Bacterial genera identified in plasma sample of PIRs and INRs.

<p>Positive (%) = # patients displaying a specific bacterial genera/total # of PCR positive PIRs (or INRs). Negative (%) = # patients negative for a specific bacterial genera/total # of PCR positive PIRs (or INRs). (<b>A–B</b>) A similar composition of bacterial genera were seen in PIRs both at T0 and T12. (<b>C–D</b>) INRs presented no variation in bacterial genera following 12 months of HAART. At baseline, significant differences between PIRs and INRs were seen for <i>Lactobacillus sp</i> *(PIRs 3/6, 50% vs INRs 0/8 0%; p = .05) and <i>Pseudomonas sp *</i>(PIRs 5/6, 83% vs INRs 1/8, 13%; p = .026). No differences between the two groups at T12.</p

Characterization of translocating microflora in PIRs and INRs.

<p>Bacterial microflora translocating in peripheral blood was evaluated in 44 HAART-naive patients with severe immune depression (CD4+≤200/µL) at baseline and after 12 months of stable HAART (T12). At T12, 29 patients were partial immunological responders -PIRs- (CD4+≥250/µL; VL<60 cp/mL) and 15 were immunological-non-responders -INRs- (CD4+<200/µL; VL<60 cp/mL). At baseline, we found a significantly higher proportion of PCR-positive INRs (INRs: 8/15, 53%; PIRs: 6/29, 20%; p = .04). At T12 a non significantly higher proportion of INRs yielded a positive 16S gene PCR amplification: INRs: 4/15 (27%); PIRs: 3/29 (10%); p = .21. PIRs and INRs displaying a similar bacterial orders both at baseline and T12, except <i>Pseudomonadales</i> order *(FRs 5/6, 83% vs 1/8, 12% p = .026). Positive (%) = # patients displaying a specific bacterial order/total # of PCR positive PIRs (or INRs). Negative (%) = # patients negative for a specific bacterial order/total # of PCR positive PIRs (or INRs).</p

Characterization of translocating microflora in HIV+ antiretroviral naive patients.

<p>Patients' plasma samples were examined before and after 12 months of HAART for the presence and identification of DNA bacterial fragments using a broad-range 16S rRNA gene PCR amplification followed by sequencing analysis. At baseline, 14/44 (32%) HIV+ patients yielded a positive PCR amplification, whereas at T12, 7/44 (16%) HIV+ patients were PCR-positive. Sequencing analysis of HIV positive patients as a whole revealed multiple bacterial orders for each patient with no differences in the bacterial composition between baseline and T12. Positive (%) = # patients displaying a specific bacterial order/total # of PCR positive patients. Negative (%) = # patients negative for a specific bacterial order/total # of PCR positive patients.</p

Patients' characteristics.

<p><b>NOTE:</b> Data are median (IQR -Interquartile range-). FRs: Full Responders; INRs : Immunological Non Responders; IDU: intravenous drug user; HAART: highly active antiretroviral therapy; NRTI: nucleoside reverse transcriptase inhibitor; NNRTI: non-nucleoside reverse transcriptase inhibitor; PI: protease inhibitor.</p>a<p>p<.01 for FRs vs INRs;</p>b<p>p<.01 for T0 vs T12.</p

Patient characteristics.

<p>DP: Double Positive; BD: Bone Disease; CD: Cardiovascular Disease; DN: Double Negative. MSM: Males Who Have Sex With Males. IVD: Intravenous Drug. HCV: Hepatitis C Virus. HAART: Highly Active Antiretroviral Therapy; PI: Protease Inhibitor; NNRTI: Non-Nucleoside Retroscriptase Inhibitor. DXA: Dual-energy X-ray absorptiometry. IMT: Intima Media Thickness. Data presented as: median (interquartile range, IQR) for continuous variables; absolute number (percentage) for categorical variables. p<0.05: ^aDP vs DN; ^bDP vs BD; ^cBD vs DN; ^dBD vs CD; ^eCD vs DN; ^fCD vs DP.</p><p>Patient characteristics.</p

Production of TNF-α (A,B), IL-10 (C,D) and IFN-γ (E,F) in PBMC cultures before (T0) and after (T1) IL-2 adjuvant treatment (A,C,E) compared to cART treatment alone in control group (B,D,F).

<p>Following IL-2 treatment significant increases in PBMC's IFN-γ production (<i>P</i> = 0.02), and substantial reduction in IL-10 levels were observed resulting in significant difference between IL-2 patients and controls at T1 (<i>P</i> = 0.03). Significant result in IL-2 treated patients are emphasized by black arrows indicating the compared groups with respective <i>P</i> value.</p

Identification of bacterial families in PIRs and INRs.

<p>Positive (%) = # patients displaying a specific bacterial family/total # of PCR positive PIRs (or INRs). Negative (%) = # patients negative for a specific bacterial family/total # of PCR positive PIRs (or INRs). PIRs and INRs presented a different profile in term of bacteria families. (<b>A</b>) PIRs displayed a similar composition of bacterial families between baseline and T12. (<b>B</b>) No major changes in bacterial families were seen in INRs after 12 months-HAART. At baseline, the significant differences between the two groups concerned <i>Lactobacillaceae</i> *(PIRs 3/6, 50% vs INRs 0/8 0%; p = .05) and <i>Pseudomonadaceae *</i>(PIRs 5/6, 83% vs INRs 1/8, 13%; p = .026). No differences between PIRs and INRs at T12.</p

Schedule of IL-2 administration.

<p>Schedule of IL-2 administration.</p

iNKT cell phenotype and function in HIV-positive “Bone Disease” (BD), “Cardiovascular Disease” (CD) and “Double Negative” (DN) patients.

<p>iNKT frequency was comparable among BD and DN groups (A). BD (n = 10) and DN (n = 10) showed similar CD161-expressing iNKT cell frequencies (B) and a significant increase in TNF production following PMA/ionomycin stimulation (p = .002 and p = .027 respectively). Despite a trend to higher spontaneous TNF release in BD patients (p = .075), comparable cytokine levels were recorded upon PMA/ionomycin (C). BD patients alone responded to PMA/ionomycin with significant IFN-γ production following stimulation (p = .0488) (D). Significantly higher TNF production was detected in BD subjects (p = .031) prior to α-GalCer stimulation. Upon α-GalCer stimulation, BD patients displayed a trend to significant increases in TNF release (p = .063), leading to higher cytokine levels in this population (p = .056) (E). No significant differences were noted in terms of IFN-γ production following α-GalCer, although BD patients tended to significant cytokine production (p = .063) (F). CD and DN showed comparable iNKT cell frequencies (G). CD (n = 10) and DN (n = 10) showed similar CD161-expressing iNKT cell frequencies (H). CD subjects showed higher TNF release both prior to (p = .005) and following stimulation with PMA/ionomycin (p = .029). Of note, DN patients alone responded to stimulation by significantly increasing TNF release from iNKT cells aspecific stimulation (p = .027) (I). In keeping with these results, the CD group displayed a trend to higher IFN-γ release after PMA/ionomycin stimulation (p = .052) (J). No statistical differences were noted between groups in terms of iNKT function following specific activation with α-GalCer (K, L). Horizontal lines indicate median values. Each symbol represents an individual.</p

iNKT cell phenotype and function in HIV-positive “Double Positive” (DP) and “Double Negative” (DN) patients.

<p>Gating strategy of flow cytometry analysis for staining of iNKT cell frequencies, phenotype and intracellular cytokine production in a representative HIV-positive individual (A); an example of staining for intracellular cytokines is also shown of a representative HIV-negative subject (B). PBMCs were gated on lymphocytes, and iNKT cells were visualized as CD3+, Vα24+ and CD1d-tetramer+. An example of CD161 surface staining is shown in the far right plot. iNKT frequency were comparable in DP and DN groups (C). iNKT cell phenotype was analyzed through the <i>ex vivo</i> expression of CD161 in DP (n = 10) and DN (n = 10) patients (D). DP subjects exhibited significantly higher levels of CD161 on iNKT cell surface compared to DN patients (p = .001). iNKT cell function was measured through the production of TNF and IFN-γ <i>ex vivo</i> (US) and following stimulation with PMA/ionomycin (n = 10 per group) (E, F) and α-GalCer (n = 5 per group) (G, H). Although DP and DN patients significantly increased TNF production upon PMA/ionomycin stimulation (p = .002 and p = .027 respectively), DP subjects showed higher TNF release both prior to (p = .049) and following PMA/ionomycin (E). Study groups exhibited similar frequencies of IFN-γ-producing iNKT cells both <i>ex vivo</i> and after stimulation with PMA/ionomycin (F). DP patients were characterized by significantly higher TNF release both prior to (p = .047) and following stimulation with α-GalCer (p = .021) (G). Similar results were obtained in terms of IFN-γ production, with a trend to higher cytokine production in DP subjects following iNKT-specific stimulation (p = .059) (H). FSC, Forward Scatter: SSC, Side Scatter. Each symbol represents an individual.</p