ASSESSMENT OF CYTOKINE VALUES IN SERUM BY RT-PCR IN HIV-1 INFECTED INDIVIDUALS WITH AND WITHOUT HIGHLY ACTIVE ANTI-RETROVIRAL THERAPY

A cross-sectional study was performed on HIV-1 infected individuals with or without antiretroviral treatment (ARV) in the AIDS Day Hospital, Botucatu Medical School, UNESP. Between August 2004 and October 2005, 73 HIV-1 infected individuals were divided into three groups: infected individuals with or without AIDS who had never received ARV (G1 = 15); patients on HAART that had had plasma HIV-1 RNA viral load (VL) equal to or greater than 50 copies/mL (G2 = 27); and patients on HAART with undetectable VL for at least the past six months (G3 = 31). There was also an additional group that comprised blood donors without any sign of the disease and with negative HIV serum tests (G4 = 20), which was the control group. Serum cytokine levels (values in pg/mL) were measured by enzyme-linked immunosorbent assay (ELISA) and specific mRNA expression by reverse transcription polymerase chain reaction (RT-PCR). Both techniques were performed on the four groups for TNF-α, IL-2, INF-γ, IL-4 and IL-10. All patients were submitted to VL determination and CD4 and CD8T lymphocyte counts. The analysis of the results revealed a significant comparison among groups for both methods and an association between the latter (> 80% – r > 0.80). There was only one exception, in control individuals for IL-2 by ELISA. The cytokine profiles, in both methods, for the three patient groups, were mature Th-0. The behaviors of IL-2 and INF-γ required emphasis due to consequent expression of dominant Th profile. Both methods showed low IL-2 and high mean values of INF-γ in the three groups. Several authors have recently drawn attention to the substantial apoptosis of infected and non-infected CD4T cells, mainly during primary infection, persisting only in those with INFγ phenotype producer and not IL-2. HIV infected individuals submitted to HAART are expected to produce IL-2 in an attempt to present Th-1 profile, but in most cases this did not occur.


INTRODUCTION
Highly active anti-retroviral therapy (HAART) causes important interference in the natural history of HIV-1 infection.Haase (9) reported the impact of this treatment on suppressing viral replication that could be observed by VL determination.However, even with this vigorous action, the treatment did not completely eliminate the virus.
Pierson et al. (20), considering the half-life of latently infected cells which are HIV-1 reservoirs, applied a mathematical model and concluded that more than 60 years of treatment would be required to eradicate this compartment.Regarding HIV surrogate markers, VL has been of great use, as the reduction in number of RNA copies per mL of plasma, often not detectable, has become an eloquent indicator of therapeutic action.The clinical evolution of concomitant patients shows increased survival, easier control of opportunistic infections, and a reduction in the number of deaths for patients under treatment (16).According to Gougeon et al. (7) the other surrogate marker, represented by CD4 + T/mm 3 , which can give information on suppression level or immune system recovery level in patients under treatment, is influenced by complex mechanisms with complicate interpretation.Carcelain et al. (2), discussing immune reconstitution during HIV infection under HAART, mentioned three mechanisms involved in CD4 + T lymphocyte recuperation, which are: redistribution of memory CD4 + T cells from tissues where they have been previously sequestered; regeneration of virgin thymus cells; and reduction of inflammatory process.In this manner, CD4 + T count does not reflect the real number of virgin, memory, or recirculating CD4 + T cells after the reduction of therapeutic viral replication.
Additionally, quantitative evaluation of these cells does not inform us which cytokines they are producing.Haase (9), referring to naïve CD4 + T cell repopulation during treatment, mentioned that they slowly increase, but only many months after therapy introduction.According to the latter author, immunity regeneration is slow, variable and partial.
The main problem concerning HIV-1 infected individuals, when they are submitted to HAART, is the evaluation of immune function under this therapy interference.It is necessary to search for other markers to notice whether or not there is immunological recovery (4,5).Cytokine levels and the knowledge of their respective profiles can be evolution markers of immune response during ARV treatment.

Patients
Between August 2004 and October 2005, 73 HIV-1 infected individuals, ill and not ill, were monitored at the Specialized Outpatient Service and Day Hospital for AIDS patients, part of the medical complex of Botucatu Medical School, UNESP.All persons had a history compatible with HIV-1 infection, with either positive ELISA or Western blot.Among them, 38 were female and 35 male and their ages ranged from 22 to 66 years old (mean: 40 years).Twenty normal blood donors from the Botucatu Hemocenter were also included, 16 were male and four female and aged between 19 and 62 years old (mean: 37 years).

• Groups
Group 1 (G 1 ): 15 HIV-1 infected individuals, with or without AIDS, who had never received ARV.These patients had not yet been indicated for ARV, or had had HIV-1 infection diagnosed a few days before inclusion in the present study.Individuals from this group were the only ones who were not on ARV treatment, thus could be considered as representatives of the natural history of HIV infection.Patients in the other two groups were under the influence of treatment.
Group 2 (G 2 ): 27 HIV-1 infected individuals, sick or not, on ARV treatment, five with two nucleoside/nucleotide reverse transcriptase inhibitors (NRTI) and one nonnucleoside reverse transcriptase inhibitor (NNRTI); and 22 on HAART with two NRTI, or one NRTI and one NNRTI, and one protease inhibitor (PI), and VL equal to or greater than 50 copies of plasma RNA/mL.
Treatment duration in this group varied between three and 145 months (mean: 53.6 months; median: 42 months).
Group 3 (G 3 ): 31 HIV-1 infected individuals on ARV treatment, 16 on HAART with two NRTI, or one NRTI and one NNRTI, and one PI, and 15 with two NRTI and one NNRTI.All G 3 patients had undetectable VL for at least the past six months.
Treatment in this group varied between five and 108 months (mean: 48.1 months; median: 42 months).In both groups G 2 and G 3 , all individuals were under ARV treatment and classified as positive (G 2 ) or undetectable (G 3 ) VL, which indicates a clear distinction between them concerning viral activity and, consequently, presents an important factor that must be considered for the interpretation of HIV-1 pathogenicity (12).Group 4 (G 4 ): 20 blood donors without any clinical complaints and negative for anti-HIV-1/2 antibodies.None of them presented any sign of the disease.

• Clinical parameters
All 73 infected individuals were submitted to clinical observation that included HIVassociated opportunistic diseases at the time of blood collection for cytokine detection.

• HIV-1 plasma viral load determination
Infected individuals were tested for HIV-1 plasma VL by the bDNA HIV-1 RNA QT system.The low limit for detection by this method was considered 50 copies/mL.

• CD4 + T and CD8 + T/mm 3 lymphocyte counts
The numbers of CD4 + and CD8 + T cells were measured from all HIV-infected patients in G 1 , G 2 , and G 3 using four-color flow cytometry and the commercially available Multitest IMK® kit (Becton Dickinson, USA) (13).

• Serum cytokine determination
We collected 8 mL of blood, in a dry tube, from 73 HIV-1 (G 1 , G 2 , and G 3 ) and 20 normal (G 4 ) individuals.The serum was immediately separated, aliquoted and stored at -70 ο C. Serum cytokine determination was performed between four and 12 weeks after storage.TNF-α, INF-γ, IL-2, IL-4 and IL-10 cytokines were determined by ELISA, with Human Quantikine® kits (R&D Systems, USA).Initially, 96 microplates were sensitized with anti-cytokine monoclonal antibody (TNF-α, INF-γ, IL-2, IL-4 and IL-10).Subsequently, 200 μL of test serum, positive and negative controls, was added (dilution 1:2) to the samples that, afterwards, were incubated at 37°C for periods ranging from 30 to 60 minutes, depending on the cytokine.Four washes were carried out with detergent solution containing 2-chloroacetamide (0.1%).This was repeated until the phase preceding substrate addition.Then, biotin marked plates received streptavidin-peroxidase.After incubation, a substrate containing hydrogen peroxide (0.02%) and tetramethylbenzene (2%) was added.The reaction was interrupted at room temperature with 2N sulfuric acid.Results were evaluated by optical density (OD) on a Multiskan analyser (EFLAB, Finland) reader at 450 nm.

• Cytokine dosage by amplification and specific cDNA detection using realtime PCR
Peripheral blood mononuclear cells (PBMC) were obtained from heparinized blood by means of standard Histopaque-1077® (Sigma-Aldrich, USA) gradient centrifugation (1).Total RNA was extracted from PBMC immediately after the collection utilizing Trizol® reagent (Invitrogen, USA) according to manufacturer   26).Thus, Th-1, mature Th-0 and Th-2 subsets were defined by their normal values according to Spellberg and Edwards (22).These authors considered mature Th-0 profile in situations where the CD4 + T cells produced elevated levels of INF-γ and IL-4.Other authors have also reported Th-0 profile (6,8,19).The proportions of PI regimens in G 2 and G 3 and treatment duration in each group submitted to HAART (G 2 and G 3 ) were compared by the χ 2 (chi-square) test (26).

• ARV treatment scheme
Twenty-two patients from G 2 received protease inhibitors associated with two NRTI or one NNRTI and one NRTI.The other five patients in this group were treated with two NRTI and one NNRTI.Sixteen G 3 patients received protease inhibitors associated with two NRTI or one NNRTI and one NRTI.The other fifteen received two NRTI and one NNRTI.There was statistical difference between groups in relation to protease inhibitor (χ 2 1 = 6.99; p < 0.01; G 2 > G 3 ).

• Treatment duration
Comparison between treatment groups showed no statistical difference with respect to duration.Seventeen G 2 patients were treated for a period of three to 60 months and the other ten, for a period of 61 to 145 months.Twenty-two G 3 patients were treated for three to 60 months and the other nine, for 61 to 145 months (χ 2 1 = 0.41; not significant).Anim.Toxins incl.Trop. Dis., 2008, 14, 4, p. 692 Comparison between groups showed no statistical difference (H = 4.371; p > 0.10; G 1 = G 2 = G 3 ).Median (Md), mean ( X ) and standard deviation (SD) values of CD8 + T lymphocytes/mm 3 were:

VL behavior
VL mean for G 1 patients was 31,231 copies/mL (log 4.49), and for G 2 patients was 8,779 copies/mL (log 3.94).G 3 , which could also be due to the antiretroviral treatment.IL-2 however, remained low, not differing from normal values (G 4 ), and presented the same median level in the four groups.This fact could be associated with a possible incapacity of CD4 + T cells, in HIV-1 infected individuals, to produce IL-2, even in patients under treatment or with undetectable VL.The latter finding suggests that a potent combined antiretroviral treatment is effective as it improves the life condition of patients, permits better control of opportunistic infections and increases survival in infected individuals.

Comparison and correlation of cytokine levels in serum (ELISA) and by amplification and specific cDNA detection using real-time PCR (RT-PCR)
However, this type of treatment is not completely efficacious, since incapacity to perform HIV-1 clearance, or increase IL-2 production, suggests an inability to induce adaptive immune recovery.Figure 1, which reveals similarity between both methods (ELISA and RT-PCR).There was a small increase in IL-2 levels in treatment virgin patients (G 1 ) when compared to persons under treatment that presented detectable VL (G 2 ), while there was a decrease in individuals under treatment with undetectable VL (G 3 ).Statistical analysis revealed that IL-2 values were equal in these groups.In this sense, IL-2 behavior was the same in both measurement methods, which displays an association between them.If its behavior in patients is judged, there is no desirable IL-2 production even with antiretroviral treatment and when VL is reduced.

DISCUSSION
Cytokines levels obtained by ELISA in serum and by RT-PCR in mononuclear cells were compared among groups.This comparison revealed statistical significances for all cytokines, except for IL-2.It was the only cytokine that, although statistically significant, did not distinguished groups by ELISA in serum; this ascertaining drew attention to IL-2.In addition, normal individuals presented lower levels of cytokines by both methods, with the exception of IL-2 by ELISA.Furthermore, significance was found for all proteins by ELISA and RT-PCR in linear correlation.However, IL-2 significance level (p) was lower.These findings suggest that similarity indeed exists between the two methods.
In an earlier publication, Meira et al. (15) exposed the serum cytokine behavior in a cross-sectional study, in which 79 HIV-1/AIDS patients were distributed into three

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D. A. Meira et al.ASSESSMENT OF CYTOKINE VALUES IN SERUM BY RT-PCR IN HIV-1 INFECTED INDIVIDUALS WITH AND WITHOUT HIGHLY ACTIVE ANTI-RETROVIRAL THERAPY (HAART).J. Venom.Anim.Toxins incl.Trop.Dis., 2008, 14, 4, p. 690 instructions.DNase digestion of RNA solution was carried with DNase (RQ1 RNase-Free DNase®, Promega, USA).The Platzer and Blankestein (21) technique was employed to obtain cDNA.Semi-quantitative RT-PCR was performed in real time (7300 Real Time PCR System®, Applied Biosystems, USA) using Lux primers (Certified Lux® Primer Set, Invitrogen, USA) for the following cytokines: IL-GenBank accession number NM 000586); IL-4 (GenBank accession number NM 000589); IL-10 (GenBank accession number NM 000572); INF-γ (GenBank accession number NM 000619); and TNF-α (GenBank accession number NM 000594); β-actin (GenBank accession number NM 001101-2) -used for housekeeping reactions.Platinum PCR Supermix® (Invitrogen, USA) was utilized for the PCR reaction.Samples were distributed on 96-well plates and amplified in real time under the following incubation conditions: 2 minutes at 50°C and 95°C, 45 cycles at 95°C for 15 seconds, 55°C and 72°C for 30 seconds during which fluorescence data were obtained.The threshold cycle (Ct) reflects the cycle number at which the fluorescence generated within a reaction cross the threshold.The Ct value assigned to a particular well thus reflects the point during the reaction at which a sufficient number of amplicons have accumulated, in that well, to be at a statistically significant point above the baseline (18).One member of the normal group (G 4 ) was randomly selected for serial dilutions of cDNA, which was used as a standard cytokine quantification reference.Results expressed as Ct values for β-actin and cytokines, for each individual, were corrected based on values from the normal individual according to the standard real-time calculation.• Statistical analysis Mean ( X ), standard deviation (SD) and median (Md) were calculated for each group.Comparison among groups G 1 , G 2 , G 3 and G 4 -regarding serum cytokines, amplification and detection of specific cDNA -was made by non-parametric Kruskal-Wallis test (26), with H and p statistics (chi-square distribution) and considering differences between group means (p < 0.05).Similarity between ELISA and RT-PCR methods was studied by linear correlation coefficient between two measurements and after the linear regression equation Y = f (X), where Y = RT -PCR and X = ELISA, with the respective coefficient of determination (r 2 Median (Md), mean ( X ) and standard deviation (SD) values of CD4 + T lymphocytes/mm 3 in the patient groups were: -G 1 : Md = 313; X = 383; and SD = 229; -G 2 : Md = 334; X = 334; and SD = 186; -G 3 : Md = 419; X = 470; and SD = 247.D. A. Meira et al.ASSESSMENT OF CYTOKINE VALUES IN SERUM BY RT-PCR IN HIV-1 INFECTED INDIVIDUALS WITH AND WITHOUT HIGHLY ACTIVE ANTI-RETROVIRAL THERAPY (HAART).J. Venom.

Figure 1
Figure 1 shows the distribution of cytokine values (TNF-α, INF-γ, IL-2, IL-4 and IL-10) obtained through serum dosage by ELISA.The graph shows individual values for each cytokine and the horizontal line in each column refers to the median values obtained from the respective study group.Analysis of this figure suggests that TNF-α values decrease in the comparison among patient groups (G 1 to G 3 ), but remain elevated in relation to controls (G 4 ).The drop may be due to the antiretroviral treatment in G 2 and G 3 .INF-γ presented opposite behavior and increased from G 1 to

Figure 2
Figure 2 presents cytokine level distribution in mononuclear cells by RT-PCR.Median values of TNF-α and INF-γ for each group (G 1 to G 3 ) show a similar behavior to
ASSESSMENT OF CYTOKINE VALUES IN SERUM BY RT-PCR IN HIV-1 INFECTED INDIVIDUALS WITH AND WITHOUT HIGHLY ACTIVE ANTI-RETROVIRAL THERAPY (HAART).J. Venom.Anim.Toxins incl.Trop.Dis., 2008, 14, 4, p. 691 Kruskal-Wallis test) were compared among groups G 1 , G 2 and G 3 (26).Serum and real-time PCR cytokine normal values were obtained by X +1SD of their respective values from the control group (G 4 ) ( ).A correlation was considered significant when r 2 > 0.60 and high when r 2 > 0.80.CD4 + T and CD8 + T/mm 3 lymphocyte counts and VL determination values (by non-parametric D. A.Meira et al.

Table 4 .
Serum and mononuclear cell cytokine subsets according to normal values, obtained by +1SD of their respective normal group values (G )* X (22)apted from Spellberg and Edwards(22).