Correlation between Abetax-40-, Abetax-42-, and Abetax-43-containing amyloid plaques and cognitive decline.

CONTEXT: Accumulation of senile plaques containing amyloid beta (Abeta)-protein is a pathologic hallmark of Alzheimer disease. Amyloid beta-peptide is heterogeneous, with carboxyterminal variants ending at residues Val40 (Abetax-40), Ala42 (Abetax-42), or Thr43 (Abetax-43). The relative importance of each of these variants in dementia or cognitive decline remains unclear. OBJECTIVE: To study whether Abeta deposition correlates with dementia and occurs at the earliest signs of cognitive decline. DESIGN, SETTING, AND PATIENTS: Postmortem cross-sectional study comparing the deposition of Abeta variants in the prefrontal cortex of 79 nursing home residents having no, questionable, mild, moderate, or severe dementia. MAIN OUTCOME MEASURES: Levels of staining of Abeta-peptides ending at amino acid 40, 42, or 43 in the frontal cortex, as a function of Clinical Dementia Rating score. RESULTS: There were significant deposits of all 3 Abeta species that strongly correlated with cognitive decline. Furthermore, deposition of Abetax-42 and Abetax-43 occurred very early in the disease process before there could be a diagnosis of Alzheimer disease. Levels of deposited Abetax-43 appeared surprisingly high given the low amounts synthesized. CONCLUSIONS: These data indicate that Abetax-42 and Abetax-43 are important species associated with early disease progression and suggest that the physiochemical properties of the Abeta species may be a major determinant in amyloid deposition. The results support an important role for Abeta in mediating initial pathogenic events in Alzheimer disease dementia and reinforce that treatment strategies targeting the formation, accumulation, or cytotoxic effects of Abeta should be pursued.

Myeloid dendritic cells stimulate both Th1 and Th2 immune responses depending on the nature of the antigen.

It has been shown that different types of pathogens induce different immune responses. Recovery from intracellular bacterial and viral infection is dependent on the secretion of Th1 cytokines, such as interferon-gamma (IFN-gamma), and on the generation of cytotoxic T cells. In contrast, responses to some parasitic invaders are of the Th2 type, characterized by secretion of interleukin-4 (IL-4). At present, it is not clear what directs this choice, and the most prevalent hypotheses are based on the dendritic cells (DC). In this work, we studied the immune responses generated in mice to a number of antigens, both replicating and nonreplicating, using bone marrow-derived DC as vehicles for immunization. We demonstrate that DC infected with influenza virus prime for a pure Th1 response in vivo devoid of IL-4 induction. This immune response correlates with the induction of DC maturation by the virus. In contrast, nonreplicating antigens, such as fetal bovine serum (FBS), beta-galactosidase, or inactivated influenza virus, do not mature the DC and prime for responses characterized by the secretion of large amounts of IL-4. These data support the hypothesis that myeloid DC are capable of eliciting both types of responses depending on the nature of the antigen.

A mouse model for immunization with ex vivo virus-infected dendritic cells.

Dendritic cells (DCs) have been demonstrated to be an important if not essential inducer of cellular immune responses. The ability to grow these cells in vitro may open up new avenues for protective immunizations. In this study we have analyzed the virus-specific memory response generated following immunization with ex vivo-infected bone marrow-derived dendritic cells. We demonstrate that mouse DCs are efficiently infected with influenza virus but do not release infectious progeny virus. Ex vivo-infected DCs secrete interleukin-12 (IL-12) and induce a potent T helper (Th)1-like immune response when injected into mice. This was demonstrated by the generation of cytotoxic T lymphocytes, the production of high levels of gamma-interferon, and undetectable levels of IL-4 upon in vitro restimulation of splenocytes from immunized animals. In addition, the virus-specific antibody response is primarily of the IgG2a isotype, consistent with the expansion of Th1 cells. Animals immunized with DCs infected with X-31 influenza virus and challenged with PR8 influenza virus cleared the infection faster than animals not vaccinated. Thus, infected DCs efficiently activate the cellular immune response and induce heterosubtypic immunity in mice.

Type I IFN modulates innate and specific antiviral immunity.

IFNs protect from virus infection by inducing an antiviral state and by modulating the immune response. Using mice deficient in multiple aspects of IFN signaling, we found that type I and type II IFN play distinct although complementing roles in the resolution of influenza viral disease. Both types of IFN influenced the profile of cytokines produced by T lymphocytes, with a significant bias toward Th2 differentiation occurring in the absence of responsiveness to either IFN. However, although a Th1 bias produced through inhibition of Th2 differentiation by IFN-gamma was not required to resolve infection, loss of type I IFN responsiveness led to exacerbated disease pathology characterized by granulocytic pulmonary inflammatory infiltrates. Responsiveness to type I IFN did not influence the generation of virus-specific cytotoxic lymphocytes or the rate of viral clearance, but induction of IL-10 and IL-15 in infected lungs through a type I IFN-dependent pathway correlated with a protective response to virus. Combined loss of both IFN pathways led to a severely polarized proinflammatory immune response and exacerbated disease. These results reveal an unexpected role for type I IFN in coordinating the host response to viral infection and controlling inflammation in the absence of a direct effect on virus replication.

Th2 responses to inactivated influenza virus can Be converted to Th1 responses and facilitate recovery from heterosubtypic virus infection.

Immunization with live influenza virus expands Th1 memory cells and facilitates more rapid recovery after heterosubtypic virus challenge. Immunization with inactivated virus generates a Th2 response and does not lead to heterosubtypic immunity. Creation of a Th1 priming environment by the inclusion of interleukin (IL)-12 with antibodies to IL-4 converted the response against inactivated virus to a Th1 response that was able to facilitate virus clearance upon heterosubtypic virus challenge. Evaluation of memory responses of mice immunized by the various protocols demonstrated that the type of immunization imprints T cell memory, dictating the nature of the response to subsequent infection. After live virus challenge, expansion of Th1 cells seems to facilitate the generation of cytotoxic T lymphocytes from naïve precursors. This latter finding may be the mechanism by which inactivated virus immunization in a Th1 cytokine context mediates heterosubtypic immunity.

Superantigen-activated T cells redirected by a bispecific antibody inhibit vesicular stomatitis virus replication in vitro and in vivo.

A bispecific Ab (BsAb) that binds the TCR on T cells and the G protein of the vesicular stomatitis virus (VSV) can redirect staphylococcal enterotoxin B (SEB)-activated T cells to kill VSV-infected cells and to inhibit VSV replication in vitro. Inhibition of virus replication in our system is dependent upon the specificity of the Ab for the viral protein. IFN-gamma does not play a very important role in this phenomenon, which is mainly mediated by the release of Pfp from CD8+ T cells. We have used a Stat1 knockout mouse model in which VSV infection is lethal. Infusion of staphylococcal enterotoxin-activated B T cells and bispecific Ab significantly slowed virus progression and prolonged the survival of VSV-infected Stat1 knockout mice in vivo.

Functional characterization of monoclonal antibodies specific to growth hormone receptor.

Upon engagement with appropriate ligands, receptors can be activated to initiate various metabolic and morphological changes in living cells. An attempt was made in this study to generate monoclonal antibodies (mAb) specific to recombinant rat growth hormone receptor (GHR) and subsequently to investigate their ability to act as biologically active ligands. Three mAbs, designated 1A9, 1H2 and 2C3, were produced and all were highly reactive with GHR in an enzyme-linked immunosorbent assay. In contrast to 1H2, 1A9 and 2C3 competed with radioactive growth hormone (GH) tracer for the binding to GHR in a radioreceptor assay, suggesting that the GH-binding sites of GHR were identical, or very close to its epitopes recognized by 1A9 and 2C3. The molecular interaction evaluated by the BIAcore technology further demonstrated the separate GHR epitopes for 1A9 and 2C3. 2C3 apparently targeted the precise GH-binding sites of GHR, while the antigenic determinants for 1A9 were not at the site, but adjacent to it. Functional analysis showed that 2C3 promoted the growth of hypophysectomized rats, whereas others failed to do so. Therefore, findings from the present study suggest that these mAbs recognize distinct GHR epitopes and are useful for investigating the structure-function relationship of GHR. Furthermore, 2C3 may prove important as a biologically active agonist for better understanding of the process of GHR activation relevant to growth.

Interleukin-4 causes delayed virus clearance in influenza virus-infected mice.

Two different subsets of T cells, Th1 and Th2 cells, have been demonstrated to secrete different profiles of cytokines and to influence various infections in different ways. Whereas cytokines secreted by Th1 cells, particularly gamma interferon, promote the generation of cell-mediated immunity, Th2 cells and their cytokines (interleukin-4 [IL-4], IL-5, IL-10, and IL-13) have been shown to function in recovery from parasitic infections and in antibody responses. In this study, we analyzed the effects of the dominant Th2 cytokine, IL-4, on immunity to virus infection. We assessed the effects of IL-4 on both secondary immune responses by an adoptive transfer assay and primary immune responses by in vivo treatment of influenza virus-infected mice with IL-4. The results demonstrated that IL-4 can function to inhibit antiviral immunity at both stages. We found that IL-4 treatment of sensitized cells during secondary stimulation in vitro had little effect on their ability to lyse virus-infected target cells in a 51Cr release assay. Nevertheless, the clearance of influenza A/PR/8/34 (H1N1) virus from the lungs of infected BALB/c mice was significantly delayed after the transfer of virus-specific T cells secondarily stimulated in the presence of IL-4 in comparison to virus clearance in recipients of cells stimulated in the absence of IL-4. In contrast to the adoptive transfer results, the treatment of PR8 virus-infected mice with IL-4 during primary infection greatly suppressed the generation of cytotoxic T-cell precursors, as assessed by secondary stimulation in vitro. In addition, culture supernatants of secondarily stimulated spleen cells from IL-4-treated mice contained significantly less gamma interferon and more IL-4 than did spleen cells from controls. More importantly, the treatment of mice with IL-4 resulted in an extremely significant delay in virus clearance. Thus, IL-4 can inhibit both primary and secondary antiviral immune responses.

A bispecific antibody recognizing influenza A virus M2 protein redirects effector cells to inhibit virus replication in vitro.

Bispecific antibodies can be used to redirect cytotoxic T cells to kill virus-infected cells, overriding the need for major histocompatibility complex restriction. We produced a bispecific antibody (3F12) which binds influenza virus M2 protein and the T-cell receptor and can redirect staphylococcal enterotoxin B-activated T cells to kill influenza virus-infected cells and inhibit virus replication in vitro.

Promotion of animal growth with a monoclonal antibody specific to growth hormone receptor.

A monoclonal antibody (mAb), designated 2C3, was raised against the growth hormone receptor (GHR) of rats. In a radioimmunoassay, 2C3 was found to compete with iodinated porcine GH (pGH) tracer for the binding to GHR, suggesting that GHR binding sites for pGH and 2C3 were identical or closely adjacent. The competition curve generated by 2C3 was identical to that generated by cold pGH, suggesting that the binding affinities of 2C3 and pGH to GHR were very similar. Administration of hypophysectomized rats with 2C3 resulted in the growth of these GH-deficient animals for a long period of time, mimicking the somatogenic effect of GH. However, this effect was abolished when 2C3 was injected into animals in the presence of exogenous GHR. A control mAb recognizing a GHR epitope distal from its binding site for GH failed to produce the growth in rats. Taken together, findings from the present study indicate that 2C3 is fully capable of engaging with GHR and subsequently triggering the growth response in rats. This mAb may also prove useful as a biologically active agonist for better understanding the initiation of the physiological process of GHR.

Staphylococcal enterotoxin B-activated T cells can be redirected to inhibit multicycle virus replication.

Cell-mediated immunity is a crucial part of recovery from virus infections. Adoptive transfer of T cells into infected animals is restricted by the need for Ag-specific and MHC-restricted T cells. One way to overcome these limitations is to use bifunctional Abs to redirect the T cells against virus-infected cells. We have demonstrated that bifunctional Abs can inhibit virus replication in the presence of activated T cells. To generate a large number of activated T cells in a short time, we tested the ability of the superantigen, staphylococcal enterotoxin B (SEB), to activate T cells. We demonstrate that SEB-activated T cells are effective killers when bridged to Fc receptor-bearing target cells using anti-CD3 Abs. SEB T cells can lyse virus-infected target cells in the presence of HHA6, a bifunctional Ab specific for the V beta 8 TCR product and the H1 hemagglutinin of influenza A/PR/8/34 virus. In addition, bifunctional Ab and SEB T cells can inhibit multicycle virus replication in vitro. In a conventional 4-h chromium release assay, SEB-activated CD8 T cells are efficient killers, whereas CD4 T cells are not. Yet both subpopulations have the ability to inhibit multicycle replication in vitro. Superantigens may represent a potent method for generation of effector cells for use in redirected immunotherapy protocols.

Promotion of animal growth with a monoclonal anti-idiotype specific to anti-porcine growth hormone antibody.

A monoclonal antibody (mAb), designated PS-7.6, was previously shown to enhance the growth-promoting activity of porcine growth hormone (pGH) in an experimental hypophysectomized (hypox) rat model. The long lasting effect of PS-7.6 was postulated to be a result of the induction of anti-idiotypic antibody (anti-id) in these treated animals. An attempt was made in this report to further explore this issue. It was demonstrated that mice following immunization with PS-7.6 were capable of producing anti-id in serum. The antibody titers of mice immunized with a mixture of PS-7.6 and pGH were much higher than that of those being immunized with PS-7.6 alone. A monoclonal anti-id, designated 2A6, was generated and found to recognize the intact PS-7.6 and its F(ab')2 fragment under non-reducing condition in Western analysis. However, it did not interact with reduced PS-7.6, suggesting the necessity of both H and L chains for the expression of a conformational idiotype. In radioimmunoassay, 2A6 competed with pGH for the binding to PS-7.6, but failed to do so with a control anti-pGH mAb recognizing a distinct pGH epitope from that of PS-7.6. Results from a biospecific interaction analysis which monitored the molecular interactions in a real-time fashion confirmed the facts that 2A6 specifically recognized the variable region of PS-7.6 and that the recognition was inhibited by the presence of pGH. Enzyme-linked immunosorbent assay provided further evidence to indicate that 2A6 bound to GH binding protein, i.e. the soluble GH receptor, and pGH prevented this interaction in a dose-dependent manner. The biological effect of 2A6 was evaluated in hypox rats and shown to promote the growth of these GH-deficient animals. Taken together, the present findings clearly demonstrate that 2A6 raised against a growth-enhancing anti-pGH mAb mimics pGH both conformationally and functionally.

Inhibition of multicycle influenza virus replication by hybrid antibody-directed cytotoxic T lymphocyte lysis.

Bifunctional antibodies with specificity for the TCR/CD3 complex as well as to a target cell-surface Ag can redirect CTL to lyse the target cell. We have produced a hybrid hybridoma, HHA6, which secretes bifunctional antibodies capable of redirecting CTL to lyse influenza virus-infected target cells. When added along with CTL to virus-infected cells, these antibodies very efficiently inhibit multicycle virus replication. Because hybrid hybridomas reassort H and L chains randomly we attempted to purify bifunctional antibody by using HPLC. The purification increased the potency of HHA6. This increase probably reflects an enrichment of the active species and the removal of inhibiting antibody species.

Antibody-mediated redirected cytolysis against murine melanoma cells in vivo.

Cytotoxic T lymphocytes can lyse Fc receptor-bearing target cells in vitro in the presence of anti-CD3 antibodies. This "redirected" lysis is not restricted by major histocompatibility antigens nor does it require nominal antigen recognition by the CTL. The efficacy of redirected lysis in vivo was assessed by comparing the survival of mice inoculated with a melanoma cell line transfected with the gene for mouse Fc receptor versus the untransfected melanoma when inoculated with syngeneic mouse CTL and anti-CD3 antibody. Survival was significantly higher in the animals given injections of Fc receptor melanoma, CTL, and anti-CD3 monoclonal antibody. Delayed injection or i.v. injection of CTL failed to significantly improve survival. Redirected lysis does not preclude the establishment of tumor immunity, since animals that rejected the tumor as a result of redirected lysis exhibited an increased resistance to reinoculation with tumor cells.

A novel technique for the production of hybrid antibodies.

Chemically linked bifunctional antibodies (heteroconjugates) composed of one antibody specific for the TcR/CD3 complex on cytotoxic T cells and another specific for viral antigens expressed on the surface of infected cells have been shown to redirect CTL to lyse virus-infected cells. Hybrid antibodies are bifunctional antibodies produced by the fusion of two hybridomas. As a result of their native dimeric immunoglobulin structure, hybrid antibodies may be more effective than heteroconjugates in vivo. We have developed a unique method for production of hybrid antibodies by infecting each hybridoma with a different retrovirus vector which confers resistance to either G418 or methotrexate. The hybridomas are fused and selected in medium containing both inhibitors. Using this technique, we have produced hybrid antibodies made up of one antibody combining site which binds to the TcR and a second specific for the hemagglutinin of X-31 influenza virus. We show that this hybrid antibody effectively mediates the lysis of virus-infected cells in the presence of appropriate CTL. Thus hybrid antibodies as well as heteroconjugates can redirect CTL to lyse virus-infected targets.

New method for titration of virus infectivity by immunostaining.

We have developed a new method for titration of viruses utilizing automated microtiter technology. Compared to existing methods such as plaque assay or hemagglutination titration of influenza virus, the new method offers distinct advantages in terms of time and effort. In addition because multiple replicates can easily be employed accuracy can be increased.

Sideways killing: the cytolysis of Fc receptor-bearing cells through bridging to cytolytic T lymphocytes by antibodies specific for the T-cell receptor-T3 complex.

Cytolytic T lymphocytes (CTL) cause cytolysis of foreign or virus-infected syngeneic cells when recognition of the target plus major histocompatibility complex (MHC) occurs via the T-cell receptor (TCR). The recognition event leads to intimate contact between the two cells and activation of the cytolytic effector. Activation and target cell lysis can also occur in the presence of antibodies to the TCR. This is accomplished by bridging the effector cell TCR to the target cell FcR by an anti-TCR monoclonal antibody (MoAb). Recent findings have placed the role of the FcR in this event in a questionable light. We confirm the importance of Fc gamma R by demonstrating that: (a) melanoma cells are killed by CTL clones in the presence of anti-TCR-CD3 antibodies only when the melanoma cells express the Fc gamma R on their surface; (b) native Ig, heat-aggregated Ig, or an Fc fragment from an antibody expressing the same isotype as the anti-TCR antibody can block the killing of high avidity Fc gamma RI-bearing cells mediated by anti-TCR antibody (F23.1); and (c) anti-Fc gamma R MoAb (2.4G2) and a truncated soluble Fc gamma RII molecule inhibit the killing of low-avidity Fc gamma RII-bearing cells mediated by anti-CD3 MoAb (145-2C11). Thus, we show that both high-avidity Fc gamma RI and low-avidity Fc gamma RII can mediate sideways killing depending upon the isotype of the anti-TCR antibody and the type of FcR present on the target cell surface.

Characterization of the fine specificity and antigen markers associated with the receptor of autoreactive T-cell hybridomas.

In this study we attempted to define the determinants on Ia molecules recognized by autoreactive hybridomas obtained from (C57BL/6 X BALB/c)F1 mice. The epitopes recognized by the T cells were characterized (a) using stimulating cells from various congenic and H-2 recombinant inbred strains and (b) by inhibition of activation with anti-Ia antibodies. Our hybridomas were strictly autoreactive and did not exhibit any alloreactivity, as is often observed for such cells. Our results show that more epitopes than previously believed are recognized by autoreactive T cells. One T-cell hybridoma (QW27.1) is unique in that it recognizes a hybrid F1 Ia determinant. Antigenic markers associated with the receptor of the T-cell hybridomas were studied with monoclonal antibodies (mAbs) specific for L3T4 and a V beta "idiotype". The results indicate that all Lyt 1.2 autoreactive T cells express L3T4 antigen in association with their receptor. One clone (QW64.14) expresses the V beta idiotype recognized by F23.1 monoclonal antibody. Moreover, this clone is activated by F23.1, linked to Sepharose 4B beads, which was believed previously to activate only Lyt 2+, L3T4 T cells. The supernatant of one clone (QW17.5) helps B cells to differentiate into antibody-producing cells without requiring direct contact with the autoreactive clone.

Methods for the selection and growth of antigen-specific cytolytic T lines and clones bearing a defined T cell receptor beta chain marker.

Murine cytolytic T lymphocyte (CTL) clones specific for type A influenza virus antigens were generated by in vitro stimulation with syngeneic virus-infected cells in the presence of T cell growth factor (TCGF). All CTL clones recognize viral determinants shared by PR8 and X31 influenza viruses in association with a class I antigen, coded either by the H-2K or H-2D end of the appropriate haplotype. All clones express the Lyt2 antigen marker. Two of five clones also express an antigenic determinant of the V beta chain of the T cell receptor (TCR) identified by F23.1 monoclonal antibody. To effectively generate F23.1+ and antigen-specific CTL clones, heterogenous CTL lines were expanded with F23.1 coated Sepharose beads in the presence of TCGF and then stimulated with PR8 virus-infected cells. Thus, both the proliferative activity to PR8 and the expression of the F23.1 marker was increased significantly. Alternatively, F23.1+ T cells were sorted from in vivo primed mice and expanded with PR8 virus-infected stimulator cells in the presence of TCFG. This F23.1+ T cell line exhibited antigen-specific cytotoxicity for PR8 virus-infected target cells. Additionally, in an 'FcR-focused killing' assay only the F23.1+ CTL line and F23.1+ clones lysed Fc receptor bearing target cells in the presence of F23.1 antibody. These findings indicate that antigen-specific and F23.1+ clones can be selected with high efficiency by alternating stimulation with influenza virus-infected cells and with F23.1-coated Sepharose beads or through the use of a cytofluorograph. The usefulness of antigen-specific and F23.1+ CTL clones and other possible strategies for their selection are discussed.