The isolation of immunogenic molecular entities from immunogenic and nonimmunogenic tumor homogenates by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

Summary YAC, a Moloney-virus-induced tumor of A-strain mice, is a nonimmunogenic tumor. Mice injected with the inactivated neoplastic cells and challenged with viable tumor cells did not survive longer than mice that received the challenge dose alone. The homogenate of this nonimmunogenic tumor was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis, the gel slices containing isolated molecular entities were injected into various groups of mice. The mice were challenged with low doses of viable tumor cells (10–30 cells) and their survival time was recorded. Small but significant numbers of mice injected with apparent 80–90 K SDS-PAGE-isolated molecular entity rejected the tumor or survived longer than the control groups of mice. Spleen cells from mice injected with 80–90 K molecular entity inhibited the YAC tumor cotransferred with them to naive recipients (Winn assay). Spleen cells from mice injected with monoclonal antibody against nonspecific T-cell helper factor and immunized with 80–90 K SDS-PAGE-isolated molecular entity failed to inhibit the tumor growth in naive recipients, indicating that helper T cells are involved in induction of the antitumor resistance. Nylon-wool-passed splenocytes from mice injected with 80–90 K inhibited tumor growth in some of the recipient mice. Spleen cells from these mice treated with anti-Thy-1 and complement also inhibited the tumor growth in some of the recipients, suggesting that the effector cells were both T and non-T cells. C57BL/6 mice immunized with apparent 20 K SDS-PAGE-isolated molecular entity of RBL5 tumor also induced in vivo resistance to the syngeneic viable RBL5 cells, but not to the syngeneic B16 melanoma cells, indicating the specificity of the protective effect. The practical and theoretical implications of these findings are discussed.     

Tumor idiotype vaccines

In this study, the contribution of idiotype-positive antitumor antibodies (anti-Id) in protective tumor immunity was investigated. We have previously shown that among various anti-Id generated and typed as the internal-image Ab2 of the tumor-associated antibody (TAA) gp52, only 2F10 antibody induces protective immunity. Increase of the 2F10 idiotope in sera of tumor-bearing mice correlated with long-term survival, while in mice with short survival the circulating 2F10 idiotype decreased. 2F10⁺ Ig were purified from sera of tumor-bearing mice with longterm survival and the amount of 2F10⁺ anti-TAA antibodies was determined. Only about 3% of 2F10⁺ antibodies are 2F10⁺ anti-TAA⁺. Hybridomas were generated from a 2F10 high mouse with spontaneous tumor regression. Only 2 out of 52 tumor-specific hybridomas were 2F10⁺. These results suggest that the protective effect induced by 2F10 vaccination may not be directly mediated by 2F10⁺ antibodies but indirectly through the stimulation of a 2F10-specific cellular immune response.Supported in part by a grant from N.I.H. no. CA51434     

Antiidiotypic vaccination against murine coronavirus infection.

Rabbit polyclonal antiidiotypic antibodies were generated against a neutralizing mAb specific for a conformational epitope on the S glycoprotein of murine hepatitis virus, strain A59 (MHV-A59). These anti-Id were directed predominantly against an Id that was undetectable in rabbit and rat anti-MHV-A59 sera and weakly represented in syngeneic and allogeneic antiviral sera. However, some partial idiotypic sharing was observed between the Id-bearing antibody and a mAb with a similar antigenic site specificity. The anti-Id inhibited the virus-binding and neutralizing activities of the immunizing antibody, demonstrating that they recognize paratope-associated idiotopes. Mice immunized with affinity-purified anti-Id developed MHV-A59-specific antibodies that neutralized viral infectivity to high titers. Moreover, these animals survived an otherwise lethal challenge with viral murine hepatitis virus, unlike control mice immunized with normal rabbit Ig. These results indicate that at least a subpopulation of the polyclonal anti-Id could induce a protective immune response directed toward a biologically important MHV-A59 epitope, and demonstrate the feasibility of antiidiotypic vaccination against a coronavirus infection.     

Chemoprotective effects of recombinant human IL-1 alpha in cyclophosphamide-treated normal and tumor-bearing mice. Protection from acute toxicity, hematologic effects, development of late mortality, and enhanced therapeutic efficacy

In this study, recombinant human IL-1 alpha (rhIL-1 alpha) was used to protect normal and tumor-bearing BALB/c mice from the acute toxicity caused by lethal doses of cyclophosphamide (Cy) and 5-fluorouracil. Pretreatment of mice for 7 days with 10,000 U/day of rhIL-1 alpha protected 70 to 100% of mice from the acute death induced by lethal doses of both Cy (380 mg/kg) and 5-fluorouracil (250 mg/kg). In contrast, post-treatment of mice with single or multiple doses of rhIL-1 alpha was not chemoprotective. Pretreatment of mice with rhIL-1 alpha increased the acute LD90 of Cy from 380 mg/kg to greater than 500 mg/kg in normal mice, an LD90 dose-modifying effect of at least 1.25, was accompanied by a more rapid recovery from neutropenia and a less severe reduction in the number of bone marrow single lineage monocyte, myeloid, or myelomonocytic colonies. Some of the mice (10 to 50%) that were successfully protected by pretreatment with rhIL-1 alpha died after day 50. These mice consistently presented with extensive pulmonary inflammation and fibrosis at death. Mice bearing murine renal cancer (Renca) were also protected from the acute toxic effects of Cy (450 mg/kg) by pretreatment with rhIL-1 alpha. Renca-bearing mice pretreated with rhIL-1 alpha and either sublethal (300 mg/kg) or lethal (450 mg/kg) doses of Cy exhibited enhanced survival times over those of untreated Renca-bearing mice. Interestingly, the cause of death in Renca-bearing mice that ultimately failed treatment with rhIL-1 alpha plus 300 mg/kg Cy was recurrent tumor, whereas most mice treated with rhIL-1 alpha plus 450 mg/kg Cy had no detectable tumor, although several died from late pulmonary inflammation and fibrosis. Thus, the dose escalation of Cy in rhIL-1 alpha-pretreated mice results in greater antitumor effects of Cy. However, the dose escalation of some cytotoxic agents allowed by the use of myelostimulatory agents can result in late fatal complications not detected in acute toxicity testing.     

Therapeutic protective effects of IL-12 combined with soluble IL-4 receptor against established infections of herpes simplex virus type 1 in thermally injured mice.

The effect of combination therapy between IL-12 and soluble IL-4R (sIL-4R) on the established infection of HSV-1 in thermally injured mice (TI mice) was investigated. All of the TI mice infected with lethal amounts of HSV-1 died when IL-12 was given therapeutically at a dose of 500 U/mouse. However, 80% of these mice treated prophylactically with IL-12 survived compared with 0% survival of the same mice treated with saline. The therapeutic administration of IL-12 to TI mice currently infected with HSV-1 caused an 80% survival of these mice when the treatment was combined with sIL-4R. Although IL-12 did not stimulate IFN-gamma production in cultures of splenic T cells from TI mice, IFN-gamma was produced by stimulation with IL-12 when the producer cells were prepared from TI mice that had been treated previously with sIL-4R. After stimulation with anti-CD3 mAb, splenic T cells from TI mice with the established infection of HSV-1 produced IL-4 into their culture fluids. However, IL-4 was not produced by splenic T cells that were prepared from the same infected mice treated with IL-12 and sIL-4R in combination. The results obtained herein indicate that the efficacies of the combination therapy against the established infection of HSV-1 may result from the IFN-gamma production stimulated by IL-12 in TI mice that are treated with sIL-4R for reducing burn-associated type 2 T cell responses.     

Synergic effect of grape seed extract with amphotericin B against disseminated candidiasis due to Candida albicans

Amphotericin B (Amp B) is considered as a drug of choice for treatment of fungal infections, but it causes severe side effects such as renal damage. To lessen the severity, it is often combined with the azole, but data reporting resistance of Candida albicans to the azole have been recently increasing. Thus, finding a new product that can reduce Amp B dose by combination seems to be important. In the present study, we investigated a synergic effect of grape seed extract (GSE) combined with Amp B against the fungus. Our results showed that the GSE alone inhibited growth of C. albicans yeast cells, and that in a murine model of disseminated candidiasis mice groups given GSE before intravenous inoculation with the yeast cells survived longer than diluent-received (control) mice groups (P<0.05). This GSE antifungal effect was dose-dependent. Upon combination of GSE plus Amp B, the combination therapy strikingly retarded the yeast growth as determined by the broth susceptibility method. Against the disseminated disease, mice given diluent (negative control), Amp B (0.5mg/kg of body weight), or GSE (2mg/kg of body weight) had mean survival times (MSTs) of approximately 11.4, 14.4, and 17.6 days, respectively. However, mice treated with the combination of the doses of Amp B and GSE had a MST value of 38.4 days, surviving an average of 24 days longer than Amp B alone-treated mice groups. This MST value from the combination-received mice group was greater than the MST value from the mice group given four times the Amp B dose (2mg/kg of body weight). All these data indicate that the combination therapy can reduce more than 75% of Amp B dose, implying that GSE has a synergic effect with Amp B.     

Vaccination with membrane-associated idiotype provides greater and more prolonged protection of animals from tumor challenge than the soluble form of idiotype

The purpose of this work was to compare the efficacy of immunizing mice with a soluble vs a cell-associated form of a tumor Ag. A murine B cell tumor (2C3), which displays an Id on its cell surface, grows progressively and gives rise to Id-negative tumor variants in nonimmunized animals. We previously reported that the tumor variants arise as a consequence of Id-specific T cell suppression of the Id+ tumor. The Id-specific effector T cells are CD4+, CD8-. Based upon these findings vaccination protocols have been designed and tested to determine whether the expansion of tumor-specific effector T cells would eliminate the Id+ tumors and prevent the subsequent generation of Id- tumor variants in vivo. MHC-restricted T cells typically recognize soluble Ag subsequent to modification by an APC, and APC may ultimately express a processed form of the Ag that is different from that expressed on the surface of the tumor cells. Based upon this assumption, the efficacy of immunizing mice with cell-associated 2C3 Id was compared to immunization with a soluble form of the same Id. Mice were immunized with either irradiated 2C3 cells or syngeneic spleen cells to which 2C3 protein was covalently linked. These immunization protocols provided a complete and lasting protection against a tumor challenge of up to 1 x 10(6) tumor cells. In contrast, most mice hyperimmunized with a soluble form of the Id did not survive this level of tumor challenge in spite of the production of significant levels of anti-Id antibodies. Mice immunized with the soluble form of the Id, which did survive, produced slowly progressing tumors expressing a 1000-fold less of the marker Id. These results illustrate the importance of understanding and properly exploiting a host's natural response to a tumor-specific Ag when designing effective immunization protocols for cancer therapy.     

Protection of mice against Clostridium chauvoei infection by anti-idiotype antibody to a monoclonal antibody to flagella

Abstract Polyclonal rabbit anti-idiotypic antibody (anti-Id) against the protective monoclonal antibody specific to the flagella of Clostridium chauvoei was produced, purified, and characterized. Anti-Id inhibited the binding of its related monoclonal antibody to the flagellar antigen, suggesting that the anti-Id bore an internal image of the flagellar antigen. When mice were immunized with anti-Id intraperitoneally, the survival rate increased significantly, compared with mice immunized with normal rabbit IgG ( P < 0.01), and specific anti-flagellar antibodies were induced.     

In vitro induction of cell-mediated immunity to murine leukemia cells

Summary An adoptive chemoimmunotherapeutic model based on the use of chemotherapy and lymphocytes specifically sensitized against tumor cells in vitro was tested in mice transplanted with syngeneic leukemia cells. C57BL/6 and A strain mice were inoculated i.p. or i.v. (day 0) with lethal doses (1×10³–1×10⁵) of EL4 and YAC leukemia cells, respectively. Leukemic mice were subsequently treated (day 1 or day 3) with partially curative doses (80–140 mg/kg) of cyclophosphamide (Cy), followed by i.p. or i.v. administration of 1–3×10⁷ cytotoxic lymphocytes (CL) induced in macro-mixed leukocyte-tumor cell cultures (MLTC). The following results were obtained: untreated mice died with tumor within 20 days; mice receiving sensitized lymphocytes only showed a modest prolongation of survival and only 5–15% of the animals were cured; treatment with Cy alone or with Cy and normal lymphocytes prolonged survival considerably and cured 20–60% of the mice; mice subjected to Cy in conjunction with in vitro-sensitized lymphoid cells, either syngeneic or allogeneic, had survival rates of 80–100% (100 days). Under the conditions employed, no severe manifestations of clinical graft-versus-host (GVH) reaction were observed. These findings imply that in vitro-sensitized immunocytes and cytoreductive drugs can operate cumulatively.     

Cellular mechanisms involved in protection and recovery from influenza virus infection in immunodeficient mice.

We investigated the role of different lymphocyte subpopulations in the host defense reaction against influenza virus infection, taking advantage of various immunodeficient mouse strains. Whereas, following immunization, wild-type animals showed complete protection against challenge with a lethal dose of A/PR8/34 (PR8) virus, mice that lack both B and T cells but not NK cells (namely, scid and RAG2(-/-) mice) did not display any protective effect in similar conditions. By contrast, J(H)D(-/-) mice devoid of B cells and immunized with virus showed a protective response after challenge with a lethal dose. The immunized J(H)D(-/-) mice that survived completely recovered from the influenza virus infection. Immunized J(H)D(-/+) mice exhibited a more complete protection, suggesting the role of specific antibodies in resistance to infection. To assess the role of natural immunity in the host defense against influenza virus, we carried out experiments with scid mice challenged with lower but still lethal doses of PR8 virus. While an increased NK activity and an increased number of NK1.1+ cells in lungs of scid mice infected with PR8 virus were noted, in vivo depletion of the NK1.1+ cells did not affect the overall survival of the mice. Our results show that specific T cells mediate protection and recovery of J(H)D(-/-) mice immunized with live virus and challenged with lethal doses of influenza virus.     

Ecdysterone Modulates Antitumor Activity of Cytostatics and Biosynthesis of Macromolecules in Tumor-Bearing Mice

The influence of therapeutic and half doses of cisplatin and adriamycin combination with the anabolic drug ecdysterone (20-hydroecdison) on development of subcutaneously and intraperitoneally transplanted P388 and L1210 leukemia and metastasizing B16 melanoma was studied. Ecdysterone significantly stimulated the chemotherapeutic effect of low doses of the cytostatics: inhibition of tumor growth, mice survival rate, their lifespan, and the antimetastatic activity index were comparable or better than after therapy with high doses of the antitumor drugs. The influence of high and low doses of cisplatin and its low dose in combination with ecdysterone on the dynamics of protein and DNA biosynthesis in the liver, pancreas, thymus, spleen, and adrenals of tumor-bearing mice were also studied. Although the therapeutic effect of 4 mg/kg cisplatin by activated protein biosynthesis and DNA repair is comparable or better than that of its low dose (2 mg/kg) in combination with ecdysterone, in terms of chemotherapy the combination looks preferable since the therapeutic dose of cisplatin is toxic for the intact tissues.     

Idiotypic intramolecular help. Induction of tumor-specific antibodies by monoclonal anti-idiotypic antibody with the help of Fc-specific T helper clones

In this study, the mechanism of anti-Id vaccination was investigated by using cloned Th cells and an anti-idiotypic mAb. 2F10, an anti-idiotypic mAb derived from an Igh1-e allotype mouse strain, which induces protection against the L1210/GZL DBA/2 tumor, was used to prime DBA/2 mice. An Fc (Igh1-e)-specific syngeneic Th clone was cocultured in the presence of 2F10 anti-Id with 2F10-Fab-primed B cells. The Th clone responded with proliferation and also provided help for 2F10-Fab-primed B cells to produce antibodies that bind to L1210/GZL and not to P815 tumor cells. Intact 2F10 anti-Id was presented to Fc-specific Th cells by Fab (or Id) primed B cells more efficiently than the fragment mixture (Fab plus Fc) of 2F10 anti-Id, indicating that 2F10-Fab (or Id)-primed B cells capture 2F10 anti-Id through surface Ig receptors. Presenting B cells are sensitive to treatment with chloroquine and must come from H-2 matched mice, indicating that the Ag presentation by Fab-primed B cells to Fc-specific Th cells requires processing and is MHC restricted. Collectively these results outline a mechanism that may operate in anti-Id therapy of tumor-bearing animals by using tumor Ag mimicking anti-idiotypic antibodies. A similar mechanism could be effective in tumor patients immunized with xenogeneic anti-idiotypic antibodies operating under the "intra(Ag) molecular help."     

Selection of antibody repertoires by anti-idiotypes can occur at multiple steps of B cell differentiation

These experiments were designed to evaluate whether alterations in Id expression after anti-Id treatments result from direct modulation of Id-producing B cells, and whether idiotypic selection operates in bone marrow or spleen B cells. By using the NPb Id model, we have studied the functional behavior of isolated LPS-reactive B cells transferred from B6 mice into histocompatible LPS-NR B10.Cr hosts and primed with LPS conjugates of anti-Id antibodies. We have found that previous anti-idiotypic manipulation of host mice by neonatal administration of suppressive doses of Ac 38 antibodies, or adult injection of enhancing doses of Ac 146 antibodies, modulated the T cell-independent Id response of either immature bone marrow or mature splenic responding cells, transferred from normal, untreated donors. These results are interpreted to suggest that selection of antibody repertoires by anti-Id may occur at multiple steps of B cell differentiation.     

Idiotype matching: correlation of expression of protective idiotype in sera with survival of tumor mice

A monoclonal anti-Id, 2F10, has previously been shown to protect against transfer of L1210/GZL tumor cells in DBA/2 mice and also to have therapeutic effects in mice with growing tumor. In this study we have measured expression of an idiotope which reacts with a tumor-protective anti-idiotypic antibody, 2F10, in the sera of mice bearing the L1210/GZL tumor. The levels of antibodies binding to 2F10, referred to as the "2F10 idiotope," are different in individual mice and also fluctuate over time. A statistical analysis demonstrated a significant correlation between these changes in 2F10 levels in mice with tumors and their survival times. Increasing 2F10 idiotope in sera of tumor mice correlated with long-term survival, whereas a decreasing trend was found in mice which died shortly after tumor transfer. Correlations between the 2F10 idiotope and survival were observed in groups of mice which had received surgery, cyclophosphamide, a combination of cyclophosphamide and anti-idiotype, or no treatment at all. No correlation between a nonrelated idiotope and survival was noted. Although 2F10 is an idiotope expressed by an anti-tumor-associated Ag antibody, the correlation between anti-tumor-associated Ag titers and survival was significantly lower than that between the 2F10 idiotope and survival. This demonstrates that 2F10 is preferentially associated with antibodies which are involved in tumor regression. Thus, the 2F10 idiotope in sera of tumor-bearing mice has predictive value for survival and tumor regression.     

Thymus-dependent antiidiotype and anti-antiidiotype responses to a dinitrophenyl-specific monoclonal antibody

BALB/c mice were inoculated i.p. with graded doses of a DNP-specific, IgM mAb (designated 57.1). Injection with unmodified 57.1 in the absence of adjuvants resulted in the generation of an anti-Id response (Ab2) and an anti-anti-Id response (Ab3). The generation of serum anti-Id antibodies was found to be thymus dependent. Nude mice immunized with 57.1 were unable to produce a serum Ab2 response above nonimmunized controls whereas euthymic mice receiving identical doses of 57.1 produced strong Ab2 responses. To examine the specificity of serum anti-Id, sera from mice receiving 57.1 were screened against a panel of mAb representing at least five distinct VH gene families. Serum titers were significantly higher against 57.1 than against any of the other antibodies in the panel. Three of the antibodies in this panel bind FD5-1, a monoclonal anti-Id (Ab2) that also binds 57.1. However, sera from mice receiving 57.1 bound 57.1 only. Thus, the serum Ab2 response appears to be highly specific for idiotopes on 57.1. The predominant isotype of these anti-Id antibodies was IgG1. The number of isotypes detected increased in a dose dependent manner with all IgG subclasses having anti-Id specificity in sera from animals receiving the higher doses of 57.1. Further analysis of the serum demonstrated that approximately 8% of the Ab2 response was paratope-specific (inhibitable by the monovalent hapten DNP-lysine). The same sera were analyzed for the presence of Ab3 by binding to the monoclonal anti-Id antibody FD5-1. Lower serum titers of Ab3 were generated in comparison to serum titers of Ab2. Analysis of the binding specificity of the Ab3 response revealed that DNP-BSA was able to partially inhibit the binding of serum IgM and IgG Ab3 to FD5-1. A subset of the Ab3 response. Ab1' that is specific for DNP was observed in a direct binding assay where detectable amounts of DNP binding IgM, IgG1, and IgG3 isotypes were present. We have thus described a complete circuit (Ab1----Ab2----Ab3) of antibodies within the Id network by immunizing animals with an unmodified mAb in the absence of Ag or adjuvants.     

Bispecific anti-idiotype/anti-CD3 antibody therapy of murine B cell lymphoma.

Retargeting of T cells by bispecific IgG which binds to both CD3 and a tumor-associated Ag can induce T cell lysis of target cells irrespective of TCR specificity. The current studies were designed to further explore the efficacy and specificity of bispecific IgG-directed therapy in an immunocompetent animal model, and to evaluate the mechanisms responsible for bispecific IgG-directed inhibition of tumor cell growth by using the 38C13 murine lymphoma system. In vitro, proliferation of activated T cells in the presence of bispecific IgG was increased when the relevant, but not the irrelevant target cells were present. Bispecific IgG specifically induced activated T cell mediated lysis of cells expressing the target Ag, but not of cells expressing an irrelevant Ag, even when the irrelevant cells were in the same cell mixture, indicating contact between target cells and T cells plays a major role in bispecific IgG-mediated lysis. Bispecific IgG was less effective than anti-Id at inducing target cell lysis when peritoneal macrophages were used as effectors, suggesting bispecific IgG Fc is not responsible for cytotoxicity in this system. In vivo, bispecific IgG was significantly superior to anti-Id, anti-CD3, or a combination of anti-Id and anti-CD3 in preventing tumor growth in immunocompetent mice inoculated with syngeneic lymphoma. Phenotypic evaluation of tumors that emerged despite therapy indicated bispecific IgG selects for the emergence of Id variant lymphoma cells. In separate studies, 38C13 tumor inocula containing cells recognized by the therapeutic antibody were supplemented with a small number of 38C13 cells which expressed a distinct Id not recognized by the therapeutic antibody. Untreated mice inoculated with this mixture developed tumors containing cells of both phenotypes, whereas tumors emerging from mice treated with bispecific IgG contained only cells expressing the nonreactive Id. These studies demonstrate bispecific IgG-directed lysis is therapeutically superior to monospecific anti-Id therapy in the 38C13 tumor model, and that tumor lysis is mediated largely by cell-cell contact. As with other forms of anti-Id based therapy, Id variants can emerge as resistant cell populations after bispecific IgG therapy.     

Immunotherapy of EL4 lymphoma with reovirus

Summary EL4 lymphoma was grown as an ascitic tumor in the peritoneal cavity of C57Bl/6 mice. Animals with different tumor burdens (either 10⁷ or 10⁹ cells) were treated with a single intraperitoneal injection of BCNU using doses from 20–40 mg/kg. Response as measured by mean survival time and percent survival was dependent on tumor burden and dose of drug. The objective of chemotherapy was to increase the mean survival time, but not the percent survival, in order to evaluate the therapeutic effect of reovirus. Mice were given 10⁸, 10⁹, or 10¹⁰ Pfu of reovirus at various times with respect to chemotherapy. The number of mice cured after treatment with both BCNU and reovirus was significantly greater compared to mice treated with BCNU only. Mice cured with combination therapy developed tumor-specific immunity as measured by cytotoxic lymphocytes and serum, and resistance to a lethal tumor challenge. The Abbreviations used are: BCNU: 1,3-bis-(2-chloroethyl)-1-nitrosourea; Saline: 0.9% NaCl solution; MEM: minimal essential medium; Pfu: plaque-forming units; FCS: fetal calf serum; BME: basal eagle's medium; SSC: sodium citrate-sodium chloride     

Tumor idiotype vaccines. VII. Analysis and correlation of structural, idiotypic, and biologic properties of protective and nonprotective Ab2

We have previously generated and used anti-Id mAb (Ab2) to induce protective immunity against the L1210 DBA/2 tumor and for immunotherapy of established tumors. Among various anti-Id that were typed serologically as internal image Ab2 of the mouse mammary tumor virus tumor-associated Ag gp52, only one induced protective immunity and was effective in immunotherapy. In this study we compared the structural, idiotypic, and network properties of the protective and nonprotective antiidiotypic antibodies. The DNA sequence of the variable regions of six anti-Id was determined. The VH sequence of four Ab2, including the protective Ab2, are highly homologous, whereas the VL sequences differ and were assigned to different Vk families. In addition, the DH sequence region of the same four Ab2 are identical, whereas one is highly homologous and another one without homology. Search for amino acid sequence homologies between the Ab2 and gp52 showed the strongest similarities in the CDR2 of the L chain from the protective Ab2. In addition, the CDR2 region also had homology with a T cell epitope on gp52. The biologic basis of effective idiotypic mimicry was studied at the level of Ab3 induced by the Ab2. Id inhibition analysis using Ab3 induced by either protective or nonprotective Ab2, revealed differences. Thus, there is evidence for differences among the Ab1-Ab2-Ab3 cascade induced by protective and nonprotective anti-Id.     

Prolongation of cardiac allograft survival by pretreatment of recipient mice with donor blood or spleen cells plus cyclophosphamide.

Using six mouse strain combinations, we attempted to prolong cardiac allograft survival by pretreatment of recipients with a single iv injection of donor-specific whole blood or spleen cells plus a single ip injection of cyclophosphamide (Cy). Significant prolongation of cardiac allograft survival occurred in a small proportion of pretreated mice of some strain combinations, with some grafts surviving for periods longer than 6–9 months. Cy injected alone did not influence the normal cardiac allograft rejection time of between 1 and 2 weeks. Depending upon the strain combination, accelerated rejection of all or some of the grafts occurred in mice pretreated with blood or spleen cells or myocardial cells alone.     

The effect of elastase-specific monoclonal and polyclonal antibodies on the virulence ofAspergillus fumigatus in immunocompromised mice

Elastase has been implicated as a potential virulence factor involved in the invasion process of the opportunistic pathogen,Aspergillus fumigatus. Monoclonal and polyclonal antibodies, known to inhibit elastase in vitro, were employed in an immunocompromised mouse model of invasive aspergillosis to determine if the antibodies could protect mice from fatal infection. Individual monoclonal antibodies, known to inhibit elastase partially (13 to 23%), or combinations of monoclonal antibodies, known to inhibit elastase 70 to 100%, were tested in the mouse model. No individual nor combination of monoclonal antibodies protected immunosuppressed, infected mice in the doses tested. Similarly, elastase-specific polyclonal antibodies, raised in mice or rabbits, did not exhibit a protective effect, nor did immunization of mice with elastase prior to immunosuppression and infection. Histological examination of the lungs of immunosuppressed, infected mice showed no amelioration of fungal invasiveness by treatment with elastase-specific monoclonal or polyclonal antibodies. However, immunocompetent mice, instilled with a spore inoculum much higher than used in the preceding studies and treated with antibodies, survived, while control mice not treated with antibodies were overwhelmed by the massive spore dose and died. Nevertheless, overall evidence suggests that elastase may not be the primary virulence factor involved in invasive pulmonary aspergillosis.