1. Introduction

Human immunodeficiency virus, the virus that causes acquired immunodeficiency syndrome has become one of the world’s most serious health and development challenges ^[9]. Since the start of the epidemic, around 78 million people have become infected with HIV and 39 million people have died of AIDS-related illnesses. In 2013, there were 35 million people globally living with HIV of which 24.7 million were from sub-Saharan Africa ^[10]. Worldwide, 2.1 million people became newly infected with HIV in 2013. There were an estimated 1.5 million new HIV infections in sub-Saharan Africa, accounting for almost 70% of the global total of new HIV infections ^[10]. The total number of people living with HIV in Kenya was estimated to be 1.6 million in 2013. Children under 15 years of age account for 12% of all people living with HIV. An estimated 58% (815,630) of all adults aged 15+ living with HIV in Kenya are women ^[12]. Worldwide, 2.1 million people became newly infected with HIV in 2013. There were an estimated 1.5 million new HIV infections in sub-Saharan Africa, accounting for almost 70% of the global total of new HIV infections ^[10], while in Kenya there were approximately 88,620 new HIV infections that occurred among adults and 12,940 among children in 2013 ^[12].

The Human Immunodeficiency Virus (HIV) targets the immune system and weakens the surveillance and defense system of the body against infections, resulting in HIV infected individuals becoming more susceptible to a wide range of infections normally cleared by the immune system of a healthy individual ^[26]. There is need to monitor HIV disease progression during the early stages of infection in order to advice and counsel individuals affected and not accepting their status, and consequently to avoid further spread and also it could guide future treatment of the disease.

Cytokines are soluble proteins that play an important role in immunity, inflammation, and hematopoiesis. They are rapidly produced by a variety of cell types and secreted in response to specific and non-specific stimuli ^[13]. They are effecter molecules that can instantly alter the quality of the immune response. The effect of a particular cytokine on a given cell depends on the cytokine, its extra cellular abundance, the presence (or absence) of the complementary receptor on the cell surface, and downstream signals activated by receptor binding ^[19].

Cytokines are regulators of host responses to infection, immune responses, inflammation, and trauma. Some cytokines act to make disease worse (pro-inflammatory cytokines), whereas others serve to reduce inflammation and promote healing (anti-inflammatory cytokines). CD4+ T helper cells play a vital role in the immune system by secreting cytokines, which regulate the immune response. Cytokines are secreted by T cells when an intracellular infection is detected as in case of HIV infection. HIV is known to infect T cells that have CD4+ receptors present on their surface. These cells, among others in the immune system, secrete cytokines that are crucial for fighting off infections and in other immune responses. However, they can become dysregulated and pathological in inflammation, trauma, and sepsis ^[3].

Cytokine profiles are highly relevant parameters of an immune response. Different cytokines possess biologically overlapping functions, and they have the ability to regulate production of other cytokines. Therefore, analysis of production and function of the complete set of cytokines expressed during acute HIV infection may be valuable in predicting disease progression. Quantification of cytokine produced is a valuable adjunct in standard immunological assays for defining several pathologic processes. There may be alterations in the synthesis of host cytokines following HIV infection, thereby favoring successful survival of the virus inside the host and enhancing the susceptibility of the host to opportunistic infections. Most cytokines exert pleiotropic and sometimes contrasting effects on the immune response ^[11]. The concentration of cytokines in blood plasma can provide valuable information about the immune status of infected individuals. Measurement of cytokine levels may also be of use in the monitoring of disease progression and / or inflammation; this type of study has not been done in this region.

2. Materials and Methods

The study was carried out in the Nakuru Provincial General Hospital (PGH), Nakuru County, Kenya, after the approval by the hospital’s administration. Consent was sought from the study group by the researcher with the assistance and guidance of the staff of the Centre for Comprehensive Care (CCC) before commencement of the study. Only those who gave consent were enrolled in the study and they completed a written informed consent form in accordance with the Helsinki Declaration ^[27].

This was a prospective cross sectional study that involved selecting consenting individuals who attended the Voluntary counseling and Testing centre (VCT) and the Centre for Comprehensive Care at the hospital. Eighty consenting male and female of different ages were sampled according to Yamane ^[28] to participate in the study and were later subdivided into three groups as recommended by Sudman ^[23]. Forty patients that included sixteen males and twenty four females were sampled from those recently diagnosed with HIV before they started antiretroviral treatment (treatment naïve HIV patients); twenty patients that included four males and sixteen females were sampled from HIV positive patients that were receiving treatment with highly antiretroviral therapy (HAART) while twenty participants that included nine males and eleven females were sampled from HIV negative individuals.

Whole blood was collected into EDTA treated tubes, stored at room temperature and then centrifuged for 10 minutes at 3000 revolution per minute (r.p.m). Plasma was pipetted and aliquoted into cryovials that were labeled with appropriate information of the individuals in the study group. The plasma was refrigerated at -20^oC until when the entire cohort had been collected in order to analyze all the samples simultaneously.

All the eighty labeled plasma samples were thawed before flow cytometry for cytokine detection. The types and quantities of cytokines were detected by flow cytometry using a multiplex assay system that included Becton and Dickinson Cytometric Bead Array (BD CBA), Human Inflammatory Cytokine kit and Becton and Dickinson Fluorescence Activated Cell Sorter (FACSCalibur) flow cytometer (BD Biosciences, U.S.A). The workflow consisted of the following steps according to the recommended procedure (BD Biosciences, U.S.A):

A vial of lyophilized Human Inflammatory Cytokine Standard was opened and the standard spheres transferred to a 1 ml polypropylene tube. The tube was labeled “Top Standard” (Tube 10). The standards were reconstituted with 2 ml of assay diluent and allowed to equilibrate for 15 minutes at room temperature. The reconstituted protein was gently mixed by pipette. Eight 12x75 mm tubes were labeled and arranged in the following order: 1:2 (Tube 9; 2,500 pg/mL), 1:4 (Tube 8; 1,250 pg/mL), 1:8 (Tube 7; 625 pg/mL), 1:16 (Tube 6; 312.5 pg/mL), 1:32 (Tube 5; 156 pg/mL), 1:64 (Tube 4;80 pg/mL), 1:128 (Tube 3; 40 pg/mL) and 1:256 (Tube 2; 20 pg/mL). 300 µl of assay diluent was pipetted in each of the tubes. Serial dilution was performed by transferring 300µl from the Top Standard to the 1:2 dilution tube and mixed thoroughly by pipette, and continued by transferring 300µl from 1:2 tube to the 1:4 tube until to the 1:256 tube. One tube was prepared containing only assay diluent to serve as the 0 pg/mL negative control. This was labeled as Tube 1 and had no standard dilution. The standard curve for each protein covers a defined set of concentrations from 20 to 5,000 pg/mL (BD Biosciences, U.S.A).

Six Capture Beads that had been bottled individually were pooled before using them in the assay. Each capture bead suspension was vortexed before mixing. 10 µl aliquot of each capture bead, for each assay tube to be analyzed, was added into a single tube labeled “Mixed Capture Beads”. Eighty assay tubes were to be analyzed and so the mixture comprised of the following capture beads: 800 µl (10 x 80) of interleukin 8 (IL-8), 800 µl of interleukin 1β (IL-1β), 800 µl of interleukin 6 (IL-6), 800 µl of interleukin 10 (IL-10), 800 µl of tumor necrosis factor (TNF) and 800 µl of interleukin 12p-70 (IL-12p70). The mixture of capture beads was vortexed thoroughly and was ready for transfer to the assay tubes.

After preparing the standards and mixing the capture beads, the next step was to perform the assay. The mixed capture beads were vortexed and 50 µl added to all assay tubes. 50 µl of the sample was added to eighty labeled tubes each containing about 12.5 µl of plasma that included forty from HIV positive patients, twenty from HIV negative and twenty from HIV positive patients on HAART. The assay tubes were incubated for 1.5 hours at room temperature. After the incubation period, 1 ml of wash buffer was added to each assay tube and centrifuge at 200g for 5 minutes; the supernatant was aspirated and discarded. 50 µl of the human inflammatory cytokine phycoerythrin (PE) detection reagent was added to all assay tubes and incubated for 1.5 hours at room temperature. After this second incubation period, again 1 ml of wash buffer was added to each assay tube and centrifuged at 200g for 5 minutes; the supernatant was aspirated and discarded from the assay tubes and finally 300 µl of wash buffer was added to each assay tube to resuspend the bead pellet.

The samples were acquired on the flow cytometer. The assay setup procedure was followed according to the protocol by the manufacturers (BD Biosciences, U.S.A) and the appropriate acquisition template was available. Each sample was vortexed for 3-5 seconds immediately before acquiring on the flow cytometer. Tube 1 (0 standard) was vortexed and ran in setup mode according to the protocol. Sample acquisition continued by running Tube 2 (20 pg/mL), followed by Tube 3 (40 pg/mL), and so on throughout Tube 10 (Top Standard). The test samples were run after the standards. Data for the detection of individual proteins was analyzed using BD Cell Quest software, according to the recommended protocol (BD Biosciences, U.S.A).

Descriptive statistics were applied. Data analyses were performed using Graph Pad Prism 6. Independent sample T tests were used to compare the cytokine means while Spearman Rank tests were used to test for correlations. Statistical analysis were done using SPSS version 17.

3. Results

Five types of plasma cytokines: Interleukin 12p70 (IL-12p70), Tumor Necrosis Factor (TNF), Interleukin 10 (IL-10), Interleukin 6 (IL-6) and Interleukin 1β (IL1β) were detectable in the study population. IL-12p70, TNF, IL-10 and IL-6 were detected in treatment naïve HIV patients; in HIV positive patients on HAART and in HIV negative patients. IL-1β was detected in treatment naïve HIV patients and in HIV positive patients on HAART but none was detectable in HIV negative patients (Table 1).

The mean concentrations of IL-12p70 (3.317±4.441 pg/mL), TNF (7.707±13.40 pg/mL), IL-10 (2.794±4.437 pg/mL) and IL-6 (6.629 pg/mL) were higher in the treatment naïve HIV patients compared to the HIV positive patients on HAART (0.5593±1.397 pg/mL, 1.975± 4.980 pg/mL, 0.6918± 1.628 pg/mL and 2.602± 4.620 pg/mL and the HIV negative patients (0.2815± 0.5145 pg/mL, 0.7895± 2.247 pg/mL, 0.1630± 0.5032 pg/mL, 0.5135± 1.206 pg/mL) respectively. The mean concentration of IL-1β (5.401±10.14 pg/mL) was higher in the treatment naïve HIV patients compared to HIV positive patients on HAART (0.7028± 4.445 pg/mL). There was no detectable IL-1β in HIV negative patients (Table 1).

Table 1. Plasma Cytokine Profiles of the Study Population

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The means of plasma cytokines of treatment naïve HIV positive patients and HIV negative patients were compared. The treatment naïve HIV patients had significantly (p<0.001) high means of IL-12p70, TNF, IL-10, IL-6 and IL-1β compared to HIV negative patients (Table 2).

Table 2. Means of blood cytokines of HIV patients and HIV negative patients and associated pvalues

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The means of plasma cytokines of treatment naïve HIV patients and HIV patients on HAART were also compared. The treatment naïve HIV patients had significantly (<0.001) high means of 12p70, TNF, IL-10, IL-6 and IL-1β compared to HIV patients on HAART (Table 3).

Table 3. Means of plasma cytokines of treatment naïve HIV patients and HIV patients on haart and associated p-values

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Finally, the means of plasma cytokines of HIV negative patients and HIV patients on HAART were compared. HIV patients on HAART had significant high means of TNF (<0.05) and IL-6 compared to HIV negative patients (<0.05; Table 4). IL-12p70 (p=0.079) and IL-10 (p=0.008) were significant in HIV patients on HAART; IL-β (p=0.154) was the least significant in this group compared to HIV negative patients (Table 4).

Table 4. Means of plasma cytokines of HIV negative patients and Hiv patients on haart and associated p-values

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4. Discussion

Plasma interleukin 12p70 (IL-12p70), tumor necrosis factor (TNF), interleukin 10 (IL-10), interleukin 1β (IL-β), interleukin 6 (IL-6), and interleukin 1β (IL-β) were detected in the study population. The treatment naïve HIV patients had higher concentration levels of all cytokines detected. Reduced concentration levels of the cytokines detected in this study was observed in patients on antiretroviral therapy, an indication that treatment of HIV with antiretroviral therapy may have had some effects on cytokine production. The HIV negative patients had the lowest levels of detectable cytokines. There was no detectable IL-1β in these patients. These individuals may have been suffering from other diseases other than HIV that made them seek medical attention. This finding indicate varied immune responses among the study population and agrees with a study that suggested that cytokine levels are indicative of the nature of the immune response in a particular individual ^[3].

The study showed that the treatment naïve HIV patients had significantly higher (p=<0.001) concentration levels of IL-12p70 compared with HIV negative individuals and with HIV patients on HAART (p=0.001). The mean concentration levels of IL-12p70 of HIV patients on HAART was higher than in the HIV negative patients though not significant (p=0.079). This observation differs from a study by Tudela et al., ^[24] who found lowered concentrations of IL-12p70 in HIV positive participants but agrees with a study by Lindi et al., ^[15] who found higher levels of IL-12p70 during acute HIV infection.

This study showed that treatment naïve HIV patients had significantly high concentration levels of IL-10 (p=<0.001) compared to HIV negative patients. The treatment naïve HIV patients also had significantly high concentration levels of IL-10 (p=0.001) compared to HIV patients on HAART. This observation is in agreement with a study by Tudela et al., ^[24] who observed significant increase in IL-10 in participants positive for HIV infection. Lindi et al., ^[15] also observed and increase in IL-10 concentration levels during acute HIV infection. Stylianou et al., ^[22] also demonstrated that HIV-infected patients had significantly higher circulating IL-10 levels; the same study found a significant fall in IL-10 levels during HAART and observed that HAART had an effect on IL-10 levels. In this study, the HIV patients on HAART had slightly higher concentration levels of IL-10 (p=0.008) compared to HIV negative patient. This is in agreement with a study by Haissman et al., ^[8] who found only slightly lower levels of cytokines in HIV uninfected compared to individuals on HAART. While IL-10 is a potent down-regulator of TNF production, TNF is in itself a stimulus for IL-10 release from various cells. The balance between these two cytokines has been suggested to be of importance in the pathogenesis of HIV infection.

In this study, the treatment naïve HIV patients were found to have significantly higher concentration of TNF (<0.001) compared to HIV negative patients. The HIV negative patients had a significant lower concentration of TNF (p=<0.05) compared with HIV patients on HAART. This observation agrees with a study by Lindi et al., ^[15] who found increased concentration of TNF in recently HIV infected individuals compared with HIV uninfected. Haissman et al., ^[8] also found elevated levels of TNF in HIV infected individuals. In this study, the treatment naïve HIV patients were also found to have significantly high concentration of TNF (<0.001) compared to HIV patients on HAART. This is in agreement with Haissman et al., ^[8] who observed a decrease in TNF levels in HIV patients upon administration of antiretroviral therapy.

Interleukin 6 (IL-6) has been found to serve as a growth factor for the HIV [24). High plasma interleukin-6 levels are associated with morbidity and mortality in HIV patients [4, 14, 6]^{[4, 14, 6]}. In this study, the treatment naïve HIV patients were found to have significantly higher levels of IL-6 (p=<0.001) compared with HIV negative patients and also compared with HIV patients on HAART (p=<0.001).The HIV negative patients were found to have significantly low concentration levels of IL-6 (p=<0.001) compared with HIV patients on HAART. This observation agrees with Tudela et al., ^[24] who found significant increase of IL-6 in participants positive for HIV infection and another one by Haissman et al., ^[8] who also observed significantly higher levels of IL-6 in HIV infected individuals compared with HIV-uninfected. Haissman et al., ^[8] also observed that IL6 decreased after administration of antiretroviral therapy. Plasma levels of IL-6 were also observed to be higher in HIV infected individuals in a study by Martyn et al., ^[17].

IL-1β plays a crucial role in triggering the immune response during various diseases. It has been shown that IL-1β is an important mediator during systemic [21, 20]^{[21, 20]} as well as local inflammation ^[2], and it may also be involved in the development of autoimmune diseases ^[1]. This cytokine is thought to have an inducing effect on Kaposi’s sarcoma in association with acquired immunodeficiency syndrome ^[16] and possibly contributes to several other secondary complications during the course of HIV disease, such as neurological ^[25] and haematological disorders ^[18]. Additionally, it has been shown that IL-1 can induce HIV viral replication ^{[5, 7]}. In this study, the detectable IL-1β concentration levels in treatment naïve HIV patients was significantly higher (p=0.001) than in the HIV negative patients and in HIV patients on HAART (p=<0.001). The HIV patients on HAART had higher IL-1β levels (p=0.154) compared with HIV negative patients. These findings agree with Lindi et al., ^[15] who found increased levels of IL-1β during acute HIV infection compared with pre-infection samples but differ with an observation by Martyn et al., ^[17] who found lower levels of Il-β in HIV infected individuals compared with controls.

5. Conclusions

Higher levels of plasma cytokines characterized the period of early human immunodeficiency virus infection, an indication of the hosts’ response to the disease. Human immunodeficiency virus (HIV) promotes plasma cytokine production that could be used to predict subsequent disease progression. Tumor necrosis factor (TNF) and interleukin – 6 (IL-6) are more informative biomarkers of HIV disease progression. Higher plasma cytokine levels were observed among the younger treatment naïve HIV patients and there was no much difference in cytokine production within the gender. Concentration levels of plasma cytokines produced were altered by administration of antiretroviral therapy.

6. Recommendations

Tumor necrosis factor (TNF), Interleukin 6 (IL-6) and Interleukin 1β (IL-1β) can be useful biomarkers in HIV disease progression.

Further Research

Investigating other biomarkers that can be detectable on other samples, other than blood, that could be useful in HIV disease progression, particularly in resource limited set ups.

Acknowledgement

I acknowledge the scholarly advice of Professor Michael Gicheru. I am grateful to the administration of the Nakuru Provincial General Hospital (PGH), Kenya, headed by Dr. John Murimi and including Dr. Beatrice Etemesi, in charge of research in the hospital. I wish to thank the staff of the Voluntary Counseling and Testing Centre and of the Centre for Comprehensive Care of the hospital, especially Rose Wairimu, for their cooperation and assistance during the initial stages of the study. I am indebted to the staff of the PGH Laboratory especially Winnie Migwi and Peter Kariuki of the Virology Laboratory without whose support and assistance this study could not have been completed. I thank them for their technical assistance. I acknowledge the technical assistance of Eva Kimani of BD Biosciences (Nairobi, Kenya) for selflessly and tirelessly guiding me through the assays. I am indeed grateful to all who assisted in one way or the other.

Conflicting Interest

The authors declare that there is no existing conflicting interest.

References

[1]	Brennan, F.M. and Feldmann, M, (1992). Cytokines in autoimmunity. Current Opinion Immunology, 4:754-9.
	In article		View Article
[2]	Dayer, J.M.and Fenner, H, (1992). The role of cytokines and cytokine inhibitors in arthritis. In: Emery, P.ed. Bailliere’s clinical rheumatology, Bailliere Tindall, London. , 6:485-91.
	In article		PubMed
[3]	Dinarello, C.A. (2000). "Proinflammatory cytokines", Chest, 118 (2): 5038.
	In article		View Article
[4]	Duprez, D.A., Neuhaus, J., Kuller, L.H., Tracy, R., Belloso, W.and De Wit, S, (2012). Inflammation,coagulation and cardiovascular disease in HIV-infected individuals. PLoS One, 7:e44454.
	In article		View Article  PubMed
[5]	Folks, T.M., Justement, J. and Kinter, A, (1987). Cytokine-induced expression of HIV-1 in a chronically infected promonocyte cell line, Science, 238:800-2.
	In article		View Article  PubMed
[6]	Fuster, D., Cheng, D.M., Quinn, E.K., Armah, K.A., Saitz, R.and Freiberg, M.S, (2014). Inflammatory cytokines and mortality in a cohort of HIV-infected adults with alcohol problems, AIDS, 28: 1059-1064.
	In article		View Article  PubMed
[7]	Granowitz, E.V., Santos, A.A. and Poutsiaka, D.D, (1991). Production of interleukin-1 receptor antagonist during experimental endotoxaemia. Lancet, 338:1423-4.
	In article		View Article
[8]	Haissman Judith, Vestergaard, Lasse S., Sembuche Samuel, Erikstrup Christian, Lemnge Martha, Gerstoft Jan and Ullum Henrik, (2009). Plasma cytokine levels in Tanzanian HIV-infected Adults and the Effect, Journal of Acquired Immune Deficiency Syndrome, 52: (4) 493-497.
	In article		View Article  PubMed
[9]	Joint United Nations Programme on HIV/AIDS (UNAIDS). GAP Report; 2013.
	In article
[10]	Joint United Nations Programme on HIV/AIDS (UNAIDS) (2014). Global Statistics, Fact sheet 2014 page 1-2.
	In article
[11]	Katsikis P.D., Mueller, Y.M. and Villinger, F. (2011). The Cytokine Network of Acute HIV Infection: A promising Target for Vaccines and Therapy to reduce viral set-point? PloS Pathogens 7(8): e1002055.
	In article		View Article  PubMed
[12]	Kenya HIV Estimates (2014). Ministry of Health: National AIDS and STI Control Programme. 4-5.
	In article
[13]	Korn, T., Bettelli, E, Oukka, M. and Kuchroo,V.K. (2009).IL-17 and Th17 cells. Annual Review of Immunology, 27:485-517.
	In article		View Article  PubMed
[14]	Kuller,L.H., Tracy, R., Belloso, W., De Wit, S., Drummond, F. and Lane, H.C. (2008).Inflammatory and coagulation biomarkers and mortality in patients with HIV infection. PLoS Med, 5:e203.
	In article		View Article  PubMed
[15]	Lindi, Roberts; Jo-Ann S. Passmore, Carolyn Williamson, Francesca Little, Lisa M. Bebell, Koleka P. Mlisana, Wendy A. Burgers, Francois van Loggerenberg, Gerhard Walzl, Joel F. Djoba Siawaya, Quarraisha Abdool Karim; and Salim Abdool Karim. (2010). Plasma cytokine levels during acute HIV-1 infection predict HIV disease progression. AIDS, 24: 1251
	In article
[16]	Louie, S., Cai, J. and Law, R. (1995).Effects of interleukin-1 and interleukin-1 receptor antagonist in AIDS-Kaposi’s sarcoma. Journal of AIDS Human Retrovirus, 8:455-60.
	In article
[17]	Martyn A. French, Alessandro Cozzi-Lepri, Roberto C. Arduino, Margaret Johnson, Amit C. Achira and Alan Landay. (2015). Plasma levels of cytokines and chemokines and the risk of mortality in HIV-infected individuals: a casecontrol analysis nested in a large clinical trial. AIDS, 29:847-851.
	In article		View Article  PubMed
[18]	Maury, C.P.J. and Labdevirta, J. (1990). Correlation of serum cytokine levels with hematological abnormalities in human immunodeficiency virus infection. Journal Internals of Medicine, 227:253-7.
	In article		View Article  PubMed
[19]	Mosmann, T.R.and Fong, T.A. (1989). Specific assays for cytokine production by T cells. Journal of Immunological Methods, 116:151-158.
	In article		View Article
[20]	Ohlsson, K., Bjork, P. and Bergenfeldt, M. (1990). Interleukine-1 receptor antagonist reduces mortality from endotoxin shock. Nature, 348:550-2.
	In article		View Article  PubMed
[21]	Okusawa, S., Gelfiand, J.A. and Ikejima, T. (1988). Interleukin-1 induces a shock-like state in rabbits. Synergism with tumor necrosis factor and the effect of cyclooxygenase inhibition. Journal of Clinical Investigations, 81:1162-7.
	In article		View Article  PubMed
[22]	Stylianou, E., Aukrust, P, and Kvale, D. (1999). IL-10 in HIV infection: increasing serum IL-10 levels with disease progression-down-regulatory effect of potent anti-retroviral therapy. Clinical Immunology, 116:
	In article		View Article
[23]	Sudman, S. (1976). Applied Sampling. New York. Academic Press.
	In article		PubMed
[24]	Tudela,E.V., Singh, M.K., Lagman, M., Ly, J., Venkeraraman, V. et al., (2014). Cytokines Levels in plasma samples of individuals with HIV infection. Austin Journal of Clinical Immunolygy, 1(1):1003.
	In article
[25]	Weiss, J.M. and Sundar, S.K. (1992). Interlleukin-1: effects in the central nervous system and relevance to AIDS. Clinical Neoropharmacol, 15:661A-2A
	In article		View Article  PubMed
[26]	World Health Organization (WHO). Global HIV/AIDS response. 2013.
	In article
[27]	59th WMA General Assembly, Seoul. (2008).World Medical Association Declaration of Helsinki: ethical Principles for Medical Research involving Human subjects. (2008).
	In article
[28]	Yamane, T. (1967). Statistics: An Introductory Analysis.2nd ED., New York, Harper and Rao. 886.
	In article