Recombinant gp120 proteins cause apoptosis in Primary Human neurons and reduce BDNF levels.

<p><b>A.</b><i>gp120 mediated neuronal apoptosis</i>. Primary Human Neurons were treated with 1 nM each recombinant gp120 s from representative US-B (HIV-1_Bal), Southern African HIV-1C (HIV-1_96ZM651) and Indian HIV-1C (HIV-1_IN16055) isolates. Percent TUNEL positive cells from 20 different fields in 3x wells per treatment were analyzed and the level of apoptosis was determined. Similar to what was determined using immuno-depletion of gp120 in HIV-1-infected supernatants, the recombinant gp120 from Indian isolate showed a significantly lower percentage of neurotoxicity. <b>B.</b><i>gp120-mediated effect on BDNF release</i>. Supernatants were collected from the primary human neurons following treatment with recombinant gp120s at 6 and 24 hours. Secreted BDNF levels were measured using ELISA and the results were plotted as a percentage of the Control (PBS). Both HIV-1_Bal (US clade-B) and HIV-1_96ZM651 (Southern African clade-C) gp120s resulted in a 40% decrease in secreted BDNF levels at 6 h and a decrease of 60% at 24 h as opposed to HIV-1 IN₁₆₀₅₅ which showed no decrease at 6 h and a 10% decrease at 24 hours compared to Control (PBS). <b>C.</b><i>Neuronal apoptosis caused by recombinant gp120 proteins.</i> Tunnel stained primary human neurons co-stained with anti-neurotubulin antibodies and DAPI (Prolong Gold anti-fade with DAPI, Invitrogen) to verify the direct effect of recombinant gp120 on neuronal death. The four panels show representative fields for neurons treated with PBS and 1 nM recombinant gp120 s from Indian HIV-1C, Southern African HIV-1C and US-B, isolates. Neurons treated with Southern African clade C gp120 and US- Clade B show greater neurotoxicity. (*p<0.05; **<i>p</i><0.01; ***<i>p</i><0.005).</p

Relative contribution of gp120 and Tat to neurotoxicity.

<p>HIV-1-infected MDM supernatants (100 µl) from representative isolates US clade B (HIV-1B:HIV-1_ADA), Southern African (HIV-1C:HIV-1_1084i) and Indian clade C HIV-1 (HIV-1C:HIV-1_IndieC1) with equal p24 loads were diluted in SH-SY5Y media and added to SH-SY5Y cultures and incubated for 24 h. Media with and without immuno-depletion of gp120 and Tat were tested. Medium indicates untreated MDM supernatants, while ‘Medium minus gp120’ and ‘Medium minus gp120 and Tat’ refer to gp120- immunodepleted and gp120 and Tat-depleted media respectively. WST-1 assay was performed to determine neuronal death. (**<i>p</i><0.01; ***<i>p</i><0.005).</p

Phylogenetic analysis of gp120 sequences.

<p>Neighbor-joining phylogenetic tree was constructed in MEGA 6.06 software using 878 HIV-1C and four HIV-1B (as outlier) gp120 sequences from India and from the Southern African Countries of South Africa and Zambia. Due to the comparatively fewer HIV-1C gp120 sequences that were available from India, we included HIV-1C gp120 sequences from China and Myanmar that were available in the database. A separate phylogenetic tree with HIV-1C gp120 sequences from China, Myanmar confirmed their Indian origin. HIV-1B sequences from US were included in the phylogenetic analysis for comparison. Bootstrap values >50% were shown in the tree. The HIV-1C gp120 sequences from India and Southern African countries are genetically distinct and cluster separately. The specific isolates used in our in-vitro analysis (HIV-1_ADA, HIV-1_Bal, HIV-1_IndieC1, HIV-1_93IN905, HIV-1_IN16055, HIV-1_1084i, HIV-1_97ZA012 and HIV-1_96ZM651) are included in this analysis (and indicated on the diagram) to show that they qualify to be used as representative isolates.</p

Predicted survival percentiles (1–50%) of second-line ART viral failure stratified by reason to therapy switch.

<p>Predicted survival percentiles of time to VF of second-line ART modeled using a Laplace regression adjusted by: Sex, type of regimen first and second-line ART, route of transmission, country of birth. Age at first-line ART initiation, CD4 cell count at first and second-line ART initiation, HIV RNA load at first and second-line ART initiation, time in first-line ART and time in second-line ART as continuous variables. DRM = drug resistance mutations.</p

Patients infected outside of Sweden - sensitivity analysis of year of HIV-1 diagnosis and the trends in subtype pattern during 1983–2012, using subtype B (HIV-1B) as reference.

<p>An univariate multinomial logistic regression model <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099390#pone.0099390-Lee1" target="_blank">[17]</a> was used. The outcome (the HIV-1 subtypes) was grouped into HIV-1B (reference group), HIV-1C, recombinants, and other pure subtypes. Relative risk ratio and 95% confidence intervals were determined. RRR represents relative risk ratio.</p

Predicted survival percentiles (1–50%) of second-line ART virological failure by category of HIV-1 RNA load at second-line ART initiation.

<p>Predicted survival percentiles of time to VF of second-line ART modeled using a Laplace regression adjusted by: Sex, type of regimen first and second-line ART, route of transmission, country of birth. Age at first-line ART initiation, CD4 cell count at first and second-line ART initiation, HIV RNA load at first and second-line ART initiation, time in first-line ART and time in second-line ART as continuous variables.</p

Demographic and clinical characteristics of the patients by category of antiretroviral therapy (ART) switch.

<p>Demographic and clinical characteristics of the patients by category of antiretroviral therapy (ART) switch.</p

Demographics of individuals diagnosed with HIV-1 during 1983–2012 in Sweden, included in the study (n = 3967).

<p>*Kruskal-Wallis equality-of-populations rank test; **Chi2 test: ***Intravenous drug users: IVDU; Hetero: heterosexually infected; MSM: men who have sex with men; Others: blood products or unknown; ****The countries were divided into categories based on the UNAIDS definition (<a href="http://www.unaids.org/en/regionscountries/countries/" target="_blank">http://www.unaids.org/en/regionscountries/countries/</a>).</p

Clinical and demographic parameters during first viral rebound for viral failure and intermittent viral rebound groups.

a<p>p-value based on t-test (d.f. = 58) for age and CD4 count and viral load, Fisher’s exact test (d.f. = 1) for male gender and adherence.</p

Patients’ demographic, clinical and laboratory parameters at baseline and whole study period of 2 years.

<p><b>Abbreviations</b>: d4T, Stavudine; 3TC, lamivudine; AZT, zidovudine; FTC, emtricitabine; TDF, tenofovir; NVP, Nevirapine; EFV, Efavirenz; SD, Standard deviation; IQR, interquartile range; N, Number; PVL, plasma viral load; VAS, Visual analogue scale.</p>*<p>Over the study period of 2 years,</p