)-negative (LIN-) cells, we showed that the LIN- population actively self-renews on top of MS-5 stromal cells, and differentiates to LIN+ cells. Removal of stroma induces apoptosis and none of the growth factors tested can prevent apoptosis. Granulocyte-macrophage colony-stimulating factor accelerates the differentiation towards the myeloid-macrophage lineage. Using this clone, we show that (1) contact with stroma induces expression of bcl-2, (2) stromal cells derived from SI/SI homozygous fetuses can support long-term growth, and (3) conditioned media of specific stromal cells contains an activity that supports proliferation and self-renewal of the clone. Myl-D-7 can thus be used as an indicator cell for unknown factors that may provide stromal cell support.

Rates of mutation to growth factor autonomy and tumorigenicity differ in hematopoietic stem and precursor cells expressing the multilineage colony-stimulating factor gene.

At least two separate but interdependent events are required to attain autonomous growth as a consequence of ectopic expression of the multilineage colony-stimulating factor gene in hematopoietic progenitor cells. The rate at which the second event occurs is more than 3 orders of magnitude higher in precursor cell lines (FDC-P1 or FDC-P2) than in stem cell lines (FDC-Pmix). Autonomous, but not density-dependent, growth is tightly coupled to tumorigenicity in precursor cells; however, neither growth-factor-independent nor autonomously growing stem cell lines are tumorigenic.

Conversion of factor-dependent myeloid cells to factor independence: autocrine stimulation is not coincident with tumorigenicity.

It has been postulated that the disruption of the normal hormonal regulation of blood cell formation and proliferation leads to the autonomous growth of hematopoietic progenitors or stem cells and thus to leukeamia. We have utilized established hematopoietic cell lines to establish the different mechanism by which growth autonomy is acquired. The analysis of thirteen spontaneous factor-independent mutants revealed that the majority (12/13) secreted a factor that stimulated growth of the parental cell line. Thus, autocrine stimulation may be a important mechanism by which normal growth control is disrupted. This is supported by the observation of Young and Griffin (1987) that some cells isolated from patients with acute myeloblastic leukemia (AML) autogenously produce growth factor. In the majority of Dind mutants more closely examined, growth factor gene activation was due to the juxtapostion of a retrotransposon. Although the exact nature of the involvement of human retroviruses in inducing leukemia has not been elucidated, one could envisage that altered growth factor regulation due to integration of the virus may play an important role. The existence of a second class of Dind mutants that have obtained factor-independence by a mechanism not involving factor production concurs with the acquisition of factor-independent growth in hematopoietic cells after introduction of some oncogenes. Several models have been proposed to explain how oncogenes may "short circuit" and thus activate the normal signal transduction pathway by mimicking the active receptor, transducer, or effector (Weinberg, 1985). To investigate more closely the role of autocrine stimulation in the induction of growth autonomy and tumorigenicity, retroviral vectors expressing either GM-CSF or IL3 were introduced into factor-dependent hematopoitic cell lines. Non-linear clonability of infected cell lines in the absence of exogenous growth factor and inhibition of proliferation by antiserum supported a model of autocrine stimulation. However, a secondary event, correlated with amount of factor released, often occurred that abrogated the requirement for secreted CSF. Growth of cells in which this alteration had occured was cell-density independent and could not be blocked by antibody. It has been postulated that autogenous factor may react with its receptor intracellularly (Lang et al., 1985). The results presented here cannot exclude that the secondary events may allow the internal interaction of receptor and factor.(ABSTRACT TRUNCATED AT 400 WORDS)

Aberrant expression of the multi-CSF gene in hematopoietic precursor and stem cell lines initiates leukemogenic progression.

Tumorigenesis of hemopoietic cells and acquisition of factor independence as a consequence of aberrant growth factor release are closely correlated. In previous work we were able to dissect two stages leading to growth factor autonomy of cells: the first step requires the secretion of the constitutively expressed CSF gene product and extracellular interaction with its cognate receptor. This requirement for external stimulation is abrogated by a second step. We were interested in characterizing the parameters that influence the conversion from nonautonomous to autonomous growth properties of hematopoietic precursor cells. The frequency with which this alteration occurs varies and correlates with the level of growth factor production. However, a significant increase of CSF production accompanying the progression to autonomy could not be detected. We thus conclude that there is no direct link between level of CSF production and acquisition of true autonomy but an indirect influence enhancing the frequency of genetic alteration(s) that lead to growth autonomy. Lang et al. have suggested that the acquisition of autonomous growth occurs due to internal receptor-ligand interaction. Indeed, Keating and Williams have claimed that PDGF may react with an intracellular PDGF receptor resulting in autocrine stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Transformation of single myeloid precursor cells by the malignant histiocytosis sarcoma virus (MHSV): generation of growth-factor-independent myeloid colonies and permanent cell lines.

Direct single-cell assays for oncogenic transformation are available for fibroblasts but not for other cell types. Using malignant histiocytosis sarcoma virus (MHSV), a member of the ras family of retroviruses, in vivo-infected granulocyte/macrophage and macrophage precursor cells lost the requirement for externally added hematopoietic growth factors. Factor-independent growth was demonstrated by colony-transfer experiments. More than 25% of the independent colonies were established as permanent macrophage cell lines following a phase of adaptation to tissue culture conditions. Factor-independent colony growth was also obtained by in vitro infection of single cells. As many as 50% of all myeloid precursor cells were target cells for MHSV as measured by this assay. About 2 x 10(-3) of these colony-forming cells acquired growth factor independence and immortality after in vitro infection. Cell lines derived from these colonies did not require adaptation to tissue culture conditions.

Multipotential hemopoietic cell lines isolated from stem cell cultures infected with Friend virus complex (MuLV + F-SFFV) show presence of MuLV but not F-SFFV.

The only factor-dependent or factor-independent hemopoietic murine stem cell lines which can be permanently maintained in vitro are cell lines which originate from bone marrow cultures of congenic mice differing in the Fv-2 locus infected with Friend spleen focus forming virus (F-SFFV) in conjunction with either Rauscher or Friend helper virus (R-MuLV or F-MuLV). We determined the viral state of these cells by restriction enzyme analysis, measurement of SFFV and MuLV related RNA, immunoprecipitation analysis of viral related proteins and biological activity to test whether integration and expression of R-MuLV or F-SFFV are obligatory. All stem cell lines (SUT, JUT, 416B) showed expression of MuLV coded proteins or virus but SFFV was not found in these cell lines. A two-fold difference of RNA hybridizing with SFFV-specific cDNAs observed in stem cells of Fv-2rr and of Fv-2ss genotype is in agreement with data published earlier.

[5-Methylcytosine content in erythroleukemic cells of rats after induction with dimethylsulfoxide]. / 5-Methylcytosin-Gehalt in Rattenerythroleukämie-Zellen nach Induktion mit Dimethylsulfoxid.

Erythroleucemic cells of rats were induced by DMSO for the terminal erythroid differentiation. The base composition and the 5 MC content of the DNA were analyzed by HPLC and a significant difference between non-induced and induced cells was not found. The analysis of labelled DNA by the restriction enzymes Hha I, Hpa II, and Msp I did not result in a different pattern before and after the induction. These results suggest that the 5 MC content of the DNA does not change during the induced terminal erythroid differentiation.

Co-expression of mouse and rat haemoglobins in interspecific hybrids of mouse and at erythroleukemia cells.

A modified technique for cell fusion with lysolecithin-lipid emulsions was used to generate hybrid erythroleukemia cell lines from Friend leukemia mouse cells (FLC) and chemically transformed rat erythroleukemia cells. Chromosome analysis of the hybrid cells showed the presence of both parental genomes even after long culture periods. The hybrids were still able to undergo erythroid differentiation after dimethylsulphoxide (DMSO) stimulation. Analysis of the globin chains from the DMSO-stimulated cells showed that both the rat and the mouse erythroid phenotypes were expressed. This demonstrates the compatibility of the regulatory genetic elements for the control of erythroid differentiation in cell hybrids of erythroleukemic populations from different species.

Erythroid differentiation and commitment in rat erythroleukemia cells with hypertonic culture conditions.

Cell cultures of 7,12-dimethylbenz[a]anthracene-induced rat erythroleukemia can be stimulated to synthesize hemoglobin when cultured in hypertonic media. During hypertonic treatment the intracellular osmotic conditions immediately readjust to those of the extracellular medium. None of the Friend virus-induced mouse erythroleukemia cell lines was inducible for differentiation with the same hypertonic culture conditions used for rat cells. Earliest commitment to erythroid terminal differentiation by hypertonic treatment of cells grown in either Eagle's or Joklik's medium occurs coincident with the commitment induced by hexamethylenebisacetamide. These results make it likely that induction of differentiation in rat erythroleukemia cells is a cell membrane-mediated event.

Dimethylsulfoxide-induced differentiation and hemoglobin synthesis in tissue cultures of rat erythroleukemia cells transformed by 7,12-dimethylbenz(a)anthracene.

Permanent cell lines from transplantable tumors from 7, 12-dimethylbenz(a)anthracene-induced erythroleukemia of the rat were established. These cell lines maintain their erythroid nature. Erythroid differentiation can be induced by dimethylsulfoxide. This is shown by decrease in cell size, appearance of red and benzidine positive cells, and induced synthesis of four out of six adult globin chains.

RNA-dependent DNA polymerase from a cell line derived from the bone marrow of a patient with polycythemia vera.

A RNA-dependent DNA polymerase was isolated from a human cell line derived from the bone marrow of a patient with polycythemia vera. The purification procedure included chromatography on phosphocellulose and oligo(dT)-cellulose, and glycerol gradient centrifugation. The enzyme could be distinguished from polymerase A by salt elution from phosphocellulose, utilization of poly(rC) - oligo(dG) and its molecular size of about 70000, as determined by centrifugation. Throughout the purification procedure ribonuclease H activity was co-purified. Upon dodecylsulfate-polyacrylamide electrophoresis on microgradient gels two main bands with molecular weights of 68000 and 66000 and three minor bands were detected. The enzyme preferentially used poly(rA) - oligo(dT) as template-primer compared with poly(dA) - oligo(dT). It incorporated dGMP into polymer on poly(rC) - oligo(dG).

Particle-associated RNA dependent DNA polymerase and high-molecular-weight RNA in a human cell line derived from polycythemia vera bone marrow.

A particle fraction with a density of 1.15-1.19 g/cm3 was isolated from the cytoplasm of a human cell line established in culture from the bone marrow of an untreated patient with polycythemia vera. Electron micrographs of cross sections of cells and cell homogenates revealed virus-like particles on which DNA could be synthesized. An RNA-dependent DNA polymerase, isolated from the particles, preferred poly(rA)-oligo(dT) over poly(dA)-oligo(dT) and was able to polymerize deoxyguanosine monophosphate in a reaction stimulated by poly(rC)-oligo(dG).

Induction of endogenous and of spleen focus-forming viruses during dimethylsulfoxide-induced differentiation of mouse erythroleukemia cells transformed by spleen focus-forming virus.

Spleen focus-forming virus-transformed erythroleukemic cell clones, which have been established by infection of N type mice with NB trophic Friend virus, continue to release biologically active tfriend virus of NB host range. Dimethylsulfoxide induces erythroid differentiation and a 10- to 100-fold increase in the release of biologically active Friend virus. The increase of Friend virus release is a function of the differentiating erythroleukemic cell. The induced Friend viurs is not the NB tropic Friend virus complex, but shows N host range. The induction of the Friend virus complex is due to simultaneous induction of both spleen focus-forming and endogenous viruses.

Induction of endogenous virus and of thymidine kinase by bromodeoxyuridine in cell cultures transformed by Friend virus.

Thymidine kinase positive (TK(+)) N type cell lines that had been transformed by spleen focus-forming virus were established by transformation with NB tropic Friend virus complex. Thymidine kinase deficient (TK(-)) cell clones were isolated. Some of these cell clones release 1000- to 100,000-fold reduced amounts of Friend virus complex as compared to the TK(+) parental cell clone. TK(-) clones were grown in medium without BrdUrd. Some of these TK(-) clones can be induced to release endogenous helper virus and transforming spleen focus-forming virus on reexposure to 10(-6)-10(-4) M BrdUrd. The induced Friend virus complex is of N host range as expected with induced endogenous virus in N-type cells. Before the induction of the endogenous virus spleen focus-forming virus complex, an induction of thymidine kinase (ATP:thymidine 5'-phosphotransferase, EC 2.7.1.75) activity is observed. The latter is possibly a prerequisite for the induction of endogenous virus in TK(-) cells. Induction of thymidine kinase activity and of endogenous virus is transient and always correlated. The role of BrdUrd and another thymidine analogue, azidothymidine, in interfering with C-type virus release in virus positive cells is discussed. Azidothymidine is unable to induce endogenous virus. Induction of endogenous virus by BrdUrd and inhibition of virus release in virus positive cells is apparently not caused by the same mechanism.