Fractionation and immunologic assessment of KCl-extracted cardiac antigens in coxsackievirus B3 virus-induced myocarditis.

Hypertonic salt extracts prepared from the heart tissues of adolescent CD-1 mice were fractionated on Sephadex G-100 columns. Two separate fractions were obtained. Fraction I, containing the antigenic immunoreactive activity, was able to inhibit the migration of CVB3-PPD immune mouse peritoneal exudate cells (IMPEC) as well as PEC from mice infected with CVB3 virus alone. Fraction II did not have antigenic activity as assessed by the agarose droplet cell migration inhibition assay. As controls, Fraction I prepared from the livers of spleens of CVB3-infected CD-1 mice was unable to inhibit the migration of CVB3 IMPEC. Unimmunized or "normal" mouse peritoneal exudate cells (NMPEC) were not inhibited by Fraction I. Antibodies prepared against Fractions I and II were unable to neutralize CVB3m virus in the plaque reduction test, and polyacrylamide gel analysis revealed multiple bands in 10% SDS gels.     

Atomic spectroscopic evidence for the absence of a low-molecular-weight (486) antigen in RNA extracts shown to transfer delayed-type hypersensitivity in vitro

Ribonucleic acid-rich extracts obtained from the spleens or lymph nodes of guinea pigs skin test sensitive to mono-(p-azobenzenearsonate)-N-chloracetyl-l-tyrosine (ARS-NAT) (MW 486) were able to convert “nonsensitive” peritoneal exudate cells (PEC) to a state of specific immunologic sensitivity, as assessed by the cell-migration-inhibition correlate of delayed hypersensitivity. Specific inhibition of migration of RNA-treated PEC by ARS-NAT antigen was observed while no inhibition of migration occurred with RNA alone or by incubation with unrelated antigens. The RNA used to transfer sensitivity was assessed for arsenic (As) content as a chemical marker for the ARS-NAT antigen utilizing two methods: a Gutzeit As assay, and atomic absorption spectroscopy (AAS). Preliminary chemical analysis utilizing the Gutzeit assay, which detects as little as 1 μg As, failed to detect As in 3200–4800 μg of RNA or in cell suspensions from the spleen, lymph nodes, and liver of immunized guinea pigs. Further attempts to detect As utilizing AAS, where the limit of As sensitivity was 0.1 ng, failed to detect As in 250 μg to 10 mg of “ARS-NAT-sensitive” RNA, suggesting that, if As is associated with the RNA-rich extracts, it could be present in an amount of no more than 5 pg in 500 μg of RNA; this corresponds to less than 0.0000065% ARS-NAT antigen. These results suggest an informational role for the RNA extracts in our delayed hypersensitivity system, paralleling similar evidence for the action of RNA extracts in antibody systems.     

Migration inhibition by an agarose microdroplet assay: monitoring of tumor-associated antigens on a simian virus 40-induced sarcoma.

Macrophage migration inhibition assays, with a direct agarose microdroplet method, were used to monitor TAA activity of preparations of SV-40-induced mKSA cells. These preparations included cell-free crude membranes, papain-solubilized and NP40 detergent-solubilized membrane extracts from mKSA tumor cells. The assay was extremely sensitive and could detect migration inhibition reactivity with all three types of antigenic preparations with concentrations as low at 250 ng protein/ml. The reactivities were quite reproducible from experiment to experiment using the same or different lots of these antigen preparations, and the reactivities were specific in that peritoneal exudate cells from BALB/c mice, immunized with antigenically unrelated but syngeneic plasmacytomas, were not inhibited by these antigens. The results demonstrated the usefulness of this assay in rapidly detecting small concentrations of partially purified TAA preparations by using small number of immune cells.     

Leucocyte migration inhibition test as an index of immunological response to measles virus. IV. Blood, spleen, lymph nodes and peritoneal exudate cell reaction.

The migration inhibition test of leucocytes isolated both from the peripheral blood, spleen and lymph nodes as well as peritoneal exudate cells of guinea pigs, immunized with the various doses of measles virus was determined. For in vitro testing of cellular immunity to measles virus, viral antigens could be used in both infective and inactivated form.     

Murine cell-mediated immune response recognizes an enterovirus group-specific antigen(s).

Splenocytes taken from mice inoculated with coxsackievirus B3 (CVB3) (Nancy) developed an in vitro proliferative response against CVB3 antigen. This response could not be detected earlier than 8 days postinoculation but could be detected up to 28 days after exposure to CB3. CVB3-sensitized splenocytes responded not only to the CVB3 antigen but to other enteroviruses as well. This response was found to be enterovirus specific in that no response was detected to a non-enteroviral picornavirus, encephalomyocarditis virus, or to an unrelated influenza virus. The generation of a splenocyte population capable of responding to an enterovirus group antigen(s) was not limited to inoculation of mice with CVB3, as similar responses were generated when mice were inoculated with CVB2. Cell subset depletions revealed that the major cell type responding to the enterovirus group antigen(s) was the CD4+ T cell. Current evidence suggests that the group antigen(s) resides in the structural proteins of the virus, since spleen cells from mice inoculated with a UV-inactivated, highly purified preparation of CVB3 virions also responded in vitro against enteroviral antigens.     

Dietary alpha-linked galacto-oligosaccharide suppresses ovalbumin-induced allergic peritonitis in BALB/c mice

To determine whether alpha-linked galacto-oligosaccharide (alpha-GOS) prevents allergic peritonitis, BALB/c mice were fed a synthetic diet with and without alpha-GOS supplementation for 7 d, and were then subcutaneously immunized with ovalbumin on days 0 and 7. The mice were challenged by intraperitoneal injection with ovalbumin on day 14, followed by peritoneal lavage on day 15. The total number of peritoneal exudate cells was significantly lower in the mice fed the alpha-GOS diet than in those fed the control diet. Peritoneal lavage fluid from mice fed the alpha-GOS diet not only had less potency to attract peripheral blood leukocytes and peritoneal exudate cells ex vivo, but also had lower concentrations of monocyte chemoattractant protein-1 (MCP-1) and eotaxin. Preincubation of the cells with alpha-GOS failed to affect the migration to peritoneal lavage fluid. We propose that dietary alpha-GOS reduces cell infiltration in allergic peritonitis by reducing antigen-induced elicitation of MCP-1 and eotaxin in mice.     

Prostaglandin-mediated suppression of delayed-type hypersensitivity to infected erythrocytes during Babesia microti infection in mice

The mechanism of suppression of delayed-type hypersensitivity (DTH) to intraerythrocytic Babesia microti which occurs during infection in mice was examined. The suppression was not specific for anti-parasite DTH; infected mice immunized and challenged with sheep red blood cells had a similar depression of anti-sheep red blood cell DTH. Sublethal or lethal irradiation did not significantly alter the suppression of the DTH response, and cyclophosphamide pretreatment of infected mice also had no effect on suppression. Multiple passive transfer experiments using serum or regional lymph node cells from immunized or infected and immunized (suppressed) donor animals failed to demonstrate any ability to transfer suppression of DTH. Adherent cells from the spleens or peritoneal exudates of suppressed mice, however, did significantly depress the ability of immunized mice to express a DTH response. The cells responsible for this suppression were Thy 1- and nonspecific esterase+. Treatment of suppressive cell populations with 10 micrograms/ml indomethacin for 24 hr in vitro abrogated their suppressive ability, and in vivo administration of indomethacin to suppressed mice also restored DTH to normal levels. By examining levels of prostaglandin E2 (PGE2) in supernates of cultured peritoneal exudate cells from immune or suppressed mice, it was shown that infected mice had peritoneal exudate cells which produced significantly more PGE2 than similar cells from immune mice. These data suggest that B. microti infection elicits synthesis of PGE2 by macrophage-like cells which results in suppression of DTH to parasite as well as heterologous antigens.     

Mechanisms of suppressed cell-mediated immunity and impaired antigen-induced interleukin 2 production in granuloma-bearing mice

Pulmonary granulomas were induced in BALB/c mice immunized with methylated bovine serum albumin in complete Freund's adjuvant by the intratracheal injection of plain agarose beads or beads conjugated to specific antigen. Large hypersensitivity granulomas developed around antigen-coupled beads in immunized animals. Smaller but still prominent granulomatous reactions developed around plain beads in immunized mice. In nonimmunized animals, both plain and antigen conjugated beads produced very small granulomas. Granuloma formation in sensitized animals was associated with suppressed delayed-type hypersensitivity reactions induced by the footpad injection of specific and nonspecific antigens. Lymph node cells from sensitized granuloma-bearing mice with cutaneous anergy showed suppressed specific and nonspecific antigen-induced proliferative responses in vitro. These cells also showed suppressed interleukin 2 production in response to specific antigen. Although no soluble suppressive factor was detected in granuloma extracts, suppressor cells were found in lymph nodes of granuloma-bearing mice, which could inhibit antigen-induced production of interleukin 2 by lymph node cells from immunized mice. Antigen-specific immunoglobulin G antibody production was not suppressed in immunized granuloma-bearing mice. Previous studies from our laboratory have demonstrated migration inhibition factor and interleukin 1 activities in aqueous extracts prepared from granuloma-bearing lungs of immunized mice. These results and the findings reported here indicate that granuloma formation and the associated anergy observed in this system are primarily expressions of cell-mediated immunity; selective suppression of in vivo and in vitro expressions of cell-mediated immunity in granuloma-bearing mice may be due to impaired antigen-induced interleukin 2 production; and such impairment is caused by suppressor cells.     

Use of preparative isoelectric focusing in a Sephadex gel slab to separate immunizing and growth facilitating moieties in crude 3 M KCl extracts of a murine fibrosarcoma

Summary Crude 3 M KCl extracts of the methylcholanthrene-induced fibrosarcoma of C3H/HeJ mice, MCA-F, were demonstrated to contain two fractions, one inducing tumor resistance and the other facilitating the outgrowth of neoplastic cell challenge. In immunoprotection tests in syngeneic C3H/HeJ mice, optimal doses of crude solubilized tumor antigen afforded only a 28% reduction in growth compared with saline-treated controls. When crude extracts were fractionated by preparative isoelectric focusing (pIEF) in a slab of superfine Sephadex G-75, significant biologic activity was demonstrated in two fractions. Fraction (Fr) 1, pI 2.5–3.6, induced potent tumor facilitation, increasing the tumor size by more than 100%, while Fr 15, pI 5.8–6.0, engendered resistance that reduced their respective biological effects to MCA-F, but not the antigenically unrelated MCA-D tumor. Thus 3 M KCl extracts contain at least two biologically active components, one immunoprotective and one tumor-facilitating. Since the weak immunoprotective activity of crude materials may represent the vectorial effect of these antagonistic components, subsequent molecular characterization of both moieties may afford insight into the complex response of hosts toward tumors. Furthermore, TSTA purified by the rapid method of isoelectric focusing may be a more suitable reagent for immunotherapy than the parent crude 3 M KCl extracts by virtue of the absence of facilitating antigens.Abbreviations CE crude 3 M KCl extract - pIEF preparative isoelectric focusing - Fr fraction from pIEF - MCA-F and MCA-D antigenically different methylcholanthrene-induced fibrosarcomas of C3H/HeJ mice - TSTA tumor specific transplantation antigens     

Delayed hypersensitivity to crude and partially purified murine tumor-specific transplantation antigens and alloantigens utilizing the footpad swelling assay

Antigen preparations extracted from C3H/HeJ methylcholanthrene-induced fibrosarcomas by the 3M KCl extraction procedure were assessed for tumor-specific and allospecific antigenicity. Specificity of crude tumor antigen preparations and of fractions from preparative isoelectric focusing was investigated by evocation of footpad swelling (FPS) in syngeneic mice immunized with irradiated fibrosarcoma cells. Tumor immune mice displayed delayed hypersensitivity as positive FPS responses to challenge with 3M KCl extract and with fraction (Fr) 15 (pH 5.7 to 6.0) from preparative isoelectrically focused 3M KCl extract. Crude extracts and Fr 15 exhibited immunoprotective activity in vivo. Immune mice demonstrated a specific FPS response only to crude antigen preparations of Fr 15 from immunizing tumors, not to materials from a noncross-reactive neoplasm. DBA/2J mice immunized with C3H/HeJ spleen cells displayed FPS to challenge with crude antigen preparations, but not with the tumor-specific Fr 15. Alloantigen activity, however, was detected by a positive FPS response in C3H-immune DBA mice in fractions from the pH range 5.1 to 5.5. These experiments demonstrated that the FPS assay provides the setting for detection of specific delayed hypersensitivity responses to crude and fractionated tumor antigen preparations and for delineation of tumor-specific and histocompatibility antigen activities in fractions from crude extracts.     

Induction of suppressor cells by a tumor-derived suppressor factor

Murine fibrosarcomas produce a factor that activates suppressor cells to inhibit expression of delayed-type hypersensitivity (DTH) responses to dinitrochlorobenzene (DNCB). This tumor-derived suppressor factor (TDSF) was partially purified by preparative isoelectric focusing of spent medium and 3 M KCl extracts of cultured methylcholanthrene-induced and spontaneous fibrosarcomas of C3H/He mice. Incubation of 1 micrograms/ml of a fraction, isoelectric pH less than 2.9, with normal syngeneic spleen cells for 1-6 hr at 37 degrees C induced suppressor cells that inhibited the primary DTH response to DNCB upon intraperitoneal transfer to normal C3H/HeJ mice. TDSF was not present in extracts of either syngeneic embryonic fibroblasts or normal spleen cells or in medium conditioned by normal peritoneal exudate cells but was present in 3 M KCl extracts of and the spent medium from four different cultured murine fibrosarcomas. TDSF activity was not restricted at the major histocompatibility complex. The suppressor cells inhibited the efferent limb of the DTH response because (1) hyporesponsive recipients of TDSF-treated spleen cells had splenic effector T cells capable of transferring DTH to DNCB into naive secondary recipients and (2) the ability of Lyt 1+,2- effector Tdth cells to transfer a secondary DTH response to DNCB was inhibited by co-incubation with macrophages or Lyt 1-,2+ T cells treated with TDSF. Preliminary biochemical analysis suggested that TDSF was an RNA- protein complex. Thus, several murine fibrosarcomas produced a soluble factor that activated splenic suppressor cells to depress the immune response to nonneoplastic antigens. These suppressor factors represent a novel group of regulatory molecules which may be ribonucleoprotein complexes.     

Neutralizing Activity Induced by the Attenuated Coxsackievirus B3 Sabin3-like Strain Against CVB3 Infection

Coxsackievirus B3 (CVB3) causes viral myocarditis, and can ultimately result in dilated cardiomyopathy. There is no vaccine available for clinical use. In the present work, we assessed whether the Sabin3-like mutant of CVB3 could induce a protective immunity against virulent CVB3 Nancy and CVB4 E2 strains in mice by both oral and intraperitoneal (IP) routes. Serum samples, taken from mice inoculated with Sabin3-like, were assayed in vitro for their anti-CVB3 neutralizing activity. CVB3 Sabin3-like was highly attenuated in vivo and was able to induce an anti-CVB3 activity of the serum. However, at 4 days post-CVB3 challenge, significant increased titers of CVB3 neutralizing antibodies were detectable in the sera of immunized mice over the next 6 days. Non-immunized mice challenged with CVB3 Nancy had no anti-CVB3 activity in their sera until 10 days post-infection. CVB3 Nancy induced higher viral titers than did the mutant strain. There was no variation of the neutralizing activity of serum taken from mice immunized with CVB3 Sabin3-like and challenged with CVB4 E2, compared to non-immunized mice. Despite the fact that CVB3 and CVB4 are closely related viruses, virus-neutralizing activity clearly distinguish between these viruses. A variable and limited amount of pancreatic inflammation was seen in some mice 10 days after Sabin3-like inoculation by IP route, whereas there was no evidence of pancreatic damage in mice inoculated by oral route. All immunized mice were protected from myocarditis and pancreatitis at 8 days post-challenge with CVB3 or CVB4 E2. These findings strongly suggest that the mutant strain could be considered a candidate for an attenuated CVB3 vaccine.     

In vitro studies on transplantation immunity. II. The migration inhibition in homograft reactions in guinea pigs

The migration of peritoneal exudate cells from guinea pigs exhibiting transplantation immunity is inhibited in the presence of donor antigens. This inhibition of migration is demonstrable whether the donor transplantation antigens are presented in the form of viable cells (peritoneal exudate cells) or as particulate subcellular antigens (spleen microsomes). A greater degree of inhibition was observed when transplantation immunity was induced with lymphoid cells in Freud's adjuvant compared to sensitization with orthotopic skin grafts. There was no inhibition of migration in mixtures of normal allogeneic cells or when peritoneal cells from guinea pigs exhibiting tuberculin hypersensitivity were mixed with similar cells from normal animals. Finally, supernatants from cultures of sensitive lymphocytes plus donor antigens inhibited the migration of normal peritoneal cells indicating the presence of migration inhibitory factor (MIF) activity.     

Macrophage immunity to influenza virus: in vitro and in vivo studies.

Using M-TUR, a macrophage-adapted avian influenza A virus (Hav1, Nav3), antiviral resistance of peritoneal macrophages obtained from specifically or nonspecifically immunized mice towards in vitro infection was assessed. M-TUR grew to high titers in macrophages from nonimmune mice thereby causing a marked cytopathic effect. In contrast, peritoneal macrophages from mice specifically immunized with TUR virus were not affected by infection with M-TUR in vitro. This antiviral immunity was specific: mice immunized with antigenetically unrelated influenza strains such as influenza A/Hong Kong/1/68 (H3, N2) or influenza B/Lee yielded susceptible macrophages. Specific macrophage immunity could be abrogated by trypsin treatment in vitro. Susceptible macrophages from nonimmune hosts became resistant following in vitro exposure to homologous anti-TUR sera. Peritoneal exudate cells from BCG-infected animals were less susceptible to in vitro challenge with M-TUR than control macrophages. In vivo treatment of mice with the unspecific immunostimulants BCG or Corynebacterium parvum did not protect the animals against lethal infection with a hepatotropic variant of TUR.     

The expression of theta-like antigen by rat peripheral lymphocytes: serologic and functional studies.

AKR/Cum mice (Thy-1b = thetaC3H) immunized with nucleated cells from WF rat thymus, Peyer's patches, peritoneal exudate, mesenteric lymph nodes, blood, bone marrow, or spleen produced antibodies cytotoxic for ADR/J (Thy-1a = thetaAKR) but not for AKR/Cum thymocytes. The specificity of these antibodies for the Thy-1.1 (theta-AKR) antigen was confirmed by tests using thymocytes from backcross mice segregating at the Thy-1 locus. This result suggested that the rat lymphocyte antgen cross-reactive with Thy-1.1 was expressed by at least some members of each of the rat lymphoid cell populations tested. AKR/Cum mice immunized with killed rat cells also produced anti-Thy-1.1 antibodies; thus indicating that further differentiation of the injected cells was not a prerequisite for the anti-Thy-1.1 response. Unexpectedly, about 9% of unimmunized adult AKR/Cum males were found to be producing antibodies against Thy-1.1. To our knowledge, natural antibodies of this specificity have not been previously reported. Finally, it was found that peritoneal exudate cells taken from WF rats previously immunized with EL-4 mouse leukemia cells were neither killed nor functionally inactivated by treatment with anti-Thy-1.1 antibodies and complement.     

Involvement of natural killer cells in coxsackievirus B3-induced murine myocarditis

The role of natural killer cells in the temporal development of coxsackievirus B3-induced myocarditis in adolescent CD-1 male mice was examined. Inoculation of purified CVB3m induced maximum NK cell activity in the splenic populations at 3 days postinoculation (p.i.) as assessed by lysis of YAC-1 cells; maximum virus titers in heart tissues were also found at day 3 p.i. Mice depleted of NK cells after injection of anti-asialo GM1 antiserum i.v. had decreased NK cell activity, increased CVB3m titers in heart tissues, and exacerbated myocarditis. Although lesion number was not increased in heart tissues of the latter mice, lesions in these mice exhibited increased myocyte degeneration and dystrophic calcification above that found in lesions of mice inoculated with CVB3m only. No alteration in interferon titers were observed in CVB3m-infected mice treated with anti-asialo GM1 antiserum as compared with normal CVB3m-infected mice. Measurements of splenic NK cell activity in mice inoculated with doses of 10(2) to 10(8) PFU of CVB3m per mouse or UV-irradiated virus suggest that replication of CVB3m is required for NK cell activation. An amyocarditic variant of CVB3m (ts5R) was shown to replicate in heart tissues and to elicit NK cell activity comparable to that elicited by CVB3m. Therefore, the data suggest that NK cell activation depends on virus replication and that these cells provide some protection against CVB3m-induced myocarditis by limiting virus replication in heart tissues.     

Inoculation of the attenuated Coxsackievirus B3 Sabin3-like strain induces a protection against virulent CVB3 Nancy and CVB4 E2 strains in Swiss mice by both oral and intraperitoneal routes

We have previously addressed the question of whether the attenuating mutations of domain V of the Poliovirus IRES were specific for a given genomic context or whether they could be extrapolated to a genomic related virus, the Coxsackievirus B3 (CVB3). Accordingly, we have described that Sabin3-like mutation (U⁴⁷³→C) introduced in the CVB3 genome led to a defective mutant with a serious reduction in translation efficiency. In this study, we assessed the protection provided by the Sabin3-like mutant against CVB3 infection. For this purpose, we analyzed, in vivo, the Sabin3-like phenotype in Swiss mice inoculated with CVB3 and CVB4 E2 prototype strains either by oral or intraperitoneal (i.p) routes and explored the capacity of this mutant to act as a vaccine vector after the challenge. The Sabin3-like RNA was detected by semi-nested PCR in different organs: heart, pancreas and intestine at 10 days post-inoculation with both oral and i.p routes. Additionally, we did not observe any histological alterations in heart and intestine tissues. RNA was detected in the different organs of all mice immunized with the Sabin3-like strain and challenged with either CVB3 or CVB4 E2 by oral route at 7 days post-challenge. In contrast, no histological alteration of heart or pancreas tissues was observed after challenge with both wild-strains. Interestingly, the detection of viral RNA in heart, pancreas and intestine of mice immunized by i.p route was negative at 7 days post-challenge with CVB3 and CVB4 E2, and mice were protected from myocarditis and pancreatitis.     

Modulation of granulomatous inflammation in murine schistosomiasis mansoni by enteric exposure to schistosome ova: in vitro characterization of a regulatory mechanism within the granuloma

Enteric immunization with schistosome ova results in a diminished granulomatous response. This study explored a mechanism by which enteric immunization may decrease granuloma size. Granulomas from livers of acutely infected mice were dissociated and the dispersed cells were depleted of macrophages. As defined by a direct in vitro migration inhibition factor (MIF) assay, the macrophage-depleted cells, composed of lymphocytes and eosinophils, inhibited the migration of normal peritoneal exudate cells when exposed to soluble egg antigens. Anti-Thy 1.2 or -Lyt 1.1, but not -Lyt 2.1, treatment of these cells abrogated MIF activity. Next, mice were exposed enterically to eggs 4 weeks prior to sacrifice. Cells from granulomas isolated from these animals demonstrated no MIF activity unless treated with anti-Lyt 2.1. When granuloma cells from enterically immunized mice were mixed with those from unimmunized animals, MIF activity by the latter was abrogated. Treatment of cells from immunized mice with anti-Lyt 2.1 or -Thy 1.2, but not -Lyt 1.1 prior to mixing once again permitted MIF activity. These results suggest that the diminished granulomatous response induced by enteric immunization could be mediated by Lyt 2+ suppressor T cells. These suppressor cells may regulate the MIF activity of Lyt 1+ T lymphocytes residing within these lesions.     

Activity and Properties of Macrophage Migration Inhibitory Factor produced by Mixed Lymphocyte Cultures

THE macrophage migration test is an in vitro demonstration of delayed hypersensitivity. Supernatant fluids of sensitive lymphocytes cultured for 24 h in the presence of specific antigen contain migration inhibitory factor (MIF) that arrests the migration of macrophages of unsensitized animals in vitro¹,². In vivo, it induces delayed skin reactions³. The use of the macrophage migration test, based on differences of transplantation antigens in donor and recipient, to show histocompatibility has been suggested⁴. The test was also recommended as an indicator of immunological reactivity after organ transplantation, to demonstrate impending rejection⁵. It can demonstrate homograft sensitivity, for migration of peritoneal exudate cells (containing lymphocytes and macrophages) of CBA mice previously sensitized by grafts from A/Jax donors was inhibited when they were mixed with peritoneal exudate cells of the donor strain. However, histocompatibility was not demonstrated, for mixtures of peritoneal exudate cells of ungrafted CBA mice and A/Jax mice migrated regularly during the 24 h test⁶.