Johnson Chia-Shen Yang

Whole blood-derived microRNA signatures in mice exposed to lipopolysaccharides

BackgroundLipopolysaccharide (LPS) is recognized as the most potent microbial mediator presaging the threat of invasion of Gram-negative bacteria that implicated in the pathogenesis of sepsis and septic shock. This study was designed to examine the microRNA (miRNA) expression in whole blood from mice injected with intraperitoneal LPS.MethodsC57BL/6 mice received intraperitoneal injections of varying concentrations (range, 10–1000 μg) of LPS from different bacteria, including Escherichia coli, Klebsiella pneumonia, Pseudomonas aeruginosa, Salmonella enterica, and Serratia marcescens and were killed 2, 6, 24, and 72 h after LPS injection. Whole blood samples were obtained and tissues, including lung, brain, liver, and spleen, were harvested for miRNA expression analysis using an miRNA array (Phalanx miRNA OneArray® 1.0). Upregulated expression of miRNA targets in the whole blood of C57BL/6 and Tlr4−/− mice injected with LPS was quantified using real-time RT-PCR and compared with that in the whole blood of C57BL/6 mice injected with lipoteichoic acid (LTA) from Staphylococcus aureus.ResultsFollowing LPS injection, a significant increase of 15 miRNAs was observed in the whole blood. Among them, only 3 miRNAs showed up-regulated expression in the lung, but no miRNAs showed a high expression level in the other examined tissues. Upregulated expression of the miRNA targets (let-7d, miR-15b, miR-16, miR-25, miR-92a, miR-103, miR-107 and miR-451) following LPS injection on real-time RT-PCR was dose- and time-dependent. miRNA induction occurred after 2 h and persisted for at least 6 h. Exposure to LPS from different bacteria did not induce significantly different expression of these miRNA targets. Additionally, significantly lower expression levels of let-7d, miR-25, miR-92a, miR-103, and miR-107 were observed in whole blood of Tlr4−/− mice. In contrast, LTA exposure induced moderate expression of miR-451 but not of the other 7 miRNA targets.ConclusionsWe identified a specific whole blood–derived miRNA signature in mice exposed to LPS, but not to LTA, from different gram-negative bacteria. These whole blood-derived miRNAs are promising as biomarkers for LPS exposure.

Profiling Circulating MicroRNA Expression in Experimental Sepsis Using Cecal Ligation and Puncture

The levels of circulating microRNAs (miRNAs) in mice with experimental sepsis induced by cecal ligation and puncture (CLP) were determined using whole blood samples obtained from C57BL/6 mice at 4, 8, and 24 h after CLP; miRNA expression analysis was performed in these samples using an miRNA array. Microarray analysis revealed upregulation of 10 miRNA targets (miR-16, miR-17, miR-20a, miR-20b, miR-26a, miR-26b, miR-106a, miR-106b, miR-195, and miR-451). The expression of these miRNA targets in the whole blood, serum, and white blood cells (WBCs) of CLP mice was quantified using quantitative real-time PCR; these values were compared to those in sham-operated C57BL/6 mice, and the results indicated that these miRNA targets were significantly up-regulated in the whole blood and serum but not in the WBCs. In addition, the levels of these 10 miRNA targets in the serum of Tlr2−/−, Tlr4−/−, and NF-κB−/− mice at 8 h after CLP did not decrease significantly., which indicated that the transcription of these miRNAs was not directly mediated by the TLR2/NF-κB or TLR4/NF-κB pathway, and pathways induced by exposure to the gram-positive or gram-negative bacteria. Immunoprecipitation with the Argonaute 2 ribonucleoprotein complex revealed significantly increased expression of the 10 miRNA targets in the serum of mice after CLP, and the levels of 6 (miR-16, miR-17, miR-20a, miR-20b, miR-26a, and miR-26b) of these 10 miRNA targets increased significantly in exosomes isolated using ExoQuick precipitation solution. In this study, we identified circulating miRNAs that were up-regulated after CLP and determined the increase in the levels of these miRNAs, and our results suggest that circulating Ago2 complexes and exosomes may be responsible for the stability of miRNAs in the serum.

Alternative reconstructive choices for anterolateral thigh flap dissection in cases in which no sizable skin perforator is available

Alternative choices were proposed to facilitate a successful reconstruction when no sizable skin perforator is encountered in anterolateral thigh (ALT) flap dissection.

Swine Hemi-Facial Composite Tissue Allotransplantation: A Model to Study Immune Rejection

OBJECTIVE Partial face composite tissue allotransplantation was recently achieved in a human subject. However, the side effects of long-term immunosuppression and chronic rejection area still need concerning. This preliminary study investigated the reproducibility of swine hemi-facial transplantation for preclinical studies. MATERIALS AND METHODS Eleven out-bred miniature swine underwent hemi-facial transplant. The hemi-facial orthotopic transplant consisted of ear cartilage, auricular nerve, parotid gland and lymphoid tissue, muscle with surrounding hemi-facial skin paddle supplied by the carotid artery, and external jugular vein transplanted to recipient swine. Three different experimental designs were studied, as follows: group I (n = 4): autologous hemi-facial transplantation as a normal control; group II (n = 4): hemi-facial allotransplantation without treatment; group III (n = 3): hemi-facial allotransplantation with cyclosporine-A treatment for 4 wk. The transplanted face was observed daily for signs of rejection. Biopsy of donor skin, gland lymphoid tissue, and cartilage were obtained at specified predetermined time (d 7, 14, 28), or at the time of clinically evident rejection. RESULTS The results indicated the survival of group I following autologous hemi-facial transplant was 100% and indefinite until sacrifice. Group II without treatment as the controls revealed allograft rejection by d 7 to 28. The allograft with short-term cyclosporine-A treatment revealed delayed rejection by d 38 to 49 postoperatively. The histological examination in group I revealed abundant lymphocyte infiltration, especially in lymphoid gland and alloskin at 1 wk and sacrifice. In contrast, the cyclosporine treatment group showed no significant rejection signs in 4 wk posttransplants. These results demonstrated that lymphoid tissue and alloskin are both susceptible to early rejection. CONCLUSION The experimental results revealed this model is suitable to investigate the new strategies for preclinical facial allotransplantation studies. Monitoring and modulation of early rejection in alloskin and gland lymphoid tissue is a useful strategy to evaluate composite tissue allotransplantation survival.

Single free anterolateral thigh flap for simultaneous reconstruction of composite hypopharyngeal and external neck skin defect after head and neck cancer ablation

Single flap for complex hypopharyngoesophageal and anterior neck skin defect reconstruction is still a challenge for reconstructive surgeons. Herein, we present five patients, with advanced hypopharyngeal cancer and anterior neck skin invasion, which received a single anterolateral thigh (ALT) fasciocutaneous flap for composite inner pharyngeal and outer skin defect reconstruction after wide composite resection. Two ALT flaps were divided into two distinct paddles supplied by two or more separate perforators, one part for reconstructing the inner pharyngeal defect and another for neck skin coverage. Three ALT flaps only supplied by one sizable perforator could not be divided and de‐epithelization of mid‐part had to be done to reconstruct both defects with the single flap. The results revealed survival of all flaps. There were no flap loss, fistulas, or bleeding complications. All patients recovered uneventfully and could eat a soft diet to regular diet postoperatively. In conclusion, one‐staged reconstruction of complex pharyngoesophageal and external skin defects after extensive oncological resection is feasible using a single ALT fasciocutaneous free flap.

The use of radial vessel stump in free radial forearm flap as flap monitor in head and neck reconstruction.

The versatile use of the free radial forearm flap for the reconstruction of pharyngoesophageal defects has been proven ideal in previous studies. However, the monitoring of flap viability when buried underneath the skin in head and neck surgery still posed technical difficulties. An innovative monitoring method for the buried free radial forearm flap by using distal radial vessel stump elevated over the skin is to be presented here. Eighteen patients received free radial forearm flap reconstruction for their pharyngoesophageal defects after tumor ablation during June 2003 to March 2005. All patients were males; ages ranged from 36 to 71 years, averaging 53.2 years old. Fourteen skin tubing and patches were designed for the defects. The pharyngoesophageal defects ranged from 6 to 12 cm in length, averaging 8.5 cm. The free radial forearm flap was designed to allow a distal radial vessel stump about 3 cm long, which was then elevated above the skin in the neck region after insetting to act as a monitor for the viability of the buried flap. The flap viability can be easily demonstrated simply by observing continuous pulsation coming from the distal radial vessel stump with naked eyes. The monitoring stump was then ligated and resected at bedside after the viability of the buried flap was insured 2 weeks postoperatively. All free flap transfers were successful. One case was found with kinking of artery during the operation and it was corrected immediately. One case with venous insufficiency was detected 13 hours after the operation and the flap was salvaged successfully by thrombectomy and venous reanastomosis. Three patients developed temporary fistula and healed spontaneously after conservative treatment. Deep neck infections were found in patients, and recovered after aggressive antibiotic treatment. Two patients had esophagocutaneous fistula and needed secondary surgical intervention. The use of distal radial vessel stump as a monitor for the buried flap is a reliable method in head and neck surgery. It not only allows easy monitoring, no further donor site morbidity, but it also eliminates the need for special monitoring device.

The posteromedial thigh flap for head and Neck reconstruction: anatomical basis, surgical technique, and clinical applications

Background: The authors present the posteromedial thigh flap as an alternative source for head and neck reconstruction, and the perforator patterns and vascular anatomy of this flap were further investigated. Methods: From March to August of 2014, 23 patients underwent head and neck reconstruction with 23 posteromedial thigh flaps. The numbers, locations, and types of perforators were measured. The surgical technique and the results after reconstruction were evaluated. Results: Most perforators were located 8 to 10 cm away from the pubic crease on the reference line between the perineum and the insertion of the semitendinosus muscle. The average number of perforators was 1.7 (range, 1 to 3), and the average pedicle length was 10.3 cm (range, 8 to 13 cm). Eighty percent of the perforators (32 of 40) were musculocutaneous, and 20 percent (8 of 40) were septocutaneous. Ninety-five percent of the perforators (38 of 40) originated from the profunda femoris artery, and 5 percent (two of 40) originated from the medial circumflex femoral artery. The flap survival rate was 95.6 percent; one flap failed due to pedicle thrombosis. The donor sites were all closed primarily. Conclusions: The location of the perforators of the posteromedial thigh flap is consistent, and the pedicle length is sufficient to reach the neck region. Different reconstruction demands can be met by incorporating various soft-tissue components. The donor-site scar is well concealed, with minimal morbidity. The above advantages make the posteromedial thigh flap an excellent option for head and neck reconstruction. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, IV.

Circulating microRNA signatures in mice exposed to lipoteichoic acid

BackgroundPreviously, we had identified a specific whole blood–derived microRNAs (miRNAs) signature in mice following in vivo injection of lipopolysaccharide (LPS) originated from Gram-negative bacteria. This study was designed to profile the circulating miRNAs expression in mice exposed to lipoteichoic acid (LTA) which is a major component of the wall of Gram-positive bacteria.ResultsC57BL/6 mice received intraperitoneal injections of 100 μg of LTA originated from Bacillus subtilis, Streptococcus faecalis, and Staphylococcus aureus were killed 6 h and the whole blood samples were obtained for miRNA expression analysis using a miRNA array (Phalanx miRNA OneArray® 1.0). Up-regulated expression of miRNA targets in the whole blood, serum and white blood cells (WBCs) of C57BL/6 and Tlr2−/− mice upon LTA treatment in 10, 100, or 1000 ug concentrations was quantified at indicated time (2, 6, 24, and 72 h) using real-time RT-PCR and compared with that in the serum of C57BL/6 mice injected with 100 ug of LPS. A significant increase of 4 miRNAs (miR-451, miR-668, miR-1902, and miR-1904) was observed in the whole blood and the serum in a dose- and time-dependent fashion following LTA injection. Induction of miRNA occurred in the serum after 2 h and persisted for at least 6 h. No increased expression of these 4 miRNAs was found in the WBCs. Higher but not significant expression level of these 4 miRNAs were observed following LTA treatment in the serum of Tlr2−/−against that of C57BL6 mice. In contrast, LPS exposure induced moderate expression of miR-451 but not of the other 3 miRNA targets.ConclusionsWe identified a specific circulating miRNA signature in mice exposed to LTA. That expression profile is different from those of mice exposed to LPS. Those circulating miRNAs induced by LTA or LPS treatment may serve as promising biomarkers for the differentiation between exposures to Gram-positive or Gram-negative bacteria.

Suprafascial Anterolateral Thigh Flap Harvest: A Better Way to Minimize Donor-site Morbidity in Head and Neck Reconstruction.

Background: The purpose of this study was to compare the clinical outcomes and donor-site morbidity between the suprafascial and subfascial harvesting of anterolateral thigh flaps. Methods: Sixty-one patients who underwent free flap reconstruction (30 suprafascial and 31 subfascial anterolateral thigh flaps) were included in this study. The patients assessed the subjective donor-site morbidity and satisfaction with the overall functional result using a self-reported questionnaire. The flap characteristics (i.e., perforator number, flap size, and harvest time) and outcomes (i.e., success rate, partial necrosis, infection, hematoma, and fistula) were compared. Results: The success rates of suprafascial and subfascial anterolateral thigh flaps were 96.7 and 96.8 percent, respectively. There were no significant differences in flap size, harvest time, or overall complication rates. The suprafascial anterolateral thigh flap group experienced fewer abnormal sensations (p < 0.001) and better subjective satisfaction at the donor site than did the subfascial anterolateral thigh flap group (p = 0.03). Conclusions: In terms of reducing donor-site morbidity, the suprafascial anterolateral thigh flap group showed fewer sensory disturbances in donor thighs and exhibited better patient satisfaction than did the subfascial anterolateral thigh flap group, but meticulous dissection of tiny perforators above the fascia is required for the former procedure. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, III.

Weight-reduction through a low-fat diet causes differential expression of circulating microRNAs in obese C57BL/6 mice

BackgroundTo examine the circulating microRNA (miRNA) expression profile in a mouse model of diet-induced obesity (DIO) with subsequent weight reduction achieved via low-fat diet (LFD) feeding.ResultsEighteen C57BL/6NCrl male mice were divided into three subgroups: (1) control, mice were fed a standard AIN-76A (fat: 11.5 kcal %) diet for 12 weeks; (2) DIO, mice were fed a 58 kcal % high-fat diet (HFD) for 12 weeks; and (3) DIO + LFD, mice were fed a HFD for 8 weeks to induce obesity and then switched to a 10.5 kcal % LFD for 4 weeks. A switch to LFD feeding led to decreases in body weight, adiposity, and blood glucose levels in DIO mice. Microarray analysis of miRNA using The Mouse & Rat miRNA OneArray® v4 system revealed significant alterations in the expression of miRNAs in DIO and DIO + LFD mice. Notably, 23 circulating miRNAs (mmu-miR-16, mmu-let-7i, mmu-miR-26a, mmu-miR-17, mmu-miR-107, mmu-miR-195, mmu-miR-20a, mmu-miR-25, mmu-miR-15b, mmu-miR-15a, mmu-let-7b, mmu-let-7a, mmu-let-7c, mmu-miR-103, mmu-let-7f, mmu-miR-106a, mmu-miR-106b, mmu-miR-93, mmu-miR-23b, mmu-miR-21, mmu-miR-30b, mmu-miR-221, and mmu-miR-19b) were significantly downregulated in DIO mice but upregulated in DIO + LFD mice. Target prediction and function annotation of associated genes revealed that these genes were predominantly involved in metabolic, insulin signaling, and adipocytokine signaling pathways that directly link the pathophysiological changes associated with obesity and weight reduction.ConclusionsThese results imply that obesity-related reductions in the expression of circulating miRNAs could be reversed through changes in metabolism associated with weight reduction achieved through LFD feeding.