Adhesion co-receptor expression and intracellular signalling in HIV disease: implications for immunotherapy.

OBJECTIVES: To investigate, in lymphocytes from HIV-1-infected individuals, the phenotypic expression of various adhesion co- or counter-receptors [lymphocyte function-associated antigen (LFA)-3, LFA-1 and intercellular adhesion molecule (ICAM)-1] involved in providing the co-stimulatory signal through the phospholipase C-gamma pathway in relation to inositol polyphosphate metabolism. DESIGN AND METHODS: Cell adhesion molecule profiles of peripheral blood lymphocytes (PBL) from 39 HIV-1-infected individuals at various stages of infection and 20 healthy laboratory controls were studied using flow cytometry. These were studied in 14 patients with late-stage disease in conjunction with their inositol polyphosphate metabolic profiles measured by high performance liquid chromatography. Levels of HIV-1 present in cell lysates were concurrently measured by a p24 antigen capture assay. In addition, the effects of a specific anti-ICAM-1 antisense oligonucleotide on the intracellular phosphatase activities of lymphocytes from a separate group of eight HIV-1-infected individuals were examined. RESULTS: The expression of LFA-1, a beta 2 integrin, was upregulated among patient PBL in parallel with disease progression, whereas that of LFA-3 (CD58) was found to be significantly reduced among the CD4+ lymphocyte subset in all stages of infection. The 5-phosphatase activity, which we previously observed to be defective in HIV disease, was found to correlate linearly with the expression of both LFA-1 and its ligand, ICAM-1. Treatment of patient lymphocytes with an antisense oligonucleotide, which reduced the cell surface expression of ICAM-1 by blocking the translation of its mRNA, resulted in further reduction of intracellular phosphatase activities. CONCLUSIONS: Our results suggest a pivotal role for adhesion co- and counter-receptors in influencing lymphocyte signalling and hence cellular response to recall antigens in HIV-1-infected individuals.

Persistent immune complexes and abnormal CD4/CD8 ratios in HIV infection.

We assessed the immunopathologic role of circulating immune complexes in human immunodeficiency virus infection by evaluating the data base and the serum bank of the San Francisco Men's Health Study, a longitudinal clinical and epidemiological investigation conducted since 1983. A group of 4,276 sera from 1,023 (including 811 homosexual/bisexual) men were tested for circulating immune complexes. We used a modification of the commercially available enzyme immunoassay test, based on monoclonal anti-C1q antibodies coupled to the solid phase, for capturing circulating immune complexes from the test serum, followed by detection of circulating immune complexes with either anti-IgG or with anti-IgM probes. Although persistent IgM and IgG circulating immune complexes were most frequently encountered in human immunodeficiency virus-seropositive homosexual/bisexual men, they were not an indicator of disease progression as assessed by abnormalities in the absolute numbers or ratios of CD4- and CD8-positive T cells, or clinical signs and symptoms of AIDS/ARC.

Nucleic acid immunization: concepts and techniques associated with third generation vaccines.

A radical change in vaccine methodology arrived nine years ago with the advent of nucleic acid immunization. Aspects such as plasmid design, gene selection, the use of immunostimulatory complexes and clinical trials are discussed in this review. Furthermore, concepts and protocols involved in the construction, evaluation and immunization of a DNA vaccine have been examined as new strategies to enhance this technology continues to grow.

Design, development, and interpretation of HIV neutralization assays.

In developing therapeutic reagents for the control of HIV infection, it is necessary to screen candidate products in vitro for their ability to reduce or neutralize viral infection. Although the current literature describes numerous neutralization assays, no universally accepted standards have been adopted. In this article, we briefly review the available neutralization assays and describe in detail the methods we have selected in our laboratory for the screening and characterization of reagents with potential anti-HIV properties. After evaluating many different technical protocols and experimental procedures, we have found the syncytium inhibition and syncytial focus assays to be particularly useful and have found p24 gag antigen production to be an excellent objective measure of HIV infection under a variety of conditions. These assays proved reproducible and sensitive and are suitable for use in the majority of laboratories.

Anti-idiotypic antisera raised against monoclonal antibody specific for a p24 gag region epitope detects a common interspecies idiotype associated with anti-HIV responses.

One potential strategy for the control of human immunodeficiency virus (HIV) infection is immune network manipulation using anti-idiotypic antibodies: this study was undertaken to demonstrate experimentally the potential of such an approach which, in a more highly evolved form, could be used for the treatment of the acquired immune deficiency virus (AIDS) and related disorders. Anti-idiotypic antibodies were generated in rabbits against a murine monoclonal antibody identifying an epitope on the p24 gag core protein of HIV. After extensive absorption on affinity columns to remove isotype- and allotype-specific antibodies, the purified anti-idiotypic antibody preparation was shown to have specific complementarity with the immunizing mouse monoclonal antibody. This anti-idiotypic antibody was also shown to recognize a common idiotype associated with HIV-specific antibodies from both humans and chimpanzees infected with the AIDS virus. In addition a group of rats immunized with the anti-Id responded with significant antibody titers to recombinant derived p24 gag. These data indicate that at least a subpopulation of these polyclonal anti-Id antibodies structurally mimics an HIV gag region epitope and suggest that immunoregulation by anti-idiotypic antibodies may have therapeutic utility for the AIDS epidemic.

Long-term observation of baboons, rhesus monkeys, and chimpanzees inoculated with HIV and given periodic immunosuppressive treatment.

Baboons, rhesus monkeys, and chimpanzees were injected with the human immunodeficiency virus (HIV) and monitored for up to 4 years. Various immunosuppressive regimens were used during this time in attempts to induce development of the acquired immune deficiency syndrome (AIDS). No infectious virus was recovered or anti-HIV antibodies detected in the baboons and rhesus monkeys. Virus has been recovered from lymphocyte cultures of all five of the chimpanzees at intermittent periods following inoculation. The chimpanzees developed anti-HIV antibodies from 1 to 5 months after virus inoculation and had circulating antibodies that neutralized HIV. All the infected animals were capable of in vitro lymphocyte blastogenic responses to recombinant envelope and core HIV antigens. Despite immunosuppressive therapies and evidence of some immunologic abnormalities, none of the five chimpanzees has yet developed AIDS or a related disorder.

Desensitization of the inflammatory response in humans: changes in response to cardiopulmonary bypass.

Although circulating levels of interleukin 8 (IL-8), a potent pro-inflammatory chemokine, and many other inflammatory mediators increase in response to cardiopulmonary bypass, only a small proportion of patients develop a clinically significant systemic inflammatory response. The natural mechanisms that control the inflammatory response are poorly understood. To investigate the role of IL-8 in a human inflammatory model, 15 adult patients undergoing cardiopulmonary bypass for elective coronary artery bypass grafting were studied. Following reperfusion, plasma IL-8 levels increased significantly from 58 pg/mL (pre-bypass) and 66 pg/mL (after 20 min of bypass) to 98 pg/mL (p = .02 and .04, respectively), but this was accompanied by a concomitant threefold decrease in the IL-8 binding affinity of circulating neutrophils (Dissociation constant (KL) post-reperfusion/KL pre-bypass = 3.2; KL post-reperfusion/KL after 20 min of bypass = 2.8). IL-8-triggered release of myeloperoxidase and elastase by peripheral blood neutrophils ex vivo was also down-regulated following reperfusion. There were no significant changes in beta 2 integrin expression or inositol polyphosphate metabolism of peripheral blood neutrophils. These changes in receptor affinity and neutrophil responsiveness to IL-8 may represent an important in vivo regulatory mechanism which serves to prevent excessive tissue injury from inflammatory triggers.

Delivery systems for molecular vaccination.

Vaccination is one of the medical success stories of the 20th century, however, there are many diseases for which no prophylactic regimes are available. A major hindrance that has prevented the development of effective mass immunization programs is the inability to induce an appropriate, protective, immune response. For example, for vaccines against intracellular pathogens there is a requirement for cell-mediated immunity as characterized by cytolytic T-lymphocyte activity. However, such a response can be extremely difficult to elicit, especially those employing recombinant, soluble protein subunits. This deficiency is due to the inability of these antigens to access the machinery of the appropriate antigen-processing pathway. Following an improved understanding of the mechanisms underlying such processing, as well as the realization that delivery systems can affect, quantitatively and qualitatively, the resulting immune response, the last decade has witnessed an intense research effort in this field. In this article we will review the major developments in the area of antigen delivery as related to vaccination.

The ultrastructure of tomatine adjuvant.

The tomatine adjuvant, consisting of tomatine, n-octyl-beta-D-glucopyranoside, phosphatidylethanolamine, cholesterol, and ovalbumin, has recently been shown to potentiate the immunogenicity of protein antigen and elicit cytotoxic T-lymphocyte responses in immunized animals. The physicochemical properties of tomatine adjuvant have not been characterized. The aim of this study was to examine the microstructure of this complex formulation, as directly related to its physicochemical properties. To elucidate the micromorphology of this system, the tomatine adjuvant was separated by isopycnic ultracentrifugation, followed by freeze fracturing and examination by transmission and scanning electron microscopy. The adjuvant mixture was shown to be composed of several micro- and nano-structures. The major fraction obtained from isopycnic separation was shown to consist of flaky needle-like microcrystals, approximately 80-160 nm in width and 2-4 microm in length. The tomatine crystals alone in 0.9% NaCl, on the other hand, were shown to be elongated hollow tubular crystals of hundreds of nanometers up to a few microns in length, along which n-octyl-beta-glucopyranoside was speculated to serve as a seeding microtemplate for gel crystallization of protein complexes. Indented marks within the gel phase were observed in the freeze fractured replicas of the adjuvant, suggesting that protein complexes may have been crystallized or precipitated within the gels. Several other forms of micro- and nano-structures were also observed, showing multiple-dispersion features with gel characteristics. The presence of gel crystalline and multiple-dispersed phases is postulated to contribute to the sustained immunopotentiation effect of tomatine adjuvant.

Autoantibodies and overlap syndromes in autoimmune rheumatic disease.

Many patients diagnosed with autoimmune rheumatic disease cannot be categorised easily into one of the established clinical entities such as systemic lupus erythematosus, dermatomyositis, or systemic sclerosis. The term "overlap syndrome" has been increasingly used to identify such patients and is useful in terms of clarifying prognosis and facilitating disease management. This article reviews overlap syndrome in autoimmune rheumatic disease, with particular emphasis on the associated serological markers.

Antiperinuclear factor in Sjögren's syndrome in the presence or absence of rheumatoid arthritis.

Serum from 91 patients with Sjögren's syndrome (SS) was examined for antiperinuclear factor (APF). Nine out of 29 patients with SS alone (31.0%) were positive as opposed to 27 out of 40 with SS + rheumatoid arthritis (67.5%) and to 3 out of 22 with SS + other autoimmune disorders (13.6%). A clear relationship was established between APF and agglutinating or non-agglutinating rheumatoid factor.

Pathogenic mechanisms in psoriatic arthritis.

Psoriatic arthritis is an inflammatory arthritis which can develop in some psoriasis patients. The absence of a serological test means that diagnosis must be based on consideration of clinical indices. This article summarizes the inflammatory mediators and immunological events which characterize psoriatic arthritis and examines evidence for involvement of an infectious agent in its aetiology.

Can soluble antigens induce CD8+ cytotoxic T-cell responses? A paradox revisited.

Cytotoxic T lymphocyte (CTL) induction usually follows processing of antigens via endogenous pathways before presentation on the cell surface in association with MHC class I molecules. Soluble antigens do not, in general, induce specific CTL responses. Here, Syamal Raychaudhuri and John Morrow suggest that a novel adjuvant formulation can elicit CTL responses to soluble antigens and discuss the implications for vaccine development.

Murine monoclonal anti-idiotope antibody breaks unresponsiveness and induces a specific antibody response to human melanoma-associated proteoglycan antigen in cynomolgus monkeys.

The mouse monoclonal antibody MEM136 (mAb1) is directed against an epitope on human melanoma-associated proteoglycan antigen (MPG). This epitope is also present on various normal human and subhuman tissues. A monoclonal murine anti-idiotope (anti-Id) antibody (mAb2), designated I-Mel-2, was generated against MEM136 and used as a surrogate antigen for the MPG molecule. I-Mel-2 was tested in cynomolgus monkeys (Macaca fascicularis) for its ability to induce anti-MPG humoral responses. All monkeys immunized with Ab2 developed specific anti-anti-idiotype (Ab3) responses that were capable of inhibiting binding of Ab2 to Ab1. Furthermore, I-Mel-2 immune monkey serum contained anti-MPG antibodies (Ab1') that bound to MPG-positive but not to MPG-negative melanoma cell lines. Monkeys immunized with Colo38 melanoma cells (membrane-bound MPG antigen) did not contain anti-MPG antibodies that inhibited the binding of two distinct anti-MPG mAb 125I-labeled MEM136 or 125I-labeled 225.28 to Colo38 cells. The induction of anti-MPG responses in monkeys did not cause any apparent side effects in animals, despite the fact that the MPG antigen is expressed by many normal tissues. The affinity-purified, I-Mel-2 idiotype-specific, Ab3 immunoprecipitated MPG antigen from melanoma cells. Furthermore, the I-Mel-2-induced Ab3 inhibited melanoma cell invasion in an in vitro assay, implying that these antibodies have biological significance.

Methyl prednisolone pulse therapy in the treatment of systemic lupus erythematosus.

Twenty patients with active systemic lupus erythematosus (SLE) were treated with methyl prednisolone pulse therapy (MPPT) and followed up for up to 24 weeks (mean 18 weeks). Beneficial effects of MPPT were observed principally on arthralgia, pleuritic pain, vasculitic skin rash, pyrexia, and lymphadenopathy. The serological tests showing the most improvement were ds DNA binding and the serum C3 level. MPPT was found to be both safe and easy to administer. It may be of value in treating patients with SLE whose disease is not controlled by moderate doses of corticosteroids and may also enable the dose of maintenance corticosteroids to be reduced appreciably.

Helicobacter pylori vaccine strategies--triggering a gut reaction.

Helicobacter pylori causes peptic ulcer disease and some forms of gastric cancer; it is one of the most common chronic bacterial infections of humans. Although several prototype protein-based vaccines have shown promising results, they have not cleared infection and/or prevented reinfection. Nucleic acid vaccination offers a useful alternative to protein immunization, especially now that two complete H. pylori genome sequences are available, and facilitates the selection of antigenic targets.