Altered intracellular expression of the chemokines MIP-1alpha, MIP-1beta and IL-8 by peripheral blood CD4+ and CD8+ T cells in mild allergic asthma.

BACKGROUND: The ability of chemokines to regulate Th1 and Th2 responses suggests a role in the pathogenesis of atopic disorders such as allergic asthma where Th2 response dominance has been observed. Although the impact of allergic asthma on local chemokine production in the lung has been the subject of investigation, little is know about the influence of disease progression on peripheral chemokine production. We now report use of whole blood culture and flow cytometry to assess the influence of mild allergic asthma on peripheral T-cell chemokine expression. METHODS: Study participants included patients with mild allergic asthma (n = 7) and nonasthmatic controls (n = 7). Following in vitro stimulation of peripheral venous blood with phorbol 12-myristate acetate (PMA) and ionomycin, flow cytometry was used to estimate the percentage of CD4+ and CD8+ T cells producing a number of chemokines, including macrophage inflammatory proteins MIP-1alpha and MIP-1beta, RANTES (regulated on activation, T-cell expressed and secreted), monocytic chemotactic protein-1 (MCP)-1, and interleukin (IL)-8, or the cytokines interferon (IFN)-gamma and IL-4. Serum levels of MIP-1alpha, MIP-1beta, RANTES, MCP-1, IL-8, IFN-gamma and IL-4 were also assessed by quantitative ELISA. RESULTS: Intracellular expression of MIP-1beta by CD4+ and CD8+ T cells from allergic asthmatics was significantly reduced in comparison to that observed for nonasthmatics (median = 2.29% (1.75-3.50) vs 4.57% (3.38-6.64), P = 0.05; 14.20% (13.18-17.88) vs 44.10% (30.38-48.70), P = 0.01). Similarly, intracellular expression of MIP-1alpha by CD8+ T cells from allergic asthmatics was also significantly lower (3.67% (1.17-5.42) vs 17.10% (4.97-20.43), P = 0.05). Conversely, IL-8 expression by both CD4+ and CD8+ T cells from allergic asthmatics demonstrated significant enhancement (9.93% (7.77-11.28) vs 4.14% (3.61-7.11), P = 0.05; 8.40% (6.97-10.04) vs 4.98% (3.37-6.08), P = 0.05). Examination of intracellular IFN-gamma and IL-4 revealed no significant difference in the expression of either cytokine by CD4+ T-cells from allergic asthmatics and nonasthmatics. In contrast, expression of IFN-gamma was significantly reduced in CD8+ T-cells from allergic asthmatics (24.60% (21.08-32.50) vs 48.40% (41.50-55.28), P = 0.01). CONCLUSIONS: The occurrence in mild allergic asthma of peripheral T-cell chemokine expression suggestive of a diminished Th1 response, coinciding with marginal change in cytokine profiles indicative of a Th2 response bias, confirms the importance of chemokine involvement in the etiology of allergic asthma. The ability to use whole blood culture to estimate chemokine expression in T cell subsets may ultimately provide a practical means to evaluate disease status and to monitor early intervention therapies which target chemokines.

Long-term evaluation of T-cell subset changes after effective combination antiretroviral therapy during asymptomatic HIV-infection.

Demonstration of long-lived HIV-reservoirs resistant to the effects of combination antiretroviral therapy raises concern over the ability of treatment to maintain long-term beneficial alterations in T-cell subset composition. To address this issue, we have examined the effect of antiretroviral therapy on T-cell subset change during early HIV-infection in a 2-year prospective open-label trial composed of treatment-naive asymptomatic HIV-infected patients with CD4+ T-cell counts > or =400 cells/microl. Therapy consisted of double (zidovudine and lamivudine) or triple (zidovudine, lamivudine, and ritonavir) combination antiretroviral therapy. Retrospective analysis based on magnitude of viral suppression was used to characterize responder and nonresponder groups. Among responders, long-term antiretroviral therapy maintained a significant increase in numbers of total CD4+, naive CD4+/CD45RA+, and memory CD4+/CD45RO+ T cells. A concomitant significant decrease in numbers of memory CD8+/CD45RO+ and both activated CD8+/HLA-DR+ and CD8+/CD38+ T cells was also maintained. In contrast, long-term antiretroviral therapy among nonresponders led only to a significant increase in the numbers of CD4+ T cells and a significant reduction in numbers of activated CD8+/HLA-DR+ T cells. The long-term ability of antiretroviral therapy during early asymptomatic HIV-infection to maintain reversal of disease-induced T-cell activation and maturation abnormalities continues to support the concept that immunologic advantage is gained by commencing early aggressive antiretroviral therapy. Nevertheless, continued management of T-cell subset recovery is significantly more effective in the presence of completely suppressed viral replication.

Quantification of in vitro retroviral replication using a one-tube real-time RT-PCR system incorporating direct RNA preparation.

The methodological and logistic benefits gained from assessing in vitro antiretroviral replication using one-tube real-time RT-PCR procedures are currently diminished by a continuing need for prior RNA isolation. We now report a simple and inexpensive modification of a commercially available one-tube RT-PCR assay, consisting of detergent-based virus lysis in the presence of a ribonuclease inhibitor, which can be used to directly quantify retroviral RNA levels in culture supernatant. This approach circumvents the potential loss of RNA inherent to RNA-isolation procedures based on prior extraction and demonstrates a dynamic range of at least 4 logs. Using in vitro culture systems incorporating either HIV-1 or FIV, we show that this ability to isolate retroviral RNA directly during the RT-PCR process can provide an equivalent alternative to one of the more time and resource-consuming steps in quantifying in vitro retroviral RNA levels.

Residual HIV-RNA levels persist for up to 2.5 years in peripheral blood mononuclear cells of patients on potent antiretroviral therapy.

The long-term response of 10 asymptomatic, antiretroviral therapy-naive HIV-1-infected patients to potent combination antiretroviral therapy was characterized by monitoring levels of HIV-1 RNA in plasma, peripheral blood mononuclear cells (PBMC), and lymphoid tissue using highly sensitive HIV-1 RNA assays. Although plasma viral loads were continuously suppressed to levels below 50 HIV-1 RNA copies/ml for up to 2.5 years (60-128 weeks), HIV-1 RNA was still detectable at very low levels (1 to 49 HIV-1 RNA copies/ml) in 25% of the samples. In corresponding PBMC specimens, residual HIV-RNA was detectable in as much as 91% of samples tested (1 to 420 HIV-1 RNA copies/microg total RNA). Similarly, HIV-1 RNA levels in lymphoid tissue also remained detectable at a high frequency (86%). A highly significant correlation was demonstrated between therapy-induced change in PBMC HIV-1 RNA levels and change in plasma HIV-1 RNA levels (r2 = 0.69; p = 0.003). These findings support the concept that measurement of HIV-1 RNA in the easily accessible PBMC compartment is relevant for evaluating the potency of current and future antiretroviral therapies.

Effects of early antiretroviral treatment on HIV-1 RNA in blood and lymphoid tissue: a randomized trial of double versus triple therapy. Swiss HIV Cohort Study.

To assess the effects of early initiation of antiretroviral therapy on cell-free and cell-associated viral load in blood and lymphoid tissue, we performed a randomized, open-label, multicenter trial comparing a double (zidovudine + lamivudine) and triple (zidovudine + lamivudine + ritonavir) drug combination in treatment-naive, asymptomatic patients with CD4 counts >400 cells/microl. HIV-1 RNA was measured in plasma, peripheral blood mononuclear cells, and sequential tonsil or lymph node biopsies (27 patients); the study follow-up was 2 years. Among 42 randomized patients, the proportion with plasma HIV-1 RNA <50 copies/ml was 16% and 74% at week 24 (p<.001) in those randomized to double and triple therapy, respectively, necessitating frequent treatment intensification in the double arm. After a rapid decline within 4 weeks in both arms, cell-associated HIV-1 RNA decreased further only in those patients with sustained suppression of plasma viral load, but remained almost always detectable at low levels, indicating persisting transcription of viral RNA. CD4 counts increased by 200 to 250 cells/microl at week 96 in both arms without significant differences (intent-to-treat analyses). Thus, even if treatment is initiated early in asymptomatic patients with preserved CD4 counts, three drugs are necessary to achieve sustained decreases of HIV load in blood and lymphoid tissue.

Highly active antiretroviral therapy during early HIV infection reverses T-cell activation and maturation abnormalities. Swiss HIV Cohort Study.

OBJECTIVES: To evaluate the impact of early initiation of highly active antiretroviral therapy (HAART) on disease-induced T-cell activation and maturation abnormalities during asymptomatic HIV infection. DESIGN: A prospective open-label trial of zidovudine, lamivudine and ritonavir in treatment-naive asymptomatic HIV-infected individuals with CD4 cells > or = 400 x 10(6)/l. METHODS: Peripheral blood CD4+ and CD8+ T cells derived from 15 asymptomatic HIV-infected individuals (median baseline CD4+ cells, 608 x 10(6)/l; CD8+ cells, 894 x 10(6)/l; plasma HIV RNA, 3.93 log10 copies/ml) undergoing therapy with zidovudine (300 mg twice daily), lamivudine (150 mg twice daily), and ritonavir (600 mg twice daily) were assessed for changes in expression of phenotypic markers of T-cell activation (HLA-DR and CD38) and maturation (CD45RA and CD45RO). At weeks 0, 2, 4, 8, 12, 16, 20 and 24, T-cell subsets were quantified by flow cytometry and plasma HIV viral loads determined using reverse transcription PCR. RESULTS: HAART-induced decrease in plasma HIV RNA levels coincided with a significant reduction in numbers of activated CD4+/HLA-DR+ (maximum change, -36%; P < or = 0.05), CD8+/HLA-DR+ (maximum change, -66%; P < or = 0.005) and CD8+/CD38+ (maximum change, -51%; P < or = 0.01) T cells. A concomitant significant increase in numbers of naive CD4+/CD45RA+ (maximum change, +12%; P < or = 0.005) and memory CD4+/CD45RO+ (maximum change, +6%; P < or = 0.05) T cells was also evident, which contrasted with a significant decrease in memory CD8+/CD45RO+ cells (maximum change, -42%; P < or = 0.005). CONCLUSION: The observed ability of HAART during early asymptomatic HIV infection to initiate rapid reversal of disease-induced T-cell activation and maturation abnormalities, while preserving pretherapy levels of immune function, supports the concept that therapeutic advantage is to be gained by commencing early aggressive antiretroviral therapy.

Improvement in immune function due to treatment with indinavir despite severe immune deficiency.

To study the virological, immunological and clinical effects of the protease inhibitor indinavir in human immunodeficiency virus (HIV)-infected patients with CD4 counts < 50 cells/mm3, indinavir was added to prior treatment with nucleoside analogues in a prospective open-label study in 23 HIV-infected patients with median CD4 count of 10 cells/mm3 and median serum HIV-1 RNA load of 27,508 copies/ml. Addition of indinavir induced a decrease in HIV-1 RNA levels to < 400 copies/ml in 15 patients that was maintained until week 36 of the study in 8 (35%) patients. The median increase in CD4 cell counts was 92 cells/mm3 (range 55-258 cells/mm3) and in CD8 counts was 245 cells/mm3 (range 51-1552 cells/mm3) at week 30. The treatment induced a significant CD8 T cell expansion, consisting in the first 6 weeks of predominantly memory CD45RO+ cells and followed by expansion of naive cells from week 12 on, and a significant decrease in the proportion of activated CD8/CD38 cells. In addition, significant increases in T cell proliferation following stimulation with phytohaemagglutinin and significant decreases in the rates of spontaneous apoptosis of CD4+ and CD8+ T cells were observed. In conclusion, the addition of indinavir induced restoration of both memory and naive CD8 T cells. Corresponding evidence of improving T cell function, as assessed by enhanced lymphoproliferative capacity and diminished propensity to undergo apoptosis, provides evidence for treatment-induced regeneration of immune function even in patients with severe immunodeficiency.

Change in circulating levels of the chemokines macrophage inflammatory proteins 1 alpha and 11 beta, RANTES, monocyte chemotactic protein-1 and interleukin-16 following treatment of severely immunodeficient HIV-infected individuals with indinavir.

OBJECTIVE: To evaluate the in vivo relationship between HIV replication and circulating levels of the chemokines macrophage inflammatory protein (MIP)-1 alpha, MIP-1 beta, RANTES (acronym for Regulated upon Activation, Normal T-cell Expressed and presumably Secreted), interleukin (IL)-16 and monocyte chemotactic protein (MCP)-1, which have recently been characterized as factors capable of regulating in vitro HIV replication. DESIGN AND METHODS: We have compared changes in plasma HIV-RNA levels and circulating levels of MIP-1 alpha, MIP-1 beta, RANTES, IL-16 and MCP-1 in 20 severely immunodeficient HIV-infected individuals (CD4+ T cells = 14 +/- 3 cells x 10(6)/l; plasma HIV RNA = 4.45 +/- 0.27 log 10 copies/ml) undergoing treatment with the HIV protease inhibitor indinavir that added to pre-existing nucleoside-based therapy. At weeks 0, 2, 6 and 12, viral load was quantified using a commercial reverse-transcription polymerase chain reaction assay, peripheral blood T-cell subpopulations assessed by flow cytometry, and chemokine levels quantified using commercial sandwich enzyme-linked immunosorbent assay kits. RESULTS: Following initiation of indinavir-based therapy, significant decreases in plasma HIV-RNA levels (change = 2.0 +/- 0.75 log 10 copies/ml) were observed in conjunction with significant increases in absolute CD4+ (change = 83 +/- 19 cells x 10(6)/l) and CD8+ (change = 293 +/- 96 cells x 10(6)/l) T-cell numbers. Concomitantly, significant increases in MIP-1 alpha (19% increase), MIP-1 beta (14% increase), RANTES (15% increase) and IL-16 (1213% increase) levels occurred. In contrast, MCP-1 levels decreased significantly (47% decrease). CONCLUSION: The in vivo demonstration of an association between diminishing plasma HIV-RNA levels and the emergence of a circulating chemokine profile capable of inhibiting HIV replication corroborates recent in vitro observations and provides evidence for the restoration of chemokine capacity by HIV protease inhibitor-based therapy.

Molecular mimicry in the pathogenesis of AIDS: the HIV/MHC/mycoplasma triangle.

The immune defects characterizing infection with the human immunodeficiency virus (HIV) and culminating in the acquired immune deficiency syndrome (AIDS) are the result of a multifactorial disease process, components of which are the occurrence of autoimmune phenomena and opportunistic infection. In this discussion, the observation that both the HIV-1 gp 120 envelope and Mycoplasma genitalium adhesin proteins share an area of significant similarity with the CD4-binding site of the class II major histocompatibility complex (MHC) proteins is placed in this perspective and mechanisms by which interaction within this triad could contribute to the T-cell dysfunction, T-cell depletion, Th1-cell-->Th2-cell shift, B-cell proliferation, hyperglobulinemia and antigen-presenting cell dysfunction observed during the development of AIDS are proposed.

Using a neural network to identify potential HLA-DR1 binding sites within proteins.

The presentation by antigen-presenting cells of immunodominant peptide segments in association with major histocompatibility complex (MHC) encoded proteins is fundamental to the efficacy of a specific immune response. One approach used to identify immunodominant segments within proteins has involved the development of predictive algorithms which utilize amino acid sequence data to identify structural characteristics or motifs associated with in vivo antigenicity. The parallel-computing technique termed 'neural networking' has recently been shown to be remarkably efficient at addressing the problem of pattern recognition and can be applied to predict protein secondary structure attributes directly from amino acid sequence data. In order to examine the potential of a neural network to generalize peptide structural features related to binding within class II MHC-encoded proteins, we have trained a neural network to determine whether or not any given amino acid of a protein is part of a peptide segment capable of binding to HLA-DR1. We report that a neural network trained on a data base consisting of peptide segments known to bind to HLA-DR1 is able to generalize features relating to HLA-DR1-binding capacity (r = 0.17 and p = 0.0001).

Looking for a 'superantigen' in AIDS: a possible role for Mycoplasma?

The extensive effort made to comprehend the complex immunopathology of human immunodeficiency virus (HIV) infection has resulted in research groups focusing attention on hypotheses as disparate as the possible 'superantigen' potential of HIV and the possible requirement for a Mycoplasma 'cofactor'. Utilizing the recent observation that a Mycoplasma species possesses 'superantigen' properties, this paper attempts to reconcile these seemingly discrepant observations in a model of the acquired immunodeficiency syndrome (AIDS) which builds on the potential contribution of a 'superantigen cofactor' to the ongoing process of HIV infection. A possible role for mycoplasma-induced T-cell proliferation, T-cell dysfunction, B-cell proliferation, and hyperglobulinaemia in the exacerbation of HIV infection is discussed. The relevance of a recent observation regarding protein sequence homology between the mycoplasma adhesion protein and several human class II major histocompatibility complex (MHC) proteins is also examined and incorporated into this model.

T cell response to staphylococcal superantigens by asymptomatic HIV-infected individuals exhibits selective changes in T cell receptor V beta-chain usage.

Recognition that the murine mammary tumor C-type retrovirus and the replication-defective murine leukemia virus have "superantigen" properties raises the specter that human immunodeficiency virus might also generate T cell impairment and destruction as a result of inherent superantigen properties. The observation that individuals with AIDS lack the expression of several T cell receptor V beta-chain genes lends support to this hypothesis. Staphylococcal exotoxins represent another class of superantigen with a similar ability to stimulate large numbers of T cells bearing specific T cell receptor V beta-chain types. To examine the hypothesis that T cells from HIV-infected individuals may be exposed to a superantigen during the infection process, we have compared the ability of T cells from asymptomatic HIV-infected donors and healthy donors to respond to stimulation with several known staphylococcal exotoxin superantigens. Following in vitro stimulation with staphylococcal enterotoxin D and staphylococcal enterotoxin E, asymptomatic HIV-infected individuals responded with a significantly different T cell receptor V beta-chain usage to that observed for healthy individuals. This skewed V beta-chain usage is likely to reflect preferential conditioning of T cells bearing specific V beta-chains as a result of HIV infection, supporting the hypothesis of superantigen involvement early in the course of infection.

Altered humoral immunoregulation during human pregnancy.

The in vitro production of immunoglobulins in response to stimulation with pokeweed mitogen (PWM) and fixed/killed Staphylococcus aureus Cowan 1 (SAC) was measured in conjunction with in vivo assays of plasma immunoglobulin levels to examine the quality and quantity of humoral immunity during human pregnancy and at parturition. Following stimulation with PWM, there is a significant enhancement of in vitro immunoglobulin-G (IgG) production during pregnancy. Following stimulation with PWM and SAC, there was a significant reduction in in vitro immunoglobulin-M (IgM) production immediately following parturition. There was a significant decrease in the plasma levels of IgG during pregnancy, although no change in the plasma levels of IgM were observed. The decrease in plasma immunoglobulin levels during pregnancy cannot be explained as the result of hemodilution and transplacental transfer. Altered humoral immunoregulation is the most likely means whereby an increase in immunoglobulin production during human pregnancy could occur. The possible effects of this on the outcome of pregnancy are discussed.

Humoral immunity in oral contraceptive users. I. Plasma immunoglobulin levels.

The worldwide acceptance of steroid-based oral contraception makes it imperative that the effect of these agents on the immune system is understood. Nevertheless, information regarding the effect of steroid-based oral contraception on plasma immunoglobulin (Ig) levels is often conflicting. In this report immunoglobulin levels in the plasma of females taking steroid-based oral contraceptives are measured in a novel manner using an enzyme-linked immunosorbent assay (ELISA). No significant alterations in the levels of IgG or IgM are reported.

PIP: Immunoglobulin IgG and IgM levels in 10 women taking a variety of oral contraceptives containing ethinyl estradiol and levonorgestrel were assayed by the sensitive and specific ELISA method. 10 women matched for age and socioeconomic status served as controls. No significant differences in IgG or IgM levels were observed. Although several reports in the literature, using less sensitive methods such as immunodiffusion and immunoelectrophoresis, also reported no difference, there have been a few conflicting reports of alterations in women taking pills.

Humoral immunity in oral contraceptive users. II. In vitro immunoglobulin production.

The worldwide acceptance of steroid-based oral contraception makes it imperative that the effect of these agents on the immune system is understood. Nevertheless, information regarding the effect of steroid-based oral contraception on humoral immunoregulation is limited. In this report the in vitro production of IgG and IgM is measured following stimulation with either the T-dependent activator pokeweed mitogen (PWM) or the T-independent activator fixed/killed Staphylococcus aureus Cowan I (StaCw). No significant differences are observed between the in vitro IgG or IgM levels following stimulation with PWM or StaCw for females taking steroid-based oral contraceptives and females not taking steroid-based oral contraceptives. We conclude that humoral immunoregulation is unaltered in steroid-based oral contraceptive users.

PIP: Production of IgG and IgM immunoglobulins in vitro by purified monocytes isolated from the blood of women taking oral contraceptives was assayed by the sensitive ELISA method. The subjects were taking pills containing ethinyl estradiol and levonorgestrel. Washed mononuclear cell suspensions containing no more than 5% polymorphonuclear cells were incubated 7 days with either formaldehyde-killed Staphylococcus aureus strain Cowan I or T-cell dependent pokeweed mitogen. No significant differences were observed in immunoglobulin production between pill users and controls.