Retrovirus-induced lymphoproliferative disease in mice undergoing graft-versus-host reaction.

The effect of graft-versus-host reaction on the course of concommitant retrovirus-induced lymphoproliferative disease was investigated. The graft-versus-host reaction was elicited by a single i.v. injection of 1.2 x 10(8) parental spleen cells into adult F1 mice. Lymphoproliferative disease was induced by a single transfusion of 0.2 ml of whole blood from donors with fully developed disease, induced by infection with retrovirus LP-BM5 MuLV. Graft-versus-host reaction and the lymphoproliferative disease each separately produced similar syndrome consisting of splenomegaly, lymphadenopathy, leukopenia, neutrophilia, reduced in vitro proliferation of spleen cells and suppression of in vivo immune responsiveness. The above symptoms were usually less pronounced during graft-versus-host reaction. Ongoing graft-versus-host reaction neither aggravated nor accelerated the course of the virus-induced lymphoproliferative disease in genetically susceptible F1 hybrids. Likewise, an ongoing graft-versus-host reaction in genetically resistant F1 hybrids did not alter their susceptibility to the retrovirus infection. The apparent lack of the effect of graft-versus-host reaction -dependent immunosuppression on the severity and the course of the concommitant retrovirus-induced lymphoproliferative disease suggests pathogenic differences between the murine syndrome and human AIDS for which the murine disease is considered by some to be an animal model.

Retrovirus-induced lymphoproliferative disease in mice: role of humoral immunity in perinatally exposed mice.

The mice born to female mice infected with LP-BM5 MuLV, the etiologic agent for lymphoproliferative disease and nursed for 4-6 weeks by them were less susceptible upon reinfection by i.v. transfusion of blood or plasma from infected donors with fully developed disease. Sera of 7 week or older perinatally exposed mice were capable of a complete in vitro neutralization of virus in plasma or blood from mice with fully developed disease. In contrast, sera from 3-week old perinatally exposed mice were ineffective. The neutralizing ability of the sera was drastically reduced or abrogated after their absorption with anti-mouse IgM. These observations are consistent with the notion that perinatally exposure results ina moderate form of the disease of the offspring. This perinatal infection is followed by a production of neutralizing antibodies of predominantly the IgM class that significantly alters the course of the lymphoproliferative disease and, in some instances, even prevents its development.

Quantative aspects of transfusion-transmitted retrovirus-induced lymphoproliferative disease in mice.

Healthy, adult C57BL/6Kh mice of both sexes were transfused with blood or blood products from syngeneic donors with retrovirus (LP-BM5)-induced lymphoproliferative disease. The disease produced in the recipients 8 weeks after transfusion was characterized by splenomegaly, disseminated lymphadenopathy, leukopenia with neutrophilia, abrogation of the primary immune response to SRBC, decreased in vitro proliferation of spleen cells co-stimulated with phorbol ester and IL-2 or ionomycin and abrogation of synergistic effect of the co stimulators. Quantitative analysis of the blood or blood products used for transfusion show that a single transfusion of 0.2 ml of PBS containing 0.2 mu 1 of whole blood or 2 microliters of plasma or 400 Ficoll-isolated peripheral blood mononuclear cells was sufficient for the inducing the disease. The results suggest that the retroviruses were present in preparations of peripheral blood mononuclear cells and plasma of mice with the disease. However, the latter was 10-fold less efficient in inducing the disease. Transfusion of 1.8 x 10(6) isolated erythrocytes failed to induce the disease suggesting a marginal role, if any, in transmission of the disease via transfusion of these cells. Thus, a simple, reliable and reproducible method for propagation of the murine lymphoproliferative disease in the laboratory has been elaborated. These results also point to some important differences with regard to blood transfusion between human and murine AIDS.

Acquired immunodeficiency in murine lymphoproliferative disease: considerations on pathogenesis.

C57BL/6Kh mice were infected with a single i.p. injection of 1 x 10(5) FFU of LP-BM5 MuLV. The development and progress of the virus-induced lymphoproliferative disease was followed for 12 weeks after infection. As anticipated, progressive splenomegaly and lymphadenopathy, as well as almost total abrogation of immune responsiveness ensued. In contrast to previous reports, there was a dramatic increase in the frequency of CD4+ cells in spleens among which over 20% expressed V beta 5 TCR, as compared with fewer than 3% in spleens of normal mice. Spleen cells from infected mice retained their in vitro ability to proliferate upon stimulation with IL-2 and anti-CD3, but were unable to respond when stimulated with phorbol ester and either a low dose of IL-2 or calcium ionophore (ionomycin). A similar pattern of in vitro proliferative responses was obtained when normal spleen cells were treated with K252a compound, a known inhibitor of protein kinase C activity. Together with the observations that viral infection impaired down-regulation of the phorbol-induced kinase activity and that the kinase inhibitor only marginally enhanced suppression of virus-infected cells proliferation, this finding suggests that disturbances of protein kinase C activity may underly the pathological effects seen after viral infection. However, since no apparent quantitative and qualitative changes in protein kinase C itself and its translocation were observed, it is more likely that the virus may interfere with either the substrate or product of kinase activity.

Blood transfusion as a means for transmission of retrovirus-induced lymphoproliferative disease in mice.

Lymphoproliferative disease was elicited in C57BL/6KH and (BALB/c x C57BL/6)F1 hybrids by a single intraperitoneal injection of 10(5) FFU of LP-BM5 virus preparation. The disease could reproducibly be transferred by a single intravenous transfusion of 0.2 ml of whole blood as well as 0.1 ml of blood cells, plasma or serum from the infected animals. F1 hybrids displayed a delayed development of the disease when an acellular virus preparation was administered, but they were fully susceptible to the disease when syngeneic blood from infected F1 donors was transfused. Blood from donors in the prodromal stage was as effective in transmission of the disease as blood from donors with fully developed disease. This indicated that in murine lymphoproliferative disease viremia develops very early in the course of the disease. It seems that using the blood transfusion one could develop a reliable semiquantitative assay for the infectiveness of the animals suffering from LP-MB5-induced lymphoproliferative disease.

Proliferative responses of peripheral blood lymphocytes of mice and humans stimulated with recombinant interleukin 2.

In vitro proliferative responses of human and murine peripheral blood lymphocytes (PBL) were studied. The cells were stimulated with recombinant interleukin 2 (IL-2) or with a mixture of phorbol-13-myristate-12-acetate with either IL-2 or calcium ionophores. Proliferation of human PBL could be induced by doses of human IL-2 or phorbol that were much lower than those required to produce responses of murine cells. However, the magnitude of the responses of human PBL was definitely lower than that of the responses of murine PBL. Murine IL-2 did not induce proliferation of human PBL, but it synergized with phorbol. The responses of murine PBL reflected the previously described strain differences in responses of spleen cells that were shown to be under complex polygenic control. The proliferation of murine spleen cells could be induced with human, as well as murine IL-2, and both species of IL-2 synergized with phorbol. While no clear-cut low and high responders could be identified among normal healthy human subjects, 2 individuals produced consistently low responses to IL-2 and 1 individual displayed low responses to phorbol and calcium ionophore A23187. Available data suggest, but do not allow a firm conclusion, that proliferative responses of human PBL are under genetic control, perhaps as complex as that reported for mice.

The interaction of Cryptococcus neoformans with primary rat lung cell cultures.

Conditions under which Cryptococcus neoformans adhered to and was internalized by primary lung epithelial cell cultures were studied. Adherence was affected by the yeast culture age, glucose concentration and growth temperature. Formalin or heat treatment did not affect yeast adherence. Trypsin treatment, however, inhibited adherence.

Postnatal development of murine spleen cell proliferative responses induced by interleukin 2 or phorbol ester and calcium ionophore A23187.

To determine the kinetics of the ontogeny of polygenically controlled proliferative responses, spleen cells from 5, 10 and 20 day-old mice, previously identified as high (B10.PL-Thy-1a, AKR.M) and low (C3H.A) responders were tested. Cells (10(5)) were simulated in vitro with human recombinant IL-2 (35 U), a mixture of either IL-2 (0.7 U) and 13-myristate 12-acetate phorbol (5 ng) or phorbol and calcium ionophore A23187 (5 ng). Two parameters were assessed; proliferation measured by 3H-thymidine incorporation and the synergistic effect expressed as a synergism coefficient (SC): the ratio of proliferation induced by a mixture to the sum of proliferations induced by each component alone. At the age of 5 days, all three strains, regardless of stimulation, produced similar proliferation and showed no synergistic effect (SC = 1.0). At the age of 10 days, proliferation of high responder cells was 3-5 times higher than that of the low responder, but only high responders displayed marginal synergistic effect (2 greater than SC greater than 1.5). At the age of 20 days, proliferative responses of the high responders increased somewhat and a definitive synergistic effect became clearly demonstrable (SC greater than 3.0). This effect increased further after the age of 20 days to reach the level of SC greater than 5.0 in adult high responders. The delay in expression of synergistic effect was due to a substantial proliferation induced by phorbol alone in 10 day-old animals. The available data allow the conclusion that phenotypic strain variation in proliferative responses is first detectable at the age of 10 days and becomes fully developed only after attaining the age of 20 days or more. Since the magnitude of proliferative responses was not associated with frequencies of IL-2R alpha + cells it seems that differences in responsiveness can not be attributed to the induction of IL-2R alpha chain, and they may reflect intrinsic abilities of cells to proliferate.

Inhibition of serum bactericidal reaction by lipopolysaccharide.

An Rc-mutant of Escherichia coli that lacks UDPgalactose 4-epimerase grows normally without galactose but makes lipopolysaccharide lacking most of its carbohydrate. Exogenous galactose overrides the mutation and results in the formation of a complete lipopolysaccharide, thereby producing a smooth phenocopy. The smooth phenocopy was much more resistant to the bactericidal activity of normal human serum than was the rough phenotype. More complement was utilized by the rough mutant in the bactericidal process than by the smooth phenocopy. An antiserum was prepared in rabbits to a specific outer membrane protein in the mutant bacterium, the lambda receptor, whose expression could be suppressed by the addition of 10 mM maltose. The effect of the O-antigen in the lipopolysaccharide produced by the smooth phenocopy on the binding of antibody to the lambda receptor was determined. The smooth phenocopy exhibited significantly less binding of antibody than did the rough phenocopy. In addition, expression of the lambda receptor had little effect on the binding of antibody to the lambda receptor in the smooth phenocopy but caused significantly increased binding in the rough mutant. The results suggest that the increased resistance to the lethal action of normal human serum shown by the smooth phenocopy may be due to the blocking of antibody binding sites by the O-antigen of lipopolysaccharide, thereby preventing activation of the classical pathway of complement.

The macrophage-phagocyte system in rabbits with experimentally induced serum sickness: functional studies.

The objective of this study was to measure the phagocytic functions of the spleen and the liver and, if possible, to correlate them with the morphology and the immunohistology of these organs in rabbits with serum sickness. Serum sickness was induced by repeated intravenous injections of bovine serum albumin (BSA). Rabbits with acute serum sickness (stage A of serum sickness), with chronic serum sickness (stage C), rabbits in a stage of immunization between stage A and stage C (stage B), and rabbits that did not respond to the antigenic challenge [nonresponders (NR)], as well as nonimmunized (NI) rabbits were used. The function of the spleen and of the liver was measured by the clearances of 51Cr-labeled heat damaged (HE), periodate-treated (PE), or immunoglobulin G (IgG)-coated (IgGE) autologous erythrocytes. In NR rabbits the functions of the spleen and liver were normal. In rabbits in stage A of serum sickness the clearance of HE, the only one measured in this stage, was decreased. In rabbits in stage B the clearances of HE and IgGE were determined; both clearances were decreased. In rabbits in stage C, the clearances of HE and IgGE were decreased, while the time of clearance of 50% (T 1/2) of PE was not significantly prolonged. In addition, in rabbits in stages A, B, and C the ratio of whole spleen vs liver radioactivity was increased. In rabbits in stage B the clearance of HE returned to normal 3 days after discontinuation of BSA injections. While all animals with detectable morphologic and immunohistologic changes of the spleen and the liver had prolonged clearances of treated erythrocytes, some animals with similarly prolonged clearances lacked evidence of pathology of these organs. The results obtained indicate that: (1) In NI and in NR rabbits, treated erythrocytes are rapidly removed by the macrophage-phagocyte system (MPS) and preferentially by the liver. (2) In rabbits in stages A, B, and C of serum sickness the clearances of treated erythrocytes are significantly prolonged. Moreover, treated erythrocytes appear to be preferentially removed by the spleen, indicating that the ability of the spleen to clear treated erythrocytes is less impaired than that of the liver. (3) In stages A, B, and C of serum sickness the nonimmunologic clearance of autologous erythrocytes is as altered as the clearance that occurs via Fc receptors. (4) In rabbits in stage B of serum sickness recovery of MPS function occurs after the interruption of BSA injections, suggesting reversible damage.(ABSTRACT TRUNCATED AT 400 WORDS)

Cryopreservation of complement-coated erythrocytes.

Cells coated with complement components (C3b-C4b and C4b cells) were prepared by various methods and stored in liquid nitrogen using a low-glycerol, rapid freeze technique. Freshly coated cells, and cells coated and then frozen were tested versus various antiglobulin and anticomplement reagents to evaluate the reactivity of such frozen, stored, complement-coated human red blood cells. Liquid nitrogen preservation of such coated cells proved to be feasible when such cells were compared with freshly coated control cells.

The production of anti-A1 agglutinins in A-like rabbits injected with human group a1 secretor saliva.

A-like and non-A-like rabbits were injected with human group A1 and A2 secretor saliva. The sera were then examined for their agglutinative capacity against human group A1, A2, B and O erythrocytes before and after sequential adsorption with group O, B and A2 erythrocytes. The A-like rabbits produced specific anti-A1 agglutinins when injected with group A1 saliva but not when injected with group A2 saliva. The non-A-like rabbits produced anti-A and anti-A1 agglutinins in response to the A1 antigen but only anti-A agglutinins when group A2 saliva was injected. These experiments support the interpretation that a qualitative difference exists between subgroups A1 and A2. The non-A-like rabbits also formed cross-reacting antibodies as shown by the increase in the agglutinins reactive with human group B red cells.

Potentiation of antibiotic bactericidal activity by normal human serum.

Combinations of certain antibiotics and normal human serum at concentrations at which there was no killing by the agents when used alone were found to be bactericidal for Escherichia coli K-12 cells. This effect was observed with tetracycline, streptomycin (SM), trimethoprim, and ampicillin, but not with chloramphenicol or nalidixic acid. Synergy between SM and human serum was also observed against four of nine smooth strains of E. coli. A plasmid-bearing strain of E. coli K-12 was also killed by combinations of tetracycline or SM plus serum, even though the plasmid conferred resistance to tetracycline and SM. Evidence is presented that the synergy between antibiotics and serum is due to a complement-mediated effect on the bacterial cells that makes the cells more susceptible to the bactericidal effects of the antibiotics.

Effects of antibiotic resistance plasmids on the bactericidal activity of normal rabbit serum.

The ability of normal rabbit serum to kill Escherichia coli J6-2 was measured. With the concentration of serum adjusted so that approximately 2% of the cells survived after 2 h of incubation, there was no killing of the same strain bearing the F-like plasmid R100. Other F-like plasmids also provided the host strain with resistance to serum bactericidal activity, whereas none of the I-like plasmids used provided the host strain with resistance. When E. coli J6-2 bore both R100 and an I-like plasmid, there was some resistance to serum but less than with R100 alone. The effects of lysozyme on E. coli J6-2, which had been treated with tris(hydroxymethyl)aminomethane and ethylenediaminetetraacetate, were not altered by the presence of R100. The plasmids from 16 clinical E. coli isolates were transferred to J6-2N, a nalidixic acid-resistant mutant of J6-2. Four of the 16 plasmids provided J6-2N with resistance to normal rabbit serum.

Influence of various physicochemical factors on hemagglutination.

A study is made of the influence on hemagglutination of various physicochemical parameters, such as: cell shape, cell distance, extracellular colloid-osmotic pressure, degree of hydration of the cell surface, and cell zeta-potential. To a varying extent these are changed by: the addition of divers soluble polymers, the action of enzymes, the presence of various salt ions, centrifugation, agglomeration at low ionic strength, complex coacervation with cationic polymers, and the interaction with different kinds of antibodies. Among the more important parameters are cell distance and cell shape (particularly when the latter tends to spiculation), while the influence of zeta-potential appears to be of fairly minor consequence. The action of dissolved polymers is mediated through polymer bridging, increase in extracellular colloid-osmotic pressure, decrease of cell surface hydration, and change in cell shape. A peculiarity of dextran is that in addition to all of the above, it causes pronounced erythrocytic spiculation.

Agglutination of group Ax erythrocytes by anti-A sera (group B).

Four random examples of group Ax erythrocytes were tested with anti-A sera (group B). Microscopic examination demonstrated a high incidence of reactivity of these cells with the anti-A sera (group B) obtained after immunization with group A substance.