Syndecan-1 increases B-lymphoid cell extravasation in response to HIV-1 Tat via αvß3/pp60src/pp125FAK pathway.

Syndecan-1 is a heparan sulfate proteoglycan (HSPG) commonly upregulated in AIDS-related B lymphoid malignancies. Tat is the main HIV-1 transactivating factor that has a major role in the pathogenesis of AIDS-related lymphomas (ARL) by engaging heparan sulfate proteoglycans (HSPGs), chemokine receptors and integrins at the lymphoid cell (LC) surface. Here B-lymphoid Namalwa cell clones that do not express or overexpress syndecan-1 (EV-Ncs and SYN-Ncs, respectively) were compared for their responsiveness with Tat: in the absence of syndecan-1, Tat induces a limited EV-Nc migration via C-X-C motif chemokine receptor 4 (CXCR4), G-proteins and Rac. Syndecan-1 overexpression increases SYN-Nc responsiveness to Tat and makes this response independent from CXCR4 and G-protein and dependent instead on pp60src phosphorylation. Tat-induced SYN-Nc migration and pp60src phosphorylation require the engagement of αvß3 integrin and consequent pp125FAK phosphorylation. This complex set of Tat-driven activations is orchestrated by the direct interaction of syndecan-1 with pp60src and its simultaneous coupling with αvß3. The Tat/syndecan-1/αvß3 interplay is retained in vivo and is shared also by other syndecan-1+ B-LCs, including BJAB cells, whose responsiveness to Tat is inhibited by syndecan-1 knockdown. In conclusion, overexpression of syndecan-1 confers to B-LCs an increased capacity to migrate in response to Tat, owing to a switch from a CXCR4/G-protein/Rac to a syndecan-1/αvß3/pp60src/pp125FAK signal transduction pathway that depends on the formation of a complex in which syndecan-1 interacts with Tat via its HS-chains, with αvß3 via its core protein ectodomain and with pp60src via its intracellular tail. These findings have implications in ARL progression and may help in identifying new therapeutical targets for the treatment of AIDS-associated neoplasia.

Detection of KI WU and Merkel cell polyomavirus in respiratory tract of cystic fibrosis patients.

In the last few years, many reports have confirmed the presence of WU, KI and Merkel cell (MC) polyomaviruses (PyV) in respiratory samples wordwide, but their pathogenic role in patients with underlying conditions such as cystic fibrosis is still debated. To determine the prevalence of MCPyV, WUPyV and KIPyV, we conducted a 1-year-long microbiological testing of respiratory specimens from 93 patients with cystic fibrosis in Brescia, Italy. We detected PyV DNA in 94 out of 337 analysed specimens. KIPyV was the most common virus detected (12.1%), followed by WUPyV (8.9%) and MCPyV (6.8%). We found an intriguing association between the presence of MCPyV and the concurrent isolation of Pseudomonas aeruginosa, as well as with the patient status, classified as chronically colonized with P. aeruginosa. Our study adds perspective on the prevalence and the potential pathogenic role of PyV infections.

A specific Toll-like receptor profile on T lymphocytes and values of monocytes correlate with bacterial, fungal, and cytomegalovirus infections in the early period of allogeneic stem cell transplantation.

BACKGROUND: Bacterial, fungal, and viral infections often affect non-relapse mortality after allogeneic stem cell transplantation (alloSCT). Recovery from infections depends on a balanced integration between innate and adaptive immune responses. In this complex interplay, a key role is played by Toll-like receptors (TLRs), which are sensors of pathogen-associated molecular patterns. To our knowledge, no previous study deals with both expression and function of all human TLRs together, in relation to infections in the setting of alloSCT. METHODS: We prospectively evaluated 9 TLRs by flow cytometry on T lymphocytes and monocytes of 35 patients in relation to infectious events from day +30 to day +120. Tumor necrois factor-alpha, interleukin-4, interferon-gamma, and monocyte chemoattractant protein-1 induction upon TLR activation was assessed by enzyme-linked immunosorbent assay on cell supernatants. RESULTS: In multivariate Cox regression analysis, levels of TLR-9 expression on T lymphocytes (P = 0.01) and values of natural killer cells (P = 0.01) correlated negatively with bacterial infections, whereas cytomegalovirus (CMV) infection resulted as a positive predictor. We observed a trend for negative correlation between TLR-7 levels on T lymphocytes and fungal infections (P = 0.07). Values of monocytes were negatively associated with CMV infection (P = 0.03), whereas levels of TLR-5 on T lymphocytes were positive predictors (P = 0.01). Age (P = 0.03) and bacterial infections (P = 0.006) negatively influenced overall survival. Monocyte values were positive predictors of survival (P = 0.003). CONCLUSIONS: Bacterial, fungal, and CMV infections were associated with a different expression of some TLRs on T lymphocytes. The protective role of TLR-7 and TLR-9 seemed dominant over other TLRs involved in recognizing fungi and bacteria. We also observed an atypical involvement of TLR-5 in CMV infection. The dominant and atypical role of some TLRs could depend on their pleiotropic functions and the changing inflammatory environment of transplanted patients. A specific TLR profile and an adequate count of monocytes could improve survival, promoting an effective control of infections, and balanced immune responses. If our findings will be confirmed by further studies, these immunological variables could be useful as parameters to predict susceptibility to infections.

The immunomodulatory molecule pidotimod induces the expression of the NOD-like receptor NLRP12 and attenuates TLR-induced inflammation.

Pidotimod (3-L-pyroglutamyl-L-thiaziolidine-4-carboxylic acid) (PDT) is a synthetic dipeptide with in vitro and in vivo immunomodulatory properties that is largely used for treatment and prevention of infections in paediatric and disease-prone patients. However, the effects of PDT on cellular immune responses are still poorly characterized and there is little information on the mechanism of action of this compound. It has been speculated that PDT action may be exerted through the interaction with a Pattern Recognition Receptor (PRR). Therefore, to gain a further understanding of the immune pathways involved by PDT, we first decided to investigate whether PDT could modify the immune response triggered by TLR ligands. Monocytic cells were exposed to PDT then stimulated with a panel of TLR agonists. Under these experimental conditions, we observed a significant decrease in the synthesis of key proinflammatory mediators in comparison to the production observed in TLR-stimulated cells that were not treated with PDT. Using RT² Profiler PCR Array we have observed that PDT specifically up-regulates the expression of the NOD-like receptor NLRP12 mRNA in the absence of any further costimulation. Increase of NLRP12 in cells treated with PDT was confirmed using specifically designed real-time quantitative PCR and western blotting assays where a clear increase in the amount of NLRP12 protein was detected. Furthermore, in myeloid/monocytic cells we demonstrated that PDT treatment counteracts the NLRP12 reduction induced by TLR agonists. Finally, the results obtained using NLRP12 silenced cells showed that down-regulation of the proinflammatory function occurring in PDT-treated cells upon interaction with TLRs is associated with the increased levels of NLRP12 induced by PDT. To our knowledge this is the first evidence of an immunomodulatory peptide that upregulates NLRP12 and, through this molecule, antagonizes the TLR-induced inflammatory response. These results pave the way for the development of innovative therapeutic approaches aimed at controlling different pathological settings such as tumorigenesis, systemic inflammatory processes and autoimmunity, where NLRP12 plays a crucial role.

Anti-inflammatory effect of 3,4-DHPEA-EDA [2-(3,4 -hydroxyphenyl) ethyl (3S, 4E)-4-formyl-3-(2-oxoethyl)hex-4-enoate] on primary human vascular endothelial cells.

Diets in which fat is significantly provided by olive oil and are relatively rich in vegetables, have been associated with a low incidence of cardiovascular diseases, mostly due to the presence of several phenolic compounds which have anti-oxidant and antiinflammatory properties. [1]. In this work, we describe the anti-inflammatory effect of 3,4-DHPEA-EDA in a cell model that we developed to mimic inflammatory injury of endothelium. This was based on the production of the proinflammatory chemokine CCL2, following in vitro stimulation of primary human endothelial cells. Pre-treatment of cells with 3,4-DHPEA-EDA resulted in a dose-dependent inhibition of CCL2 secretion. The effect of 3,4-DHPEA-EDA on CCL2 expression was observed at the transcriptional level. Functional data have shown that 3,4-DHPEA-EDA diminished monocyte adhesion to HUVECs. These results point on the use of 3,4- DHPEA-EDA as a novel drug aimed to prevent or reduce inflammation of endothelium.

A partially humanized monoclonal antibody to human IFN-gamma inhibits cytokine effects both in vitro and in vivo.

The mouse monoclonal antibody (MoAb) IGMB17 (muIGMB17) is a high-affinity antibody- neutralizing human interferon (IFN)-gamma and, accordingly, is a potential therapeutic agent for patients suffering from various diseases in which the cytokine is abnormally expressed. The clinical usefulness of mouse antibodies is limited, however, owing to their immunogenicity in humans. MuIGMB17 antibody was partially humanized by engrafting a small portion of mouse light chain (LC) in a human framework and by engineering its heavy chain (HC) in a chimeric version. The engineered IGMB17 (huIGMB17) was able to replicate a range of functional properties of the original muIGMB17, namely, specific binding to IFN-gamma, inhibition of histocompatibility complex (HLA-DR) expression in response to IFN-gamma induction, reversion of IFN-gamma antiproliferative activity on sensitive cell lines. We have hypothesized that as huIGMB17 was able to block IFN-gamma binding to its receptor as well as its murine counterpart, huIGMB17 could neutralize all cytokine activity, also in vivo. Indeed huIGMB17 was capable of interfering with delayed-type hypersensitivity reaction in humans, thus demonstrating its effectiveness in neutralizing IFN-gamma-mediated reactions in vivo.

Adult human heart microvascular endothelial cells are permissive for non-lytic infection by human cytomegalovirus.

OBJECTIVE: Human cytomegalovirus (CMV) infection has been linked to chronic heart disease. The mechanism of CMV dissemination to the heart remains unknown. CMV antigens and nucleic acid sequences have been detected in endothelial cells (ECs) in vivo, and ECs are fully permissive hosts to CMV replication in vitro. This report examines the characteristics of CMV replication in primary cultures of human heart microvascular ECs (HHMECs). METHODS: Capillary ECs were isolated from heart tissue biopsies of six patients at the time of heart surgery. HHMECs were infected with CMV and viral antigens were detected by immunofluorescence assay using monoclonal antibodies as specific reagents. Cytokine and chemokine release in the supernatant of sham- and CMV-infected cells was quantitated by ELISA. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to analyse expression of mRNA for adhesion molecules. RESULTS: CMV was found to productively infect HHMECs without cytolytic effects. Infected cultures released high levels of pro-inflammatory chemokines and enhanced their adhesion molecule expression. CONCLUSIONS: Our data provide new insights into the mechanism of CMV dissemination to the heart, signalling the need for further investigation of the pathogenetic role of this virus in cardiac disorders.

CD11b expression identifies CD8+CD28+ T lymphocytes with phenotype and function of both naive/memory and effector cells.

A previously unreported CD8(+)CD28(+)CD11b(+) T cell subset occurs in healthy individuals and expands in patients suffering from primary viral infections. In functional terms, these cells share the features of naive/memory CD8(+)CD28(+)CD11b(-) and terminally differentiated effector CD8(+)CD28(-)CD11b(+) subpopulations. Like CD28(-) cells, CD28(+)CD11b(+) lymphocytes have the ability to produce IFN-gamma, to express perforin granules in vivo, and to exert a potent cytolytic activity. Moreover, these cells can respond to chemotactic stimuli and can efficiently cross the endothelial barrier. In contrast, like their CD11b(-) counterpart, they still produce IL-2 and retain the ability to proliferate following mitogenic stimuli. The same CD28(+)CD11b(+) subpopulation detected in vivo could be generated by culturing naive CD28(+)CD11b(-) cells in the presence of mitogenic stimuli following the acquisition of a CD45RO(+) memory phenotype. Considering both phenotypic and functional properties, we argue that this subset may therefore constitute an intermediate phenotype in the process of CD8(+) T cell differentiation and that the CD11b marker expression can distinguish between memory- and effector-type T cells in the human CD8(+)CD28(+) T cell subset.

Expansion of rare CD8+ CD28- CD11b- T cells with impaired effector functions in HIV-1-infected patients.

The decline in the number of CD4+ T cells in HIV-1-infected patients is known to be related to the increased number of CD8+CD28- T cells. In this paper, we show that CD8+CD28- T cells from HIV-positive patients have an impaired capability to interact with human endothelial cells. This is due to the dramatic expansion, within this subset, of rare CD11b- cells lacking cell-cell adhesion functions. In 50 HIV-positive patients, 19.5% +/- 6.5% of all T cells were CD8+CD28-CD11b-, whereas only 0.8% +/- 0.4% of all T cells from healthy donors showed this uncommon phenotype. The percentage of circulating CD8+CD28-CD11b- T cells was strongly related to the percentage of CD4+ T cells (r = -0.82). This population is peculiar in terms of HIV infection and was found to possess some characteristics associated with effector functions but its cytotoxic properties were impaired. The percentage of target cells lysed by CD8+CD28-CD11b- was significantly lower than that of cells lysed by its CD11b- counterpart (p <.05) both at low (5:1) or at relatively high (20:1) effector/target ratios. CD8+CD28-CD11b- T cells, which lack the ability to interact with endothelial cells, are likely to accumulate and persist in circulation. The biologic properties of CD8+CD28-CD11b- T cells suggest that these cells might be endstage or aberrant differentiated effector cells. Lack of cell-cell adhesion and impaired cytolytic functions favor the hypothesis of a role for CD8+CD28-CD11b- T cells in the development of immunodeficiency.

Restriction of T-cell receptor repertoires in idiopathic CD4+ lymphocytopenia.

We report that alpha/beta and gamma/delta T-cell repertoires of three patients with idiopathic CD4+ lymphocytopenia, who showed different clinical manifestations and outcomes over time, were highly restricted. The disruption of T-cell repertoires does not influence the susceptibility to infections: the first patient was unable to attain a protective response to mycobacterium, the second showed clinical improvement and the third did not develop opportunistic infections. These results indicate that idiopathic CD4+ lymphocytopenia could give rise to mono-/oligoclonal T-cell expansions, but the degree of repertoire disturbance is not indicative of the severity of disease progression.

Differential production of IFN-gamma, analyzed at the single-cell level, by specific subsets of human NK and T cells from healthy and HIV(+) subjects.

BACKGROUND: Interferon gamma is a cytokine that plays a central role in immunity, and is physiologically secreted by T and NK cells under appropriate stimuli during the immune response. By means of flow cytometry, we performed a single cell analysis of interferon gamma producing NK cells and their surface phenotype in normal and HIV(+) individuals that show several defects of cytokine production and cellular immunity. METHODS: PBMC or purified NK cells were stimulated for 1-12 h with PMA/ionomycin in the presence of monensin, subsequently stained for surface CD56 and CD3 or CD8, and for intracytoplasmic IFN-gamma, and analysed by flow cytometry. RESULTS: Our results show that CD56(+) NK cells are more efficient interferon gamma producers than T cells. Moreover, within the CD56(+) NK cell population, those that co-express low density CD8 are the best producers. Finally, we show that NK cells during HIV infection are more massively recruited to interferon gamma production than those from normal subjects. CONCLUSIONS: Both in the normal and HIV(+) subjects, a higher percentage of NK cells than T cells can produce IFN-gamma although differences can be identified within the NK cells subset in terms of IFN-gamma production. The production of IFN-gamma is fully achievable in the HIV(+) subjects, which is consistent with their elevated plasmatic levels of the cytokine. The possibility that NK cells that produce interferon gamma could represent a functionally distinct population committed to the production of this cytokine, is discussed.

Generation of CD28- cells from long-term-stimulated CD8+CD28+ T cells: a possible mechanism accounting for the increased number of CD8+CD28- T cells in HIV-1-infected patients.

According to CD28 molecule expression, CD8+ T cells can be classed as CD28bright, CD28dim, and CD28-. The CD28dim T cells were found to derive from mitogenic stimulated CD28-T cells but also from CD28bright T cells through a mechanism of CD28 down-modulation. Moreover, after prolonged in vitro interleukin-2 stimulation, clonal CD28bright, cells showed a CD28dim expression before further evolution to a stable CD28-phenotype. This loss was concomitant with the disappearance of CD28 mRNA. A study of the cytokine production pattern revealed that CD28dim and CD28- T cell clones produced similar levels of type 1 and type 2 cytokines, which differed from those produced by the CD28bright T cell clones. A high percentage of CD28dim and CD28- cells, with similarities in their cytokine production pattern, were found in the blood samples of HIV-infected patients, as compared to healthy donors. The CD28 down-modulation may account for the increased number of CD8+CD28- T cells in HIV-infected patients.

Reduced production of both Th1 and Tc1 lymphocyte subsets in atopic dermatitis (AD).

An imbalance of interferon-gamma (IFN-gamma)-bearing CD4+ T (Th1) cells in the pathogenesis of AD is well recognized; however, a possible role in AD for CD8+ T cells secreting Th1-like cytokines (Tc1) has not been properly addressed. In this study, two- and three-colour FACS analysis allowed us to discriminate the Th1 from the Tc1 subset. AD patients had half the number of IFN-gamma-producing circulating T cells (P < 0.005; 13.6 +/- 1.9% (mean +/- s.d.)) compared with normal donors (25.0 +/- 2.4%). Specifically, both Th1 (4.8 +/- 0.7%) and Tc1 (8.1 +/- 1.1%) cells in AD were decreased compared with Th1 (8.8 +/- 0.8%) and Tc1 (15.0 +/- 1.5%) cells in controls. Moreover, at the mRNA level, the ratios of IFN-gamma/IL-4 and IFN-gamma/IL-10 were lower in cells from AD patients compared with controls. In conclusion, the decrease of IFN-gamma-producing T lymphocytes in AD is due to a reduction in both Th1 and Tc1 IFN-gamma-secreting cells; this may not only contribute to the over-production of IgE, but also explain the high incidence of cutaneous infections observed in AD patients.

Expression of inducible nitric oxide synthase in human granulomas and histiocytic reactions.

Inducible nitric oxide synthase (iNOS) is required in immune response against infections and is involved in granuloma formation in animals; in murine macrophages, iNOS is induced by lipopolysaccharide and interferon-gamma. In contrast, the role of iNOS in human immune response against infections is still questioned, and its expression in granulomas is poorly investigated. Using Western blotting and immunohistochemistry, we investigated iNOS expression in human lymph nodes with nonspecific reactions and in tissues containing granulomas caused by mycobacteria, Toxoplasma, Cryptococcus neoformans, Leishmania, Bartonella, noninfectious granulomas (sarcoidosis, foreign body), and other hystiocitic reactions (Kikuchi's disease, Omenn syndrome). iNOS was undetectable in nonspecific reactive lymphadenitis, foreign-body granulomas, and Omenn syndrome, whereas it was strongly expressed in infectious granulomas, sarcoidosis, and Kikuchi's diseases. Immunohistochemistry demonstrated that iNOS was selectively expressed by the epithelioid and multinucleated giant cells within the granulomas. Use of an anti-nitrotyrosine antibody, recognizing nitrosilated amino acid residues derived from nitric oxide production, revealed a consistent positivity within the cells expressing iNOS, thus suggesting that iNOS is functionally active. Detection of cytokines by reverse transcriptase-polymerase chain reaction demonstrated that tissues that were positive for iNOS, also expressed the Thl-type cytokine interferon-gamma mRNA, but not the Th2-type cytokine interleukin-4. Taken together, these results indicate that iNOS is involved in different human immune reactions characterized by histiocytic/granulomatous inflammation and associated with Th1-type cytokine secretion.

Phenotypic and functional characteristics of tumour-derived microvascular endothelial cells.

We recently developed a method for the isolation and purification of tumour-derived endothelium. In this study the phenotypic and functional properties of human tumour-derived microvascular endothelial cells (TdMEC) were examined. Endothelium obtained from human adrenal gland specimens (HAMEC) was used as a reference microvascular endothelial cell population. TdMEC formed a confluent monolayer with the typical morphological appearance of endothelium and were positive for endothelial markers such as Ulex-1 lectin, CD31 antigen, von Willebrand Factor and VE-cadherin. The addition of acidic Fibroblast Growth Factor (aFGF), basic FGF (bFGF) or Vascular Endothelial Growth Factor (VEGF) substantially improved proliferation of TdMEC; and kidney carcinoma derived endothelial cells were more responsive to FGFs, whereas glioblastoma derived endothelial cells greatly responded to VEGF TdMEC expressed high levels of the VEGF receptors, KDR/flk-1 and Flt-1, as shown by northern blot analysis. TdMEC expressed the adhesion molecules ICAM-1, VCAM-1 and E-selectin that could be further increased by exposing TdMEC culture to interleukin-1. All the TdMEC expressed interleukin-8 mRNA. These findings show that TdMEC in vitro maintain several of the features described for microvasculature. Thus, TdMEC represent a useful tool to study markers for tumor vasculature.

Segregation of type 1 cytokine production in human peripheral blood lymphocytes: phenotypic differences between IFN-gamma and IL-2-producing cells in the CD8+ T cell subset.

T cell clones are classified as type 0, 1 or 2 depending on the lymphokines they produce. However, it has remained unclear whether single cells of a given type produce one or several cytokine species. Flow cytometric analysis of peripheral blood lymphocytes (PBL) obtained from 20 healthy donors for the production of the type 1 cytokines IFN-gamma and IL-2 revealed very few cells that co-expressed both cytokines independently of the mitogenic stimulus used for PBL activation. Similarly, kinetic studies of cytokine synthesis indicated a low percentage of IFN-gamma/IL-2 double-positive T cells at all time points. Reverse transcription-PCR analysis of sorted IL-2- and IFN-gamma-positive T cells showed the presence of IL-2- or IFN-gamma-specific mRNA only in those cells expressing the corresponding cytokine. This segregation of the two type 1 cytokines was lost in long-term cultured T cells and in T cell clones. A high percentage of cells expressing only IL-2 or IFN-gamma was observed even when the production of these cytokines was evaluated on CD4- and CD8+ subsets. Moreover, in some healthy individuals, IFN-gamma and IL-2 production by CD8+ T cells was related to CD8+ expression levels and cell size, i. e. IL-2-expressing cells were generally smaller with more intense CD8+ staining as compared with IFN-gamma-producing T cells. These data indicate that activated T lymphocytes are strongly committed in vivo to produce IFN-gamma or IL-2 and emphasizes the independent regulation of the two cytokine genes.

HIV p17 enhances lymphocyte proliferation and HIV-1 replication after binding to a human serum factor.

OBJECTIVE: To analyse the role of recombinant HIV-1 protein p17 in the modulation of cell activity. METHODS: Peripheral blood mononuclear cells (PBMC) obtained from healthy donors were cultured in the presence or absence of p17 with mitogens such as phytohaemagglutinin or interleukin-2 and their response assayed by cell proliferation. Cross-linking experiments were employed to investigate the presence of a binding between p17 and factor(s) present in human serum. An immunoenzymatic assay for p24 antigen detection was used to analyse the effect of the addition of exogenous p17 to cultures of PBMC infected with HIV-1 in vitro. RESULTS: Purified recombinant p17 protein at a concentration of 0.25 microg/ml significantly increased the proliferation of preactivated PBMC obtained from healthy donors. This effect was obtained by binding p17 to factor(s) present in human serum and observed on both CD4+ and CD8+ T cells. Recombinant p17 also induced an increased rate of HIV-1 replication, probably due to enhanced T-cell proliferation. The activity of p17 protein was inhibited by anti-p17 antibodies generated by injecting recombinant p17 in rabbits, but not by human antibodies generated during the natural course of HIV infection. CONCLUSION: Characterization of the human factor(s) and identification of the interacting p17 epitope(s) will improve our understanding of the mechanisms used by HIV to efficiently replicate in our organisms.

Lack of polarized type 1 or type 2 cytokine profile in asymptomatic HIV-1-infected patients during a two-year bimonthly follow-up.

The production of type 1 (interferon or IFN-gamma) and type 2 (interleukin or IL-4 and IL-10) cytokines by mitogen-stimulated peripheral blood mononuclear cells (PBMCs) obtained from asymptomatic human immunodeficiency virus-seropositive (HIV+) patients untreated with any antiviral, antibacterial or antimycotic drugs, and from healthy individuals, was evaluated by quantitative ELISA. Patients who were HIV+ were characterized by the absence of abnormal cytokine production. The level of each cytokine differed among individuals in the same group with intersubject variations greater for HIV+ patients than for healthy individuals. The longitudinal evaluation of IFN-gamma, IL-4 and IL-10 production showed intrasubject variations which were particularly marked in HIV+ patients. Accordingly, HIV+ patients and, to a lesser extent, healthy individuals were characterized by a wide spectrum of possible profiles, which were confined to type 0 phenotype. In HIV+ patients no correlation was found between each cytokine level and the number of CD4+ T cells, not even in those with a falling CD4+ T-cell count and clinical symptoms.