Immunoglobulin isotype distribution of malaria-specific antibodies produced during infection with Plasmodium chabaudi adami and Plasmodium yoelii

The antibody response of mice to Plasmodium chabaudi adami and Plasmodium yoelii has been compared using a solid phase isotype-specific radioimmunoassay and sodium dodecyl sulfatepolyacrylamide gel electrophoresis. Serological cross-reactivity between these parasites was substantial. Studies using a radioimmunoassay detecting all classes of malaria-specific antibody demonstrated that during the early part of infection it was not possible to distinguish between homologous and heterologous reactions. Immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that 50% or more of the protein antigens detected were apparently shared by both parasites although the intensity of bands was always greater with homologous reactions. However, the distribution of isotypes in the antibody (Ab) response differed in the two infections. P. chabaudi infections were characterized by a predominant and persistent IgM response, moderate IgG₂ and IgG₃ and little significant IgG₁ response during a primary infection. By contrast, IgM antibodies were transient in P. yoelii infection, IgG₂ was the predominant isotype, and both IgG₁ and IgG₃ antibodies were present during a primary infection. These differences in isotypes were also detected when sera were tested on the heterologous antigen extracts suggesting that antigens shared by P. chabaudi and P. yoelii do not necessarily induce similar antibody responses in the two infections.     

Capillaria hepatica: granuloma formation to eggs. II. Peripheral immunological responses

Peripheral immunological responses were assessed by indirect hemagglutination, passive cutaneous anaphylaxis, gel diffusion, and delayed dermal reactivity in mice with experimental primary and secondary Capillaria hepatica egg granulomas. Agglutinating and homocytotropic antibodies as well as delayed dermal reactivity, but not precipitating antibodies, were detected in animals with primary and secondary granulomas. The demonstration of circulating antibody during the course of granuloma formation indicates a possible role for antibody in the response and is cause for a reassessment of the etiology of experimental helminth egg granulomas.     

Ascaris suum: homocytotropic antibody responses in mice.

Homocytotropic antibodies in the sera of CD-1 and DBA/1 mice infected with larval A. suum were titered by PCA reactions. IgG₁ and reaginic antibody responses were similar in both strains of mice. With a dose of 8000 to 10,000 embryonated eggs, reaginic antibody was detected during the second week and IgG₁ antibody during the third week of infection. Doses of 1500 to 5000 eggs gave delayed antibody responses or did not induce a detectable response, although an anamnestic response followed a challenge inoculation even when no detectable antibody was observed in initial infection. Larval A. suum infections in two strains of mice did not potentiate a reaginic response to ovalbumin.     

Acetylcholinesterase secretion by parasitic nematodes. IV. Antibodies against the enzyme in Trichostrongylus colubriformis infected sheep

Sheep infected with the nematode parasite Trichostrongylus colubriformis showed anti-T. colubriformis acetylcholinesterase. (AChE) antibodies in the IgG₁ but not the IgG₂ or IgM fractions prepared from their serum. Using the fluorescent antibody technique with representative sera, antibodies in the IgG₁ fraction exhibited specificity for antigens in the subventral glands of the worm excretory system. IgA antibody specificity for antigens in the excretory glands and intestine of the worm was also demonstrated.     

Schistosoma mansoni: identification of immunoglobulins associated with the tegument of adult parasites from mice.

Immunocytochemical and immunodiffusion studies were conducted in an attempt to identify the immunoglobulins associated with the tegumental surfaces of Schistosoma mansoni. Peroxidase-labeled monospecific rabbit anti-mouse immunoglobulin class or subclass sera revealed the presence of mouse IgG₁, IgG₂, IgG₃, IgA, and IgM on the surface of adult parasites recovered from mice. These observations were confirmed by double gel diffusion of the various rabbit antisera against an eluate obtained from mouse worms.     

Evidence for histamine-induced auto-anti-idiotypic antibody immunoregulation in vivo

The in vivo effects of histamine injection in LAF₁ male mice on the immune reactivity to trinitrophenylated bovine γ-globulin was studied using plaque-forming cell (PFC) responses and their avidity distributions. Splenic anti-trinitrophenyl (anti-TNP) PFC responses of mice treated with histamine (5 × 10^?6 mol or 1 mg, intravenously) were significantly reduced in number and restricted in heterogeneity and characterized by a preferential loss of high-avidity IgG PFCs. The reduced PFC response in histamine-treated mice was dose and time dependent. No evidence of suppressor cell activity in the spleens from histamine-treated mice was demonstrable. Only histamine-treated mice produced a significantly high percentage of anti-idiotype-blocked, hapten-augmentable IgG PFCs, suggesting the presence of auto-anti-idiotypic activity. Immune sera taken from histamine-treated mice caused an inhibition of anti-TNP PFC in vitro. This PFC-inhibiting factor in immune sera of histamine-treated mice was an antibody of the IgG₁ and IgG_2a class, lacked anti-TNP antibody activity, but reacted with anti-TNP antibody of LAF₁ origin. Passive hemagglutination study of this sera showed anti-(anti-TNP F(ab′)₂-IgG) titer. Thus, the results of this study suggest that histamine in combination with antigen induces auto-anti-idiotypic antibody which, in turn, is involved in the normal regulation of the immune response to trinitrophenylated bovine γ-globulin in vivo.     

Fasciola hepatica: Comparative studies on fascioliasis in rats and mice

Chapman C. B. and Mitchell G. F. 1982. Fasciola hepatica: comparative studies on fascioliasis in rats and mice. International Journal for Parasitology12: 81–91. Certain characteristics of infection differ between rats and mice exposed to metacercariae of the trematode parasite, Fasciola hepatica. Rats develop a degree of age-related resistance (and infected older females contain fewer parasites than older males), resistance to reinfection in infected rats is demonstrated readily though is partial, and a comparable degree of resistance can be obtained in recipients of infected rat serum provided the serum is given at about the time of challenge. None of these features of F. hepatica infection is seen in mice. Rats also differ from mice in that they can be vaccinated against infection (although again, resistance is incomplete) using larval antigen mixtures in adjuvants. Mice do respond to infection by production of antilarval antibodies and a slight IgG₁ hypergammaglobulinaemia and larvae will sensitize mice for delayed hypersensitivity. The results of this study indicate that sera from infected rats versus infected mice will be useful in pinpointing antigens of F. hepatica larvae which are involved in expression of partial host protection.     

Mechanisms of protective immunity against Schistosoma mansoni infection in mice vaccinated with irradiated cercariae: I. Analysis of antibody and T-lymphocyte responses in mouse strains developing differing levels of immunity

The kinetics of cellular and humoral responses directed against schistosomula were examined in mice of three inbred strains which demonstrate differences in the degree of resistance induced by immunization with irradiated cercariae. T-Cell reactivity was observed during the first 4 weeks after vaccination but declined to control levels thereafter. Anti-schistosomulum antibody was first detected 2 weeks after vaccination, peaked by 6 weeks, and persisted as late as 15 weeks. In sera obtained at 6 weeks, antibody activity was detected in affinity chromatography-purified fractions containing IgM, IgA, IgG₁, IgG_2a, and IgG₃ immunoglobulins. In general, the cellular and humoral responses observed in C57B1/6J mice, which consistently developed a high level of immunity after vaccination, were not significantly different from those observed in C3H/HeJ or CBA/J mice, which achieved only low to moderate levels of immunity. Thus, although antibody production appears to correlate more closely than T lymphocyte responsiveness with the typical long-term resistance pattern observed in this model, the absence of striking differences in parasite-specific antibody levels between mice of these different strains suggests that additional mechanisms may be involved in the development of immunity after vaccination.     

Granulomatous hypersensitivity and antibody production in response to antigens of Capillaria hepatica eggs

Soluble egg antigens of Capillaria hepatica were fractionated by gel filtration chromatography into two major protein peaks of 300,000 and 14,000 mol.wt. The unfractionated antigen and both peaks were capable of sensitizing mice to produce quantitatively larger liver granulomas upon experimental innoculation of eggs; the increase in granuloma size was related to the amount of antigen used to sensitize. The antibody response in C. hepatica infected mice was primarily directed toward the high molecular weight components. Comparison of these findings with those reported for the Schistosoma mansoni egg granuloma suggests a diversity in the mechanisms of granuloma formation among helminth parasites.     

Characterization of sheep antibodies involved in precipitate formation with surface antigens of Fasciola hepatica in vitro

Sandeman R. M. and Howell M. J. 1982. Characterization of sheep antibodies involved in precipitate formation with surface antigens of Fasciola hepatica in vitro. International Journal for Parasitology12: 467–471. The role of sheep antibodies which precipitate with surface antigens of Fasciola hepatica is unclear. In an attempt to clarify their function these antibodies were characterized as to their immunoglobulin class and ability to affect the survival of fluke in rats. The ability of fluke antigens complexed with sheep antibody to vaccinate rats against infection was also tested. IgM antibodies were involved in precipitate formation on the teguments of fluke 3 weeks after infection but IgG₁ predominated at later stages of infection. The decreased survival of fluke in rats after culture with increasing levels of sheep antibodies suggests that the antibodies exert some deleterious effect on the fluke in vitro. The fluke antigen-sheep antibody complex failed to immunize rats against infection. Since sheep antibodies to F. hepatica can impair the ability of fluke to resist further attack in rats but not sheep, it is suggested that some effector mechanism other than antibody is defective in the latter.     

Trypanosoma lewisi: antibody classes which mediate precipitation, agglutination, and the inhibition of reproduction

Sera from Trypanosoma lewisi-infected and uninfected rats were applied to Protein A-Sepharose CL-4B columns. The absorbed fractions of antisera which contained only IgG molecules were reacted in microimmunodiffusion analyses with the exoantigens of T. lewisi in plasma collected from irradiated infected rats, and formed one precipitin line. These sera were also applied to T. lewisi extract immunoabsorbent columns and bound proteins were eluted and analyzed by immunodiffusion against antisera specific for rat immunoglobulins. IgG₁, IgG_2a, IgG_2b, IgG_2c, and IgM were absorbed by the immuno-absorbent columns. Absorption of the rat antisera with anti-rat IgG or anti-rat IgM removed one of the two precipitin lines against extracts prepared from parasites collected from irradiated infected animals. The absorbed IgG fractions and nonabsorbed fractions of antisera which were collected after Protein A-Sepharose CL-4B column chromatography agglutinated trypanosomes. After treatment of antisera with 2-mercaptoethanol, the agglutinin titers were lower than those of the control antisera suggesting both IgG and IgM are involved in the agglutination. The ablastic activity of the fractions eluted from Protein A-Sepharose CL-4B Chromatographic columns was assayed in cultures of bloodstream forms ofT. lewisi. Ablastic activity of proteins of antisera absorbed by the columns was demonstrated indicating they belonged to the IgG class of antibodies.     

Taenia crassiceps: Serum and surface immunoglobulins in metacestode infections of mice

Serum levels of IgM, IgA, IgG1, IgG2a, IgG2b, and IgG3 were measured weekly for 8 weeks by radial immunodiffusion in pooled sera from female BALB/c and BDF1 mice with primary and secondary Taenia crassiceps infections and age-matched normal control mice of each strain. Although increases in levels of all immunoglobulin classes occurred during primary and secondary infections in both strains of mice, the only consistent changes common to both strains of mice were higher levels of IgG1 and IgG3 in early weeks of secondary infections as compared to primary infections, and high levels of IgG1 late in primary infections. High levels of IgG3 occurred late in primary infections in BDF1 mice but not in BALB/c mice. It was not possible to correlate increased levels of any one immunoglobulin class either with cytotoxic activity of early immune serum or with the onset of the cellular encapsulation response in secondary infections. IgM, IgA, IgG1, IgG2a, IgG2b, and IgG3 could be demonstrated on the surface of washed fixed larvae from long-term infected donor mice by the indirect fluorescent antibody method. Living T. crassiceps larvae were capable of shedding fluorescent label within 1 hr at room temperature, but not at 4 C after staining with either rabbit anti-T. crassiceps serum or rabbit anti-mouse immunoglobulin serum and fluorescein-conjugated goat anti-rabbit globulin.     

Direct and indirect plaque forming cells in extrapulmonary lymphoid tissue following local vs systemic injection of soluble antigen

AKR strain mice were immunized with solubilized SRBC stroma either by direct injection into the lower respiratory tract or intravenously via the tail vein. The number of plaque forming cells (PFC) in the draining plumonary lymph node (tracheobronchial node) and spleen were determined by direct (IgM) and indirect IgG₁, IgG_2b, IgA) plaque assays.Intravenously administered antigen induced an initially strong IgM response in the spleen which was subsequently followed by antibody of the IgG₁, IgG_2b, and IgA classes of immunoglobulins. The tracheobronchial lymph node contained a minimal number PFC representing all four types of immunoglobulins studied. Conversely, following a single local injection of antigen directly into the lower respiratory tract, the tracheobronchial node responded with relatively high concentrations of PFC of all classes. The response in the spleen, although higher than background, was barely detectable. The splenic response to locally administered antigen was, however, considerably augmented as a result of a second local injection given 45 days after the initial stimulation. Under these conditions, IgG₁ IgG_2b, and IgA were represented in both tissue sites by sharp increases in the number and a decrease in the time of appearance of their respective antibody forming cells. Comparable changes were not noted for the case of IgM.Serum hemagglutination titres following a single injection by either route did not vary significantly during the time course of the experiment (28 days). The sera from locally immunized mice were treated with the reducing agent dithiothreitol and hemagglutination titres, before and after treatment, were compared. The major serum activity observed during the first 10 days following injection was affected by reduction and could therefore be assigned to high molecular weight antibody (19S, 13S). Subsequent titres (Days 13–26) were less susceptible to DTT and are considered to represent low molecular weight immunoglobulins (7S).     

Granuloma formation to Capillaria hepatica eggs. I. Descriptive definition

The course of the cellular response in the liver to nonembryonated Capillaria hepatica (Bancroft, 1893) eggs given by intravenous injection into the portal circulation of unsensitized and sensitized mice was studied qualitatively and quantitatively. A gradual infiltration of predominantly mononuclear cells occurred around the eggs in the liver, leading to the formation of distinct granulomatous lesions characterized by macrophages and lymphocytes. This was followed by an infiltration of eosinophils. Previous intraperitoneal sensitization led to an earlier and an enhanced reaction to an intravenous challenge with eggs. Specificity of the cellular response was suggested by the lack of an enhanced reaction to presensitization with eggs of a closely related species, Trichuris muris. These studies indicate that granuloma formation to C. hepatica eggs has an immunological basis and furthermore the cell composition of the granuloma would suggest that a cell-mediated component may be involved as part of the specific response.     

In vitro inhibition of the helper activity of GAT-specific T-cell lines by a syngeneic anti-idiotypic serum: preferential effect on the IgG response

We described in this paper the characteristics of a syngeneic anti-idiotypic serum made in BALB/c against BALB/c anti-poly (Glu⁶⁰ Ala³⁰ Tyr¹⁰) (GAT) antibodies. This serum recognizes idiotypic determinants present in all anti-GAT sera whatever the allotypic markers of the mice used to prepare the sera. The functional effect of this serum on two helper cell lines is also described. Cell line BDF₁/52 was obtained from GAT immunized lymph node cells (LNC). Cell line BDF₁/E₃ was selected from splenic T-cells educated in vitro on GAT-pulsed adherent cells. Both lines were propagated in presence of filler cells, antigen, and medium containing T-cell growth factor(s) from splenic cells activated with concanavalin A. Both cell lines exhibit a helper activity as measured by the plaque-forming cell (PFC) response they induce in vitro in the presence of DNP-GAT and DNP sensitized B cells. Their helper activity is specific and they require a hapten-carrier bridge to activate B cells. These lines are able to induce IgG₁, IgG_2a and IgG_2b anti-TNP PFC. Syngeneic anti-idiotypic serum B 658 inhibits specifically the function of these two lines but does not affect the helper activity of an OVA-specific T-cell line. The blocking activity of the serum can be adsorbed on a hybridoma protein with anti-GAT activity. This inhibition affects more dramatically the IgG₁ response than the IgG_2a and IgG_2b responses.     

Heligmosomoides polygrus (equals nematospiroides dubius): humoral and intestinal immunologic responses to infection in mice

Measurements of serum IgG₁, IgG_2a, IgM, and IgA levels and antibody titers in these immunoglobulin classes were made at intervals after initial infection and challenge infection of mice immunized by two or three previous infections. Identical measurements were made on the content of the small intestine in mice which had been exposed to the same infection schedule. Sections of small intestine taken after initial infection and challenge infection were examined by the fluorescent antibody technique for changes in populations of immunoglobulin-containing cells and by routine histologic procedures for histopathologic changes.In serum, only IgG₁ was consistently increased after initial infection, and antibody in IgG₁ was detected within the first 2 wk of infection. In immunized animals, only IgG₁ and antibody of this class always responded to challenge infection, although antibody in other immunoglobulin classes was detected.IgA concentration of the intestinal content did not differ significantly after initial infection or challenge infection of immunized mice. Immunized mice had about twice the IgG₁ concentration in intestinal content as singly infected animals. No intestinal antibody was detected after initial infection; only IgG₁ antibody was detected in the intestinal content of immunized and challenged mice.Cell infiltrates in the intestinal mucosa and submucosa of immunized animals contained numerous IgG₁-containing cells. Mast cells and globular leukocytes were observed in the intestine of immunized animals.     

Trichinella spiralis: anaphylactic antibody formation and susceptibility in strains of inbred mice.

The anaphylactic antibody response of various strains of inbred mice of different H-2 specificities was investigated using the passive cutaneous anaphylactic technique (PCA) for the detection of the antibody response. Neither IgC₁ nor reaginic antibody were detected in serum samples obtained at the end of the first week of infection with Trichinella spiralis. Subsequently, all animals had detectable IgG₁ antibodies, although in some strains the titers were very low. Reaginic antibody was detected in relatively high titers in C57L, A, and DBA/1 mice. Two other strains were very poor responders (SJL and AKR). In most strains, reagin and IgG₁ remained detectable for 14 wk or longer. The pattern of response of all strains was very reproducible, indicating genetic control of the anaphylactic antibody production to the infection. In F₁ hybrids obtained from crosses between good and poor anaphylactic antibody responders, intermediate levels of both antibody classes were detected.Adult worm recovery rates were established at various points during the intestinal phase of infection, and no correlation between worm numbers and reaginic antibody titers in the various strains of mice could be demonstrated. There were noticeable differences in larval yields obtained after muscle digestion of mice belonging to the different inbred strains. In fact, we generally observed an inverse relationship between the number of larvae recovered from a given strain and their reaginic antibody titer.The intravenous injection of newborn larvae (NBL), obtained upon in vitro incubation of adult worms, produced detectable antibodies only in mice of the DBA/1 strain. These antibodies were consistently of low titer and became detectable only after the administration of two additional injections of NBL. This contrasted with the results observed after “per os” infection of DBA/1 mice, where high titers of these antibodies were always obtained, in spite of comparable ratios of muscle larval yield.     

Adjuvant and immunogenic properties of bacterial lipopolysaccharide in IgE and IgG1 antibody formation in mice

The ability of Gram-negative lipopolysaccharide (LPS) to function as an adjuvant and as an antigen in IgE and IgG₁ immune responses in mice was investigated. LPS failed to induce LPS-specific IgE or IgG₁ under a variety of experimental conditions. Both isolated LPS and whole heat-killed bacteria were capable of enhancing IgE and IgG₁ antibody formation to a protein antigen, egg albumin (EA). The LPS-induced anti-EA, IgE, and IgG₁ antibody titers exhibited a cycling phenomenon with time. In the presence of LPS, IgE, and IgG₁ antibodies specific for EA did not occur in athymic nude (BALB/c-nu/nu) mice, demonstrating the inability of LPS to substitute for the stringent requirement for T cells in homocytotropic antibody formation.     

Modulation of mouse anti-SRBC antibody response by placental extracts

Mouse placental extracts (PE) and corresponding Sephadex G-200 fractions were administered to isogeneic CBA mice along with an optimal immunizing dose of SRBC. Spleen cells were harvested 8 days later and transferred to CBA recipients, subsequently immunized with SRBC. The immunoregulatory activity of spleen cells from PE-treated donors was compared to cells from liver extract (LE)-treated controls or from mice immunized with SRBC only, using Cunningham's PFC direct and indirect tests. Within the dose range used, selective modulatory activities were obtained with cells from PE, but not from LE, treated mice, the latter being comparable to cell transfer effects from donors immunized with SRBC only. Spleen cells from animals injected with low doses of PE (0.25 to 4 mg per mouse) added to immunizing SRBC had a suppressive effect on the primary IgM response of recipients immunized against SRBC. In contrast, when SRBC were given to donor animals with higher doses of PE (8 to 13 mg), transferred spleen cells potentiated the IgM response of the recipients. These opposite suppressive and potentiating activities were found in distinct Sephadex G-200 fractions of 40 and 60 kDa, respectively. When the effect of PE treatment was tested within the same animal, the indirect secondary PFC response following a challenge with SRBC was significantly modified. We observed an overall suppression of the different isotypes after treatment with lower doses of PE or with its 40-kDa fraction. PE doses of 0.5 to 2 mg resulted in a stronger inhibition of IgM than IgG₁ production. This phenomenon was also obtained with the 40 KDa fraction. IgG₂ responses were significantly reduced by all doses of this fraction. In contrast, all doses of the 60-kDa fraction gave a strong stimulation of IgG₂ and IgM responses and a constant suppression of the IgG₁ response. This shows a clear dissociation between IgG₁ and C'-fixing (IgM, IgG₂) antibody classes as far as the influence of placental substances is concerned in their regulation. These data emphasize the relevance of isogeneic placental products as a useful physiological material capable of modulating xenogeneic immune responses (as well as allogeneic systems).     

Tumor localization of Lewis lung carcinoma with radiolabeled monoclonal antibodies

Summary Mouse 6D6 IgG_2a and 5B5 IgM monoclonal antibodies which specifically bind murine lung carcinoma cells (3LL cells) were injected to healthy and tumor-bearing mice. In vivo localization was analyzed by counting the tissue radioactivity and by external gamma ray scintigraphy at various times after IV injection of ¹²⁵I- or ¹³¹I-labeled antibodies. The clearance of the two monoclonal antibodies was not modified by the presence of the tumor, and the 6D6 IgG_2a was cleared at a rate slower than the 5B5 IgM. Both antibodies gave a high specific uptake at the tumor level; the tumor-to-healthy tissue ratios were higher with the 6D6 IgG_2a than the 5B5 IgM; unspecific mouse immunoglobulins (IgG₂) did not localize in the tumor. The amount of 6D6 IgG_2a antibody still associated with the tumor after 2 days following IV injection was 10 times higher than that of 5B5 IgM, and was still high enough to localize the tumor after 5 days.Imaging experiments confirmed the ability of 6D6 IgG_2a to detect the presence of tumor cells. The targeting kinetics determined by computer analysis of camera images indicated a rapid targeting of antibodies in tumor with a maximal concentration after 4–6 h; after 48 h the background was quite low and the 6D6 IgG_2a appeared to be specifically localized in the tumor.