Serum C-peptide level correlates with the course of muscle tissue healing in the rabbit model of critical limb ischemia.

AIM: The therapeutic potential of adipose-derived stem cell conditioned medium (ASC-CM) was studied in the rabbit model of critical limb ischemia (CLI). METHODS: Rabbits received treatment with ASC-CM or placebo. Gastrocnemius muscle tissue was collected 35 days after ischemia induction. Ischemic changes were evaluated in hematoxylin-eosin stained tissues for early (necrotic lesions/granulation tissue) and late (fibrous scars) phases of tissue repair. The expression of proangiogenic miR-126 was also evaluated using in situ hybridization. The levels of cytokines, insulin, and C-peptide were measured in blood. RESULTS: Early repair phases were observed more often in placebo-treated samples (45.5%) than in ASC-CM-treated ones (22.2%). However, the difference was not statistically significant. We demonstrated a statistically significant positive correlation between the early healing phases in tissue samples and C-peptide levels in peripheral blood. The expression of proangiogenic miR-126 was also shown in a number of structures in all phases of ischemic tissue healing. CONCLUSION: Based on our results, we believe that treatment with ASC-CM has the potential to accelerate the healing process in ischemic tissues in the rabbit model of CLI. The whole healing process was accompanied by miR-126 tissue expression. C-peptide could be used to monitor the course of the tissue healing process.

The Role of von Willebrand Factor, ADAMTS13, and Cerebral Artery Thrombus Composition in Patient Outcome Following Mechanical Thrombectomy for Acute Ischemic Stroke.

BACKGROUND The aim of the study was to investigate the role of von Willebrand factor (vWF), the vWF-cleaving protease, ADAMTS13, the composition of thrombus, and patient outcome following mechanical cerebral artery thrombectomy in patients with acute ischemic stroke. MATERIAL AND METHODS A prospective cohort study included 131 patients with ischemic stroke (<6 hours) with or without intravenous thrombolysis. Interventional procedure parameters, hemocoagulation markers, vWF, ADAMTS13, and histological examination of the extracted thrombi were performed. The National Institutes of Health Stroke Scale (NIHSS) score was used on hospital admission, after 24 hours, at day 7; the three-month modified Rankin Scale score was used. RESULTS Mechanical thrombectomy resulted in a Treatment in Cerebral Ischemia (TICI) score of 2-3, with recanalization in 89% of patients. Intravenous thrombolysis was used in 101 (78%). Patients with and without intravenous thrombolysis therapy had a good clinical outcome (score 0-2) in 47% of cases (P=0.459) using the three-month modified Rankin Scale. Patients with a National Institutes of Health Stroke Scale (NIHSS) score ≥15 had significantly increased vWF levels (P=0.003), and a significantly increased vWF: ADAMTS13 ratio (P=0.038) on hospital admission. Significant correlation coefficients were found for plasma vWF and thrombo-embolus vWF (r=0.32), platelet (r=0.24), and fibrin (r=0.26) levels. In the removed thrombus, vWF levels were significantly correlated with platelet count (r=0.53), CD31-positive cells (r=0.38), and fibrin (r=0.48). CONCLUSIONS In patients with acute ischemic stroke, mechanical cerebral artery thrombectomy resulted in a good clinical outcome in 47% of cases, with and without intravenous thrombolysis therapy.

Serum Cytokine and Growth Factor Levels in Children with Autism Spectrum Disorder.

BACKGROUND The immune system may have a role in the pathogenesis of autism spectrum disorder (ASD), including typical and atypical autism. The aim of this study was to determine whether a cytokine and growth factor panel could be identified for the diagnosis and prognosis in children with ASD, including typical and atypical autism. MATERIAL AND METHODS This study included 26 children with ASD (typical or atypical) and 11 of their siblings who did not have ASD. A panel of ten serum cytokines and growth factors were investigated using addressable laser bead assay (ALBIA) and enzyme-linked immunosorbent assay (ELISA) kits. Results were correlated with scores using the Childhood Autism Rating Scale (CARS) and Autism Diagnostic Observation Schedule (ADOS) for the children with ASD and compared with the findings from their siblings without ASD. RESULTS There were no statistically significant differences in serum cytokine and growth factor levels between children with ASD and their siblings. The scores using CARS and ADOS were significantly greater in children with typical autism compared with children with atypical autism as part of the ASD spectrum. Serum levels of cytokines and growth factors showed a positive correlation with CARS and ADOS scores but differed between children with typical and atypical autism and their siblings. CONCLUSIONS The findings of this study showed that serum measurement of appropriately selected panels of cytokines and growth factors might have a role in the diagnosis of ASD.

The Role of miR-126 in Critical Limb Ischemia Treatment Using Adipose-Derived Stem Cell Therapeutic Factor Concentrate and Extracellular Matrix Microparticles.

BACKGROUND Paracrine factors secreted by adipose-derived stem cells can be captured, fractionated, and concentrated to produce therapeutic factor concentrate (TFC). The present study examined whether TFC effects could be enhanced by combining TFC with a biological matrix to provide sustained release of factors in the target region. MATERIAL AND METHODS Unilateral hind limb ischemia was induced in rabbits. Ischemic limbs were injected with either placebo control, TFC, micronized small intestinal submucosa tissue (SIS), or TFC absorbed to SIS. Blood flow in both limbs was assessed with laser Doppler perfusion imaging. Tissues harvested at Day 48 were assessed immunohistochemically for vessel density; in situ hybridization and quantitative real-time PCR were employed to determine miR-126 expression. RESULTS LDP ratios were significantly elevated, compared to placebo control, on day 28 in all treatment groups (p=0.0816, p=0.0543, p=0.0639, for groups 2-4, respectively) and on day 36 in the TFC group (p=0.0866). This effect correlated with capillary density in the SIS and TFC+SIS groups (p=0.0093 and p=0.0054, respectively, compared to placebo). A correlation was observed between miR-126 levels and LDP levels at 48 days in SIS and TFC+SIS groups. CONCLUSIONS A single bolus administration of TFC and SIS had early, transient effects on reperfusion and promotion of ischemia repair. The effects were not additive. We also discovered that TFC modulated miR-126 levels that were expressed in cell types other than endothelial cells. These data suggested that TFC, alone or in combination with SIS, may be a potent therapy for patients with CLI that are at risk of amputation.

Therapeutic Potential of Adipose-Derived Therapeutic Factor Concentrate for Treating Critical Limb Ischemia.

Transplantation of adipose-derived stem cells (ADSCs) is an emerging therapeutic option for addressing intractable diseases such as critical limb ischemia (CLI). Evidence suggests that therapeutic effects of ADSCs are primarily mediated through paracrine mechanisms rather than transdifferentiation. These secreted factors can be captured in conditioned medium (CM) and concentrated to prepare a therapeutic factor concentrate (TFC) composed of a cocktail of beneficial growth factors and cytokines that individually and in combination demonstrate disease-modifying effects. The ability of a TFC to promote reperfusion in a rabbit model of CLI was evaluated. A total of 27 adult female rabbits underwent surgery to induce ischemia in the left hindlimb. An additional five rabbits served as sham controls. One week after surgery, the ischemic limbs received intramuscular injections of either (1) placebo (control medium), (2) a low dose of TFC, or (3) a high dose of TFC. Limb perfusion was serially assessed with a Doppler probe. Blood samples were analyzed for growth factors and cytokines. Tissue was harvested postmortem on day 35 and assessed for capillary density by immunohistochemistry. At 1 month after treatment, tissue perfusion in ischemic limbs treated with a high dose of TFC was almost double (p < 0.05) that of the placebo group [58.8 ± 23 relative perfusion units (RPU) vs. 30.7 ± 13.6 RPU; mean ± SD]. This effect was correlated with greater capillary density in the affected tissues and with transiently higher serum levels of the angiogenic and prosurvival factors vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF). The conclusions from this study are that a single bolus administration of TFC demonstrated robust effects for promoting tissue reperfusion in a rabbit model of CLI and that a possible mechanism of revascularization was promotion of angiogenesis by TFC. Results of this study demonstrate that TFC represents a potent therapeutic cocktail for patients with CLI, many of whom are at risk for amputation of the affected limb.

ANCA in the diagnosis of neutrophil-mediated inflammation.

Inconsistencies in ANCA (anti-neutrophil cytoplasm autoantibodies) and other NSA (neutrophil-specific autoantibodies) terminology frequently cause incorrect indications, choices, applications and interpretations of ANCA diagnostics in routine practice, except for ANCA-associated vasculitis. A review of the current knowledge and the authors' personal experiences based on routine assessments of ANCA and other NSA are documented and presented. A better understanding of the principles and mechanisms of ANCA and other NSA responses and determination, as well as unification of their terminology could result in improvements in indications, applications and the interpretation of ANCA diagnostics in diseases other than vasculitis, especially in IBD (inflammatory bowel diseases), AILD (autoimmune liver diseases), CTD (connective tissue diseases) and other chronic neutrophil-mediated inflammatory diseases.

[Low response rate to a vaccination against hepatitis B in patients with end-stage liver disease]. / Malá úcinnost vakcinace proti virové hepatitide B u pacientu s pokrocilým jaterním onemocnením.

Hepatitis B vaccination was started in 41 patients with end-stage liver disease who were liver transplant candidates. Patients received three 20 microg or, starting from 1999, 40 microg doses of recombinant vaccine at 0, 2 and 4 weeks. Blood samples were obtained 4 weeks after vaccination or each revaccination; patients without protective hepatitis B surface antibodies (anti-HBs) were once or repeatedly revaccinated. Thirty-eight patients received at least 3 doses of vaccine. Protective anti-HBs level (≥ 10 IU/l) was detected in 17 of 34 patients (50 %) after the third up to eight dose. No case of chronic HBsAg carrier status was detected. Immunisation against hepatitis B in persons with liver cirrhosis is associated with a poor response and new vaccines should be considered for these patients.

Multiplex assays to diagnose celiac disease.

Patients with celiac disease are sensitive to the gluten fractions of wheat. Symptoms include gastrointestinal problems and a failure to thrive in children, but may range from headaches to general malaise in adults. Thus, it is difficult to diagnose celiac disease by symptoms alone. The standard diagnostic criteria include the presence of the characteristic anti-gliadin or anti-tissue transglutaminase antibodies (anti-tTG) in serum, flattened mucosa on intestinal biopsy, and improved symptoms on a gluten-free diet. Because of the ease of use of the tTG enzyme-linked immunosorbent assay (ELISA) compared to endomysial by indirect immunofluorescence assay, there has been much more screening for celiac disease in recent years. This increased screening showed that celiac disease was more prevalent than previously believed. We compared a new multiplex assay that includes a novel form of deamidated gliadin and recombinant human tTG as the antigens to other assays using standard antigens. In addition, the new assay detects the presence of selective IgA deficiency, which shows a 10-fold increase in prevalence in patients with celiac disease compared to the general population. The combination of sensitivity and specificity of the new multiplex assay was equal or better than those for standard assays. Thus the performance, ease of use, and ability to measure three clinically important parameters in a single test make the new multiplex assay a viable alternative to standard assays in a clinical lab.

A multicenter evaluation of the Biotest legionella urinary antigen EIA.

OBJECTIVES: To undertake a multicenter study to evaluate the Biotest legionella urinary antigen enzyme immunoassay (EIA) performance against those EIAs already in use in 14 European laboratories. METHODS: Each laboratory examined urine specimens from appropriate patients using both their current assay and the Biotest EIA. Each examined: a standard panel of 12 coded urine samples (distributed by Biotest); a panel of 10 coded urine samples provided as part of a European external quality assurance (EQA) scheme; urine samples from patients with proven legionnaires' disease (LD); urine samples from patients with pneumonia of microbiologically proven cause other than LD; and urine samples submitted for routine examination. Thus, the performance of the Biotest assay (in comparison with current EIAs), its specificity and utility, and the inter-laboratory agreement were assessed. RESULTS: Inter-laboratory agreement was excellent, with all participants obtaining the expected results for 20 of 22 coded urine specimens. Specificity, determined using 123 specimens from patients with infections of known etiology, was 100%. The Biotest EIA gave positive results in 86% of specimens which had been positive in the laboratories' current EIAs, and in 94.6% of those specimens which were positive for Legionella pneumophila serogroup 1. CONCLUSION: The Biotest EIA is simple to use and specific and the results obtained in different laboratories show excellent agreement. The assay compares well existing EIAs, at least for L. pneumophila serogroup 1