Dysregulation of the IgE/Fc epsilon RI network in HIV-1 infection

Serum IgE levels are increased in adults and children with HIV-1 infection and could be a marker of poor prognosis. Allergic reactions and adverse reactions to drugs are also increased in HIV-1-infected individuals. An imbalance between a T(H)1-like and a T(H)2-like cytokine profile has been documented in HIV-1 infection. We have found that HIV-1 gp120 from different clades is a potent stimulus for histamine and cytokine (IL-4 and IL-13) release from basophils. Gp120 acts as a viral superantigen, interacting with the V(H)3 region of IgE to induce mediator release from human Fc epsilon RI(+) cells. Human basophils and mast cells express the chemokine receptor CCR3, which binds the chemokines eotaxin and RANTES. By interacting with the CCR3 receptor on Fc epsilon RI(+) cells, HIV-1 Tat protein is a potent chemoattractant for human basophils and lung mast cells. Tat protein also induced IL-4 and IL-13 release from basophils. Preincubation of basophils with Tat protein upregulated the surface expression of the CCR3 receptor. Extracellular Tat can influence the directional migration of human Fc epsilon RI(+) cells, the expression of chemokine receptor CCR3, and the release of T(H)2 cytokines. Because Tat protein is actively released by HIV-1-infected cells, our results indicate a novel mechanism by which Fc epsilon RI(+) cells are rendered more susceptible to infection with CCR3-tropic HIV-1 isolates; that is, two HIV-1 proteins, gp120 and Tat, trigger the release of cytokines critical for T(H)2 polarization from Fc epsilon RI(+) cells, and Tat upregulates beta-chemokine receptor CCR3 on these cells.     

Mechanisms of IgE elevation in HIV-1 infection

Serum IgE levels are high in adults and children with HIV-1 infection and could be a marker of poor prognosis. Allergic reactions and adverse reactions to drugs also tend to increase in HIV-1-infected individuals. An imbalance between a "T(H)1-like" and a "T(H)2-like" cytokine profile has been documented in HIV-1 infection. We have demonstrated that HIV-1 gp 120 from different clades is a stimulus for histamine and cytokine (IL-4 and IL-13) release from basophils. Gp 120 acts as a viral superantigen, interacting with the V(H)3 region of IgE to induce mediator release from human Fc epsilonRI+ cells. Human basophils and mast cells express the chemokine receptor CCR3, which binds the chemokines eotaxin and RANTES. By interacting with the CCR3 receptor on Fc epsilonRI+ cells, HIV-I Tat protein is a potent chemoattractant for human basophils and lung mast cells. Preincubation of basophils with Tat protein upregulates mRNA CCR3 and the surface expression of this chemokine receptor. Tat also induces IL-4 and IL-13 release from basophils. Extracellular Tat can influence the directional migration of human Fc epsilonRI+ cells, the expression of chemokine receptor CCR3, and the release of T(H)2 cytokines. Our results indicate two novel mechanisms by which two HIV-1 proteins, gp120 and Tat, trigger the release of cytokines critical for T(H)2 polarization from human Fc epsilonRI+ cells.     

Mycobacterium avium infection in HIV-1-infected subjects increases monokine secretion and is associated with enhanced viral load and diminished immune response to viral antigens.

The complex interaction between HIV-1 infection and Mycobacterium avium was studied. Viral burden was assessed, as well as immune response to HIV-1 in the context of Myco. avium infections. We also examined serum cytokine levels and cytokine release by blood mononuclear cells in HIV-1-infected subjects, infected or not with Myco. avium. Undetectable serum levels of IL-1, tumour necrosis factor-alpha (TNF-alpha) and IL-6 were found in normal controls and in groups I, II and III of HIV-1-infected subjects. Moderate levels of TNF-alpha, IL-1 and IL-6 were found in the sera of group IV patients. When group IV was subdivided into subjects with and without Myco. avium infections, subjects with Myco, avium infections were shown to have higher serum levels of TNF-alpha, IL-1 beta and IL-6 than those with other infections. Blood mononuclear cells from controls and HIV subjects were stimulated with bacterial lipopolysaccharide, and cytokine levels assessed. Cells from group II patients were shown to secrete normal levels of TNF-alpha and IL-6, and lower levels of IL-1 beta; group III subjects released higher levels of IL-6. Patients in group IV had blood cells that released elevated levels of IL-6 and TNF-alpha, and lower levels of IL-1 beta. Group IV subjects with Myco. avium infections had blood cells that released higher levels of TNF-alpha, IL-6 and IL-1 than group IV subjects with other infections. Assessment of viral burden in cells of HIV-1-infected subjects revealed that Myco. avium-infected subjects had a higher level of virus burden and a lower level of lymphoproliferative response to an inactivated gp120-depleted HIV-1 antigen than AIDS subjects with other infections. These data suggest that Myco. avium infections in HIV-1-infected subjects hasten the progression of viral disease, enhance cytokine release and contribute to the anergy to viral antigens.     

Production of Interleukin-4, Interferon (IFN)-γ and IFN-α in Human Immunodeficiency Virus-1 Infection: An Imbalance of Type 1 and Type 2 Cytokines may Reduce the Synthesis of IFN-α

Interferon-α (IFN-α) is an important molecule in the antiviral response, but cells from HIV-1-infected individuals show a reduced ability to secrete IFN-α. We investigated an association between an imbalance of type 1/type 2 cytokines and the production of IFN-α in HIV-1 infection. We used whole blood culture to study the cytokine production profile, interferon-γ (IFN-γ) and interleukin-4 (IL-4), in response to HIV-1 antigens and to study the Sendai Virus and HSV-1-induced-production of IFN-α in seven HIV-1-infected patients. An impaired synthesis of IFN-α was obtained in patients with a predominant IL-4 production (IL-4 > IFN-γ), and we found a positive correlation between the ex vivo production of IFN-α and the IFN-γ/IL-4 ratio but not with the HIV RNA copy number in plasma. We investigated the role of T-cell-derived cytokines in the in vitro production of IFN-α by PBMC from eight healthy donors, activated with Sendai Virus or HSV-1. Whereas type 2 cytokines (IL-4, IL-13) inhibited virus-induced IFN-α synthesis, on the contrary, type 1 cytokines (IL-2, IFN-γ) enhanced it. A disarray in the T-cell-derived cytokine response may play a role in the defect of IFN-α production in HIV-1-infected individuals. Further investigations are needed to explore this hypothesis.     

Chronic Chagas' disease cardiomyopathy patients display an increased IFN-gamma response to Trypanosoma cruzi infection

One-third of all Trypanosoma cruzi -infected patients eventually develop chronic Chagas' disease cardiomyopathy (CCC), a particularly lethal inflammatory dilated cardiomyopathy, where parasites are scarce and heart-infiltrating mononuclear cells seem to be the effectors of tissue damage. Since T. cruzi is a major inducer of interleukin-12 production, the role of inflammatory cytokines in the pathogenesis of CCC was investigated. We assayed cytokine production by peripheral blood mononuclear cells (PBMC) from CCC and asymptomatic T. cruzi -infected (ASY) individuals, as well as by T cell lines from endomyocardial biopsies from CCC patients. PBMC from CCC and ASY patients produced higher IFN-gamma levels than normal (N) individuals in response to B13 protein and phytohaemagglutinin PHA; IFN-gamma high responders (> or =1 ng/ml) were 2-3 fold more frequent among CCC patients than ASY individuals. Conversely, IL-4 production in response to the same stimuli was suppressed among T. cruzi -infected patients. The frequency of PHA-induced IFN gammaproducing cells on PBMC was significantly higher among CCC than ASY and N individuals. IFN-gamma and TNF-alpha were produced by ten out of ten PHAstimulated T cell lines from CCC patients; IL-2 and IL-10 were produced by four out of ten and one out of ten lines, respectively; IL-4, IL-1alpha, IL-1beta, IL-6 and IL-12 were undetectable. Our results suggest that CCC and ASY patients may respond differentially to the IFN-gamma-inducing stimulus provided by T. cruzi infection. Given the T(1)-type cytokine profile of heart-infiltrating T cell lines from CCC patients, the ability to mount a vigorous IFN-gamma response may play a role on the differential susceptibility to CCC development.     

Role of IL-15 in HIV-1-associated hypergammaglobulinaemia

IL-15 is a novel cytokine, produced by monocytes/macrophages, with biological activities similar to IL-2 but with no significant sequence homology. IL-15 also stimulates human B cells to proliferation and immunoglobulin secretion. We measured serum levels of IL-15 in 84 HIV-1-infected individuals at different stages of disease in reference to 41 healthy blood donors. Our results show a marked elevation of IL-15 serum levels during HIV-1 infection. Moreover, we found that this increase correlated with serum levels of IgG (r=0.376, P<0.0001), and partly with serum IgM (r=0.265, P<0.0015). A significant increase of IL-15 production by cultured peripheral blood mononuclear cells (PBMC) and purified monocytes in the presence of HIV-1 virus suggests that monocytes/macrophages may be a source of higher IL-15 serum levels in HIV-1-infected individuals. These findings indicate a participation of IL-15 in the hypergammaglobulinaemia frequently associated with HIV-1 infection.     

Expansion of restricted cellular immune responses to HIV-1 envelope by vaccination: IL-7 and IL-12 differentially augment cellular proliferative responses to HIV-1

The failure of immune effector mechanisms to control HIV-1 infection has important consequences for the human host. In a randomized cohort of HIV-infected patients, there was striking in vitro restriction of the proliferative response to HIV-1 envelope protein (Env), gp160; only 34% of patients recognized Env. Therapeutic vaccination with recombinant gp160 or gp120 (rgp160, rgp120) reversed the restriction in vitro, with Env recognition rising to 81%. Peripheral blood mononuclear cells (PBMC) from HIV-infected vaccine recipients, placebo recipients, and seronegative volunteers were cultured with exogenous IL-7 or IL-12 and either tetanus toxoid (TT) or gp160. IL-7 significantly augmented proliferative responses to TT and gp160, whereas IL-12 only affected proliferation to gp160. IL-7, but not IL-12, increased the number of HIV-infected placebo recipients who recognized rgp160. IL-12 had its greatest effect in the induction of rgp160-specific responses from seronegative individuals. The data suggest that these two cytokines have differential activity in the relief of restricted cellular immunity to Env; the predominant effect of IL-7 is in individuals who have been primed by exposure to antigen, while the effect of IL-12 is most evident in seronegative, unprimed individuals. Modification of restricted proliferative responses to Env by vaccination or cytokines in vitro suggests that strategies incorporating IL-7 or IL-12 as adjuvants may selectively boost cellular reactivity to HIV-1.     

Recombinant human IL-16 inhibits HIV-1 replication and protects against activation-induced cell death (AICD)

The chemoattractant cytokine IL-16 has been reported to suppress lymphocyte activation and to inhibit HIV-1 replication in acutely infected T cells. We have cloned and expressed human IL-16 in Escherichia coli and investigated whether the recombinant protein could regulate the level of lymphocyte apoptosis from HIV-1-infected subjects. After purification and refolding, only 2–10% of the recombinant cytokine was present in a biologically active homotetrameric form. This could explain the need for high concentrations of the bacterially derived IL-16 to induce significant inhibition of HIV-1 replication. Addition of IL-16 to unstimulated peripheral blood mononuclear cell (PBMC) cultures from HIV-1-infected subjects did not modify the observed level of spontaneous lymphocyte apoptosis. In contrast, IL-16 added to PBMC cultures stimulated with anti-CD3, anti-CD95 or dexamethasone reduced significantly the percentage of lymphocytes undergoing AICD. This effect was found to correlate with the ability of the cytokine to decrease CD95 expression on activated CD4⁺ T cells. Comparative studies on PBMC from healthy individuals indicated that the regulation of apoptosis levels by IL-16 is a complex phenomenon and could depend on the nature of the activator used and/or the immune status of lymphocytes tested. The outcome of CD4 cross-linking on T cells by various ligands is discussed in the context of the observed beneficial activities of IL-16 and its potential role in the treatment of HIV disease.     

Autoproliferation in HIV-1-infected patients undergoing active HIV-1-specific immunotherapy.

We have observed a treatment-associated autoproliferative response in cultured peripheral blood mononuclear cells (PBMC) of asymptomatic HIV-1-infected subjects receiving a gp120-depleted, inactivated HIV-1 antigen in incomplete Freund's adjuvant (IFA; HIV-1 Immunogen). The frequency and magnitude of the autoproliferative response appeared to be dose-related (P < 0.05), and was not observed in subjects receiving IFA alone. Immunophenotyping of the proliferating cells demonstrated the presence of both CD4+ and CD8+ lymphocytes, with the CD4+ blasts almost exclusively expressing the CD45RO+ phenotype. A comparison of this response with the HIV-1-specific antigen stimulation responses in this cohort revealed a significant correlation between increases in HIV-1-specific cell-mediated immunity and autoproliferation (r2 = 0.61, P < 0.001). These findings suggest that immunization with the HIV-1 Immunogen induces an autoproliferative response that may reflect changes in HIV-1-specific cell-mediated immunity in infected individuals.     

HIV-1 induced AIDS is an Allergy and the Allergen is the Shed gp120 – a Review,Hypothesis, and Implications

The possibility that the induction of T helper 2 (Th2) cytokine synthesis and the gradual increase in interleukin 4 (IL-4) and IgE levels during HIV-1 infection are an allergic response to HIV-1 proteins was raised in the author's previous article [Becker, Virus Genes 28, 1-4, 2004]. The present review extends this hypothesis by citing experimental reports which indicate that HIV-1 shed gp120 virions share a striking resemblance with the allergens that bind to IgE molecules bound to Fc epsilon receptor I-positive (Fc epsilon RI) cells (mast cells, basophils, monocytes, and dendritic cells, DC) and then induce them to release and synthesize the IL-4 cytokine. In the earlier review, it was established that the IL-4 cytokine is responsible for the following processes: IgE synthesis by B cells and the inhibition of antiviral IgG synthesis; the inactivation of T helper 1 (Th1) cells; and the inhibition of the antiviral cytotoxic T cell (CTL) response. The binding of the shed gp120 to B cell-bound immunoglobulin (Ig) depletes these cells. Moreover, it was reported that the viral regulatory proteins Tat and Vpr also stimulate IL-4 release from basophils. The mode of action used by HIV-1 gp120 in the induction of IL-4 [Becker, Virus Genes 28, 1-14, 2004] revealed that the viral protein contains a superantigen (SAg) domain that is capable of binding to the V(H)3 domain of IgE and Ig; namely, the shed gp120 protein act as endogenous and environmental allergens that bind to IgE molecules, which are bound to the Fc epsilon RI receptors on hematopoietic cells. Based on these findings, it is hypothesized that gp120 is an allergen. Consequently, it can be inferred that the active replication of HIV-1 in infected individuals constantly exposes the immune system to an increase in the allergen content until the host immunity is eventually compromised. These finding suggest that HIV-1 engage in a two-pronged attack of the human immune system: it infects Th2 cells, macrophages, and DCs and then replicates at these venues; and the shed gp120 cells cause an allergy. This allergic condition consequently prevents the induction of Th1 cells, cytokine synthesis, and antiviral CTLs that are needed to clear the infecting virus and thus devitalizes the antivirals that are used to treat the virus infection. It was hypothesized [Becker, Virus Genus 28, 1-14, 2004] that the HIV-1 induced allergy can be treated with IL-4 inhibitors or immune response modifiers. A treatment that employs both antivirals and anti-allergen drugs may very well defeat the AIDS syndrome. Another implication of the present hypothesis is the need to improve the viral antigen used for vaccinating healthy individuals against HIV-1 infection. It is thus suggested harmful domains be eliminated from the gp120.     

HIV-1 Inhibits Leishmania-lnduced Cell Proliferation but not Production of Interleukin-6 and Tumour Necrosis Factor Alpha

The immune response of normal human peripheral blood mononuclear cells (PBMC) after stimulation with human immunodeficiency virus-1 (HIV-1) antigens plus Letshmania donovani promastigotes in vitro was investigated. HIV-I-antigen stimulation of PBMC did not induce the intracellular accumulation of interleukin-6 (IL-6),. tumour necrosis factor-alpha (TNF-α), or Interferon-gamma (IFN-γ). However, cells stimulated with L. donovani antigens exhibited the production of IL-6 and TNF-α, but not IFN-γ. Furthermore, co-stimulation of PBMC with HIV-1 antigen plus L. donovani resulted in the intracellular accumulation of IL-6 and TNF-α comparable to that of cells that were activated with L. donovani antigen alone. Heat-inactivated HIV-1 antigen did not appear to induce or suppress cytokine production by PBMC. However, the same HIV antigens did suppress L. donovani-induced proliferation as well as PPD-induced proliferation in a dose-dependent fashion. Elevated levels of serum cytokines have been demonstrated in patients with HIV infection indicating their role in the pathogenesis of HIV-associated immunosuppression. The results may partially support the idea that the abnormally increased cytokine levels in the sera of HIV-infected subjects is due to the various opportunistic pathogens that these patients contract, rather than a response to HIV antigens. As cytokines have been shown to up-regulatc HIV replication, the data suggest a role for opportunistic infections in cytokine-induced transactivation of HIV-1 and disease progression.     

Immunomodulatory effects of peptide T on Th 1/Th 2 cytokines.

Peptide T is an octapeptide from the V2 region of HIV-1 gp120. It has been shown to resolve psoriatic lesions--an inflammatory skin disease. The mechanisms of anti-inflammatory actions of peptide T are not well understood. Th1 cytokines such as IL-2, and IFN-gamma are upregulated in psoriasis. These cytokines play a key role in the inflammatory and proliferative processes of psoriasis. The effects of peptide T on Th1 and Th2 cytokines were studied in order to elucidate the mechanisms of antiinflammatory actions of peptide T. It was observed that peptide T at 10(-8) M induces IL-10 production by the human Th2 cell line and PBMC (P < 0.05, ANOVA). Also peptide T at 10(-9) M concentration significantly inhibited IFN-gamma production by PBMC (P < 0.001, ANOVA). Anti IL-10 antibody inhibited the anti-IFN-gamma effect of peptide T (P < 0.05, t-test). Our study shows that peptide T induces IL-10 production and inhibits IFN-gamma production. IL-10 is a potent anti-inflammatory cytokine. It inhibits IL-2 and IFN-gamma production from the T cells and downregulates the expression of TNF-alpha in the antigen presenting cells. Recently, IL-10 has been shown to resolve psoriatic lesions. The effects of peptide T on IL-10 and IFN-gamma production provides a plausible explanation for its clinical efficacy in psoriasis.     

中国HIV/AIDS患者保守表位的2F5抗体与疾病进展关系

目的 研究中国HIV/AIDS患者gp41保守表位中和抗体2F5与疾病进程的关系.方法 应用合成4(ELDKWA)-C24肽及gp41抗原肽包被96孔酶标板,ELISA方法检测特异性抗体水平.提取正常人外周血单个核细胞(PBMC),应用HIV-1 SF33毒株进行病毒多轮感染PBMC中和试验.结果 缓慢进展者(slow progressor,SP)的2F5样中和抗体水平显著高于典型进展者(typical progressor,TP)(P<0.05);2F5样中和抗体水平与gp41特异性抗体水平正相关(r=0.406,P<0.05).结论 中国HIV.-缓慢进展者体内具有高水平的2F5样抗体,2F5抗体可能是疾病不进展的保护性因素之一.     

In vitro cytokine production by mononuclear cells exposed to bone cement extracts

The authors evaluated the ability of bone cement to modify the profile of pro-inflammatory cytokines secreted by the immune cells. Peripheral blood mononuclear cells (PBMC) collected from healthy individuals were cultured with cement extracts and tested to assess the release of IL-1beta, TNFalpha, GM-CSF and IL-6 in both unstimulated and PHA-stimulated PBMC. The cytokine release of unstimulated PBMC was very poor, and in particular the IL-1beta was undetectable: the addition of cement extract increased both TNFalpha and GM-CSF release and decreased IL-6, sometimes significantly. The most recurrent observation in PHA-stimulated PBMCs exposed to bone cement extract was the increase in both IL-1beta and IL-6 release, while both the mean concentration and the index of release of TNFalpha and GM-CSF were changeable. In conclusion our results showed that leachable components of some bone cements can induce in vitro the release of pro-inflammatory cytokines which are known to be involved in the bone resorption associated with aseptic loosening of hip prostheses. These findings allowed us to identify materials endowed with the highest inflammatory power.     

Temporal relationship of cytokine release by peripheral blood mononuclear cells stimulated by the streptococcal superantigen pep M5.

We undertook this study to determine the quality, quantity, and temporal relationship of pep M5-induced cytokine release. The ability of pep M5 to stimulate interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-alpha) production by a T-cell-depleted, monocyte- and B-cell-enriched cell population was dependent on the presence of T cells. The requirement for T cells could be met by addition of exogenous gamma interferon (IFN-gamma). In the presence of IFN-gamma, pep M5 induced the release of TNF-alpha, IL-1, and IL-6, TNF-alpha levels peaked at 24 h, while IL-1 and IL-6 levels peaked at 48 h. pep M5 induced T cells to produce IFN-gamma, which may have accounted for the ability of the super antigen to induce the production of IL-1, IL-6, TNF-alpha, and TNF-beta by peripheral blood mononuclear cells (PBMC). The addition of excess IFN-gamma to cultures of pep M5 and PBMC did not further increase the release of these cytokines at 24 and 48 h but resulted in sustained higher levels at 72 h. Interestingly, TNF-beta production occurred only in the presence of pep M5 and exogenous IFN-gamma. The ability of pep M5 to induce cytokine production was compared with that of a potent super antigen, staphylococcal enterotoxin B (SEB). SEB was a 2- to 14-fold-more-potent inducer of IFN-gamma production. Furthermore, the profile of cytokine released by PBMC in response to this super antigen mimicked that seen with pep M5 in the presence of exogenous IFN-gamma. In conclusion, pep M5 induces the production of cytokines that are involved in immune regulation and inflammation. These cytokines also play a major role in human T-cell responses to this super antigen.     

Immunological dysfunction in HIV-1-infected individuals caused by impairment of adenosine deaminase-induced costimulation of T-cell activation

The cell surface association between CD26 and adenosine deaminase (ADA) has a costimulatory function during T-cell activation. Several studies have revealed correlations among CD4⁺ CD26⁺ T-cell depletion, increased serum levels of ADA, and the evolution of human immunodeficiency virus (HIV) infection, implicating CD26 and ADA in HIV disease progression. In this context, we aimed to determine whether ADA costimulation could be altered during HIV infection. ADA costimulation was investigated in cells from HIV-infected patients (n = 36) in terms of proliferation and cytokine secretion. An effect of ADA on T-cell proliferation was found in HIV-1-infected patients and correlated positively with the CD4⁺ percentage and the nadir CD4 count and negatively with viral load, demonstrating that the response depends on the immunological status of the patient. The robust ADA-induced increase in cytokine production [interferon (IFN)-γ, interleukin (IL)-6 and IL-10] was markedly reduced in T cells from HIV-1-infected subjects. To eliminate some of the variables associated with immunological defects in HIV-1-infected patients, anti-CD3 plus ADA assays with T cells from healthy volunteers were performed in the presence of recombinant glycoprotein 120 (gp120). It was found that gp120 was responsible for the impairment of the ADA–CD26 interaction and consequently of the ADA-induced effect on both costimulation and cytokine production. The gp120-mediated disruption of the CD26–ADA interaction is a novel mechanism that might explain, at least in part, the altered immunological features observed in HIV-1-infected patients and may have significant relevance in AIDS pathogenesis.     

Cytokine gene expression occurs more rapidly in stimulated peripheral blood mononuclear cells from human immunodeficiency virus-infected persons

Evaluation of cytokine gene expression following in vitro stimulation is one means of examining the dysregulation of the immune system in human immunodeficiency virus (HIV) infection. We have assessed differences in the immune status of non-HIV-infected (HIV-) and HIV-infected (HIV+) individuals by evaluating the kinetics of the expression of cytokine genes. We compared detailed time courses of cytokine mRNA expression in HIV- and HIV+ peripheral blood mononuclear cells (PBMC) and found that there is a significant shift (P<0.01) for all cytokines examined (interleukin 2 [IL-2], IL-6, IL-10, gamma interferon, and tumor necrosis factor alpha [TNF-alpha]) to an earlier time of mean peak mRNA expression by HIV+ PBMC (between 4 and 8 h) compared to HIV- PBMC (8 h) in response to either phytohemagglutinin (PHA) or anti-CD3 stimulation. Additional studies showed that although PHA-stimulated HIV+ PBMC showed decreased median IL-2, IL-4, and TNF-alpha mRNA levels, they typically demonstrated more rapid kinetics (increased mean 4-h/24-h cytokine mRNA ratios), with significant differences for IL-4 (P<0.05) and TNF-alpha (P<0.005), compared to HIV- PBMC. The use of fresh or frozen cells gave comparable cytokine mRNA data; however, the secretion of some cytokine proteins (IL-2 receptor, IL-10, and TNF-alpha) appeared to be reduced in HIV+ PBMC that had been frozen and thawed. Our studies demonstrate that the kinetics of cytokine gene expression can reveal additional dysregulation of the immune system in HIV infection, suggesting that PBMC of HIV-infected persons exist in an activated state in vivo that permits them to express cytokine genes more rapidly than a normal PBMC.