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

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

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

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

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

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

Baseline characteristics of patients enrolled in the study.

<p><b>IQR</b> Inter Quartile Range; <b>PI</b>: Protease Inhibitors; <b>NRTI</b>: Nucleoside Reverse Transcriptase Inhibitors; <b>NNRTI</b>: Non-Nucleoside Reverse Transcriptase Inhibitors.</p

Mycobacterial growth (CFU: Colony Forming Unit) before (T0) and after (T1) IL-2 adjuvant treatment (A,C) compared to cART treatment alone in control group (B,D).

<p>CFU growth has been tested in different aliquots of PBMC cultures of 1×10⁶ cells/well (A,B) and 3×10⁶ cells/well (C,D). In IL-2 treated patients a significant reduction in mean residual mycobacterial growth was observed in both experimental conditions of PBMC cultures (<i>P</i><0.01). Significant results are emphasized by square brackets with respective <i>P</i> values.</p

Activated HLA-DR+CD4+ and CD8+ T-cells according to EVR and SVR.

<p><b>a</b>)<b>-b</b>) Activated HLA-DR+CD4+ and CD8+ T-cells were compared between patients with early virological response [EVR, i.e. undetectable serum HCV-RNA (<50 IU/mL) or ≥2 log₁₀ reduction from baseline after 12 weeks of therapy], and Null Responders (NR) (i.e. serum HCV-RNA ≥50 IU/mL and <2 log₁₀ reduction from baseline). <b>c</b>)<b>-d</b>) Activated HLA-DR+CD4+ and CD8+ T-cells were compared between patients with sustained virological response [SVR, i.e. undetectable serum HCV-RNA (<50 IU/mL) 24 weeks after the end of a full course of 48 or 72 weeks of anti-HCV treatment, according to genotype], and N-SVR subjects. Each point represents the value from one subject's plasma. Activated HLA-DR+CD4+ and CD8+ T-cells % values are presented. p-values were assessed by Mann Whitney U test. p>0.05 was considered non significant (NS).</p

Higher microbial translocation is associated with HCV genotypes 1–4 and cirrhosis.

<p><b>a</b>)<b>-b</b>) sCD14 and LPS were compared between patients with advanced fibrosis (AF) and non advanced fibrosis (N-AF). <b>c</b>)<b>-d</b>) sCD14 and LPS were compared between patients with cirrhosis and absence of cirrhosis (N-Cirrhosis). <b>e</b>)<b>-f</b>) sCD14 and LPS were compared between patients with HCV genotypes 1–4 and genotypes 2–3. Each point represents the value from one subject's plasma. sCD14 and LPS were measured in plasma samples; sCD14 µg/mL, LPS pg/mL. AF = advanced fibrosis – N-AF = non advanced fibrosis. p-values were assessed by Mann Whitney U test. p>0.05 was considered non significant (NS).</p