A new cell surface marker of aging in human diploid fibroblasts

The relationship of cell surface changes to proliferative decline of human diploid fibroblasts was investigated using the concanavalin A-mediated red blood cell adsorption assay. The amount of the red blood cells adsorbed to human diploid fibroblasts via concanavalin A increased continuously from the early phases of cell passage up through cell senescence, while the amount of 3H-concanavalin A binding did not change to a significant extent. The red blood cell adsorption is not a function of cell cycle phase and time spent in culture. Cocultivation of young cells with old cells also did not affect the adsorption capacity of respective cells. Thus, the concanavalin A-mediated red blood cell adsorption can be expected to serve as a new cell surface marker for aging in vitro. Using this marker, it was revealed that transient cell size or 3H-thymidine incorporating capacity di not have a direct relationship with the division age of a cell. Small rapidly dividing cells in old populations resemble large slowly dividing or nondividing cells of the same populations and differ from small rapidly dividing cells in young populations, in terms of cell surface properties.     

Cell surface and clonal proliferative property of aging human diploid fibroblasts

We have described previously how concanavalin A (conA)-coated red blood cell (RBC) adsorption to human diploid fibroblasts could serve as a marker of the in vitro aging of these cells. Since the heterogeneity in RBC adsorption and the proliferative property of young and old cell populations was observed, the correlation of this cell surface property with the proliferative behavior of individual clones was examined as a function of cell age. The RBC adsorption capacity of cells in both large and small colonies changed with the aging of the parent cell populations; the cells from early passage populations did not adsorb RBCs, but those from late passage populations adsorbed them well. Thus, the amount of RBC adsorption was not a function of colony size, but was related to the age of the culture.     

Cell surface changes accompanying aging in human diploid fibroblasts : III. Division age and senescence revealed by concanavalin A-mediated red blood cell adsorption

The relationship between cell size, [³H]thymidine incorporation capacity, and cell surface property of human diploid fibroblasts was investigated using the concanavalin A (ConA)-mediated red blood cell (RBC) adsorption assay. Small cells in late passage populations adsorbed RBCs well with the RBC coating method (in which ConA-coated RBCs are adsorbed to fibroblasts) as did large cells of this population, while small cells in early passage populations did not. The RBC adsorption capacity of rapidly dividing cells with this method differed among young, middle-aged and old cell populations. The results suggest that temporal cell size and [³H]thymidine incorporating capacity is not a measure of the division age of human diploid cells at the individual cell level. On the other hand, RBC adsorption with the fibroblast coating method (in which RBCs are adsorbed to ConA-coated fibroblasts) occurred to non-dividing cells of the populations. Thus, the increase in RBC adsorption with this method is considered to be a reflection of the increase in non-dividing cells at phase III. Our results support the hypothesis that RBC adsorption with the RBC and fibroblast-coating methods represents a cell surface marker for division age and senescence of human diploid cells, respectively, at the individual cell level.     

Plasmodium falciparum: the behavior of clinical isolates in an in vitro model of infected red blood cell sequestration

An in vitro model of Plasmodium falciparum-infected red blood cell sequestration which uses C32 amelanotic melanoma cells as targets has been used to examine the binding capacity of infected red blood cells from subjects with naturally acquired P. falciparum infections of varying severity. The binding of infected red blood cells (IRBCs) to melanoma cells was specific to cells containing mature parasites. Variations in target cell density and in conditions of growth had significant effects on binding. Binding was pH dependent, being maximum at a pH of 6.9. Using standardized conditions the binding capacity of individual isolates of P. falciparum could be measured with a high degree of reproducibility. Binding capacity of IRBCs from 51 subjects between the ages of 6 months and 15 years varied between 12 and 1254 IRBCs per 100 melanoma cells when RBC suspensions at a 1% parasitemia and 4% hematocrit were used. Variation in binding was not related to the level of peripheral parasitemia of the isolate or to differences in adaptation to culture conditions. The binding capacity of parasitized cells from subjects with cerebral malaria did not differ from that of IRBCs from subjects with less serious clinical manifestations.     

Cell surface changes accompanying aging in human diploid fibroblasts : II. Two types of age-related changes revealed by concanavalin A-mediated red blood cell adsorption

Age-related changes in cell surfaces of human diploid fibroblasts (TIG-1) were investigated using the concanavalin A (ConA)-mediated red blood cell (RBC) adsorption assay. When ConA-coated RBCs were adsorbed to fibroblasts (RBC coating method), the amount of RBCs adsorbed per mg of fibroblast protein increased continuously from the early phases of cell passage up through cell senescence. On the other hand, when RBCs were adsorbed to ConA-coated fibroblasts (fibroblast coating method), RBC adsorption did not occur throughout phase II and increased with the advance of phase III. [³H]ConA binding to fibroblasts, however, did not change with aging to the extent that could explain the observed changes in RBC adsorption. These age-related characteristics in RBC adsorption and [³H]ConA binding were also observed for WI38 and IMR-90 cells. In addition, SV40- and ⁶⁰Co-transformed WI38 cells showed a close resemblance in their RBC adsorption capacity to early phase III cells.RBC adsorption with both the RBC and fibroblast coating methods was not a function of cell cycle phase and time spent in culture (metabolic time). Co-culturing of young cells with old or transformed cells did not affect the RBC adsorption capacity of respective cells. These results suggest that RBC adsorption with the RBC and fibroblast coating methods may represent cell surface markers for division age and senescence of aging human diploid cells.     

Red cell distribution and the recruitment of pulmonary diffusing capacity.

The distribution of red blood cells in alveolar capillaries is typically nonuniform, as shown by intravital microscopy and in alveolar tissue fixed in situ. To determine the effects of red cell distribution on pulmonary diffusive gas transport, we computed the uptake of CO across a two-dimensional geometric capillary model containing a variable number of red blood cells. Red blood cells are spaced uniformly, randomly, or clustered without overlap within the capillary. Total CO diffusing capacity (DLCO) and membrane diffusing capacity (DmCO) are calculated by a finite-element method. Results show that distribution of red blood cells at a fixed hematocrit greatly affects capillary CO uptake. At any given average capillary red cell density, the uniform distribution of red blood cells yields the highest DmCO and DLCO, whereas the clustered distribution yields the lowest values. Random nonuniform distribution of red blood cells within a single capillary segment reduces diffusive CO uptake by up to 30%. Nonuniform distribution of red blood cells among separate capillary segments can reduce diffusive CO uptake by >50%. This analysis demonstrates that pulmonary microvascular recruitment for gas exchange does not depend solely on the number of patent capillaries or the hematocrit; simple redistribution of red blood cells within capillaries can potentially account for 50% of the observed physiological recruitment of DLCO from rest to exercise.     

The receptors ensuring Staphylococcus aureus fixation on fibronectin-containing surfaces

The role of protein A and other components of S. aureus cell wall in binding fibronectin on the surface of formulated sheep red blood cells was studied. 41 out of 89 fibronectin-binding clinical isolates lost their capacity for agglutinating fibronectin-sensitized red blood cells after the treatment of such cells with the solution of commercial purified protein A. These strains were also shown to have pronounced direct relationship between the levels of binding of fibronectin and IgG. Other isolates in the collection possessed the protein A-independent receptor capable of binding fibronectin. The receptor was seemingly common for this group of strains, and its presence significantly increased the capacity of staphylococci for binding fibronectin.     

Effect of Adsorbed Proteins on the Rheological Properties of Erythrocytes

Impedance spectroscopy in the radio-frequency band was used to probe the adsorptive properties of red blood cells. Fluidity assays of concentrated erythrocyte suspensions revealed a close relationship between the protein adsorption on erythrocyte membranes and the extent of cell interaction in the flow. Patients with impaired peripheral circulation displayed an increase in adsorption of high-molecular-weight proteins and a dramatic decrease in fluidity of erythrocyte suspensions. Hence, adsorption was assumed to contribute to rheological disorders.     

Tolerance-like condition developing in mice under the effect of antigen of erythrocyte lysate]

Lysate of sheep red blood cells obtained by the treatment of these cells with distilled water and purified by ultracentrifugation in cold possessed a weak immunogenicity. Its administration to mice caused the state of hyporeactivity to sheep red blood cells (a reduction of the immune response level to 10-25% of control. The capacity of the mise spleen cells to respond by immune reaction to the red blood cells following adoptive transfer was not disturbed. At the early periods after the lysate administrations the mouse spleen cells possessed a weak supressive activity in case of their transfer to the intact animals. The blood serum of mice treated with the lysate possessed a blocking activity which disappeared after the serum absorption with sheep red blood cells. A conclusion was drawn that hyporeactivity originating in mice after the lysate administration was caused by the presence in the serum of antibodies inhibiting the immune response.     

Red blood cell orientation in pulmonary capillaries and its effect on gas diffusion.

When alveoli are inflated, the stretched alveolar walls draw their capillaries into oval cross sections. This causes the disk-shaped red blood cells to be oriented near alveolar gas, thereby minimizing diffusion distance. We tested these ideas by measuring red blood cell orientation in histological slides from rapidly frozen rat lungs. High lung inflation did cause the capillaries to have oval cross sections, which constrained the red blood cells within them to flow with their broad sides facing alveolar gas. Low lung inflation stretched alveolar walls less and allowed the capillaries to assume a circular cross section. The circular luminal profile permitted the red blood cells to have their edges facing alveolar gas, which increased the diffusion distance. Using a finite-element method to calculate the diffusing capacity of red blood cells in the broad-side and edge-on orientations, we found that edge-on red blood cells had a 40% lower diffusing capacity. This suggests that, when capillary cross sections become circular, whether through low-alveolar volume or through increased microvascular pressure, the red blood cells are likely to be less favorably oriented for gas exchange.     

Comparative studies on removal of Congo red by native and modified mycelial pellets of Trametes versicolor in various reactor modes

Aerated and rotated mode adsorption experiments were carried out for the removal of Congo red from aqueous solution using native and pre-treated mycelial pellets/biomass of Trametes versicolor. The effect of process parameters like contact time, dosage of adsorbent, adsorbate concentration and pH on adsorption was investigated. Higher the dye concentration lower was the adsorption. Equilibrium time was attained at 90 min. Increase in biomass dosage increased the adsorption. Experimental data were analyzed by the Langmuir and Temkin isotherms. Adsorption capacity (Q(0)) of autoclaved biomass was 51.81 mg/g, which was higher than other biomass studied. The second order kinetic model by Ho and Mckay described well the experimental data. Acidic pH was favorable for the adsorption of Congo red. Studies on pH effect and desorption show that chemisorption seems to play a major role in the adsorption process. Among the native and pre-treated biomass studied, autoclaved biomass showed a better adsorption capacity. Utilization of autoclaved biomass is much safer as it does not pose any threat to environment. Aerated mode showed a better adsorption capacity when compared to rotated mode.     

Effect of Human Serum Albumin Upon the Permeabilizing Activity of Sticholysin II, a Pore Forming Toxin from Stichodactyla heliantus

Sticholysin II (St II) is a haemolytic toxin isolated from the sea anemone Stichodactyla helianthus. The high haemolytic activity of this toxin is strongly dependent on the red cell status and the macromolecule conformation. In the present communication we evaluate the effect of human serum albumin on St II haemolytic activity and its capacity to form pores in the bilayer of synthetic liposomes. St II retains its pore forming capacity in the presence of large concentrations (up to 500 μM) of human serum albumin. This effect is observed both in its capacity to produce red blood cells haemolysis and to generate functional pores in liposomes. In particular, the capacity of the toxin to lyse red blood cells increases in the presence of human serum albumin (HSA). Regarding the rate of the pore forming process, it is moderately decreased in liposomes and in red blood cells, in spite of an almost total coverage of the interface by albumin. All the data obtained in red cells and model membranes show that St II remains lytically active even in the presence of high HSA concentrations. This stubbornness can explain why the toxin is able to exert its haemolytic activity on membranes immersed in complex plasma matrixes such as those present in living organisms.     

Regulation of the cytotoxic effect of human ‘normal killer cells’ on tumor cell lines by neuraminidase-treated T-lymphocytes

Summary Subpopulations of peripheral blood lymhocytes (PBL) from healthy individuals were separated according to their capacity to form various rosettes and tested for their cytotoxic activity on cell lines of urinary bladder and breast carcinomas. The subpopulation exerting the highest natural cytotoxic activity was characterized by the presence of cell surface Fc-receptors and by the lack of receptors for sheep red blood cells and for C'3 on their surface. Treatment with vibrio cholera neuraminidase (VCN) increased the cytotoxicity of unseparated PBL to a level twice as high as that of untreated PBL. The attachment of T-lymphocytes to tumor monolayers was increased several fold after VCN-treatment, while the attachment of other lymphocyte subpopulations was not. Evidence is presented that the augmentation of the cytotoxicity of PBL following VCN-treatment results from the interaction of VCN-treated T-lymphocytes, attached to target cells, with normal killer cells. It is suggested that augmentation of the activity of killer cells by T-lymphocytes may play a role in antitumor defense mechanisms.Abbreviations CMC Cell-mediated cytolysis - E-rosettes Rosettes formed with sheep red blood cells - EA-rosettes Rosettes formed with red blood cells coated with antibody - EAC'-rosettes Rosettes formed with red blood cells coated with antibody and complement - FCS Heat inactivated fetal calf serum - PBL Peripheral blood lymphocytes - RBC Red blood cells - RF-TAL E-rosette forming, target-attached lymphocytes - SRBC Sheep red blood cells - VCN Vibrio cholera neuraminidase     

Solubilization of a human lymphocyte factor for autorosette

With incubation at 45 degrees C, human peripheral blood lymphocytes (PBL) loose 80% of their capacity to form 1-hr autorosettes (AR). However, the addition of supernatant from heated lymphocytes (SHL) restores 93% of their rosette-forming capacity, while producing an inhibitory effect on nonincubated lymphocytes. A soluble factor present in SHL is active to a 1/5000 dilution; is absorbable on autologous red blood cells but not on sheep red blood cells; is RNase and DNase resistant and sensitive to trypsin and pronase; and acts variably on allogenic cells.     

Mutant of Polyoma Virus with Impaired Adsorption to BHK Cells

A mutant of polyoma virus PY235 has an impaired adsorption to guinea pig red blood cells and BHK-21 hamster cells. Adsorption to 3T3 mouse cells is much less inhibited. These altered adsorption properties are responsible for the apparent inability of PY235 to cause cell transformation or hemagglutination.     

Hemolytic activity of Vibrio cholerae eltor and V. cholerae O139 toxigenic and nontoxigenic strains

A total of 20 ctx- and 16 ctx+ V. cholerae eltor strains, 20 ctx- and 22 ctx+ V. cholerae O139 strains were under study. Hemolytic activity was tested in modified Greig test with sheep, guinea pig and rabbit red blood cells. The comparative study of the hemolytic properties of V. cholerae O1 and O139 under different conditions of cultivation demonstrated their capacity of lysing sheep red blood cells (SRBC) irrespective of the presence of toxigenic properties. A wider spectrum of lytic activity of ctx- strains in Greig test with respect to red blood cells of different animals and the capacity of lysing SRBC, most resistant to the action of toxin, may be due to a considerably greater content of Hly+ clones in their population.     

Opsonins and erythrophagocytosis

The author studied the capacity of granulocytes and reticular cells of rat bone marrow to phagocytize mouse red blood cells in the action of opsonins against them. A comparative assessment of phagocytosis of fresh and formaldehyde-treated mouse red blood cells was conducted. A moderate phagocytosis observed with the use of fresh erythrocytes and immune noninactivated rat serum was less pronounced when inactivated serum was used. No phagocytosis occurred with rat nonimmune serum. On the contrary, when formaldehyde-treated erythrocytes were used with the same sera phagocytosis was much stronger.     

Phagocytic capacity of Kupffer cells during hepatic amoebiasis in guinea pigs.

Very few studies have been carried out on the role of liver macrophages (Kupffer cells) during the course of hepatic amoebiasis. The kinetics of phagocytic activity of Kupffer cells and blood monocytes was studied in guinea pigs intra-mesenterically infested with Entamoeba histolytica. The phagocytic capacity of blood monocytes of normal animals was comparatively lower than Kupffer cells for both latex and haemolysin coated sheep red blood cells. Significant decline in phagocytic response of Kupffer cells and blood monocytes of infected animals was observed right from 2nd post infection day and it kept on decreasing with the progress of infection. Depression in phagocytic response of Kupffer cells and blood monocytes was more marked in those animals who had higher grades of pathological lesions. Hence, an inverse correlation was obtained between the phagocytic capacity and severity of amoebic lesions (P less than 0.01). The significance of depression in phagocytic response of Kupffer cells and blood monocytes may be responsible for the development of hepatic lesions.     

The effect of host blood in the in vitrotransformation of bloodstream trypanosomes by tsetse midgut homogenates

Abstract. Midgut homogenates prepared from Glossina morsitans morsitans , that had previously been fed on different host blood samples, were tested for their abilities to transform bloodstream Trypanosoma brucei into procyclic (midgut) forms in vitro. Compared to rat and goat blood samples, eland blood had the least capacity to support trypanosome transformation, whereas buffalo blood showed intermediate capacity. Fractionation of rat blood showed the importance of the cellular portion since both rat and eland red blood cells (RBCs) supported the process. Virtually no transformation was observed in rat and eland plasma or serum fractions. Suspending rat blood cells in eland plasma led to a reduction in parasite transformation rates. Further experiments showed that the RBC membranes were also capable of supporting the process. These results clearly show the important role played by blood, especially the red blood cells, in the transformation of bloodstream trypanosomes. In addition, the low transformation rates observed in eland blood is due to an inhibitory factor(s) present in the plasma fraction.     

Tannic acid as a stabilizer of liposomal lipids in electron microscopic research

Using electron microscopy the action of tannic acid on the morphology of positively charged liposomes were studied in suspension and after adsorption of the surface membrane of human red blood cell in vitro. After treatment with tannic acid and glutaraldehyde followed by postosmication, an irreversible reconstruction of liposome lipid material with formation of many layers with tight packed lamellae was shown. Similar structures were found on the surface of red blood cells. Monolayer microvesicles 30 nm in diameter were seen after glutaraldehyde fixation postosmication, and final treatment with tannic acid. Similar microvesicles were seen adsorbed on the plasma membrane.