Resting TcPO2 levels decrease during liner wear in persons with a transtibial amputation

BACKGROUND In our clinic, a substantial number of patients present with transtibial residual limb pain of no specific somatic origin. Silicone liner induced tissue compression may reduce blood flow, possibly causing residual limb pain. Thus, as a first step we investigated if the liner itself has an effect on transcutaneous oxygen pressure (TcPO2). METHODS Persons with unilateral transtibial amputation and residual limb pain of unknown origin were included. Medical history, including residual limb pain, was recorded, and the SF-36 administered. Resting TcPO2 levels were measured in the supine position and without a liner at 0, 10, 20 and 30 minutes using two sensors: one placed in the Transverse plane over the tip of the Tibia End (= TTE), the other placed in the Sagittal plane, distally over the Peroneal Compartment (= SPC). Measurements were repeated with specially prepared liners avoiding additional pressure due to sensor placement. Statistical analyses were performed using SPSS. RESULTS Twenty persons (9 women, 11 men) with a mean age of 68.65 years (range 47-86 years) participated. The transtibial amputation occurred on average 43 months prior to study entry (range 3-119 months). With liner wear, both sensors measured TcPO2 levels that were significantly lower than those measured without a liner (TTE: p < 0.001; SPC: p = 0.002) after 10, 20 and 30 minutes. No significant differences were found between TcPO2 levels over time between the sensors. There were no significant associations between TcPO2 levels and pain, smoking status, age, duration of daily liner use, mobility level, and revision history. CONCLUSION Resting TcPO2 levels decreased significantly while wearing a liner alone, without a prosthetic socket. Further studies are required to investigate the effect of liner wear on exercise TcPO2 levels

Resting TcPO2 levels decrease during liner wear in persons with a transtibial amputation

Background In our clinic, a substantial number of patients present with transtibial residual limb pain of no specific somatic origin. Silicone liner induced tissue compression may reduce blood flow, possibly causing residual limb pain. Thus, as a first step we investigated if the liner itself has an effect on transcutaneous oxygen pressure (TcPO2). Methods Persons with unilateral transtibial amputation and residual limb pain of unknown origin were included. Medical history, including residual limb pain, was recorded, and the SF-36 administered. Resting TcPO2 levels were measured in the supine position and without a liner at 0, 10, 20 and 30 minutes using two sensors: one placed in the Transverse plane over the tip of the Tibia End (= TTE), the other placed in the Sagittal plane, distally over the Peroneal Compartment (= SPC). Measurements were repeated with specially prepared liners avoiding additional pressure due to sensor placement. Statistical analyses were performed using SPSS. Results Twenty persons (9 women, 11 men) with a mean age of 68.65 years (range 47–86 years) participated. The transtibial amputation occurred on average 43 months prior to study entry (range 3–119 months). With liner wear, both sensors measured TcPO2 levels that were significantly lower than those measured without a liner (TTE: p < 0.001; SPC: p = 0.002) after 10, 20 and 30 minutes. No significant differences were found between TcPO2 levels over time between the sensors. There were no significant associations between TcPO2 levels and pain, smoking status, age, duration of daily liner use, mobility level, and revision history. Conclusion Resting TcPO2 levels decreased significantly while wearing a liner alone, without a prosthetic socket. Further studies are required to investigate the effect of liner wear on exercise TcPO2 levels.ISSN:1932-620

Lyn Delivers Bacteria to Lysosomes for Eradication through TLR2-Initiated Autophagy Related Phagocytosis

<div><p>Extracellular bacteria, such as <i>Pseudomonas aeruginosa</i> and <i>Klebsiella pneumoniae</i>, have been reported to induce autophagy; however, the role and machinery of infection-induced autophagy remain elusive. We show that the pleiotropic Src kinase Lyn mediates phagocytosis and autophagosome maturation in alveolar macrophages (AM), which facilitates eventual bacterial eradication. We report that Lyn is required for bacterial infection-induced recruitment of autophagic components to pathogen-containing phagosomes. When we blocked autophagy with 3-methyladenine (3-MA) or by depleting Lyn, we observed less phagocytosis and subsequent bacterial clearance by AM. Both morphological and biological evidence demonstrated that Lyn delivered bacteria to lysosomes through xenophagy. TLR2 initiated the phagocytic process and activated Lyn following infection. Cytoskeletal trafficking proteins, such as Rab5 and Rab7, critically facilitated early phagosome formation, autophagosome maturation, and eventual autophagy-mediated bacterial degradation. These findings reveal that Lyn, TLR2 and Rab modulate autophagy related phagocytosis and augment bactericidal activity, which may offer insight into novel therapeutic strategies to control lung infection.</p></div

Autophagy is important for Lyn-dependent elimination of Pa in macrophages.

<p>(A) MH-S cells were transfected with Ctrl, Atg5 or Beclin1 siRNA for 24 h, respectively. Immunoblotting was performed to verify the knock down of these two genes. (B) After Atg5 or Beclin1 was knocked down, MTT assay was used to determine the cell viability upon PAO1 infection (MOI = 10, 1 h). (C) CFU assay was performed to test the phagocytosis ability from above samples. (D) Cells above were collected and lysed for immunoblotting to measure the phosphorylation level of Lyn. (E) Quantification of pLyn and LC3-II level in D is shown. (F) MH-S cells were transfected with LC3-RFP for 24 h. Cells were infected with PAO1-GFP (MOI = 10, 1 h). Immunostaining was used to detect pLyn. Pa surrounded by LC3 and pLyn was detected by confocal microscope. Scale bar = 5 μm. (G) LC3 puncta in each cell were counted. The percentage of LC3⁺/Pa⁺/pLyn⁺ events (cell with colocalized puncta of LC3-RFP, PAO1-GFP, and pLyn) is shown. Data are derived from 100 cells in each sample. (H) MH-S cells were infected with PAO1 as above. After 1 h, cells were washed and subjected to polymyxin B for another 1 h. 10 h later, cell viability was determined by MTT assay. (I) Clearance assays were performed by counting CFU. (J) MH-S cells were infected with PAO1 for 1 h or 12 h as above. Cell lysates were performed for immunoblotting to test the invaded bacteria using Pa antibody. (K) Quantification of Pa protein in J is shown. All data are representative as means+SD of three independent experiments. One-way ANOVA (Tukey’s post hoc); *, p<0.05; **, p<0.01.</p

Lyn regulate infection-induced autophagy through Rab5.

<p>(A) MH-S cells were transfected with Rab5-RFP for 24 h. Cells were infected with PAO1-GFP (MOI = 10, 1 h). Immunostaining was used to detect pLyn. Pa surrounded by Rab5 and pLyn was detected by confocal microscope. Scale bar = 5 μm. (B) The percentage of Rab5⁺/Pa⁺/pLyn⁺ colocalization (Rab5-RFP, PAO1-GFP, and pLyn puncta) is shown. Data are derived from 100 cells in each group. (C) MH-S cells were infected with PAO1 (MOI = 10, 1 h). Co-IP was performed to detect the protein interactions between Lyn and Rab5. (D) MH-S cells were pretreated with PP2 (5 nM, 30 min). Phagosomes were isolated and lysed for immunoblotting to detect pLyn and Rab5. (E, F) MH-S cells were co-transfected with Rab5-RFP and LC3-GFP as well as Lyn siRNA for 24 h, respectively. Cells were infected with PAO1 as above. CLSM imaging showed the localization (different puncta). Data are derived from 100 cells for each sample. Scale bar = 5 μm. (G, H) MH-S cells were transfected with Rab5-RFP or Rab5-DN-RFP plasmid for 24 h. Cells were infected with PAO1-GFP as above. Cell viability was determined using MTT assay and phagocytosis was performed by CFU assay. (I, J) CLSM imaging showed the localization of Rab5 and Pa. Scale bar = 5 μm. Rab5 puncta were counted in each cell. The percentage of Rab5⁺/Pa⁺ events (cell with colocalized puncta of Rab5-RFP and PAO1-GFP) is shown. Data are derived from 100 cells in each group. (K) MH-S cells were transfected with Ctrl or Lyn siRNA for 24 h, respectively. Whole cell lysates were immunoprecipitated (IP) with beads coated with Lyn antibody and immunoblotted with actin antibody. (L, M) Cells were infected with PAO1-GFP (MOI = 10) for different time. Polymerized actin was stained with rhodamine-phalloidin. Confocal microscopy showing rhodamine-phalloidin staining (red) around GFP-expressing Pa. Phagosomes containing degraded bacteria are heavily labeled for polymerized actin (white arrowheads). Scale bar = 20 μm. Internalized Pa and colocalized puncta were counted in each cell. Data are derived from 100 cells in each group. One-way ANOVA (Tukey’s post hoc); *, p<0.05.</p

Lyn plays an important role in recruitment of LC3 upon Pa infection.

<p>(A) MH-S cells were pretreated with CD (2.5 μg/ml, 30 min), and then infected with PAO1 (MOI = 10, 1 h). Cell viability was tested by MTT assay. (B) Cells treated as above were lysed for CFU assay. (C) Cells lysates were performed for immunoblotting of pLyn, Lyn and LC3. (D, E) MH-S cells were transfected with LC3-RFP plasmid for 24 h and pretreated with CD (2.5 μg/ml, 30 min). The cells were then infected with PAO1-GFP (MOI = 10, 1 h). Confocal microscopy images were used to detect LC3 puncta upon Pa infection. LC3 puncta in each cell were counted and the percentage of LC3⁺/Pa⁺ colocalization (LC3-RFP and PAO1-GFP) is shown. Data are derived from 100 cells in each sample. (F) MH-S were pretreated with CD (2.5 μg/ml, 30 min) or rapamycin (500 nM, 12 h) and cells were lysed for immunoblotting of pLyn, Lyn and LC3. (G) MH-S cells were transfected with Ctrl or Lyn siRNA for 24 h, and treated with rapamycin (500 nM, 12 h). Cells lysates were performed for immunoblotting of pLyn, Lyn and LC3. (H, I) MH-S cells were co-transfected with LC3-RFP and Ctrl or Lyn siRNA for 24 h. The cells were treated with rapamycin (500 nM, 12 h). Confocal microscopy images were used to show LC3 puncta. LC3 puncta in each cell were counted. The percentage of LC3⁺ events (cell with more than five LC3 puncta was considered as positive) is shown. Data are derived from 100 cells in each sample. (J) MH-S cells were treated with Zymosan (10 μg/ml, 1 h). Cell lysates were performed for immunoblotting of pLyn, Lyn and LC3. (K) MH-S cells were co-transfected with Lyn-GFP and LC3-RFP for 24 h. The cells were treated with Zymosan (10 μg/ml, 1 h). Confocal microscopy images were used to show Lyn or LC3 puncta. (L) LC3 puncta in each cell were counted, and the percentage of LC3⁺ events is shown as above. Data are derived from 100 cells in each sample. (M, N) MH-S cells were transfected with Ctrl, Rubicon or ULK1 siRNA for 24 h. The cells were infected with PAO1 (MOI = 10, 1 h). Cell viability and phagocytic abilities were tested by MTT or CFU assays as above. (O) Cell lysates from above were used for immunoblotting to detect pLyn, Lyn and LC3, respectively. (P) MH-S cells were co-transfected with Ctrl, Rubicon or ULK1 siRNA, respectively along with LC3-RFP for 24 h. The cells were infected with PAO1-GFP (MOI = 10, 1 h). LC3 puncta in each cell were counted, and the percentage of LC3⁺ events is shown as above. Data are derived from 100 cells in each sample. All data are representative as means+SD of three independent experiments. One-way ANOVA (Tukey’s post hoc); *, p<0.05; **, p<0.01. Scale bar = 5 μm.</p