BACKGROUND Adipose-derived stem cells (ASCs) are mesenchymal stem cells that have recently been applied to tissue repair and regeneration. Keratinocytes and dermal fibroblasts play key roles in cutaneous wound healing. OBJECTIVE We investigated the paracrine effects of ASCs on HaCaT cells (i.e., immortalized human keratinocytes) and human dermal fibroblasts to explore the mechanism of the effects of ASCs on cutaneous wound healing. METHODS HaCaT cells and primary cultured human dermal fibroblasts were treated with 50% conditioned medium of ASCs (ASC-CM). Viability, in vitro wound healing, and fibroblast-populated collagen lattice contraction assays were conducted, and reverse transcription-polymerase chain reaction (RT-PCR) for the type I procollagen α1 chain gene was performed. RESULTS The proliferation of HaCaT cells and fibroblasts was increased by ASC-CM in the viability assay. ASC-CM promoted in vitro wound healing of HaCaT cells and increased the contraction of the fibroblast-populated collagen lattice. RT-PCR showed that the transcription of the type I procollagen α1 chain gene in fibroblasts was upregulated by ASC-CM. CONCLUSION The stimulatory effect of ASC on cutaneous wound healing may be partially mediated by paracrine effects of ASCs on other skin cells. Application of ASCs or ASC-derived molecules could be an innovative therapeutic approach in the treatment of chronic wounds and other conditions.

Development of neutralizing antibodies to blood coagulation factor VIII (FVIII) provides a major complication in hemophilia care. In this study we explored whether modulation of the uptake of FVIII by antigen presenting cells can reduce its intrinsic immunogenicity. Endocytosis of FVIII by professional antigen presenting cells is significantly blocked by monoclonal antibody KM33, directed towards the C1 domain of FVIII. We created a C1 domain variant (FVIII-R2090A/K2092A/F2093A), which showed only minimal binding to KM33 and retained its activity as measured by chromogenic assay. FVIII-R2090A/K2092A/F2093A displayed a strongly reduced internalization by human monocyte-derived dendritic cells and macrophages, as well as murine bone marrow-derived dendritic cells. We subsequently investigated the ability of this variant to induce an immune response in FVIII-deficient mice. We show that mice treated with FVIII-R2090A/K2092A/F2093A have significantly lower anti-FVIII antibody titers and FVIII-specific CD4(+) T cell responses when compared to mice treated with wild type FVIII. These data show that alanine substitutions at positions 2090, 2092 and 2093 reduce the immunogenicity of FVIII. Based on our findings we hypothesize that FVIII variants displaying a reduced uptake by antigen-presenting cells provide a novel therapeutic approach to reduce inhibitor development in hemophilia A.

OBJECTIVES: The risk of disastrous bleeding during pharyngeal surgery is increased in cases of an internal carotid artery (ICA) that is medially displaced due to its anomalous course. We attempted to assess the distance between the ICA and the pharyngeal wall (DIP) and to evaluate the predisposing factors associated with ICA variation. METHODS: The course of ICA was studied in 509 CT scans, and a retrospective chart review was performed. The course of ICA and DIP were evaluated at each level of the pharynx: nasopharynx (NP), oropharynx (OP), and hypopharynx (HP). RESULTS: The mean DIP value was greatest (15.8±4.6mm) at NP, decreased at OP (15.8±4.6mm), and was shortest at HP (13.5±6.0mm). DIP was significantly shorter in females compared with males at all three pharyngeal levels. Age was inversely correlated with DIP at NP and OP. Tortuous ICA was most common (51.4%), followed by straight (41.2%), kinking (6.9%), and coiling (0.5%) types. DIP was longest in the straight type and decreased as the curvature of ICA increased. The most common ICA type differed between younger (<60 years; 56.2% having the straight type) and older groups (≥60 years; 66.2% having the tortuous type). Females older than 60 years displayed a higher incidence of kinking ICA compared with males. CONCLUSIONS: Hypopharynx, old age, female gender, and tortuous or kinking ICA types were risk factors for a decreased distance between the ICA and the pharyngeal wall. Meticulous examination of the pharyngeal wall should therefore be performed prior to pharyngeal surgery in patients with these associated risk factors.

Thrombotic thrombocytopenic purpura (TTP) is primarily caused by immunoglobulin G (IgG) autoantibodies against A Disintegrin And Metalloprotease with ThromboSpondin type 1 repeats, 13 (ADAMTS13). Nearly all adult idiopathic TTP patients harbor IgGs, which bind the spacer domain of ADAMTS13, a region critical for recognition and proteolysis of von Willebrand factor (VWF). We hypothesize that a modification of an exosite in the spacer domain may generate ADAMTS13 variants with reduced autoantibody binding while preserving or enhancing specific activity. Site-directed mutagenesis was used to generate a series of ADAMTS13 variants, and their functional properties were assessed. Of 24 novel ADAMTS13 variants, 2 (ie, M4, R660K/F592Y/R568K/Y661F and M5, R660K/F592Y/R568K/Y661F/Y665F) exhibited increased specific activity approximately 4- to 5-fold and approximately 10- to 12-fold cleaving a peptide VWF73 substrate and multimeric VWF, respectively. More interestingly, the gain-of-function ADAMTS13 variants were more resistant to inhibition by anti-ADAMTS13 autoantibodies from patients with acquired idiopathic TTP because of reduced binding by anti-ADAMTS13 IgGs. These results shed more light on the critical role of the exosite in the spacer domain in substrate recognition. Our findings also help understand the pathogenesis of acquired autoimmune TTP. The autoantibody-resistant ADAMTS13 variants may be further developed as a novel therapeutic for acquired TTP with inhibitors.

A magnetic, sensitive, and selective fluorescence resonance energy transfer (FRET) probe for detection of thiols in living cells was designed and prepared. The FRET probe consists of an Fe(3) O(4) core, a green-luminescent phenol formaldehyde resin (PFR) shell, and Au nanoparticles (NPs) as FRET quenching agent on the surface of the PFR shell. The Fe(3) O(4) NPs were used as the core and coated with green-luminescent PFR nanoshells by a simple hydrothermal approach. Au NPs were then loaded onto the surface of the PFR shell by electric charge absorption between Fe(3) O(4)@PFR and Au NPs after modifying the Fe(3) O(4)@PFR nanocomposites with polymers to alter the charge of the PFR shell. Thus, a FRET probe can be designed on the basis of the quenching effect of Au NPs on the fluorescence of Fe(3) O(4)@PFR nanocomposites. This magnetic and sensitive FRET probe was used to detect three kinds of primary biological thiols (glutathione, homocysteine, and cysteine) in cells. Such a multifunctional fluorescent probe shows advantages of strong magnetism for sample separation, sensitive response for sample detection, and low toxicity without injury to cellular components.

BACKGROUND/AIMS We investigated the frequency of occult hepatitis B virus (HBV) infection in anti-hepatitis C virus (HCV)-positive individuals and the effects of occult HBV infection on the severity of liver disease. METHODS Seventy-one hepatitis B virus surface-antigen (HBsAg)-negative patients were divided according to their HBV serological status into groups A (anti-HBc positive, anti-HBs negative; n=18), B (anti-HBc positive, anti-HBs positive; n=34), and C (anti-HBc negative, anti-HBs positive/negative; n=19), and by anti-HCV positivity (anti-HCV positive; n=32 vs. anti-HCV negative; n=39). Liver biopsy samples were taken, and HBV DNA was quantified by real-time PCR. RESULTS Intrahepatic HBV DNA was detected in 32.4%(23/71) of the entire cohort, and HBV DNA levels were invariably low in the different groups. Occult HBV infection was detected more frequently in the anti-HBc-positive patients. Intrahepatic HBV DNA was detected in 28.1%(9/32) of the anti-HCV-positive and 35.9%(14/39) of the anti-HCV-negative subjects. The HCV genotype did not affect the detection rate of intrahepatic HBV DNA. In anti-HCV-positive cases, occult HBV infection did not affect liver disease severity. CONCLUSIONS Low levels of intrahepatic HBV DNA were detected frequently in both HBsAg-negative and anti-HCV-positive cases. However, the frequency of occult HBV infection was not affected by the presence of hepatitis C, and occult HBV infection did not have a significant effect on the disease severity of hepatitis C.

PURPOSE To determine the diagnostic utility of a frozen section biopsy in patients undergoing endoscopic submucosal dissection (ESD) for early gastric neoplasms with obscure margins even with chromoendoscopy using acetic acid and indigo carmine (AI chromoendoscopy). MATERIALS AND METHODS The lateral spread of early gastric neoplasms was unclear even following AI chromoendoscopy in 38 patients who underwent ESD between June 2007 and May 2011. Frozen section biopsies were obtained by agreement of the degree of lateral spread between two endoscopists. Thus, frozen section biopsies were obtained from 23 patients (FBx group) and not in the other 15 patients (AI group). RESULTS No significant differences were observed for size, histology, invasive depth, and location of lesions between the AI and FBx groups. No false positive or false negative results were observed in the frozen section diagnoses. Adenocarcinoma was revealed in three patients and tubular adenoma in one, thereby changing the delineation of lesion extent and achieving free lateral margins. The rates of free lateral resection margins and curative resection were significantly higher in the FBx group than those in the AI group. CONCLUSIONS Frozen section biopsy can help endoscopists perform more safe and accurate ESD in patients with early gastric neoplasm.

Spinal epidural lipomatosis (SEL) is an overgrowth of the normally encapsulated adipose tissue in the epidural space around the spinal cord in the thoracic and lumbar spine causing compression of the neural components. Idiopathic SEL in non-obese patients is exceptional. Idiopathic SEL can result in thoracic myelopathy and lumbar radiculopathy. A thoracic radiculopathy due to idiopathic SEL has not been reported yet. We report a case of idiopathic SEL with intractable chest pain and paresthesia. We suggest that idiopathic SEL should be considered as a cause of chest pain.