Mouse Red Cells Research Articles

Characterisation and separation of human lymphocytes forming mouse red cell rosettes

Rosette formation between human lymphocytes and mouse red blood cells (MRBC) was examined as a marker for B lymphocytes and as a method of B lymphocyte separation. A small proportion of lymphocytes formed spontaneous rosettes with MRBC (mean value 6%) and the number was considerably increased by pretreating the lymphocytes with neuraminidase (mean value 16%). Double marker tests demonstrated that lymphocytes forming MRBC rosettes were immunoglobulin (Ig) bearing cells, with a high proportion of IgM bearing cells, but not all Ig bearing cells formed MRBC rosettes. Lymphocyte populations enriched with T or B lymphocytes, by SRBC rosette sedimentation and nylon column filtration, gave values for MRBC rosettes consistent with a subpopulation of Ig bearing cells. Separation of MRBC rosette-forming cells gave a relatively poor degree of separation and rerosetting with MRBC produced variable results.

An Improved Micro-Method for Enumeration of Human B-Cell Rosettes with Mouse Red Blood Cells

A micro-method for enumerating B-lymphocytes in human peripheral blood is described, using formation of rosettes with mouse red blood cells as a B-cell marker. The optimal conditions for the enumeration of mouse red cell rosette forming cells have been defined. By this method, it was possible to utilize less than 1ml of peripheral blood (venous or capillary) for simultaneous enumeration of both T-cell and B-cell rosettes. Our study of human lymphoid cell lines, thymic cells, lymphocytes from agammaglobulinemia, and severe combined immunodeficiency has provided additional evidence that lymphocyte rosetting with mouse red blood cells represent B-lymphocytes.

Significance of mouse red cell rosette-forming lymphocytes in chronic lymphocytic leukaemia.

Increased mouse red cell (M) rosetting lymphocytes were demonstrated in the peripheral blood of patients with chronic lymphatic leukaemia. The range was wide, and patients showed considerable variation not only in the number of M cells but also in T and B rosetting lymphocytes. Treatment reduced M rosette lymphocytes proportionately as the total white count fell, and differential removal occurred only when the patients became leucopaenic. If we assume the M rosetting cells are the abnormal 'leukaemic' cells, treatment does not preferentially remove these. The M rosetting capacity appeared to be related to the presence of an immunoglobulin factor previously demonstrated on the cells and in the serum of patients with CLL which enhances in vitro viability of the leukaemic cells.

Kinetics of Antibody and Immunoglobulin-Producing Cells Appearing in Popliteal Lymph Nodes of Mice Stimulated with Horseradish Peroxidase

Abstract Mice were given injections in their hind footpads of peroxidase emulsified in complete Freund's adjuvant. Kinetics of cells producing antibodies and cells producing immunoglobulins without detectable antibody function were monitored in the draining popliteal lymph nodes by using enzyme-immunocytochemical procedures, a local hemolysis plaque assay, and immune cytoadherence tests. These last two techniques were performed with sheep red blood cells (SRBC) coated with either peroxidase or anti-mouse Ig antibody. The kinetics observed with the immunocytochemical and plaque assay procedures were comparable although at each interval tested a higher number of positive cells was scored with the first procedure. With these two techniques, the following observations were made: 1) After antigen administration, the first detectable antibody- (Ab+) producing cells appeared on day 7 or 8. They were preceded 4 days earlier by the appearance of cells producing immunoglobulins without detectable antibody function (Ig+Ab-). The peak response of Ab+-producing cells occurred on day 14, 4 days after the maximum response of Ig+Ab--producing cells. 2) The vast majority of these Ig+Ab--producing cells was related to the injection of peroxidase and not to the complete Freund's adjuvant. Ig+Ab--secreting cells were found not to represent cells secreting antibodies directed against SRBC, DNP determinants, or bromelain-treated mouse red cells. 3) After a second injection, Ab+ and Ig+Ab--producing cells appeared rapidly and simultaneously in approximately equal numbers; the peak number of Ab+-producing cells was seen on days 5 to 7 whereas the Ig+Ab--producing cells disappeared 3 days after challenge. 4) The ratio of secreting versus synthetizing cells remained constant throughout the response, around 2:10 for Ab+ cell population and 5:10 for Ig+Ab- cell population. With the immune cytoadherence test, cells forming rosettes with peroxidase-coated SRBC were not found in lymph nodes of unstimulated mice although they appeared 4 days after antigen injection. Their number increased exponentially until day 8 and then more slowly until day 14. Although after a secondary injection the number of rosette-forming cells was increased, the peak value attained only 20% of the peak value observed during the primary response. The above results indicate that after injection of mice with peroxidase in complete Freund's adjuvant, the following sequence occurs: a) appearance of an immunocyte population bearing peroxidase receptors, b) appearance of an immunocyte population either synthesizing and secreting, or not, immunoglobulins without detectable antibody function, d) appearance of an immunocyte population either synthetizing and secreting, or not, antibody.

Time-Dependent Loss of Invasive Ability of Plasmodium berghei Merozoites In vitro

The invasive ability of Plasmodium berghei merozoites in vivo was studied following their artificial removal from parasitized mouse red cells using complement-mediated immune lysis in vitro and in vivo. Time-course experiments revealed that lysed preparations contained two components contributing to the parasites' infectivity in mice. One component, presumed to be free merozoites released from mature schizont-infected cells, rapidly lost infectivity with time at 1 to 2 C. A second minor component appeared to have more stability at this temperature, and could be accounted for as intact parasitized cells containing mature schizonts not lysed by the complement in vitro, but lysed by the recipients' plasma complement in vivo. Further experiments revealed that suspension of parasitized cells in an isotonic diluent and centrifugation at moderate speeds substantially removes the number of invasive free merozoites insolable from a given sample of infected blood by immune hemolysis. merzoites, either contained within the confines of mature schizont-infected cells, or artificially removed from host cells, rapidly lose the ability to invade susceptible erythrocytes in vivo when suspended in an isotonic medium and held at 1 to 2 C in vitro.

A heterophile carbohydrate moiety common to mammalian IgM and erythrocytes detected by chicken IgM antibody.

Sequential treatment of chicken lymphocytes with normal sheep serum (NSS), chicken antiserum against sheep erythrocytes (SE) and formaldehyde results in rosette formation with SE. The identity of reactive components and the mechanism of rosette formation has been elucidated. The binding activity of NSS to chicken lymphocytes was ascribed to "natural" IgM anti-species antibodies. Subsequently, chicken IgM anti-SE antibody reacted with sheep IgM bound to the lymphocyte surface, and, following formaldehyde fixation, rosettes were formed by the addition of SE. The reaction of chicken anti-SE antibody was competitively inhibited with a hog blood group substance suggesting that it had binding specificity for a carbohydrate moiety which is shared by sheep IgM and SE. This specificity represented only a fraction of the bulk of chicken IgM anti-SE antibodies and was not manifested by antibodies of the IgG class. The analysis by the above-described "sandwich rosette assay" was extended to chicken antisera against horse, rat and mouse red cells and to normal sera from these species. Cross-matching of chicken antisera, with the normal sera and red cells from the tested species revealed a distinct pattern of cross-reactivities. These results imply that the saccharide epitope which is shared by IgM and red cells has a wide distribution between various mammalian species and may be classified as a new system of heterophile antigens.

Effect of cyclophosphamide on synthesis of antibodies and nonspecific immuno-globulins in mouse spleen cells

Intraperitoneal injection of cyclophosphamide (CP) into mice in a dose of 200 mg/kg body weight leads after 2–3 weeks to a marked acceleration of the formation of nonspecific immunoglobulins (NIG) by spleen cells in vitro. In the early period (3–7 days) after injection of CP the acceleration of NIG production was due mainly to synthesis of macroglobulins; after 2 weeks the formation of both macroglobulins and IgG was increased. Normal NIG synthesis was not restored until at least 1 month later. Injection of antigen (sheep's red cells) against the background of hyperproduction of NIG led to a decrease in the latter. The formation of antibodies against red cells in mice treated with CP was at a lower level at all times of the investigation than in control immunized animals.

Purine phosphoribosyltransferase activities in mouse tissues.

Purine phosphoribosyltransferases were assayed in crude extracts of mouse liver, spleen, kidney, heart, lung, cerebrum and red cells, which also contain xanthine oxidase, guanase, 5'-nucleotidase and adenosine deaminase, by the method which was based on the isolation of the products by electro-phoresis on cellulose acetate membrane. Purine phosphoribosyltransferase activities were found in all tissues examined. Among them, the activities in the cerebrum were higher than those of other tissues whereas activities in the red cells were very low. The activity ratios of hypoxanthine-guanine phosphoribosyltransferase with hypoxanthine and guanine as the substrates to adenine phosphoribosyltransferase were 0.9-2.1 and 1.8-4.1, respectively, in the liver, spleen, heart, lung and kidney. In the cerebrum these were 7.4 and 19.2, and were 0.2 and 0.2 in the red cells. The significance of these results has been discussed.

Drug-induced immunological tolerance to sheep's red cells in mice of different strains

Immunological tolerance to sheep's red cells was induced in mice of various strains (CBA, C57BL/6, CC57BR, C3H, DBA/2) by combined injection of a large dose of antigen and cyclophosphamide into the animals. After a test injection of erythrocytes the number of 19S-antibody-forming cells was determined in the spleens of the mice by the local hemolysis in gel method. The degree of tolerance developing was shown to depend on the animals' genotype, and mice of strain DBA/2 were found to be the most sensitive to its induction. No correlation was found between the level of immunological reactivity to sheep's red cells in intact mice of different strains and the degree of its inhibition as a result of the induction of tolerance.

Stimulation of B-lymphocyte colony formation in vitro by sera from patients with leukaemia or lymphoma.

Studies were made on the effects of 665 sera, from normal donors or patients with various diseases, on B-lymphocyte colony formation in agar by mouse spleen cells. Undiluted serum from most normal donors inhibited colony formation, but 43-53% of sera from patients with histiocytic lymphoma, lymphocytic lymphoma or Hodgkin's disease stimulated colony formation, serum activity correlating with the stage of the disease. Moderate colony-stimulating activity was observed with serum taken from patients with acute lymphoid or myeloid leukaemia following, but not prior to, chemotherapy. Colony stimulating activity was not correlated with the blood group of serum donors and could not be ascribed to the presence of endotoxin, red cells or mouse red cell haemagglutinins in the active sera. Elevated colony stimulating activity was not observed in sera from patients with non-neoplastic disorders ot haemopoiesis or with diseases of other organ systems.

Immune response of New Zealand mice to trinitrophenylated syngeneic mouse red cells.

NZB and NZB/W mice have reduced anti-sheep red cell (SRC) and 2,4,6-trinitrophenyl-plaque-froming cell (TNP-PFC) responses with age after injection of either the thymus-dependent antigen TNP-SRC or the thymus-independent antigen TNP-mouse red cells (MRC). However, the thymus-dependent response diminished much faster than the thymus-independent response. As a consequence, young New Zealand mice have a higher anti-TNP response after injection of TNP-SRC than after injection of TNP-MRC, while old New Zealand mice have a higher anti-TNP response after injection of TNP-MRC than after injection of TNP-SRC. The PFC avidity of NZB/W mice injected with TNP-SRC diminished with age, while the PFC avidity of mice injected with TNP-MRC did not change with agrc or TNP-SRC. Old NZB/W mice had few spontaneous anti-MRC-PFC. The number of anti-MRC PFC in old mice was increased 4 to 10 times after injection with either TNP-SRC or TNP-MRC. It is suggested that surveillance mechanisms are responsible for suppressing the autoimmune response to modified self-antigens. The unregulated immune system of NZB and NZB/W mice appears to be an expression of impairment of such a hypothetical surveillance mechanism.

The abundance of ferritin in yolk-sac derived red blood cells of the embryonic mouse.

Circulating red blood cells formed early in development have several distinctive properties which include retention of the nucleus (mammals), large size and characteristic haemoglobin type (mammals, birds, amphibia). The primitive or embryonic red cells of early development are replaced by the definitive red cells which contain fetal or adult haemoglobin; a second developmental change occurs in the haemoglobin of some mammals (man, cattle, sheep) but does not involve a cell replacement. Circulating yolk-sac derived red cells from embryonic mice are siderocytes; elevated ferritin levels are associated with the circulating red cells of bullfrog tadpoles, but not with those of the adult frog, again indicating that red cell iron metabolism can change during development. In order to extend the observations made on an amphibian to a mammal, the ferritin content of circulating red cells from embryonic mice was determined and found to be 0.65 mg/100 mg of soluble protein; no ferritin (less than or equal to 0.007 mg/100 mg of soluble protein) was detected in adult mouse red cells. Elevated ferritin levels appeared to be specifically associated with the yolk-sac derived population of red cells since a decline in red-cell ferritin content coincided with the replacement of yolk-sac derived red cells by definitive red cells derived from the liver. Fractionation of mixtures of yolk-sac derived and liver derived red cells showed that fractions rich in the definitive red cells contained less ferritin than the mixture. The results suggest that elevated ferritin levels may be a general characteristic of the circulating, haemoglobinized red cells formed early in development.

Mouse Red‐Cell Rosettes in B‐Lymphoproliferative Disorders

A high proportion of peripheral-blood lymphocytes formed spontaneous rosettes with mouse red cells in 22 out of 23 cases of chronic lymphocytic leukaemia (CLL); the proportion was significantly higher than in 19 cases of other B-lymphoproliferative disorders (non-CLL group) and in 19 normal controls. Intermediate findings were obtained in 10 cases of "hairy" cell leukaemia. Blast cells from various types of acute leukaemia did not bind mouse red cells. Pre-treatment of the lymphocytes with neuraminidase led to a significant increase in the proportion of rosettes in CLL only. This test may prove useful in distinguishing CLL from other B-lymphoproliferative disorders, particularly prolymphocytic leukaemia.

Formation of mouse red cell rosettes by lymphocytes from patients with chronic lymphatic leukemia and assessment of T cell function

Peripheral-blood lymphocytes from 16 patients with chronic lymphatic leukemia (CLL) were studied for B cell markers by the mouse red blood cell (MRBC) rosette formation test and for T cell markers by the sheep red blood cell (SRBD) rosette formation test. In 14 of the 16 CLL patients there was a high percentage of mouse red blood rosette formation (mean value, 59 ± 16.7%). In all 16 patients the percentage of T cells was very low (mean value, 9.7 ± 5.3%). There seems to be a correlation between the percentage of MRBC rosette formation and the clinical condition of the patients; the two patients with the highest and the two with the lowest percentages were in the worst clinical condition and refractory to therapy. Peripheral-blood lymphocytes from 10 normal control donors demonstrated a low percentage of MRBC rosette formation (mean value, 8 ± 4%) and a high percentage of SRBC rosette formation (60 ± 10%). The local graft versus host reaction (GVHR), which measures the functional capacity of the T lymphocytes, resulted in a positive reaction for all the normal donors when it was performed with 20 × 10 6 lymphocytes, whereas the lymphocytes of 13 of the 16 patients mounted a negative reaction. When the test was repeated with 100 × 10 6 lymphocytes in 9 of these 13 patients the reaction became positive. It would seem that the negative GVHR of the CLL patients' lymphocytes is primarily due to the overwhelming numerical superiority of B cells and the consequent small percentage of T cells in the inoculum used. These findings demonstrate that the MRBC rosette formation test can be used as a B cell marker for diagnostic purposes in CLL and supports our previous finding that in CLL the T lymphocyte defect is not a functional but rather a quantitative one.

Lack of requirement for thymocytes for efficient antibody formation to trinitrophenylated mouse red cells in mice: role for thymocytes in suppression of the immune response.

The requirement of thymus-derived cells for the immune response to trinitrophenylated syngeneic mouse red cells (TNP-MRC) was investigated. In three sets of experiments the following results were obtained: a) irradiated mice which were reconstituted with bone marrow cells alone showed a better anti-TNP response after injection with TNP-MRC than those reconstituted with both bone marrow cells and thymocytes. b) Anti-thymocyte serum augmented the mouse anti-TNP response to TNP-MRC. c) Nude thymusless mice showed a better anti-TNP response to TNP-MRC than their normal littermate controls. These results indicate that the anti-TNP response of mice to TNP-MRC does not require thymus-derived helper cells. Moreover, thymus-derived cells have a suppressive effect on the anti-TNP response.

Guanine aminohydrolase in rat and mouse red cells: A potent inhibitor of guanylation of tRNA

1. 1.|The red blood cells of mice and rats contained guanine aminohydrolase (EC 3.5.4.3). This enzyme was not present in rabbit, sheep or human red blood cells. 2. 2.|The enzyme from rat blood cells was separated into two activities by column chromatography on DEAE-cellulose. Both isozymes were labile but it was possible to show that the more abundant enzyme followed Michaelis-Menten kinetics, had an apparent K m of 4.0·10 −6 M and was not activated by GTP nor inhibited by allantoin. 3. 3.|We believe, therefore, that guanine aminohydrolase was the protein in rat and mouse red blood cells that inhibited the enzyme (in rabbit reticulocytes) responsible for guanylation of tRNA.

Studies in lipogenesis by red cells of mice.

Summary. The incorporation of[1‐14C]acetate,[U‐14C]glucose and [1‐14C]palmitate into lipids of red cells from 5‐, 30‐ and 180‐day‐old mice has been studied. The results show that the cells from 5‐ and 180‐day‐old mice have greater capacity for lipid synthesis than those from 30‐day‐old animals. Ghost preparations exhibit a similar pattern of incorporation but the levels of incorporation are higher than those of intact cells. There arc age‐dependent differences in the incorporation of the precursors into various types of lipids. The results show that there are changes in the levels of free fatty acids and triglycerides of red cells as a function of animal age. No quantitative changes were observed in phospholipids and cholesterol.

Transaminations of amino donors with α-ketoglutarate in normal and neoplastic mouse tissues measured by a simple radiometric procedure

In the present work the estimation of aminotransferase activities with α-ketoglutaric-1- 14C as acceptor has been simplified to the extent that it is not necessary to achieve separation of α-ketoglutarate or glutamate from the reaction mixture in order to determine the extent of transamination. The determination consists of a two-step procedure. The first step is the transamination of α-ketoglutarate-1- 14C with an amino donor to produce glutamic acid-1- 14C. The second step is the enzymatic decarboxylation of the labeled glutamate and the measurement of the 14CO 2 produced. This method affords a rapid and variably sensitive procedure for multiple determinations of those aminotransferase activities in which α-ketoglutarate is the acceptor. It has been applied in our present work to the determination of aminotransferase activities of several normal mouse tissues and red cells and serum and to two transplantable mouse tumors. The amino donors employed were 19 amino acids commonly found in proteins, 17 aminophosphonic acids, and several amines and diamino monocarboxylic acids. A possible application of this method to clinical problems is mentioned.

Differential induction of anti-trinitrophenyl plaque-forming cell responses to lightly and heavily conjugated trinitrophenylated heterologous and autologous erythrocytes in mice.

AbstractVarious anti‐TNP PFC (2,4,6‐trinitrophenyl plaque‐forming cell) responses were obtained by injecting C3H mice with different conjugates of trinitrophenylated sheep red cells (TNP‐SRC). Mice which were injected with lightly conjugated TNP‐SRC (TNP0.14SRC) produced indirect anti‐TNP PFC 4 days after injection, while mice which were injected with heavily conjugated TNP‐SRC (TNP14SRC) produced both direct and indirect anti‐TNP PFC or direct anti TNP PFC only.Heavily conjugated trinitrophenylated autologous mouse red cells elicited a low direct anti‐TNP PFC response 4 days after injection. The system is another example of the capacity to modify antibody responses by modification of the antigen.

Some properties and susceptibility to inhibitors of partially purified acid proteases from Plasmodium berghei and from ghosts of mouse red cells

1. 1. An acid proteinase from erythrocytic stages of Plasmodium berghei was partially purified and some of its properties were compared with those of an acid protease from ghosts of mouse red cells. 2. 2. The two enzymes gave similar elution patterns upon filtration through Sephadex G-100, had similar Michaelis constants towards hemoglobin, were inhibited by high concentrations of substrate, and degraded bovine serum albumin much more slowly than they did hemoglobin. The pH optimum was 3.0–3.6 for the parasite enzyme and 2.5–3.0 for the red cell enzyme. However, the pH-response curves were quite similar. 3. 3. Both enzymes were inhibited by phenylmethane sulfonyl fluoride, and were unaffected by p- chloromercuribenzoate , iodoacetamide, dithiothreitol, and EDTA. 4. 4. Both enzymes were completely inhibited by pepstatin, chymostatin, antipain and leupeptin. ID 50's for the parasite enzyme were (in ng/ml) 0.25, 0.5, 70, and 200, respectively.