An epitope on VLA-6 (alpha6beta1) integrin involved in migration but not adhesion is required for extravasation of murine melanoma B16F1 cells in liver.

VLA-6 (alpha6beta1) integrin represents the major receptor for interaction with laminin substrate. It has been proposed that VLA-6 mediates tumor cell adhesion to the endothelium during extravasation. We have further explored this possibility using mouse melanoma B16F1 cells, which express VLA-6 as the principal laminin receptor, and two VLA-6 monoclonal antibodies (mAbs), MA6 and GoH3. Adhesion is a prerequisite of cell movement on matrix proteins. Thus, GoH3, which inhibited VLA-6-mediated adhesion, blocked cell movement on laminin. The recently prepared alpha6 integrin-specific mAb MA6 bound to an epitope in close proximity to GoH3, but it had no effect on VLA-6-mediated cell adhesion. We report here that although MA6 did not affect adhesion, it blocked mouse melanoma B16F1 cell movement on laminin to the same extent as GoH3. Results therefore demonstrate an active role of VLA-6 in providing cell movement as well as the initial adhesive event on laminin. In addition, mAb MA6 had no effect on the induction of tyrosine phosphorylation of focal adhesion kinase upon adhesion of B16F1 cells to laminin. Therefore, inhibition of cell movement by MA6 involved mechanism(s) other than an interference of VLA-6 signaling events leading to phosphorylation of focal adhesion kinase. The epitopes of GoH3 and MA6 may represent spatially and temporally related sites on VLA-6 that are involved during cell movement, or, alternatively, MA6 may inhibit the interaction of VLA-6 with associated cell surface molecules required for cell movement. In vivo videomicroscopy experiments also revealed that an inhibition of VLA-6 migratory function by MA6 resulted in a reduction in the ability of B16F1 to extravasate during hematogenous metastasis in the liver.

Integrin VLA-6 (alpha 6 beta 1) mediates adhesion of mouse bone marrow-derived mast cells to laminin.

The development of mast cells from bone marrow precursors and their function as the mucosal- or connective-tissue-type mast cell are critically dependent on microenvironmental factors. Extracellular matrix proteins, such as collagen, fibronectin, and laminin, may represent insoluble components of the microenvironment. Recent studies have described multiple isoforms of laminin isolated from different tissues. In the present study, adhesion of mouse bone marrow-derived mast cells (BMMC) and long-term mast cell lines to Engelbreth-Holm-Swarm (EHS) tumor laminin, rat laminin, human merosin, and human placental laminin was compared. The greatest level of adhesion was found with human laminin as the substrate. By use of a newly prepared mouse VLA-alpha 6 integrin-specific mAb (MA6) together with the previously described mAb GoH3, VLA-6 (alpha 6 beta 1) integrin was found to be expressed and utilized by BMMC and long-term mast cell lines. VLA-6 has been described as a major laminin receptor with roles in diverse cell functions including cell growth and differentiation. BMMC have been shown to express a 32/67-kDa laminin receptor. Therefore, in addition to the 32/67-kDa laminin receptor described in early studies, BMMC also express VLA-6 integrin, which may have roles in the regulation of their development.

Differential utilization of VLA-4 (alpha 4 beta 1) and -5 (alpha 5 beta 1) integrins during the development of mouse bone marrow-derived mast cells.

Cytokines have been shown to have major roles in the development of mast cells from bone marrow progenitors. Immature mast cells derived from bone marrow thus leave the blood system to complete their course of maturation within tissues. However, it is now clear that VLA (beta 1) integrins with function in mediating cell-cell and cell-extracellular matrix protein interactions have effects on the growth and differentiation of diverse cell types. At present, the involvement of VLA integrins during mast cell development is still unclear. In this study, we report the preparation of a new monoclonal antibody (mAb) against mouse VLA-5 (alpha 5 beta 1) integrin. Together with mAb R1-2, we characterized the expression of VLA-4 (alpha 4 beta 1) and VLA-5 integrins, the two major fibronectin receptors, on two long-term cultured mast cell lines, CFTL-15 and MC/9. CFTL-15 cells were found to express both VLA-4 and -5 integrins whereas MC/9 cells expressed only VLA-5 but not VLA-4. We speculated that VLA integrin expression may be related to mast cell development. Thus bone marrow-derived mast cells (BMMC) were characterized after varying periods of development induced by IL-3. During the first 3 weeks the expression of VLA-4 and VLA-5 increased progressively and both were involved in mediating adhesion of BMMC to fibronectin. At time periods of greater than 3 weeks, the expression of VLA-4 declined gradually to little, if any, by week 13. In comparison, VLA-5 remained stably expressed and functioned as the major receptor for fibronectin. Results from this study therefore suggest that BMMC differentially utilize VLA-4 and VLA-5 integrins during IL-3-induced development. Differential expression of VLA integrins may have effects on the recirculation properties, tissue distribution and eventual maturation of progenitors to fully matured mast cells.

Integrin VLA-2 (alpha2beta1) function in postextravasation movement of human rhabdomyosarcoma RD cells in the liver.

It is now known that members of the selectin and integrin families are critical in the initial interaction of cells in circulation with endothelial surfaces. Also, platelet/endothelial cell adhesion molecule-1 has been shown to be involved in transendothelial migration of extravasating cells. Little is known about adhesion molecules involved in subsequent postextravasation events. In this study, the significance of VLA-2 (alpha2beta1) integrin in the movement of human rhabdomyosarcoma RD cells in the liver was characterized by in vivo videomicroscopy. Results show that after extravasation, the mock-transfected RDpF cells were able to migrate to the subcapsular region of the liver. Although the RDX2C2 transfectant expressing VLA-2 integrin extravasated equally well, a majority of RDX2C2 cells remained in close proximity to blood vessels and failed to reach the subcapsular region. The functional involvement of VLA-2 in affecting the ability of RD cells to reach the subcapsular region was verified by the preparation of an RD transfectant [RDX2C2(I-)] expressing a nonfunctional variant of VLA-2 lacking the inserted (I)-domain of alpha2 subunit. In vivo microscopy showed that RDX2C2(I-) cells migrated in a manner similar to control RDpF cells. To demonstrate that RDX2C2 cells that remained in dose proximity to blood vessels were due to VLA-2 function, a blocking monoclonal antibody against VLA-2 (BHA2.1) was prepared. Mice were injected with BHA2.1 or control monoclonal antibody P3 at the time when RDX2C2 cells completed their extravasation. Treatment with BHA2.1 increased the number of RDX2C2 cells that reached the subcapsular region and subsequently formed tumor foci. Therefore, VLA-2 integrin expression has major roles in postextravasation movement and affects tumor foci formation at the liver surface.

VLA-beta 1 integrin subunit-specific monoclonal antibodies MB1.1 and MB1.2: binding to epitopes not dependent on thymocyte development or regulated by phorbol ester and divalent cations.

We report here the isolation of two new monoclonal antibodies (MB1.1 and MB1.2) against mouse VLA-beta 1 integrin subunit. Characterization by flow cytometry demonstrated binding of MB1.1 and MB1.2 to freshly isolated thymocytes, primary bone marrow mast cell lines, as well as cell lines of distinct lineage each expressing different combination of VLA integrins. The specificity of MB1.1 and MB1.2 was determined by (1) their binding to antigen with M(r) about 120 kDa, and (2) the ability of antiserum against the carboxyl terminal of VLA-beta 1 subunit to deplete antigens for MB1.1 and MB1.2 in sequential immunoprecipitation experiments. The epitopes for MB1.1 and MB1.2 were in close proximity to each other since preincubation of cells with one MAb inhibited the binding of the other. However, MB1.1 and MB1.2 differed in their affinity for the beta 1 subunit. In addition, neither MAbs had any effect on cell adhesion to matrix proteins indicating that the epitopes involved are distant from VLA integrin ligand-binding sites. MB1.1 and MB1.2 appear to differ from the two MAbs so far reported against mouse VLA-beta 1 subunit, KMI6 and 9EG7. Thus, the epitopes for MB1.1 and MB1.2 were readily detectable on unfractionated thymocytes whereas KMI6 has been reported to bind only a fraction of CD4-8- and CD4-8+ thymocytes. Phorbol ester and Mn2+, which have been shown to regulate the binding of 9EG7, had no effect on MB1.1 and MB1.2 binding to VLA-beta 1 integrin subunit.

Jumping translocation in a phenotypically normal female.

"Jumping translocation" jt refers to a rare type of chromosome mosaic, in which the same portion of a (donor) chromosome is translocated to different (recipient) chromosome sites. Jt have mainly been observed in lymphocyte cultures of patients with hematologic malignancies. We report a phenotypically normal female carrying a mosaic of two cell lines with the Xq26-qter segment translocated to the short arm of chromosomes 15 or 21 in peripheral blood lymphocytes. In skin fibroblasts, only the X/21 translocation was detected. We speculate that recombination between homologous repetitive sequences on non-homologous human acrocentrics may be the cause of such chromosomal rearrangements.

Rearrangement of chromosome 1 is a frequent finding in endometrial carcinoma. An in situ hybridization study in nine endometrial carcinomas.

Nine endometrial carcinomas were examined for numerical aberrations of the chromosomes 1,7, and X by fluorescence in situ hybridization using highly repetitive chromosome-specific probes. In addition, a combination of a centromeric and a telomeric chromosome 1 probe was applied to detect structural chromosome 1 aberrations. Chromosome aberrations were found in six tumors. In four of these, an imbalance between 1q12 and 1p36 was detected, indicating the presence of an extra 1p- chromosome. In regard to the chromosomes 7 and X, monosomies and trisomies were found. Intratumoral genetic heterogeneity in endometrial carcinomas was detectable by FISH and flow cytometry. In conclusion, our findings confirm that chromosome 1 is frequently involved in structural chromosome changes, indicating chromosome 1 to be of importance in the evolution of endometrial carcinoma.

[Correlation of cotinine level in amniotic fluid, umbilical artery blood and maternal blood]. / Korrelation des Cotininspiegels in Fruchtwasser, Nabelarterienblut und mütterlichem Blut.

The aim of the present study was to determine the correlation of cotinine levels in amniotic fluid and in fetal and corresponding maternal blood samples. Amniotic fluid samples (N = 130) were taken during second trimester amniocentesis, umbilical artery blood samples (N = 75) at birth, both together with corresponding maternal blood samples. Self-reported smokers showed maternal serum cotinine levels > 15 ng/ml in 93%, self-reported nonsmokers levels < 15 ng/ml in 89%. Correlation of corresponding values for cotinine was 0.81-0.92. Cotinine values were increased in fetal blood and amniotic fluid in comparison to maternal serum levels. Despite the fact that pathophysiology is not fully understood, an accumulation of nicotine and its metabolites both in the fetus and in the amniotic fluid appears to be evident.

[Antepartum monitoring and peripartal findings in mono- and dichorial twin pregnancy]. / Antepartale Uberwachung und peripartale Befunde bei mono- und dichorialer Zwillingsschwangerschaft.

In a group of 65 twin pregnancies the difference of perinatal findings and antepartal test results was evaluated in relation to amnionicity and chorionicity. Monochorionic placentation was found in 33% of the pregnancies. The rate of foetal malformation (11%), neuromuscular dysfunction (6%), perinatal mortality (11%) and duration of neonatal intensive care was increased in those cases. The most useful diagnostic tool was B-Mode-ultrasound (first detection and surveillance of multiple pregnancy, especially diagnosis of inter-twin growth discordancy). Non stress test and Doppler sonography were found to be of value as additional tests for detection of functional differences between both twins. There were no differences between findings in first and second twin as well as between findings in pregnancies with mono- or dichorionic placentation.

[Premature separation of the placenta--clinical findings and fetal monitoring]. / Vorzeitige Plazentalösung--klinische Befunde und fetale Uberwachungsmethoden.

In a study group of 41 pregnant women of postpartally confirmed placental abruption, the prognostic value of clinical and diagnostic findings was investigated. The incidence of placental abruption was 1.4% of all deliveries within a three-year interval. 51% of patients showed vaginal bleeding before delivery. Retroplacental haematoma was found in 49% of cases ultrasonographically. A total of 75% had a pathological CTG test. More than 95% of these findings occurred within 3 days before delivery. Abnormal Doppler flow findings in foetal vessels more than 3 days before delivery were seen in 62% of cases. In the last three days before delivery, 86% were abnormal. Preterm delivery before 37 weeks of gestation was registered in 82% of patients. Perinatal mortality amounted to 12%. The rate of severely dystrophic newborn was 30%. Even in cases with lack of the clinical and/or sonographical findings, the possibility of placental abruption should be considered, if an acute deterioration of cardiotocographic or Doppler-sonographic findings.

[Evaluation of the antepartum CTG course in cases of highly pathologic Doppler flow findings]. / Auswertung des antepartalen CTG-Verlaufs in Fällen mit hochpathologischen Doppler-Flow-Befunden.

In a group of 79 pregnancies with highly abnormal Doppler-flow findings in foetal vessels the value of antepartal CTG criteria was evaluated in detail. In cases with absent enddiastolic flow (AEDF) the prognostical value was not further increased by antepartal pathological CTG findings. In cases with permanently normal antepartal CTG's, however, foetal outcome was fairly good (median duration of pregnancy 38 + 6 weeks, median foetal birth weight 2,545 grams, arterial pH < 7.20 in 9%). This was true also in the presence of pathological Doppler-flow findings except AEDF. In cases with pathological antepartal CTG findings in the antepartal course of pregnancy the loss of acceleration was most frequent (82%), followed by decreased frequency (53%) or narrowed amplitude (38%) of the foetal heart rate variation. Combination of both methods (Doppler sonography and CTG) is recommended clinically because the rate of uncertain findings can be reduced. Especially in cases with AEDF an active management would be justified before CTG's become pathological.

[Doppler ultrasound of fetal blood vessels--optimizing the diagnostic value by a combined diagram]. / Doppler-Sonographie der fetalen Gefässe--Optimierung der Aussagekraft durch ein Kombinationsdiagramm.

The dopplersonographic data of 1926 pregnant women in the 3. trimester of pregnancy was used to evaluate a graphic system for the analysis of A/B-ratio in fetal vessels (pulsed wave duplex system; descending aorta and umbilical arteries). The basis of the newly developed combination diagram were dopplersonographic standard values (single cut off and pregnancy duration related percentiles). One of the results was the fact, that the combined measurement and analysis of both fetal vessels increased the positive predictive value compared to single measurements. The value of dopplersonographic examinations in the aorta was slightly better than those in the umbilical arteries. The combined diagram showed a marked improvement in graphicness, especially in follow-up cases, and a partial improvement in statistical values.

[Pathologic Doppler flow findings and cardiotocography results]. / Pathologische Doppler-Flow-Befunde und kardiotokographische Ergebnisse.

Of 1950 pregnant patients (2870 Doppler ultrasound measurements) we observed, in a study group with highly abnormal Doppler-flow findings (n = 66, Feb. 1990), a correlation of Doppler flow and FHR-recordings. Among these 66 patients we retained 60 (91%) in the hospital. They had at least 2 FHR-recordings a day. The results of Doppler flow measurements in the fetal aorta and umbilical artery correlated well with diagnosis of IUGR. The comparison of the overall results of both fetal vessels did not indicate any significant difference. In 21% of all patients with highly abnormal Doppler flow findings, was no abnormal FHR record until delivery. 26% already showed an abnormal non-stress test before the first pathological Doppler assessment, in 44% abnormal FHR-recordings were observed later than the first abnormal Doppler flow finding in the course of pregnancy. The median interval was 13.5 days in cases with increased Doppler flow parameters but with detectable end-diastolic blood flow and was reduced to 8 days in cases with absent end-diastolic blood flow. In 9% of all cases, abnormal results were found with both methods on the same day. In 32% we observed a reproducible notch in Doppler flow velocimetry of uteroplacental vessels. The rate of congenital malformations was 14%. Thus abnormal Doppler flow signals can be estimated as "early" prognostic criterias for a compromised fetus at risk.