BY: SHAN AND BITA

Background: MIF= cytokine macrophage migration inhibitory factor gp120= HIV-1 envelop glycoprotein p24= HIV-1 antigen PBMCS= peripheral blood mononuclear cells HIV-1 leads to immunosupression and makes the individual more susceptible to opportunistic infections and tumors. Pro-inflammatory cytokines such as TNF-α and IL-6 are considered to up- regulate HIV-1 replication while anti- inflammatory cytokines like IFN, IL- 10, IL-27 decrease it. LPS is a strong inducer of MIF secretion and it can be found in the serum of HIV-1 infected individuals MIF is an upstream activator of innate immunity and triggers release of TNF-α and IL-6. Elevated level of MIF in serum has been observed in other diseases such as west Nile virus, Dengue fever, rheumatoid arthritis. So far no studies has been done for the role of MIF in HIV-1 infection biology.kenyon.edu/.../ Lentiviral/hiv_image.jpg

ObjectiveObjective: Study whether MIF is involved in the pathogenesis of HIV-1 and if it influences HIV-1 replication upload.wikimedia.org/.../ 180px-PDB_1mif_EBI.jpg Main materials usedMain materials used: CCR5-dependent isolate HIV-1 Ba-L and CXCTR-4- dependent isolate HIV-1 type PBMCS derived from healthy donors cultured in human serum, antibiotics, and IL-2 Macrophages derived from the PBMC cell cultures CD4+T Jurkat cell line transfected with luciferase MethodsMethods: Looked for the presence of MIF in HIV-1 infected patients Looked for the presence of MIF in PBMC (and macrophages) amongst uninfected and infected cultures Looked for the presence of MIF in HIV-1 infected PBMC treated with probenecid Looked for the presence of viral replication in HIV-infected PBMC treated with anti-MIF antibodies, and the presence of HIV-1 transcription in CD4+T cells treated with PHA, Tat, or rhMIF

Main Findings and Experimental approach:Main Findings and Experimental approach: Fig.1: HIV- infected present higher plasma level of MIF Plasma collected from a selected group of HIV infected individuals (n=30), and a control group of healthy volunteers (n=10). Evaluation of MIF levels by Elisa The HIV-1-infected patients who were not under anti- retroviral treatment and did not present clinical signs of co- infections had higher plasma levels of MIF than the healthy individuals

Main Findings and Experimental approach:Main Findings and Experimental approach: Fig. 2A: Infection with HIV-1 doubled the release of MIF Infection of human primary PBMCs with R5-tropic HIV-1 isolate. Evaluation of MIF secretion in the culture supernatants by Elisa, 7 days after infection Experiments done in triplicates per donor PBMCs infected with R5-tropic HIV-1 isolate, X4-tropic HIV-1, or HIV-1 gp120 secreted MIF Fig. 2D: HIV-1 infection of macrophages didn’t change secretion of MIF Infection of macrophages with Ba-L HIV- 1 isolate. MIF secretion assessed by Elisa, 7 and 14 days after infection Experiments done in triplicates per donor

Main Findings and Experimental approach:Main Findings and Experimental approach: Fig. 3. MIF release upon HIV-1 infection is dependent of ABC transporters pre-treatment of cells with probenecid (10 μM, for 45 min), infection with HIV-1, and reapplication of probenecid to infected cells. Measurement of MIF at day 7 post- infection Experiments done in triplicates of three donors HIV-1 infected PBMC cells, when treated with probenecid (an ABC transporter inhibitor) had less MIF secretion

Fig. 4: MIF inhibition down-regulates HIV-1 replication Treatment of HIV-1 infected PBMCs with neutralizing anti- MIF polyclonal antibodies immediately after infection. Evaluation of viral replication by Elisa, 7 days after infection Experiments done in triplicates of 3, 5, and 5 donors. Main Findings and Experimental approach:Main Findings and Experimental approach: Infected PBMCs, when treated with anti-MIF antibodies, had a reduction in HIV-1 replication, while infected CD4+T cells, when treated with rhMIF, had an increase in HIV-1 transcription Fig. 6: rhMIF increases HIV-1 replication via induction of direct HIV-1 transcription Addition of rhMIF to cultures of Jurkat CD4+ T derivative cell line containing integrated HIV-LTR luciferase construct. Evaluation of luminescence by luminometer

HIV-1 (gp120) HIV-1 (gp120) Greater MIF release Via ABC transporters Greater MIF release Via ABC transporters macrophages HIV-1 transcription Via HIV-1 LTR HIV-1 replication Anti-MIF antibodies decrease Discussion: Critiques: Experiments could have been done beyond 7 days. Small sample of volunteers; the number of HIV positive patients may be 20 instead of 30. There are limitations to an in vivo study, and no cultures came from HIV positive patients. Overall a good clear research paper; many controls were made. MIF is mainly restricted to secretion by HIV-1 infected PBMCS cells, and can contribute to viral replication. Soluble viral protein gp120 may contribute to MIF secretion at a systemic level. Opportunistic pathogens may induce high levels of MIF by secondary LPS stimulation. Anti-MIF antibodies decrease viral replication; therefore, MIF could be used as a target molecule for anti-HIV-1 therapy. PBMCs NOyes