Search

Objective: Tissue perfusion measurement remains a fundamental challenge in management of peripheral arterial disease (PAD) patients. Better measurement of baseline and dynamic changes after revascularization would aid clinical judgement. The Wireless Lumee® Oxygen System represents a novel approach to tracking tissue oxygen via a subcutaneous hydrogel that provides noninvasive optical oxygen measurements. This study’s objective was to assess the correlation of the Lumee sensor with transcutaneous oximetry (tcpO2) in PAD patients. Methods: The Lumee® Oxygen System is a subcutaneously placed hydrogel (0.5x0.5x5mm). Optical signals are captured with a skin surface reader sensing extravascular oxygenation. A pilot study assessed in vivo performance with inclusion criteria of peripheral arterial disease (Rutherford I-IV). Sensors were injected in the arm and foot and oxygenation measured before, during, and after proximal blood pressure cuff inflation (Figure) and Buerger’s Test modulation. The correlation (R) of oxygenation between tcpO2 and Lumee® was assessed following sensor placement at 2, 10, 90, 180, and 365 days. Results: Eleven subjects had 148 occlusion modulations over 199 days. Mean age was 67 years (range: 55-88), with 9% females, 45% White and 55% Black Race, Rutherford class I: 36%, II: 36%, III:27%. The median ABI was 0.83 (IQR0.69-1.14). In the arm, modulation was 56.9±24.6% (Lumee) and 84.7±9.2% (tcpO2), R:0.81. In the foot, modulation was 63.4±22.7% (Lumee) and 80.4±15.5% (tcpO2), R:0.79. No injection related adverse events occurred. Conclusion: Occlusion related changes in tissue oxygen can be detected by both tcpO2 and the Lumee sensor which strongly correlate in both the arm and foot. This pilot phase study supports expansion of the trial. The Lumee sensor shows promise for future evaluation of tissue oxygenation in PAD patients, which could be used to assess changes in tissue oxygenation over time and following revascularization.

Additional file 6. Poly-functional profiles of CM CCR6+ subsets upon long-term culture under Th17 versus Th1 polarizing conditions in vitro. FACS-sorted CM subsets were analyzed for the expression of lineage-specific cytokines upon Th17/Th1-polarization in vitro (Fig. 5). Shown are bar graph representations generated with SPICE software for all possible combinations of one (blue), two (green), three (orange), four (yellow), and five (red) or no (purple) cytokines produced by T-cell subsets upon culture in vitro (mean ± SEM), together with pie charts summarizing the polyfunctional profiles (median relative contribution, n = 3).

Additional file 4. Superior Th17-lineage commitment of CM versus EM Th17 and Th1Th17-subsets. FACS-sorted Th17, Th1Th17 and Th1 subsets with CM (CD45RA−CCR7+) and EM (CD45RA−CCR7−) phenotype were cultured for 14 days under Th17-polarizing conditions, as in Fig. 5. Shown are the statistical analyses of the intracellular expression of IL-17A, IL-17F, IL-22, IFN-γ and TNF-α by distinct Th17-polarized CM (black bars) versus EM (white bars) Th17, Th1Th17 and Th1 subsets. Results (mean ± SEM) were generated with matched samples from n = 3 different subjects. Paired t-test p-values are indicated in the figures (CM versus EM).

Additional file 9. The four CCR6+ T-cell subsets isolated from HIV-infected subjects receiving ART preserve their Th17-polarizing profiles upon long term culture in vitro. (A) FACS-sorted Th17, Th1Th17 and Th1 subsets were stimulated with CD3/CD28 for four days then cultured in the presence of IL-2 (5 ng/ml) for an additional 9 days. At day 13, cells were stimulated with PMA and Ionomycin in the presence of Brefeldin A for 6 h. Intracellular staining was performed with cytokine-specific Abs. (B) Shown are flow cytometry dot plots illustrating the co-expression of IL-17A and IFNγ (n = 3). (C) Shown is the frequency of cytokine-expressing T-cells cultured long-term in vitro. Results (mean ± SEM) were generated with matches samples from n = 3 different donors. Paired t-Test p-values are indicated on the figures.

Additional file 3. GO classification of differentially expressed genes in 3 CCR6+ subsets. (A-B) Shown are Venn diagram representation of GSVA-generated canonical pathways (A) and biological functions (B), illustrating the number of differentially expressed pathways common or unique between Th17, CCR6+DN, and CCR6+DP. (C-F) Further, differentially expressed genes between (pÂ

Additional file 7. Phenotypic stability versus plasticity of the four central memory CCR6+ T-cell subsets under Th17- or Th1-polarizing conditions. FACS-sorted CM subsets were cultured under Th17- and Th1-polarizing conditions for 14 days, as described in Fig. 5A, stained with CCR6, CCR4, and CXCR3Abs and analyzed by FACS. Shown are the statistical analyses for the expression of (A) CCR6 (B) CCR4, and (C) CXCR3, and the co-expression of (D) CCR6 and CCR4 (R6+R4+ phenotype) and (E) CCR6 and CXCR3 (R6+X3+ phenotype) on each subsets at day 0 (white bars) versus day 14 in culture under Th17- (black bars) and Th1-polarizing (grey bars) conditions. (A-E) The positivity gates where defined based on fluorescence minus one (FMO) controls. Results (mean ± SEM) were generated with matched subsets from n = 3 distinct subjects. Paired t-Test p-values are indicated on the graphs.

Additional file 5. Flow cytometry analysis of cytokine co-expression at single-cell level by CM subsets upon long-term culture under Th17 versus Th1 polarizing conditions. Central memory (CM) Th17, Th1Th17, CCR6+DN, CCR6+DP, and Th1 subsets were sorted and cultured for 14 days under Th17 or Th1 polarizing conditions, as described in Fig. 5. Shown are flow cytometry dot plots illustrating the co-expression of IL-17A with IL-17F, IL-22, IFN-γ or TNF-α for each Th17- or Th1-polarized subset. Results are from one donor representative of results generated with cells from three different donors. The positivity gates where defined based on fluorescence minus one (FMO) controls.

Additional file 1. Purity of flow cytometry-sorted memory CD4+ T-cell subsets. Total CD4+ T-cells were isolated from PBMCs of healthy individuals by negative selection using magnetic beads (Miltenyi). Cells were stained with a cocktail of fluorochrome-conjugated Abs and analyzed by polychromatic flow cytometry (see Supplemental Experimental Procedure). Memory (CD45RA−) cells lacking the lineage-specific markers CD8 (CD8+ T-cells), CD19 (B cells), and CD56 (NK cells) and with differential expression of CCR6, CCR4, and CXCR3 were sorted by flow cytometry (BDAria II) as follows: CCR6+CCR4+CXCR3− (Th17), CCR6+CCR4−CXCR3− (CCR6+DN), CCR6+CCR4+CXCR3+ (CCR6+DP), CCR6+CCR4−CXCR3+ (Th1Th17) and CCR6−CCR4−CXCR3+ (Th1). A viability staining was used to exclude dead cells. The positivity gates where defined based on fluorescence minus one (FMO) controls. Shown is (A) the gating strategy for the identification of different subsets on CD4+ T-cells sorted by MACS and (B) purity upon FACS sorting of different memory T-cell subsets. The percentage of each subset is indicated on the figures. Results are from one donor representative of experiments performed with cells from >10 different donors. The positivity gates where defined based on fluorescence minus one (FMO) controls.

Additional file 10. All four CCR6+ T-cell subsets carry integrated HIV-DNA in viremic untreated HIV-infected subjects. The four memory CCR6+ subsets as well as naive cells from PBMCs of (A) HIV+ recently infected untreated (RI) (n = 3) and were sorted by FACS. Levels of integrated HIV-DNA were quantified by nested real-time PCR (mean ± SD of triplicate wells).

Additional file 11. Poly-functional profiles of HIV-p24+ CCR6+ T-cell subsets of HIV-infected individuals receiving ART upon viral reactivation in vitro. HIV reservoir reactivation was performed as described in Suppl. Figure 6 legend. Briefly, at day 13, cells were stimulated with PMA/Ionomycin in the presence of Brefeldin A and intracellular staining was performed with cytokine (IFN-γ, IL-17A, IL-22, TNF-α) and HIV-p24 Abs. Shown are bar graph representations generated with SPICE software for all possible combinations of one (blue), two (green), three (orange), and four (yellow), or no (purple) cytokines produced by HIV-p24+ Th17, Th1Th17, CCR6+DN, CCR6+DP and Th1 subsets (n = 3 CI on ART subjects). In contrast to CI on ART 5 and 6, for donor CI on ART 07 HIV reactivation was observed only for CCR6+DN (red).

Additional file 10. All four CCR6+ T-cell subsets carry integrated HIV-DNA in viremic untreated HIV-infected subjects. The four memory CCR6+ subsets as well as naive cells from PBMCs of (A) HIV+ recently infected untreated (RI) (n = 3) and were sorted by FACS. Levels of integrated HIV-DNA were quantified by nested real-time PCR (mean ± SD of triplicate wells).

Additional file 8. Table S2. Clinical parameters of chronically HIV-infected subjects under long-term viral suppressive ART (CI on ART). Table S3. Clinical parameters of the longitudinal cohort of HIV-infected subjects.

Additional file 6. Poly-functional profiles of CM CCR6+ subsets upon long-term culture under Th17 versus Th1 polarizing conditions in vitro. FACS-sorted CM subsets were analyzed for the expression of lineage-specific cytokines upon Th17/Th1-polarization in vitro (Fig. 5). Shown are bar graph representations generated with SPICE software for all possible combinations of one (blue), two (green), three (orange), four (yellow), and five (red) or no (purple) cytokines produced by T-cell subsets upon culture in vitro (mean ± SEM), together with pie charts summarizing the polyfunctional profiles (median relative contribution, n = 3).

Additional file 3. GO classification of differentially expressed genes in 3 CCR6+ subsets. (A-B) Shown are Venn diagram representation of GSVA-generated canonical pathways (A) and biological functions (B), illustrating the number of differentially expressed pathways common or unique between Th17, CCR6+DN, and CCR6+DP. (C-F) Further, differentially expressed genes between (pÂ

Additional file 1. Purity of flow cytometry-sorted memory CD4+ T-cell subsets. Total CD4+ T-cells were isolated from PBMCs of healthy individuals by negative selection using magnetic beads (Miltenyi). Cells were stained with a cocktail of fluorochrome-conjugated Abs and analyzed by polychromatic flow cytometry (see Supplemental Experimental Procedure). Memory (CD45RA−) cells lacking the lineage-specific markers CD8 (CD8+ T-cells), CD19 (B cells), and CD56 (NK cells) and with differential expression of CCR6, CCR4, and CXCR3 were sorted by flow cytometry (BDAria II) as follows: CCR6+CCR4+CXCR3− (Th17), CCR6+CCR4−CXCR3− (CCR6+DN), CCR6+CCR4+CXCR3+ (CCR6+DP), CCR6+CCR4−CXCR3+ (Th1Th17) and CCR6−CCR4−CXCR3+ (Th1). A viability staining was used to exclude dead cells. The positivity gates where defined based on fluorescence minus one (FMO) controls. Shown is (A) the gating strategy for the identification of different subsets on CD4+ T-cells sorted by MACS and (B) purity upon FACS sorting of different memory T-cell subsets. The percentage of each subset is indicated on the figures. Results are from one donor representative of experiments performed with cells from >10 different donors. The positivity gates where defined based on fluorescence minus one (FMO) controls.

Additional file 11. Poly-functional profiles of HIV-p24+ CCR6+ T-cell subsets of HIV-infected individuals receiving ART upon viral reactivation in vitro. HIV reservoir reactivation was performed as described in Suppl. Figure 6 legend. Briefly, at day 13, cells were stimulated with PMA/Ionomycin in the presence of Brefeldin A and intracellular staining was performed with cytokine (IFN-γ, IL-17A, IL-22, TNF-α) and HIV-p24 Abs. Shown are bar graph representations generated with SPICE software for all possible combinations of one (blue), two (green), three (orange), and four (yellow), or no (purple) cytokines produced by HIV-p24+ Th17, Th1Th17, CCR6+DN, CCR6+DP and Th1 subsets (n = 3 CI on ART subjects). In contrast to CI on ART 5 and 6, for donor CI on ART 07 HIV reactivation was observed only for CCR6+DN (red).

Additional file 5. Flow cytometry analysis of cytokine co-expression at single-cell level by CM subsets upon long-term culture under Th17 versus Th1 polarizing conditions. Central memory (CM) Th17, Th1Th17, CCR6+DN, CCR6+DP, and Th1 subsets were sorted and cultured for 14 days under Th17 or Th1 polarizing conditions, as described in Fig. 5. Shown are flow cytometry dot plots illustrating the co-expression of IL-17A with IL-17F, IL-22, IFN-γ or TNF-α for each Th17- or Th1-polarized subset. Results are from one donor representative of results generated with cells from three different donors. The positivity gates where defined based on fluorescence minus one (FMO) controls.

Additional file 4. Superior Th17-lineage commitment of CM versus EM Th17 and Th1Th17-subsets. FACS-sorted Th17, Th1Th17 and Th1 subsets with CM (CD45RA−CCR7+) and EM (CD45RA−CCR7−) phenotype were cultured for 14 days under Th17-polarizing conditions, as in Fig. 5. Shown are the statistical analyses of the intracellular expression of IL-17A, IL-17F, IL-22, IFN-γ and TNF-α by distinct Th17-polarized CM (black bars) versus EM (white bars) Th17, Th1Th17 and Th1 subsets. Results (mean ± SEM) were generated with matched samples from n = 3 different subjects. Paired t-test p-values are indicated in the figures (CM versus EM).

Additional file 9. The four CCR6+ T-cell subsets isolated from HIV-infected subjects receiving ART preserve their Th17-polarizing profiles upon long term culture in vitro. (A) FACS-sorted Th17, Th1Th17 and Th1 subsets were stimulated with CD3/CD28 for four days then cultured in the presence of IL-2 (5 ng/ml) for an additional 9 days. At day 13, cells were stimulated with PMA and Ionomycin in the presence of Brefeldin A for 6 h. Intracellular staining was performed with cytokine-specific Abs. (B) Shown are flow cytometry dot plots illustrating the co-expression of IL-17A and IFNγ (n = 3). (C) Shown is the frequency of cytokine-expressing T-cells cultured long-term in vitro. Results (mean ± SEM) were generated with matches samples from n = 3 different donors. Paired t-Test p-values are indicated on the figures.

Additional file 7. Phenotypic stability versus plasticity of the four central memory CCR6+ T-cell subsets under Th17- or Th1-polarizing conditions. FACS-sorted CM subsets were cultured under Th17- and Th1-polarizing conditions for 14 days, as described in Fig. 5A, stained with CCR6, CCR4, and CXCR3Abs and analyzed by FACS. Shown are the statistical analyses for the expression of (A) CCR6 (B) CCR4, and (C) CXCR3, and the co-expression of (D) CCR6 and CCR4 (R6+R4+ phenotype) and (E) CCR6 and CXCR3 (R6+X3+ phenotype) on each subsets at day 0 (white bars) versus day 14 in culture under Th17- (black bars) and Th1-polarizing (grey bars) conditions. (A-E) The positivity gates where defined based on fluorescence minus one (FMO) controls. Results (mean ± SEM) were generated with matched subsets from n = 3 distinct subjects. Paired t-Test p-values are indicated on the graphs.