Temperature alters lipopolysaccharide-induced cytokine secretion by RAW 264.7 cells.

Local and systemic temperature change is associated with the immune response to infection, but the role of temperature remains poorly understood. To study the effect of temperature on macrophage activation by lipopolysaccharide (LPS), RAW 264.7 cells were incubated with LPS at different temperatures and secretion of three cytokines was measured. Incubation at 31 degrees C increased tumour necrosis factor (TNF) secretion when compared with 37 degrees C, while cells exposed at 39 degrees C secreted less TNF. Interleukin-6 (IL-6) secretion was less at 31 degrees C than at 37 degrees C and remained unchanged at 39 degrees C. Interleukin-10 secretion was depressed on either side of 37 degrees C. Only IL-6 secretion was sensitive to preincubation temperature effects. The kinetics of cytokine secretion and steady-state mRNA analysis indicated potentially different mechanisms of temperature regulation for TNF and IL-6.

Effect of peptide-carrier coupling on peptide-specific immune responses.

Synthetic peptides are covalently linked to immunogenic carrier proteins to enhance the anti-peptide immune response. To investigate whether the method of conjugation influences the immune response, we evaluated two distinctly different choices of linker for a peptide-carrier construct. HPG-30, a synthetic peptide derived from the p17 gag protein of human immunodeficiency virus 1, was covalently linked to keyhole limpet hemocyanin by either glutaraldehyde or a maleimide ester. Glutaraldehyde linkage enhanced the anti-peptide antibody and native protein response compared to maleimide. The maleimide-linked conjugate was more effective at inducing a peptide-specific cellular response. Thus, manipulation of the conjugation method can modify the magnitude and character of the immune response to a synthetic peptide vaccine.

The predominance of beta (CC) chemokine transcripts in idiopathic inflammatory muscle diseases.

Cytokines and chemokines that upregulate major histocompatibility complex class I antigens, recruit lymphocytes, and enhance T-cell-mediated myotoxicity may be important in the pathogenesis of dermatomyositis and polymyositis. We searched for cytokine and chemokine transcripts in inflammatory muscle specimens from 14 newly diagnosed or treated patients. Control specimens from six patients without inflammatory muscle disease were analyzed for transcripts of interleukins-1 beta, -2, -4, -6, -10, and -15, and interferon-gamma, tumor necrosis factor-alpha, transforming growth factor-beta 1, macrophage inflammatory proteins-1 alpha and -1 beta (MIP-1 alpha, MIP-1 beta), and the chemokine "regulated on activation, normally T expressed and secreted" (RANTES). Surprisingly, the proinflammatory and lymphocyte cytokines were detected only sporadically in myositis muscle specimens, and their presence did not correlate with disease activity or treatment status of the patient. In contrast, MIP-1 alpha and MIP-1 beta were detected in 13 and 6 myositis biopsies, respectively, and RANTES, another beta (CC) chemokine, was detected in eight myositis biopsies. This study and other reports of low levels of acute-phase cytokines in myositis patients suggest that the proinflammatory cytokines do not play a major role in ongoing muscle damage. The CC chemokines studied here, in particular MIP-1 alpha, might contribute to ongoing muscle inflammation, and the pathogenesis of inflammation in myositis may follow a previously unrecognized pathway.

Adjuvant properties of montanide CSA 720 with a recombinant HIV P17 gag protein and synthetic peptide antigens.

The adjuvant properties of Montanide CSA 720 were assessed in a comparison with alum. BALB/c mice were immunized with recombinant HIV-1 gag protein p17 administered in either of the two adjuvants. The serum antibody response to p17 with Montanide CSA 720 appeared faster and reached a higher titre than with alum. The serum antibody response to p17 in Montanide CSA 720 was further characterized by a higher titre antibody directed against a 30 amino acid segment from the entire protein. The Montanide CSA 720 adjuvant was sufficiently strong to induce an antibody response against a weak synthetic peptide immunogen after two immunizations, while immunization with the peptide in alum generated no detectable serum antibody. The p17-specific proliferative response of splenocytes from animals immunized with recombinant protein in either adjuvant was similar.

Specific antibody responses to synthetic peptides of HIV-1 p17 correlate with different stages of HIV-1 infection.

Antibodies were determined against five synthetic peptides (epitopes) of HIV-1 p17 in the sera of an immunologically and clinically well-characterized cohort (N = 292) of HIV-1 seronegative and HIV-1 seropositive high-risk homosexual men, HIV-1 seropositive i.v. drug abusers (IVDA), and AIDS patients. The synthetic peptides, representing the entire HIV-1 p17 protein sequence were: HGP-33 (aa 1-33), HGP-19 (aa 34-52), HGP-35 (aa 51-85), HGP-30 (aa 85-114), and HGP-17 ala (aa 114-131). The presence of one or more peptide-specific antibodies in the sera of all of the HIV-1 p17-positive subjects indicated that all five peptides contain B-cell epitopes. No antibodies were found in the sera of heterosexual controls, HIV-1 seronegative high-risk men, or asymptomatic HIV-1 seropositive but p17 antibody-negative study subjects. Significant differences in antibody recognition profiles to the peptide epitopes were found among the various study groups. A significantly higher proportion of HIV-1 seropositive IVDA had antibodies specific to HGP-17 ala (aa 114-131), HGP-35 (aa 51-85), and HGP-33 (aa 1-33) compared to the HIV-1 p17-positive asymptomatic homosexuals. The epitope-specific antibody responses reflected the clinical status of the HIV-1-infected study subjects, and declined to nondetectable levels as the patient progressed to ARC/AIDS. This decline preceded by several months the reduction in the antibody titer against the intact HIV-1 p17 and p24 proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Preclinical and clinical studies on immunogenicity and safety of the HIV-1 p17-based synthetic peptide AIDS vaccine--HGP-30-KLH.

Immunization with a synthetic HIV-1 p17 peptide analog (HGP-30; aa 85-115 of HIV p17), coupled to a carrier protein (KLH, keyhole limpet hemocyanin) given with alum as the adjuvant induces antibodies which cross-react with both HGP-30 and HIV p17 and clones of cytotoxic and helper T-cells which recognize HGP-30 and HIV p17. Proliferation of lymphocytes in response to HGP-30 has been observed in mice, in HIV-infected individuals and in healthy HIV-seronegative volunteers vaccinated with the p17-based synthetic peptide construct. Cytotoxic T-cell responses against EBV transformed, recombinant p17 pulsed targets were observed using antigen-expanded PBLs from HGP-30-KLH immunized individuals. These results are consistent with predictions that the HGP-30 domain of HIV p17 contains both T- and B-cell epitopes that are recognized by animals and humans. In preclinical toxicology studies in animals and in initial clinical trials in humans the synthetic peptide construct (HGP-30-KLH/alum) has been shown to be safe. This paper summarizes the preclinical immunogenicity and safety data for HGP-30-KLH and presents the initial results from the first Phase 1 clinical trial.

Monoclonal antibody definition of multiple polymorphic epitopes on human leukocyte antigen-DRw52.

Monoclonal antibodies have revealed complexity within the human leukocyte antigen class II antigens. We have studied epitopes present on a DR3 homozygous B-lymphoblastoid cell line using five polymorphic monoclonal antibodies produced and characterized in our laboratory. Serological analysis on a panel of B-cell lines revealed that the antibodies have different, but related, specificities (NDS9-anti-DR3, NDS10-anti-DR5, less than 3, less than w6, NDS11-anti-DR3, 5, w6, NDS12-anti-DR3, 5, w6, w8, NDS13-anti-DR3, 5, w6, w8+). Competitive radioimmunoassays and two-dimensional gel analyses demonstrated that whereas the epitopes recognized by the broadly reactive antibodies NDS10, 11, 12, and 13 reside on the same molecule, the epitope detected by NDS9 is present on a molecule with different electrophoretic mobility. Thus, using polymorphic monoclonal antibodies, we have defined multiple epitopes associated with DR3, which have different distributions at the population level.

A monoclonal antibody recognizing HLA-DR2 on malignant and activated cells.

A monoclonal antibody, designated NDS15.38, which recognizes a polymorphic determinant of HLA-DR, was produced from a fusion in which mice were immunized with the human B lymphoblastoid cell line GIR2 (HLA type A1, B8, 27, Cw2, DR2,7). NDS15.38 functions efficiently as an affinity column and purifies a two-chain complex of molecular weight 33 000 and 30 000 under reducing conditions. The monoclonal antibody reacts with HLA-DR2-positive B lymphoblastoid cell lines and B lymphocytes from patients with chronic lymphatic leukemia in an indirect radioactive binding assay. However, NDS15.38 does not appear to react with peripheral blood B lymphocytes from normal individuals. Using a peroxidase staining technique, NDS15.38 was shown to react with phytohemagglutin (PHA)-stimulated lymphocytes and with apparently activated B cells in the germinal centers of lymph nodes from individuals who were tissue typed as HLA-DR2. Thus it appears that NDS15.38 recognizes a polymorphic determinant of HLA-DR on malignant and stimulated cells, but not on resting cells.

Monoclonal antibody to a human leukocyte-specific membrane glycoprotein probably homologous to the leukocyte-common (L-C) antigen of the rat.

The monoclonal antibody (F 10-89-4) described in this study recognizes an antigen which by quantitative absorption analysis is absent from human brain, kidney, liver, heart, erythrocytes, platelets and normal serum, but is present on spleen, lymph node, chronic lymphatic leukemia cells, bone marrow, thymus and granulocytes at a ratio of 1:1:0.8:0.3:0.1, respectively. Analysis with the fluorescence-activated cell sorter showed that 100% of thymocytes, lymph node lymphocytes, blood mononuclear cells and granulocytes carry the antigen, while 83% of bone marrow cells are positive. There was marked heterogeneity in the amount of labeling of thymocytes, with 3 major peaks. There was also heterogeneity of labeling of blood mononuclear cells and lymph node lymphocytes, with a weakly staining hump containing approximately 20% of the cells in the case of lymph node lymphocytes. Double labeling experiments demonstrated that the weakly staining cells of blood and lymph node were B lymphocytes, while frozen sections of thymus showed that the antigen was expressed most weakly in subcapsular cortical thymocytes, and most strongly on medullary thymocytes. Biochemical studies established that the antigen bound to lentil lectin columns, and sodium dodecyl sulfate polyacrylamide gel electrophoresis studies using NaB3H4-labeled blood mononuclear cells established that the antigen was a major glycoprotein of lymphocytes, and that its molecular weight was in the region of 190000 to 215000.

Monoclonal antibody to a human brain-granulocyte-T lymphocyte antigen probably homologous to the W 3/13 antigen of the rat.

The monoclonal antibody (F 10-44-2) described in this report recognizes an antigen which by quantitative absorption analysis is found predominantly on spleen, lymph node, bone marrow, thymus, granulocytes and brain, the amount of antigen on these tissues being approximately the same within a factor of 2 or 3. Analysis with the fluorescence-activated cell sorter showed that 29% of thymus cells, 61% of bone marrow cells, 95% of blood mononuclear cells, 98% of lymph node lymphocytes and 100% of granulocytes carried the antigen. With blood mononuclear cells and lymph node lymphocytes, there were two distinct peaks, with one peak labeling very weakly. Double labeling experiments established that the weakly labeled peak contained the B lymphocytes. Studies on frozen sections of thymus established that positive thymocytes were found only in the medulla indicating that the antigen appears late in T lymphocyte maturation. The lymphatic nodules (B lymphocyte areas) of spleen and lymph node appeared virtually negative on frozen sections showing that there was too little antigen on the B lymphocyte surface for confident detection by fluorescence microscopy. Sodium dodecyl sulfate polyacrylamide gel eletrophoresis of NaB3H4-labeled blood mononuclear cells established that the antigen was a major glycoprotein of the leukocyte membrane and that its mol. wt. was 105000. This antigen shows a striking similarity in biochemistry and tissue distribution to the W 3/13 antigen of the rat and is likely to be the human homologue of this antigen.