Interlaboratory validation of a CD71-based flow cytometric method (Microflow) for the scoring of micronucleated reticulocytes in mouse peripheral blood

An interlaboratory study was performed to validate an anti-CD71/flow cytometry-based technique for enumerating micronucleated reticulocytes (MN-RETs) in mouse peripheral blood. These experiments were designed to address International Workshop on Genotoxicity Test Procedures validation criteria by evaluating the degree of correspondence between MN-RET measurements generated by flow cytometry (FCM) with those obtained using traditional microscopy-based methods. In addition to these cross-methods data, flow cytometric MN-RET measurements for each blood sample were performed at two separate sites in order to evaluate the reproducibility of data between laboratories. In these studies, groups of male CD-1 mice were treated with vehicle (saline or vegetable oil), a negative control (saline or vegetable oil), or four dose levels of five known genotoxicants (clastogens: cyclophosphamide, benzo[a]pyrene, 5-fluorouracil, methotrexate; aneugen: vincristine sulfate). Exposure occurred on 3 consecutive days via intraperitoneal injection, and blood samples were obtained approximately 24 hr after the final treatment. MN-RET frequencies were determined for each sample based on the analysis of 2,000 (microscopy) and 20,000 (FCM) reticulocytes. Regardless of the method utilized, each genotoxic agent was observed to cause statistically significant increases in the frequency of MN-RETs, and each response occurred in a dose-dependent manner. Spearman's correlation coefficient (rs) for FCM versus microscopy-based MN-RET measurements (nine experiments, 252 paired measurements) was 0.740, indicating a high degree of correspondence between methods. The rs value for all flow cytometric MN-RET measurements performed at the two independent sites was 0.857 (n = 248), suggesting that the automated method is highly transferable between laboratories. Additionally, the flow cytometric system offered advantages relative to microscopy-based scoring, including a greater number of cells analyzed, much faster analysis times, and a greater degree of objectivity. Collectively, data presented in this report suggest that the overall performance of mouse peripheral blood micronucleus tests is enhanced by the use of the flow cytometric scoring procedure.     

Direct action of genistein on CFTR

 Human cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels were expressed in oocytes from Xenopus laevis after injection of CFTR cRNA and studied with the two-electrode voltage-clamp and the giant patch techniques. The tyrosine kinase inhibitor genistein alone activated a small chloride current in whole oocytes expressing CFTR and substantially increased the chloride current obtained upon stimulation with forskolin and isobutyl methylxanthine (IBMX). In giant excised patches, genistein was unable to open protein-kinase-A-phosphorylated CFTR channels in the absence of ATP, but increased the ATP-induced CFTR channel currents by a factor of 3.8 ± 1.7. This genistein-mediated potentiation in excised patches is independent of protein phosphatase activity, as it is readily reversible, even after complete inhibition of protein kinase A activity. Involvement of protein tyrosine kinases also seems unlikely, because this effect of genistein is not antagonized by high concentrations of the tyrosine phosphatase inhibitor ortho-vanadate. We, therefore, propose a direct interaction of genistein with CFTR, probably at a nucleotide binding site, which leads to a higher open probability. Received: 10 March 1997 / Received after revision and accepted: 22 April 1997     

Gap junction communication between cells aggregated on a cellulose-coated polystyrene: influence of connexin 43 phosphorylation

The appropriate functioning of tissues and organ systems depends on intercellular communication such as gap junctions formed by connexin (Cx) protein channels between adjacent cells. We have previously shown that Swiss 3T3 cells aggregated on hydrophilic cellulose substratum Cuprophan (CU) establish short linear gap junctions composed of Cx 43 in cell surface plaques. This phenomenon seems to depend on the high intracellular cyclic AMP (cAMP) concentration triggered by attachment of the cells to CU. We have now used a cellulose-coated polystyrene inducing the same cell behaviour to analyse the gap junction communication between aggregated cells. The transfer of the dye Lucifer Yellow (LY) between cells showed that cells aggregated on cellulose substratum rapidly (within 90 min) establish functional gap junctions. Inhibitors of cAMP protein kinase (PKI) or protein kinase C (GF109203X) both inhibited the diffusion of LY between neighbouring cells. Western blot analysis showed that this change in permeability was correlated with a decrease in Cx 43 phosphorylation. Thus, cellulose substrata seem to induce cell-cell communication through Cx 43 phosphorylation modulated by PKA and PKC. To understand the mechanisms by which a substratum regulates gap junctional communication is critically important for the emerging fields of tissue engineering and biohybrid devices.     

Migrating anterior mesoderm cells and intercalating trunk mesoderm cells have distinct responses to Rho and Rac during Xenopus gastrulation.

Rho GTPases have been shown recently to be important for cell polarity and motility of the trunk mesoderm during gastrulation in Xenopus embryos. This work demonstrated that Rho and Rac have both distinct and overlapping roles in regulating cell shape, and the dynamic properties, polarity, and type of protrusive activity of these cells. Overexpression of activated or inhibitory versions of these GTPases also disrupts development of the head in Xenopus embryos. In this study, we have undertaken a detailed analysis of Rho and Rac function in migrating anterior mesendoderm cells. Scanning electron micrographs of these cells in situ revealed that their normal shingle arrangement is disrupted and both the cells and their lamellipodia are disoriented. Anterior mesendoderm explants plated on their natural blastocoel roof matrix, however, still migrated towards the animal pole, although the tendency to move in this direction is reduced compared to controls. Analysis of a number of parameters in time-lapse recordings of dissociated cells indicated that Rho and Rac also have both distinct and overlapping roles in the motility of the prospective head mesoderm; however, their effects differ to those previously seen in the trunk mesoderm. Both GTPases appear to modulate cell polarization, migration, and protrusive activity. Rho alone, however, regulates the retraction of the lagging edge of the cell. We propose that within the gastrulating Xenopus embryo, two types of mesoderm cells that undergo different motilities have distinct responses to Rho GTPases.     

Hemoglobin Beth Israel. A mutant causing clinically apparent cyanosis.

We found that an abnormal hemoglobin with a very low oxygen affinity was responsible for overt cyanosis in an otherwise healthy adolescent. Hemoglobin Beth Israel, in which serine replaces the asparagine residue normally present at position 102 (G4) of the beta-polypeptide chain, was associated with normal blood counts and no apparent exercise intolerance in the heterozygous carrier. Cyanosis resulted from a drastically right-shifted oxygen dissociation curve, whose position and shape could account for the absence of "physiologic" anemia. The whole-blood oxygen tension at 50 per cent oxygen saturation was 88 mm Hg (normally 26 +/- 1 mm Hg), and the arterial blood was only 63 per cent saturated with oxygen despite a normal oxygen tension of 97 mm Hg. The hemolysate showed a low oxygen affinity but normal Bohr effect. Unexplained cyanosis, particularly in association with normal arterial oxygen tension should prompt a search for an abnormal hemoglobin, which may obviate the need for invasive diagnostic procedures.     

Description of a hybridoma bank towards Bordetella pertussis toxin and surface antigens

This paper describes the development of a murine bank of monoclonal antibodies against Bordetella pertussis toxin, filamentous hemagglutinin (FHA), pili, lipopolysaccharide (LPS), or outer membrane proteins (OMPs). Subunits S1, S2, S3 of pertussis toxin (PT) bound immunoglobulins and glycoproteins such as fetuin and haptoglobin in an unspecific manner. The specificity of monoclonal antibodies towards subunits S1, S2, S3 or S4 of PT could be demonstrated by using purified immunoglobulins or their Fab2 fragments. A set of FHA-specific monoclonal antibodies could be differentiated on the basis of their binding to the various breakdown products present in FHA preparations. Pili-specific monoclonal antibodies reacted with either native pili or denatured pilin, and both demonstrated serotype specificity. Monoclonal antibodies to Bordetella pertussis OMPs were directed to either the virulent phase-regulated trypsin-sensitive, detergent-extractable OMPs 92 kDa, 32 kDa, and 30 kDa or the non-virulent phase-expressed, not-trypsin sensitive OMPs 38 kDa, 33kDa, and 18 kDa.     

Membrane knobs are required for the microcirculatory obstruction induced by Plasmodium falciparum-infected erythrocytes.

We have studied the pathophysiology of the vascular obstruction induced by Plasmodium falciparum-parasitized erythrocytes with the use of an ex vivo microcirculatory preparation perfused with red cells infected with knobless and knobby clones of the FCR-3 strain. We find that parasitized erythrocyte membrane knobs are indispensable for the generation of the circulatory obstruction. Uninfected erythrocytes incubated in culture and erythrocytes infected with early or late forms of the knobless clones or the early forms of the knobby clone all failed to obstruct the microcirculation, although exhibiting various effects on bulk viscosity and peripheral resistance during flow. In contrast, late forms of the knobby clone produced significantly higher peripheral resistance during flow and significant obstruction as detected by changes in time of pressure flow recovery as well as by direct videorecorded microscopic observation. Optical and electron microscopy showed that the adherence of parasitized cells to the endothelium was limited to the venules and involved the knobs in junctions. In addition, we were able to follow the sequence of events during obstruction: initial red-cell adherence to the venular endothelium (sometimes only transitory) followed by progressive recruitment at the venule surface, finally leading to total obstruction that involved parasitized and nonparasitized erythrocytes. Sometimes, retrograde aggregation would extend the obstruction to the capillaries or even precapillary arterioles. These results show that knobs are necessary and sufficient to produce vascular obstruction and that other factors (spleen, immunological, etc.) can only have a modulating role. These results also exclude the possibility that the exclusive adherence to venules is the consequence of "plasma factors" found in the malaric patients.     

Conditional Disease-Free and Overall Survival of 1,858 Young Women with Non-Metastatic Breast Cancer and with Participation in a Post-Therapeutic Rehab Programme according to Clinical Subtypes

BackgroundBreast cancer in young women is associated with unfavourable tumour biology and is the main cause of death in this group. Conditional survival analysis estimates survival rates under the pre-condition of already having survived a certain time.ObjectivesTo describe conditional disease-free and overall survival of female breast cancer patients according to clinical subtypes and age.MethodsThis study analyses information from 1,858 breast cancer patients aged between 21 and 54 years, who were taking part in a post-therapeutic rehab programme (time between diagnosis and rehab start: maximum 24, median 11 months). Mean follow-up time was 3.6 years. We describe biological, clinical and pathological features in regard to different age groups (<40 and ≥40 years) and report conditional 5-year survival rates for overall and disease-free survival, and Cox proportional hazard models.ResultsVery young and young patients differed in regard to hormone receptor negativity, tumour grade, lymphovascular invasion, and molecular subtypes. Young women bore triple-negative and HER2-like disease more frequently. Conditional 5-year overall survival did not differ substantially between women <40 and 40–54 years of age (95 vs. 96%). It was highest for women with cancer of the luminal A subtype (98%) and lowest for the triple-negative subtype (91%). Lymphangiosis was a significant predictor of death. Results for disease-free survival were comparable.ConclusionsConditional 5-year overall survival after non-metastatic breast cancer was as high as 95.5%, and disease-free survival was 85.2%. When controlling for time between diagnosis and rehab start, molecular subtypes influenced overall and disease-free survival prospects. When additionally controlling for clinical characteristics, this effect only remained stable for disease-free survival.