Immune tolerance induction to enzyme-replacement therapy by co-administration of short-term, low-dose methotrexate in a murine Pompe disease model.

Clinical investigations of recombinant human acid alpha-glucosidase for the treatment of Pompe disease often reveal the appearance of therapy-specific antibodies. These antibodies could potentially interfere with recombinant human acid alpha-glucosidase efficacy and induce immunological consequences. Several immunosuppressive agents, including methotrexate, mycophenolate mofetil and cyclosporin A with azathioprine, were evaluated for their potential to induce immune tolerance to recombinant human acid alpha-glucosidase. Methotrexate was the only agent that reduced recombinant human acid alpha-glucosidase-specific antibody responses in acid alpha-glucosidase knock-out mice. A 3-week, low-dose methotrexate regimen controlled recombinant human acid alpha-glucosidase-specific antibody levels throughout 8 months of weekly recombinant human acid alpha-glucosidase treatment. The success of this methotrexate regimen appears to require methotrexate administration within the first 24 h of recombinant human acid alpha-glucosidase treatment. In an attempt to understand the benefit of methotrexate within the first day of recombinant human acid alpha-glucosidase administration, the immune response 24 h following intravenous recombinant human acid alpha-glucosidase treatment was investigated. A consistent expansion of peritoneal B1 B cells was observed. Control over this B1 B cell response may be part of the complex mechanism of action of methotrexate-induced immune tolerance.

Methotrexate reduces antibody responses to recombinant human alpha-galactosidase A therapy in a mouse model of Fabry disease.

Therapeutic enzymes are often recognized as foreign by the immune system of patients undergoing enzyme replacement therapy. The antibodies that develop may alter pharmacokinetics and biodistribution of the therapeutic protein, may be able to neutralize the activity of the enzyme, or may cause immune reactions in certain patients. We have explored treatment regimens to reduce the antibody response to human alpha-galactosidase A (r-halphaGAL) in Fabry (alphaGAL knock-out) and normal BALB/c mice. A wide variety of treatment modalities were tested, including high dose tolerance induction, increased frequency of therapeutic doses and immunosuppressive drugs in combination with administration of enzyme. The most substantial effects were observed in mice injected intravenously with r-halphaGAL in combination with methotrexate (MTX), which significantly lowered r-halphaGAL-specific serum antibody levels. A short course of treatment with MTX was able to reduce antibody and spleen cell proliferative responses to long-term r-halphaGAL treatment. MTX was able to suppress the development of r-halphaGAL-specific IgG in antigen-primed mice. However, MTX was not effective in dampening robust ongoing antibody responses. These experiments provide a framework for the design of clinical protocols to prevent the drug-specific antibody responses of patients undergoing enzyme replacement therapy.

Prediction of obligatory exercise by exercise-related imagery.

Obligatory exercise is a compulsive behavior pattern in which exercise dominates daily life at the expense of other activities and lack of exercise produces withdrawal symptoms. This study examined the hypothesis that obligatory exercise is motivated similarly to eating disorders (cf. S. P. Coen & B. M. Ogles, 1993) and would be predicted by appearance-related imagery. Obligatory exercise (J. K. Thompson & L. Pasman, 1991) and exercise imagery (H. A. Hausenblas, C. R. Hall, W. M. Rodgers, & K. J. Munroe, 1999) were assessed before and after a 10-week exercise program in 243 adult exercisers. Regression analyses revealed that imagery accounted for 20% of the variance in obligatory exercise. Appearance-related imagery did not predict significantly obligatory exercise. Energy-related imagery was the strongest predictor. Obligatory exercise may not be as associated with appearance-related concerns as eating disorders, suggesting different motivational bases for these 2 behavioral patterns.

Major antigenic proteins of the agent of human granulocytic ehrlichiosis are encoded by members of a multigene family.

Western blot analysis of proteins from a cell culture isolate (USG3) of the human granulocytic ehrlichiosis (HGE) agent has identified a number of immunoreactive proteins, including major antigenic proteins of 43 and 45 kDa. Peptides derived from the 43- and 45-kDa proteins were sequenced, and degenerate PCR primers based on these sequences were used to amplify DNA from USG3. Sequencing of a 550-bp PCR product revealed that it encodes a protein homologous to the MSP-2 proteins of Anaplasma marginale. Concurrently, an expression library made from USG3 genomic DNA was screened with granulocytic Ehrlichia (GE)-positive immune sera. Analysis of two clones showed that they contain one partial and three full-length highly related genes, suggesting that they are part of a multigene family. Amino acid alignment showed conserved amino- and carboxy-terminal regions which flank a variable region. The conserved regions of these proteins are also homologous to the MSP-2 proteins of A. marginale; thus, they were designated GE MSP-2A (45 kDa), MSP-2B (34 kDa), and MSP-2C (38 kDa). The PCR fragment obtained as a result of peptide sequencing was completely contained within the msp-2A clone, and all of the sequenced peptides were found in the GE MSP-2 proteins. Recombinant MSP-2B protein and an MSP-2A fusion protein were expressed in Escherichia coli and reacted with human sera positive for the HGE agent by immunofluorescence assay. These data suggest that the 43- and 45-kDa proteins of the HGE agent are encoded by members of the GE MSP-2 multigene family.

Molecular cloning and sequencing of three granulocytic Ehrlichia genes encoding high-molecular-weight immunoreactive proteins.

Granulocytic Ehrlichia was isolated from canine blood obtained from animals challenged with field-collected Ixodes scapularis and propagated in HL60 cells. PCR primers specific for the 16S ribosomal DNA (rDNA) of the Ehrlichia genogroup comprising E. equi, E. phagocytophila, and the agent of human granulocytic ehrlichiosis (HGE) amplified DNA from extracts of these cells. Sequence analysis of this amplified DNA revealed that it is identical to the 16S rDNA sequence of the HGE agent. A genomic library was constructed with DNA from granulocytic Ehrlichia and screened with pooled sera from tick-challenged, granulocytic Ehrlichia-infected dogs. Several clones were isolated and sequenced. Three complete genes encoding proteins with apparent molecular masses of 100, 130, and 160 kDa were found. The recombinant proteins reacted with convalescent-phase sera from dogs and human patients recovering from HGE. This approach will be useful for identifying candidate diagnostic and vaccine antigens for granulocytic ehrlichiosis and aid in the classification of genogroup members.

Induction of antigen-specific killer T lymphocyte responses using subunit SIVmac251 gag and env vaccines containing QS-21 saponin adjuvant.

Subunit vaccines based on recombinant proteins have proved useful for inducing antibody responses and they are safe for widespread use because they do not contain any live components. Unfortunately, they do not typically induce the types of cell-mediated immune responses required to control viral pathogens; specifically, they do not induce CD8+ cytotoxic T lymphocyte (CTL) responses. To increase the immunogenicity of recombinant proteins, we have used the QS-21 saponin adjuvant in subunit vaccine formulations. In the current study, experimental subunit vaccine formulations containing recombinant p55gag or gp120env proteins from the mac251 strain of the simian immunodeficiency virus (SIVmac251) and the QS-21 adjuvant were used to immunize rhesus macaques. These formulations induced SIV gag- or env-specific cellular immunity that was detectable in vitro and included killer cell activity. The induction of killer cells required prior vaccination and the responses were antigen specific for the immunogens contained in the vaccine formulations. Autologous target cells were required to detect these responses, suggesting genetic restriction, and effector cells appeared to be present in both the CD4+ and CD8+ T lymphocyte subpopulations. These data suggest that the vaccine-induced killer cell activity that was detected was mediated by both CD4+ and CD8+ lymphocytes. Despite the presence of these killer cells, all of the animals became infected with the SIVmac251 on experimental challenge. These findings demonstrated that antigen-specific killer cell responses could be induced by a subunit vaccine formulated with the QS-21 saponin adjuvant. The characteristics of the responses suggested that the effector cells were T lymphocytes, expressing either CD4 or CD8.(ABSTRACT TRUNCATED AT 250 WORDS)

Use of fixed autologous stimulator cells to correctly present human immunodeficiency virus type 1 viral peptides to nonhuman primate lymphocytes in proliferation and cytotoxic T-lymphocyte assays.

Autologous, virus-transformed lymphoblastoid cell lines were established by using peripheral blood lymphocytes from rhesus monkeys that were previously immunized with recombinant human immunodeficiency virus type 1 strain IIIB glycoprotein 160. These autologous cell lines were used to present human immunodeficiency virus type 1 viral antigens in a processed and cell-associated manner to T lymphocytes. This was accomplished by either infecting the cells with recombinant vaccinia viruses or pulsing them with synthetic peptides and then subjecting them to a mild fixation step with glutaraldehyde. Fixed antigen-presenting cells were then used as stimulator cells in vitro to measure cell-mediated immune responses. Both the vaccinia virus-infected and peptide-pulsed autologous cells stimulated antigen-specific cellular proliferative responses. The magnitude of the responses correlated with the immunization histories of the animals and other measures of immunity, such as antibody titers. Autologous vaccinia virus-infected cells were also capable of inducing the in vitro maturation of CD4+ and CD8+ precursor cytotoxic T lymphocytes into antigen-specific mature cytotoxic T lymphocytes. The use of stimulator cells to present viral peptides in a cell-associated manner appeared to be a very sensitive and versatile manner in which to measure cell-mediated immune responses with peripheral blood lymphocytes from nonhuman primates. It is likely that a similar approach will function with peripheral blood lymphocytes from humans.

Pharmacy services in family medicine residencies. Survey of clinics associated with Canadian residency programs.

Surveys were mailed to 82 family medicine clinics associated with residency programs in Canada to ascertain the extent, if any, of pharmacy involvement in the programs. Eight of the 58 (13.8%) usable returns had pharmacists directly involved. They provided pharmacy-based services, offered clinical services, and participated in research.

Use of particulate forms of protein antigens to increase the sensitivity of antigen-specific proliferation assays.

Different forms of recombinant HIV-1 gp160 and tetanus toxoid were adsorbed onto latex microspheres and this particulate form of the proteins was used to measure antigen-specific proliferation in vaccinated rhesus macaques. Proliferative responses to proteins bound to microspheres were significantly greater and allowed for the detection of antigen-specific responses that were not detected using soluble proteins. The responses were antigen-specific and required prior immunization of the animals. Additionally, the presence and magnitude of the proliferative responses was associated with antibody responses to the same proteins suggesting the results were representative of in vivo responses and that the assay format did not induce in vitro artifacts.

Saponin adjuvant induction of ovalbumin-specific CD8+ cytotoxic T lymphocyte responses.

The ability of a saponin adjuvant, QS-21, to induce OVA-specific, class I MHC Ag-restricted CTL was investigated using different forms of soluble OVA and OVA adsorbed onto alum as immunogens. C57BL/6 mice were immunized with soluble native or denatured OVA in formulations that contained increasing quantities of QS-21, and CTL responses were measured using EL4 and E.G7-OVA cells as targets and splenic mononuclear cells as effectors. Ag-specific CTL responses were produced but only if the QS-21 adjuvant was used. Similar responses were induced using alum-adsorbed OVA when mixed with the QS-21 adjuvant but not when used alone. The CTL were specific for an epitope present on the OVA258-276 synthetic peptide, which contains the dominant CTL epitope recognized by C57BL/6 mice. The CD8+ subpopulation of lymphocytes in immune mice was not increased in spleens but increased significantly in vitro after culture with soluble OVA. The CTL activity of splenic mononuclear cell preparations was totally destroyed by treatment with mAb specific to the CD8 Ag plus complement. The ability of the QS-21 adjuvant to induce class I MHC Ag-restricted CTL after immunization with soluble proteins is a characteristic unique to saponin adjuvants.