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A comparative study has been made of human precipitating and co-precipitating anti-tetanus toxin antibodies. IgO co-precipitating antibody represented 10% of the total antibodies in the serum and had immunological and biological properties similar to those described for co-precipitating antibodies of other animal species. Human precipitating and co-precipitating anti- bodies had the same electrophoretic mobility and were localized in the same immunoglobulin fraction. By immunoprecipitation it was not possible to find antigenic differences between precipitating and co-precipitating antibodies. Both antibodies were localized in the IgG1 and IgG3 subclasses and neither were in the IgG4 subclass. Only the precipitating antibody can form insoluble complexes with antigen. Precipitating and co-precipitating antibodies agglutinated sensitized sheep red cells, however, only the precipitating antibody agglutinated human red cells. Eight to ten times more co-precipitating antibody was required to obtain a positive reaction in PCA. Precipitating antibody activated the complement system while co-precipitating antibody lacked this capacity. This difference in behaviour could not be attributed to localization of both antibodies in different IgO subclasses. Precipitating and co-precipitating antibodies were cytophilic. Only the former activated phagocytosis and increased clearance of antigen from the blood. These results are not surprising since co-precipitating antibody does not fix complement. Competition between human precipitating and co-precipitating antibodies in opsonization was analysed. In this test competition of both antibodies for the antigen depends on their respective amounts The K= 0.18 diminished to 005 when the ratio of pp:cop. antibody changed from 10:30 to 30:70. The fact that co-precipitating antibody was isolated from the sera of vertebrates other than man indicate that this antibody could possibly play a role in some immune mechanisms. Taking into account that in previous papers we have demonstrated that co-precipitating antibody functions as a molecule with one combining site of high affinity and one of low affinity, we have proposed that this antibody could function univalently and blocks the antigen. This could facilitate chronic parasitic, bacterial and viral infections, tumour growth and other chronic infections. [ABSTRACT FROM AUTHOR]

Plaque-forming cell responses and antibody titres to sheep red cells (SRC) were elevated in normally-fed irradiated adult mice reconstituted with spleen cells from young mice which had received a 4% albumin diet, compared with those given cells from normally-fed littermates. This was independent of the dose of cells transferred (5 × 106-4 × 107), the day of assay (3–10) and the duration of protein-deficiency (1 or 3 weeks). It suggests that the depressed responses produced by protein-deficient (donor) mice reported in earlier papers resulted from an impairment of reticuloendothelial function or a shortage of protein reserves, not directly associated with the spleen. As protein-deficiency (of the donor) accelerated the initiation of the adoptive response and increased the cell yields of the recipients it is considered likely that an enhancement of cell proliferation during the first 3-5 days after transfer was responsible for the elevated responses. [ABSTRACT FROM AUTHOR]

The cell-surface glycoproteins CD44 and CD58 are involved in cell adhesion reactions. In this paper 12 monoclonal antibodies in CD44 and two in CD58 are described. Competitive binding assays using CD44 antibodies identified three distract epitope groups. Antibodies in Group I and, with one exception (BRIC 214), antibodies in group 2, but not antibodies in Group 3, recognized epitopes that are sensitive to reduction and to trypsin or chymotrypsin treatment of intact erythrocytes, and so these epitopes probably reside on the N-terminal disulphide-bonded domain of CD44. Antibodies in CD44 did not inhibit the binding of CD58 antibodies to erythrocytes or vice versa. Quantitative binding studies using radioiodinated IgG measured 1888–5592 copies of CD44 and 1772–3290 copies of CD58 on normal erythrocytes. Similar measurements with radioiodinated Fab fragments gave values of 6508–10,450 (CD44) and 3457–7622 (CD58). Immunocytochemical studies indicated that CD44 is much more widely expressed in non-haemopoietic tissues than CD58. Comparison with previously described CD44 antibodies suggests that antibodies m our Group I encompass Hermes 2 and that those in Group 2 encompass Hermes I. All the CD44 antibodies gave weakened reactions with Lu(a-b-) erythrocytes of the In(Lu) type by one or more methods. BRIC 214 and antibodies in epitope Group 3 were used to demonstrate that CD44 on these variant cells gives membrane-bound trypsin and chymotrypsin cleavage fragments of similar molecular weight to those obtained with normal erythrocytes. [ABSTRACT FROM AUTHOR]

This paper describes the induction of immunosuppression by multiple injections of carrageenan-lambda (cgn) in BALB/c mice. While cgn alone induced non-specific suppression that persisted for up to 25 days, priming with sheep red blood cells (SRBC) of mice within l0 days of cgn treatment prolonged the suppressive state. Also, antigen (SRBC)-specific suppression was observed when these mice were siren a secondary challenge 25–30 days after priming. Thus multiple cgn treatment along with priming SRBC generated non-specific and specific suppressive states which were transferable by Thy-l+ Lyt-2+ splenocytes. [ABSTRACT FROM AUTHOR]

Red cells sensitized with naturally occurring or 'immune' fowl antibody were lysed by fresh fowl serum. This fowl complement could not be replaced by that of human, rabbit or guinea pig. Both naturally occurring and 'immune' antibodies to red cells in the mammalian species tested did not lyse with fowl complement. Lysis by fresh fowl serum of sheep red cells sensitized with mammalian antibody is due to the presence of naturally occurrent antibody to these cells in the fowl serum; the enhancement of this lysis by the mammalian antibody will be described more fully in the next paper. Naturally occurring antibody to sheep red cells present in turkey serum and 'immune' antibody to fowl red cells were not lytic with fresh turkey serum, with fowl complement, with guinea-pig complement or with any combination of these. Some properties of fowl complement are given. [ABSTRACT FROM AUTHOR]

Whilst the ability of isoantibodies to agglutinate mouse red cells in saline medium does depend in part upon the properties of the antibody molecules, the concentration of antigen upon the red cell and certain genetically determined properties of the cell surface are of even greater importance. The red cells of mice of any strain will give positive results in the human serum:dextran system whilst reactions in a saline medium are unusual. In this paper the term ‘incomplete antibody’ is used to denote antibodies that can only be detected in conjunction with other antibodies in the human serum:dextran system. The time of appearance of antibodies to homografts of an ascites sarcoma, an ascites leukosis and a solid mammary carcinoma has been studied. Incomplete antibody as defined above has been detected by two methods. In the blocking test incomplete antibody is allowed to react with red cells before contact with complete antibody, a positive result being observed as a fall in titre when the treated red cells are subsequently exposed to complete antibody. In the second type of test, the synergic test, incomplete antibody is mixed with suitably diluted complete antibody and fitrated against suitable red cells, a positive result being a significant rise in titre as compared with complete antibody mixed with normal serum. Antibodies produced by the antigen donors did not give significant blocking or synergic effects. Incomplete antibodies are sometimes detectible on the third day and with regularity on the fourth day. Antibodies ‘complete’ for A strain red cells may appear at any time from the fifth day onwards. There is sometimes a drop in fitre of varying duration on the sixth day. This corresponds with the commencement of marked inflammatory changes on the graft bed. The maximum titre is attained at about two weeks. Antibodies may disappear in under three months or persist as long as a year. The function of antibodies in homograft reactions varies with the target tissue. However, they appear before the onset of anatomical signs of homograff response regardless of the type of target cell. [ABSTRACT FROM AUTHOR]

The reactivity of mouse antibodies against bromelain-treated mouse erythrocytes (BrMRBC) with mouse platelets before and after thrombin treatment was assessed by flow cytometry. Anti-BrMRBC antibodies could bind to thrombin-treated platelets, although normal platelets were also weakly reactive with the antibodies. The binding of anti-BrMRBC antibodies to platelets was confirmed by complement-dependent lysis. It is suggested that thrombin-activated platelets may be a real target for anti-BrMRBC antibodies. [ABSTRACT FROM AUTHOR]

The value of sera containing immuno-conglutinin or conglutinin in demonstrating complement-binding by human blood group iso-antibodies was investigated. Provided that a suitable complement was used, positive results were obtained with all human sera which had been shown by other methods to contain complement-binding antibodies. When human red cells were sensitized with human complement-binding blood group antibody and then treated with horse serum as a source of complement, the strongest reactions were obtained with rabbit immuno-conglutinin; the reactions with bovine serum (containing conglutinin) were distinctly weaker, and the reactions with human sera containing immuno-conglutinin were weaker still. When rabbit complement was used instead of horse complement, stronger reactions were obtained, but it was difficult to avoid ‘false positive’ reactions. When human complement was used, good reactions were obtained with rabbit immuno-conglutinin, but the reactions with bovine conglutinin and human immuno-conglutinin were completely negative. [ABSTRACT FROM AUTHOR]

The effect of an horse anti-mouse thymocyte globulin (ALG) preparation on the primary and secondary immune responses to sheep erythrocytes (RBC) was studied in 2-3-month-old male A/HeJ, C57B1, BALB/c, DBA/1, CBA and C3H mice. A wide variation in the primary and secondary responses of the individual strains was noticed but in ail cases these were significantly suppressed by ALG pretreatment. The capacity of the ALG to suppress the primary immune response was not overcome by increasing the antigen dose from 3 × 107 to 3 × 109 sheep RBC. [ABSTRACT FROM AUTHOR]

Irradiated (NZB × BALB/c)F1 mice were injected with syngeneic bone marrow cells, syngeneic or parental thymus cells, and sheep red cells. The antibody plaque-forming cell response depended on the number of sheep cells and the age and strain of the thymus cells. Young or adult BALB/c thymus, and young hybrid thymus, responded best to low numbers of sheep cells; with higher numbers they became tolerant. Adult hybrid thymus, and young or adult NZB thymus, responded better to high numbers of sheep cells. Hybrid mice irradiated and restored with BALB/c bone marrow developed thymus cells with the reactivity of BALB/c thymus. It is argued that NZB mice, and older hybrids, may develop autoimmunity because of an abnormality of tolerance induction manifested in their thymus cells, but of bone marrow origin. [ABSTRACT FROM AUTHOR]

The red cell uptake of IgG on the basis of absorption experiments appears to be due to two populations of IgG molecules. One population is readily absorbable by red cells. The quantity of this type of IgG varies from preparation to preparation and appears to depend on the method used for preparing the IgG. It appears that this population of IgG molecules represents denatured or aggre- gated IgG, although no direct evidence of aggregation was obtained in these studies. The other population of red cell absorbable IgG appears to be proportional to the IgG concentration and cannot be depleted by successive red cell absorption of the IgG solution. A similar pattern of reactivity with red cells was found using [131I] IgM. Labelled subunits of IgM and fragments of IgG (Fab and Fc), differed from IgG and IgM in that they did not contain a population consisting of a variable quantity of readily absorbable molecules. [ABSTRACT FROM AUTHOR]

This article presents the study of canine erythrocyte antibodies. An adaptation of the Polybrene technique for typing of canine erythrocytes is reported in this paper. Dog whole blood was anticoagulated with acid-citrate-dextrose in a proportion of 1:9 The samples were stored at 40, and used within 10 days. Manual typing and titrations were performed. Adaptation of this technique to canine blood study required two modifications: 15 per cent bovine serum albumin was substituted for homologous normal serum; the concentration of Polybrene was reduced to 0.033 mg/ml. Results demonstrated that the Polybrene technique is from 12.5 (anti-A) to 3200-fold (anti-He) more sensitive than the manual technique.

It is widely accepted that antibodies and CD4 T cells play critical roles in the immune response during the blood stage of malaria, whereas the role of CD8 T cells remains controversial. Here, we show that both CD8 and CD4 T cells robustly responded to an acute self-limiting blood-stage infection with Plasmodium yoelii. Similar to antigen-specific T cells, both CD8 and CD4 T cells showed dynamic expression of the surface proteins interleukin (IL)-7R and programmed death-1 (PD-1). Additionally, activated CD8 T cells showed differences in the expression of Killer cell lectin-like receptor G1, L-selectin and B cell lymphoma-2 and produced granzyme B, indicating cytotoxic activity, and the initially high expression of T-box transcription factor TBX21 in malaria-activated CD4 T cells indicated an early T helper type 1 (Th1)-skewed immune response. Our data demonstrate that blood-stage malaria infection results in a striking T-cell response and that activated CD8 and CD4 T cells have phenotypic and functional characteristics that are consistent with conventional antigen-specific effector and memory T cells. Therefore, a better understanding of the CD8 and CD4 T-cell response induced by blood-stage infection may prove to be essential in the development of a vaccine that targets the erythrocytic stage of the malarial parasite. [ABSTRACT FROM AUTHOR]

CD5 (Ly-1) B cells are a minor subpopulation in mouse spleen and are thought to be responsible for the production of natural autoantibodies to bromelain-treated autologous erythrocytes (Br-RBC). Here it is shown that substantial numbers of conventional, CD5-negative, splenic B cells also secrete these antibodies in CBA and (NZB × NZW)F1 mice, whereas in NZB and BALB/c mice they are all produced by the CD5 B-cell population. However, stimulation with bacterial lipopolysaccharide in vivo preferentially activates the CD5 B-cell group to anti-Br-RBC antibody secretion. [ABSTRACT FROM AUTHOR]

The effects of immune complex infusion on the clearance of antibody (R3/13)-coated and NEM- treated rat erythrocytes by the splenic component of the rat mononuclear phagocyte system (MPS) were investigated. Equivalence complexes of USA-anti-BSA produced a significant delay in the clearance of the NEM cells (pre-infusion T½ 19.7± 3.9 mm, post-infusion T½ 26.5 ± 3.8 min, mean ± SE, n = 6, P < 0.01), but this effect could be abolished by agents that prevented the changes in the splenic blood flow that followed complement activation. The immune complexes formed in 10- fold antigen excess (mean size 1 5S) did not delay the clearance of the NEM cells. Clearance of R3/13- coated cells was impaired by the infusion of immune complexes prepared at equivalence, 10-fold antigen excess or complexes prepared with F(ab')2 fragments of rabbit anti-USA antibody. The inhibition of red cell clearance was independent of changes in blood flow, but the degree of inhibition produced did not correlate well with the dose of immune complex injected. [ABSTRACT FROM AUTHOR]

Complement-mediated lysis of mouse erythrocytes (MRBC) by whole pigeon antisera was found to occur in the presence of magnesium ions alone. The underlying basis for this observation was demonstrated to be the ability of IgM antibodies to activate the alternative pathway of pigeon complement, whereas IgG activates a calcium-dependent pathway possessing an unusually low lytic capacity. In addition to differing from the alternative pathway by requiring both calcium and magnesium ions, the calcium-dependent pathway exhibited higher activity at low temperature and more rapid kinetics of haemolysis. The presence of early acting Cl in pigeon serum was inferred by the selective depletion of calcium-dependent activity which occurred as a result of incubating serum containing only calcium ions with MRIC sensitized with IgG. Under the same conditions, MRBC sensitized with IgM failed to deplete complement activity, indicating that Cl does not participate in complement activation by this isotype. Interestingly, the calcium-dependent pathway detected in pigeon serum appears to more closely resemble the Cl-bypass pathway rather than the classical pathway of mammalian complement. [ABSTRACT FROM AUTHOR]

When normal mice are immunized with rat RBC, autoantibodies to mouse red cells and antigen-specific suppressor cells are generated. Suppressor cell activity is found in T-enriched populations and suppresses only the induction of autoantibody, not ongoing or secondary autoantibody responses. Using anti- bodies lytic for either B cells or distinct T-cell subpopulations, we are able to show that suppression is dependent on the presence of Lyt 1 + T cells. [ABSTRACT FROM AUTHOR]

Either alone or in combination with antibodies having specificity for the carrier erythrocyte, TNP-ORBC were injected i.p. into CBA/J mice. Five days later, their spleens were removed and evaluated for TNP-specific PFC. The spleens from animals receiving 19S antibody (IgM) with carrier specificity displayed 3-A-fold more direct and indirect hapten-specific PFC than control animals receiving the TNP-erythrocyte conjugate only. Animals receiving 7S antibodies (IgG) with carrier specificity displayed very little change in their direct PFC response to the hapten. However, the indirect response to the hapten was suppressed as much as 16-fold by carrier specific IgG. Evaluation by haptenic inhibition of the relative avidity of the antibodies being secreted by these cells revealed the following: the relative avidity of antibodies secreted by indirect PFC in the spleens of animals receiving TNP-ORBC only was approximately 20-fold higher than antibodies secreted by the direct PFC. The 3-4-fold potentiation of the hapten-specific PFC response by carder-specific IgM antibody did not result in a change in relative avidity of direct or indirect PFC. IgG with carrier specificity did not change the relative avidity of the antibodies secreted by direct PFC having specificity for the hapten. However, evaluation of the remaining PFC in spleens from animals whose indirect hapten-specific PFC response had been suppressed by carrier-specific IgG revealed that the remaining PFC had a lower avidity than indirect PFC from animals receiving TNP-ORBC only. In other words, carrier-specific IgG selectively induced suppression of high avidity hapten-specific IgG antibody secreting cells. [ABSTRACT FROM AUTHOR]

Hybridomas secreting anti-mouse macrophage antibodies were obtained by fusing a routine plasmacytoma with lymphocytes of a rat immunized against mouse macrophages. An IgM, monoclonal antibody (3 A 35) reacted with mouse monocytes, macrophages and polymorphonuclear leucocytes. It did not bind appreciably to erythrocytes, platelets and unstimulated T or B lymphocytes. However, 3 A 35 bound to various murine T-derived leukaemic cells and to a small proportion of Con A-stimulated thymocytes. Cross absorption experiments confirmed the existence of a common antigenic determinant on macrophages and leukaemic cells. The possiblility that 3 A 35 identified a previously described antigen common to macrophages and normal or leukaemic T cells was investigated. The antibody was tested against thymocytes and macrophages of various mouse strains, some congenic for H-2 or Tla. The 3 A 35-detected antigen was found to be different from Ly5, Tla and Qa and did not represent a I-J encoded allotypic specificity. [ABSTRACT FROM AUTHOR]

In this study we followed the in vivo fate and distribution of hapten-coupled sheep red blood cells (TNP-SRBC) coated with 125I-labelled anti-hapten antibody in mice. The majority of these complexes was rapidly taken up by the liver, and by macrophages in the marginal zone of the spleen. Within a few hours, however, marginal zone labelling diminished and label appeared in the corona of lymphoid follicles. After 1–7 days the only remaining label was associated with areas of follicular dendritic cells This follicular trapping was abolished by decomplementing mice with cobra venom factor. These results, therefore, demonstrate that immune complexes made with a large, particulate autigen are retained for long periods in lymphoid follicles in an analogous fashion to soluble complexes. We assume (but have not proved) that the complexes retained by dendritic cells contain soluble erythrocyte antigens released from macrophages, rather than intact RBC. This possibility is strengthened by the presence of significant amounts of free, functional antibody in the circulation of mice given TNP-SRBC coated with antibody in vitro. The possible significance of immune complex trapping in antibody-induced suppression of anti-RBC responses is discussed. [ABSTRACT FROM AUTHOR]

When SJL mice are hyperimmunized with rat red blood cells (RBC), tolerance to self is readily broken and these animals develop a high autoantibody response to their own RBC. However, these mice also fail to generate those antigen-specific suppressor cells which normally regulate this induced autoantibody response. [ABSTRACT FROM AUTHOR]

Incorporation of lipid A into liposomes markedly enhances the plaque-forming cell (PFC responses against model membranes that are sensitized with synthetic amphipathic antigens 2,4-dinitrophenyl-6-N-aminocaproyldipalmytoylphosphatidylethanolamine (DNP-Cap-DPPE) and fluorescein thiocarbamyldipalmytoylphosphatidylethanolamine (FI-DPPE). The enhancing effect was pronounced only when lipid A and haptenic determinants were presented on the same liposome. Lipid A was incorporated into heterologous erythrocytes and syngeneic tumour cells. Significant and selective increase of in-vitro PFC responses was observed against heterologous erythrocytes, and immunization of C578L/6 mice with lipid A treated X-irradiated tumour cells (EL-4) markedly prolonged the survival of mice after challenge with homologous viable tumour cells. These data suggest that passively incorporated lipid A will be an effective method for selectively augmenting antibody formation against membrane-associated antigens. [ABSTRACT FROM AUTHOR]

Antigen-antibody complexes (Ag-Ab) prepared from 125I-bovine serum albumin (BSA) and guinea-pig anti-BSA were (i) incubated at 37° for 4 min with undiluted normal human serum and autologous red blood cells (RBC) together, or (ii) incubated first at 37° for 30 min with serum diluted optimally for binding and then with RBC. Reactions were stopped by dilution and cooling, and RBC bearing antigen-antibody-complement complexes (Ag-Ab--C) were washed and resuspended in either untreated normal human serum (undiluted or diluted), serum treated with zymosan (SZYM), ethylenediamine tetracetic acid (SEDTA) or ethyleneglycol tetracetic acid-Mg++ (SEGTA), serum heated at 56° for 30 or 120 min (SΔ30 or SΔ120), or buffer alone. The mixtures were placed at 37° and the percentage of Ag-Ab-C dissociated from RBC after progressively increasing times determined. (i) Ag:Ab:C bound to RBC with undiluted serum dissociated more rapidly following resuspension in SZYM, SEDTA, or SΔ30 than following resuspension in untreated serum. Rate of dissociation in SEGTA paralleled that in untreated serum. (ii) Ag-Ab-C bound to RBC with diluted serum dissociated following resuspension in SZYM, SEDTA, or SΔ30, but rebound and dissociated a second time following resuspension in untreated serum or SEGTA. Initial dissociation occurred in < min in undiluted serum, took place at 0° as well as 37°, and was diminished but detectable in >8- and > 64-fold- diluted serum, respectively. Rebinding required 37°, factors B and D and C3, and was maximal at 4-8 min. Subsequent dissociation had similar complement requirements to initial dissociation, but occurred only at 37° and was 90% complete at 15 min. No dissociation of Ag-Ab-C bound to RBC in (i) or in (ii) occurred following resuspension in SΔ120 or in buffer. These findings suggest that after initial binding, release of experimental immune complexes from RBC in whole serum involves concurrent dissociation and alternative pathway-dependent rebinding. [ABSTRACT FROM AUTHOR]

Approximately half of the rosettes formed by rabbit Ig+ lymphocytes (B cells) and anti-Ig-coated erythrocytes or glutaraldehyde-fixed bacteria are dissociated upon the addition of rabbit serum. Rabbit serum was fractionated and the rosette-dissociating activity was found in purified preparations of rabbit α1- and α2-macroglobulins. Studies designed to elucidate the mechanism of rosette dissociation suggested that the α-macroglobulins dissociated rosettes by causing the release or proteolytic cleavage of the membrane proteins complexed with the anti-Ig-coated particles. These data suggest that the α-macroglobulins may have a role in the interaction of B lymphocytes with particulate antigens. [ABSTRACT FROM AUTHOR]

We have made a detailed investigation to determine which of the B-cell subsets could be stimulated by Staphylococcus aureus Cowan 1 bacterium (SpA CoI). B-cell subsets were separated from peripheral blood and tonsil lymphocytes by means of rosette formation with E, EAIgG, anti-immunoglobulin (Ig) conjugated ox erythrocytes (OE-anti-Ig), and Protein A conjugated OE (OE-Pro A) or by separation on a bovine serum albumin (BSA) discontinuous density gradient. The cells responding to SpA Col included E receptor negative (E-), C3 receptor positive (C3R+), and surface Ig positive (SIg+) B-cell subsets. Among these B-cell subsets, FcR- cells were more responsive than FcR+ cells. These B-cell subsets responded alone to SpA CoI and significantly proliferated, although they failed to respond alone to pokeweed mitogen (PWM) and Protein A of S. aureus (Protein A). Among the SIg+ B-cell subsets stimulated with SpA CoI, IgM+ and IgG+ B cells showed a marked response and IgA+, and IgD+ B cells showed much less response. Both Protein A receptor positive (Pro A · R+) and negative (Pro A · R-) cells responded well to SpA CoI. Fractionation of B cells on a BSA gradient revealed that comparatively small sized and denser B-cell subsets responded well to SpA CoI. From these criteria, it is suggested that B cells responding to SpA Col are capable of stimulating not only mature B cells, but can also stimulate immature B cells. [ABSTRACT FROM AUTHOR]

Inclusion of 14% Ficoll in the assay of rosette formation of human lymphocytes with mouse red blood cells (RBC) markedly enhances the cellular interaction. Both the strength of the rosettes (number of attached RBC) and the number of reacting lymphocytes are increased significantly, irrespective of the lymphocyte source—blood, lymph node and tonsil. In the presence of Ficoll, most of the sIg-bearing B lymphocytes [expressing receptors for Fc(IgG) and/or C3] and a significant number of the sheep RBC-rosetting T lymphocytes (particularly the nylon wool non-adherent subset from the tonsil), express receptors for the mouse erythrocyte. [ABSTRACT FROM AUTHOR]

The aim of the present study was to prepare a monoclonal antibody against human C3 receptors. The monoclonal antibody termed C3R To5, which is characterized in this study, inhibited the ligand bind- ing of C3b receptors of human erythrocytes, neutrophils and lymphocytes, but did not block the ligand binding of C3bi and C3d receptors. C3RTo5 reacted only with cells that express C3b receptors. It did not react with cells that do not express C3 receptors or with cells that express C3 receptors other than C3b receptors. The reactivity of C3RTo5 against C3b receptors of human erythrocytes, neutrophils, glomerular cells, tonsil cells, or spleen cells could be removed by absorption with human erythrocytes or tonsil cells, whereas absorption with human peripheral I cells or sheep erythrocytes had no effect. In immunoprecipitation studies, a glycoprotein with a mol. wt of 205,000 could be isolated with C3RTo5 from non-ionic deter- gent lysate of tritiated tonsil cells. A rabbit antiserum prepared against this glycoprotein was able to stain C3b receptor-positive cells, inhibit C3b receptor ligand-binding activity and, furthermore, to precipitate a 205,000 mol. wt component. The results of this study indicate that C3RTo5 is a monoclonal antibody with selective reactivity to C3b receptors and presume ably to the binding sites within the receptor molecule. Using C3RTo5 further strong evidence was obtained that membrane-bound C3b receptors have a mol. wt of 205,000. [ABSTRACT FROM AUTHOR]

Hybridoma antibodies were produced by fusing spleen cells from mice immunized with rat alveolar macrophages (AM) with P3 × 63Ag8/653 myeloma cells. Some of the hybridomas were processed to achieve monoclonality. Reactivity was tested in an indirect membrane immunofluorescence assay. One of the monoclonal antibodies, called VEP6 antibody, reacted with rat AM but not with peritoneal or splenic macrophages and not with peripheral blood monocytes of this species. It was also unreactive with cells from thymus, bone marrow, non-adherent spleen cells, granulocytes and erythrocytes, but it reacted with thrombocytes. Corynebacterium parvum activated AM were slightly less reactive than 'resident' AM, as determined in a fluorescence activated cell sorter. The VEP6 antigen was studied in polyacrylamide gel electrophoresis after immunoprecipitation from 125I -surface-labelled-macrophage NP40-lysates. Four specific radioactive bands were seen in the precipitates corresponding to molecular weights of 31, 33, 35 and 37,000. A slurred band was seen in this molecular weight range when immunoprecipitates of surface labelled thrombocytes were analysed. This study confirms the heterogeneity of macrophages by showing that certain antigens may be expressed only on a subpopulation of macrophages. [ABSTRACT FROM AUTHOR]

C3b receptor protein was purified from human erythrocytes by 2 M KBr solubilization and affinity chromatography on C3-coated sepharose. This material served as antigen for raising monoclonal antibodies. To investigate the distribution and anti- genetic relationship between the receptors for C3b on human erythrocytes, lymphoid and phagocytic cells, as well as kidney cells three monoclonal antibodies were selected which inhibited the binding of EAC14°23b to complement receptor-bearing cells. This could be shown for human erythrocytes by inhibiting the immune adherence reaction, for tonsil lyme phocytes, Raji cells, and guinea-pig spleen cells by inhibition of rosette formation of these cells with EAC14°23b, and for human renal &omeruli by blocking of the adherence of EAC14°23b to kidney sections. In contrast, these monoclonal antibodies were not capable of inhibiting rosette formation of human granulocytes and monocytes with EAC14°23b. The antibodies only interfered with the rosette formation, of EAC14°23b and EAC 14°23d with Raji cells and tonsil lymphocytes--if at all--at high concentrations, whereas the rosette formation of Raji cells and tonsil lymphocytes with EAC14°23b was influenced by supernatants of the selected clones up to a dilution of 1:10³ to 1:105. [ABSTRACT FROM AUTHOR]

Normally circulating human leucocytes were found to consistently lyse rabbit erythrocyte (RRBC) target cells within 24 hr in culture. The reaction is referred to as the naturally-occurring cell-mediated cytotoxic, or NOCC, reaction. Characteristics of this cytotoxic reaction are as follows. (i) The cytotoxic reaction described here can be distinguished from the antibody-dependent cellular cytotoxic (ADCC) reaction as conventional Fc receptors do not appear to play a role. Although the cytotoxic reaction occurs optimally in medium fortified with normal human serum to a final concentration of 1%, neither antibodies nor immunoglobulins in general appear to play a role. Agamma human serum or purified human Serum albumin without detectable immunoglobulins are as capable of facilitating and sustaining the NOCC cytotoxic reaction as is normal human serum. Furthermore, foetal calf serum, which promotes the ADCC cytotoxic reaction, inhibits the NOCC cytotoxic reaction. (ii) The NOCC reaction is not a phagocytic one as phagocytosis of the RRBC could not be detected during the culture period. (iii) Monocytes and neutrophils, but not lymphocytes, are capable of lysing the target cells in the NOCC reaction. The effector mononuclear cell is a monocyte with receptors for Fc and C′3. (iv) The NOCC cytotoxic reaction is dependent upon direct effector cell-target cell contact and not upon soluble mediators secreted by the effector cells since third-party bystander erythrocytes are not lysed in the presence of rabbit erythrocyte target cells and human mononuclear cells. Supernatants from lysed RRBC target cells could not lyse fresh RRBC. It is concluded that the NOCC cytotoxic reaction utilizing rabbit erythrocytes as target cells permits the identification of cytotoxic subclasses of monocytes and neutrophils. [ABSTRACT FROM AUTHOR]

Anti-red blood cells (RBC) and anti-hapten antibody synthesis were studied in the goldfish, Carassius auratus. Spontaneous haemagglutination titres were found against all the antigens tested. A weak secondary response was observed in RBC-primed fish boosted during the end-phase of the primary antibody production. However, when the second antigenic challenge was performed during the early exponential phase of a primary stimulation, an important amplified response was obtained. The antibody production and immunological memory can be dissociated: no antibody synthesis occurred in glutaraldehyde-fixed RBC (F-RBC) primed animals but a characteristic amplified response was obtained when untreated or F-RBC were given to F-RBC primed animals. The amplified response to sheep red blood cells (SRBC) was significantly inhibited when fish were primed with a mixture of SRBC and Xenopus red blood cells (SRBC). demonstrating an antigenic competition phenomenon. Studies on anti-trinitrobenzene responses confirm the efficiency of E. coli lipopolysaccharide as a carrier for fish anti-hapten immunization. The kinetics and regulation of antibody synthesis in fish are discussed in relation to the described results. [ABSTRACT FROM AUTHOR]

The component of bovine serum (coagglutinogen) responsible for coagglutination of guinea-pig red cells has been shown to be C4, as anticipated from our previously reported findings on human serum. To effect coagglutination, the C4 needs to be aggregated by antibody; thereby probably increasing the avidity of the C4 for the receptors on guinea-pig red cells. The coagglutinating activity is lost if univalent Fab anti-C4 is used, but it can be restored by adding IgG antibody to the Fab. A procedure is described for producing antibody reagents to human or bovine C4 by injecting guineapigs with well-washed coagglutinated guinea-pig red cells. [ABSTRACT FROM AUTHOR]

The appearance of antibody responsiveness against sheep red blood cells (SRBC) was studied in young mice treated with hydrocortisone hemisuccinate (HHC) either during gestation or around delivery (last 5 days of gestation or day of birth). The plaque forming cell (PFC) response was normal at 2 weeks and depressed at 4 weeks in animals treated during the last 10 days of pregnancy. Conversely, when HHC was only given around delivery. PFC response was depressed at 2 weeks and normal at 4 weeks. The presence of suppressor cells in unimmunized 1-2-week-old spleen cells was evaluated by co-culturing them with adult spleen cells from mice immunized against SRBC. Suppressive activity was found at high level at 8 days, and had already declined at 15 days in non-treated mice. Spleen cells from mice treated with HHC around delivery showed reduced suppressive activity at 8 days but this activity was very high on the 15th day, greater than in normal mice. Such highly suppressive activity correlated with a high level of Lyt123 T cells. These data on suppressor cells indicate that the paradoxical effect of HHC in mice treated during gestation or around birth is probably explained by the differential effect of HHC on the kinetics of suppressor cells in ontogeny. [ABSTRACT FROM AUTHOR]

After immunization of carp with sheep red blood cells, the spleen accounts for only 5% of the total number of plaque-forming cells (PFC). In addition, thymus, peripheral blood and heart contained low numbers of PFC (« 0.5, 1 and 0.5%, respectively). Pronephros and mesonephros were the major antibody-forming organs (53 and 40% of total PFC, respectively). The temperature dependence of the antibody-forming cell response in spleen, pronephros and mesonephros was studied in animals kept at 12–24°. Lowering temperatures induced a delay in the peak of the primary response but had no effect on the magnitude of the response. The temperature-peak day relationship indicated that there are steps in the primary immune response of carp differing in temperature sensitivity. The anamnestic character of the secondary response was clearly demonstrated at 24 and 20° but lost at 18°. [ABSTRACT FROM AUTHOR]

Erythrocyte autoantibodies can be elicited in mice by injections of rat RBC which are cross-reactive with mouse RBC. This report shows that reduction of autoantibodies is dependent, in part, on gene(s) outside the H-2 complex. Using CBA mice congenic for Ig allotype and the F1 and F2 hybrids, a higher incidence of autoantibody production was observed in mice bearing the Ig allotype 1b (1bb' or 1ab) in contrast to mice homozygous for the allotype Ig-1a. Serum haemagglutination titres against rat RBC were not reduced in the groups of mice with the lower incidence of autoantibody production. A probable explanation for these observations is that the change in Ig allotype is associated with some change in the variable region determining autoimmune specificity that is governed by VH genes linked to allotype genes. The transfer of 30× 106 spleen cells from Coombs' positive mice to syngeneic recipients before starting the immunization regime with rat RBC suppressed autoantibody production and enhanced antibody production against rat RBC. These suppressor cells were effective in congemic mice and in F1 hybrids, which suggest that the Ig allotype is not a crucial site for the effector stage of suppression of this autoimmune response. [ABSTRACT FROM AUTHOR]

Mice injected with rat erythrocytes developed anti-erythrocyte autoantibodies which reached a plateau at 4 12 weeks, then gradually declined until at about 24 weeks the majority of mice were negative. In such recovered mice re-challenge with rat erythrocytes produced an accelerated peak of autoantibody and a much more rapid return to a Coombs' negative state. The auto-antibody response was distinguished from the anti-rat response in being more radio-sensitive. Purified autoantibody reacted to higher titre with rat than with syngeneic erythrocytes. Lymphoid cells, from mice given rat erythrocytes (but not sheep, rabbit or guinea-pig erythrocytes) transferred to normal syngeneic recipients given rat erythrocytes suppressed autoantibody production in the recipients. This suppression was much more effective against the autoantibody response than against the response to the inducing cross-reactive antigen; and the degree of suppression was related to the number of cells transferred and to their time of administration relative to the injection of rat erythrocytes, The induction of autoantibody and the generation of suppressor cells in donor animals was unaffected by adult thymectomy. A comparison of the effect of anti-rat erythrocyte antibodies and spleen cells from rat-immunized donors on recipients responses to rat erythrocytes revealed that whereas anti-rat antibodies suppressed both the autoantibody and the anti-rat responses, the spleen cells suppressed only the autoantibody response. Populations of spleen cells, from rat immunized donors, depleted of B cells retained their suppressive activity, whereas the suppressive efficacy of T-cell depleted populations was reduced but not abolished. It is suggested that T cells can specifically interfere with the response of autoreactive B cells, although non-T cells (possibly B cells acting by an antibody-feedback mechanism)can also suppress their response. [ABSTRACT FROM AUTHOR]

Treatment of rat erythrocytes with vibrio cholerae neuraminidase (VCN) exposed antigenic determinants on the cell surface membrane, and autologous rat serum contained antibodies which reacted with these determinants and with VCN-treated human erythrocytes. The antibodies were not present in sera of animals less than 8 weeks of age. Human serum also contained antibodies which reacted with the VCN- treated rat erythrocytes. Lectin isolated and purified from Arachis hypogoea agglutinated VCN-treated rat erythrocytes. This system has the characteristics of the T antigen which had been described initially in humans. Absorption studies showed that the VCN-treated rat erythrocyte membrane antigen cross-reacted with the VCN-treated human erythrocyte membrane antigen. Absorption of rat sera and the lectin solution with VCN-treated rat or human erythrocytes removed all of the molecules capable of reacting with both rat and human erythrocytes. Absorption of human sera with VCN-treated human cells removed their reactivity with both cell types, but absorption with VCN-treated rat cells did not remove completely their reactivity with VCN-treated human erythrocytes. These results indicate that the VCN-treated rat cells contain antigenic determinants which are identical to some on the VCN-treated human ceils and that the human sera contain antibodies to additional antigenic specificities not shared with rat red blood cells. [ASA TEXT GREATER THAN 2000 CHARS, CONTINUATION DELETED AND SAVED IN AIW-P-IMAGE\TIFFINPUT] [ABSTRACT FROM AUTHOR]

The capacity of purified lymphocytes from human peripheral blood to bind the Fc portion of IgG was investigated by the rosette technique using ox erythrocytes sensitized with rabbit anti-ox IgG (EAox) and human erythrocytes sensitized with anti-CD IgG (EACD). With unfractionated lymphocytes EAox always detected more rosette-forming cells (RFC) than did EACD; however, in lymphocyte populations specifically depleted of B lymphocytes by passage through copolymer styrene bead columns, the proportion of rosettes formed with EAox and with EACD was the same. Double labelling for Fc receptors and surface immunoglobulin (SIg) demonstrated that most of the lymphocytes which formed rosettes with either EAox or EACD also carried SIg. Pre-incubation of lymphocytes at 37° to remove heat-labile SIg did not affect their ability to form EA rosettes but reduced the proportion of SIg-bearing cells. Following pro-incubation a significant proportion of EAox RFC still remained SIg positive but the lymphocytes which formed rosettes with EACD no longer carried SIg. These studies suggest that rosette formation with EACD detects only ‘K’ lymphocytes (non-T, non-B cells bearing heat-labile SIg) while EAox detects some B lymphocytes as well. By reducing the amount of IgG bound to ox erythrocytes sensitivity was reduced to the point where EAox no longer formed rosettes with B lymphocytes but still detected ‘K’ lymphocytes indicating either a qualitative or quantitative difference between the Fc receptors on B and ‘K’ lymphocytes. Treatment of lymphocytes with trypsin decreased the percentage of rosettes formed with EAox but not with EACD supporting the conclusion that there is a structural difference between the Fc receptors on B and ‘K’ lymphocytes although a difference in receptor density is not excluded. When EACD and fluorescein-labelled ox erythrocytes sensitized with a low concentration of rabbit anti-ox IgG were mixed, most RFC bound both the indicator erythrocytes simultaneously suggesting that the Fc receptors on ‘K’ lymphocytes do not exhibit species specificity. [ABSTRACT FROM AUTHOR]

A test is described which is capable of differentiating and measuring by titration the individual classes of antibody reacting with a bacterial suspension. The serum or fluid under test is incubated with the bacteria which are then very well washed and added to indicator red cells linked with specific antiglobulin reagents. Sensitization of the bacteria by a particular class of antibody is shown by hemagglutination (passive) of the appropriate red cells. Agglutination of the bacteria themselves does not preclude an analysis. The reaction, which has been developed on a brucella system, has been designated Mixed Reverse Passive Antiglobulin Hemagglutination (or MRPAH for short). [ABSTRACT FROM AUTHOR]

Herpesvirus saimiri inoculated into owl monkeys (Aotus trivirgatus) causes leukaemia and lymphoma. Peripheral lymphocytes from leukaemic monkeys grown in culture are predominantly T lymphocytes. The supernatants from these cultures contain a factor which enhances the antibody response of murine B cells to sheep red blood cells (SRBC). The factor has been partially characterized by ammonium sulphate precipitation, DEAE-chromatography, gel filtration over Sephadex G-150 and disc-gel electrophoresis. The enhancing factor is a protein with a molecular weight of approximately 40,000. [ABSTRACT FROM AUTHOR]

The receptor for erythrocyte-antibody (EA) complexes on the surface of chicken lymphoid cells was investigated using a rosette assay. The chicken EA receptor binds chicken immunoglobulin of the IgG class but not the IgM class. Binding to the EA receptor is dependent upon the Fc region of the immunoglobulin. No receptor for complement analogous to the mammalian C3b receptor was demonstrated on chicken lymphoid cells using the rosette assay. Inhibition studies utilizing immunoglobulins from several species demonstrated that chicken spleen cells do not bind mammalian immunoglobulin but may bind immunoglobulin of other avian species (turkey and duck) and a reptilian species (turtle). The chicken EA receptor is distinct from cell membrane bound immunoglobulin light chains, bursa-specific antigens and thymus-specific antigens. The receptor for EA complexes on chicken lymphoid cells is compared with the Fc receptor on mammalian lymphoid cells in the light of these observations. [ABSTRACT FROM AUTHOR]

Affinity chromatographic experiments demonstrated that IgG was the class of antibody involved in the in vitro lysis of chicken erythrocytes by antibody and normal mouse effector cells. Further experiments designed to investigate the subclass of IgG involved, indicated that IgG2a was active and provided further evidence for the inactivity of IgM, either in the native 19S or chemically degraded 7S form. From the results obtained, the possibility of ADCC-inducing activity in the other IgG subclasses could not be excluded. [ABSTRACT FROM AUTHOR]

A method for the isolation of large numbers of almost pure human eosinophils from peripheral blood using hypaque-ficoll density gradients, gelatin sedimentation and carbonyl iron is described. Eosinophils were obtained from patients with parasitic infections, allergic reactions, undiagnosed eosinophilias and normal volunteers. Erythrocyte (E) and erythrocyte-antibody-complement (EAC) rosettes were formed by a number of eosinophils. [ABSTRACT FROM AUTHOR]

The number of plaque-forming cells (PFC) per spleen is reduced in mice experimentally infected with 200 Trichinella larvae at 14 and 28 days after infection, whereas no difference is shown at days 7 and 56. The worms of the three different phases of the life cycle of Trichinella have been isolated and kept alive in vitro at 37° in the inner compartment of a Marbrook chamber. The outer compartment of the chamber contained normal spleen cells and sheep erythrocytes. After 4 days, the spleen cells were removed and assayed for PFC. The results indicate that the new-born larvae are capable of producing substances which can diffuse through a Millipore filter and affect the spleen cells forming antibody to sheep erythrocytes. The suppression of antibody to sheep red blood cells in mice infected with Trichinella is a transitory phenomenon and can be related to the migrating phase of the life cycle of the parasite. [ABSTRACT FROM AUTHOR]

Other double PFC were micromanipulated from the original revealing medium into two successive media containing only one indicator and the homologous (second medium) or the heterologous third medium) soluble inhibitor (TNP-BSA or soluble SRBC antigen), in order to see whether a soluble inhibitor suppresses only the corresponding specific lysis. Out of 186 double PFC transferred into the media containing the indicator and the corresponding inhibitor 147 (79 per cent) were specifically inhibited, whereas out of 176 double PFC transferred mb the third medium (in which about 20 per cent of the cells cease to function) containing one indicator and the unrelated inhibitor, 117 (66.6 per cent) lysed the indicator in spite of the presence of the unrelated inhibitor. Since a specific inhibitor suppresses the lysis of the corresponding indicator, whereas its presence does not interfere with the lysis of the unrelated indicator in the majority of double PFC, it can be concluded that these cells secrete two types of antibody molecules possessing different specificities.

The experiments described demonstrate that the in vitro lysis of chicken erythrocytes (CRC) by allergized mouse spleen cells (ASC) is an antibody-dependent phenomenon. Supernatants obtained from cultures of ASC were shown to contain antibody which could effect antibody-dependent cell-mediated cytotoxicity. The cytolysis observed with ASC was inhibited by both an IgG fraction and a F(ab')2 portion of rabbit anti-mouse IgG. No inhibition was obtained by pretreating the ASC with AKR anti-C3Hθ serum and guinea-pig complement. [ABSTRACT FROM AUTHOR]

The reactions of two monoclonal IgM lambda cold agglutinins, Sch and Sher, have been studied in detail with human and animal erythrocyte antigens. Although they were unusual in having lambda and not kappa polypeptide chains, they could be assigned to the anti I and anti-Pr1 groups of cold agglutinins. The findings with serum Sher indicated that the Pr1 antigen may be more complex than previously thought. The occurrence of unexpectedly large numbers of specificities among monoclonal anti-I, anti-i and anti-Pr antibodies is discussed and it is suggested that each monoclonal antibody may recognize only a limited portion of a complex red cell antigen. [ABSTRACT FROM AUTHOR]

Surface immunoglobulin can be demonstrated on almost all sheep erythrocyte rosette-forming cells in CBA and AKR mice, either by inhibition of antigen binding by anti-immunoglobulin or by immunofluorescence. When surface immunoglobulin on a rosette-forming cell is `capped' by anti-immunoglobulin, the bound red cells migrate with the immunoglobulin, demonstrating that the receptor-combining site and immunoglobulin antigenic determinants share the same mobile unit in the membrane. These observations strongly support the concept that the antigen receptor is surface immunoglobulin (at least for B cells), and illustrate the usefulness of the `capping' technique for studying the associations between membrane elements. [ABSTRACT FROM AUTHOR]

The regulation of the immune responses by antibody administered passively on peritoneal exudate cells (PEG) was studied in guinea-pigs. The immunogenicity of sheep red blood cells (SRBC) associated with PEC for the induction of delayed hypersensitivity (DH) to soluble erythrocyte antigen was enhanced when PEG were incubated with anti-SRBG antibody. In contrast, the antibody response to SRBC was depressed. This phenomenon was only observed with specific antibody and was partially blocked when PEG covered with anti- SRBC antibody were incubated with rabbit anti-guinea-pig globulin serum. Comparable amounts of anti-SRBC antibody injected separately had no enhancing effect. Anti-SRBC sera from which the cytophilic antibody activity had been removed were inactive. PEG-associated SRBC were usually more immunogenic than the same amount of `free' SRBC for the induction of delayed hypersensitivity and always more immunogenic for antibody production. These observations suggested that macrophage cytophilic antibody might be involved in the regulation of the immune response and play a role in the preferential induction of delayed hypersensitivity in the studied system. [ABSTRACT FROM AUTHOR]

Lymphokines have a number of effects in vitro, which may be related to their role as mediators of delayed hypersensitivity. These mediators are also released in vivo. Authors of the article have described the effect of Lymphokines on in vivo and in vitro antibody production. The present article shows the effect of supernatants which contain lymphokines on the development of delayed hypersensitivity to unrelated antigen. Animals were injected into the ear vein with 1ml of 25 percent suspension of three tines washed sheep red blood cells with or without various supernatants.