Long-term stability of endogenous B-type natriuretic peptide (BNP) and amino terminal proBNP (NT-proBNP) in frozen plasma samples.

The aim of the present study was to assess the long-term stability of endogenous B-type natriuretic peptide (BNP) and amino terminal proBNP (NT-proBNP) in plasma samples stored at -20 degrees C without addition of protease inhibitors (e.g., aprotinin). Stability of BNP and NT-proBNP was tested in 60 EDTA plasma samples with BNP values between 30 and 420 pg/ml. Initial BNP and NT-proBNP plasma concentrations were determined within four hours after blood collection using the AxSYM BNP and the Elecsys NT-proBNP assays. Subsequently, all samples were stored at -20 degrees C and were thawed for the second BNP and NT-proBNP determination on the two instruments after one day, 30 days, 60 days, 90 days and 120 days, respectively. Mean recovery (i.e., residual immunoreactivity) of BNP and NT-proBNP expressed in percent of the initial value for the given time interval of storage was calculated. Mean recovery of BNP was less than 70% after one day of storage at -20 degrees C and decreased to less than 50% after two to four months of storage (e.g., recovery of endogenous BNP after three months of storage at -20 degrees C ranging from 0% to 71%). In contrast, mean recovery of NT-proBNP was generally greater than 90%, irrespective of the duration of storage at -20 degrees C (e.g., recovery of endogenous NT-proBNP after three months of storage at -20 degrees C ranging from 91% to 112%). In conclusion, the determination of endogenous BNP with the AxSYM assay using frozen plasma samples may not be valid under the conditions tested. In contrast, NT-proBNP as measured by the Elecsys assay may be stored at -20 degrees C for at least four months without a relevant loss of the immunoreactive analyte.     

HER-2/neu gene copy number quantified by real-time PCR: comparison of gene amplification,heterozygosity, and immunohistochemical status in breast cancer tissue

BACKGROUND: Amplification of the oncogene HER-2/neu influences breast cancer pathogenesis, and therapy and prognosis may be affected by the degree of amplification. The extent of amplification or protein overexpression typically is analyzed by fluorescence in situ hybridization or immunohistochemistry (IHC), but quantitative PCR techniques have been described that may provide alternatives to these methods. METHODS: We developed a rapid-cycle, real-time PCR assay for quantification of HER-2/neu gene status. We compared results obtained with this assay with short tandem repeat findings by capillary electrophoresis (CE) and with protein overexpression assessments by IHC. Accuracy and linearity were tested on cell lines and with simulation experiments. We analyzed the amplification of HER-2/neu in 51 clinical tissue samples from patients with suspected breast cancer. RESULTS: The intra- and interrun CVs for HER-2/neu quantification by real-time PCR were 12% and 18%, and the CV for different simulated amplification and deletion experiments was <7%. The results for HER-2/neu gene status in cell lines matched the values reported in literature. We detected HER-2/neu amplification by real-time PCR in 11 samples, all from patients with invasive ductal carcinoma. Allelic imbalances were found by CE analyses in three samples and by protein overexpression in six samples; five of these were also detected by real-time PCR. Comparison of the quantification results with known prognostic indices yielded results similar to those reported in several other published studies. CONCLUSIONS: The assay is suitable for accurate and precise quantification of HER-2/neu copy numbers in tumor tissue samples obtained in routine clinical practice.     

耐力训练过程中大鼠体内糖储备及胰岛激素变化与黄芪、生脉穴位注射的干预效应

目的:穴位注射疗法在临床应用较多,但在运动医学领域研究不多。观察穴位注射黄芪、生脉对耐力训练大鼠糖储备和运动能力的影响。方法:实验于2004-07在陕西师范大学完成。①实验分组:健康雄性SD大鼠32只,体质量180～220g,随机抽签法分为安静对照组、训练对照组、生理盐水组、药物注射组,每组8只。②实验方法:建立穴位注射黄芪、生脉大鼠的耐力跑台训练实验模型,安静对照组安静笼饲养。训练对照组、生理盐水组、药物注射组先于动物跑台上进行5周适应性训练,之后跑速每周递增,5d/周,共5周;然后进行2周的大强度耐力训练,30min/d,7d/周,共2周。训练对照组、生理盐水组、药物注射组第8周第1天以速度为35m/min运动至力竭。③实验评估:7周后取材测定肝糖原、肌糖原、血清胰岛素、胰高血糖素的变化。实验中对动物处置符合动物伦理学标准。结果:纳入大鼠32只,均进入结果分析。①通过大强度耐力训练,药物注射组与其他3组相比,肝糖原含量均升高(P<0.05);训练对照组肌糖原比安静对照组降低(P<0.05),生理盐水组与训练对照组相比则显著性升高(P<0.01)。②训练对照组胰岛素比安静对照组明显降低(P<0.01);生理盐水组及药物注射组都能抑制这种降低的趋势(P<0.01);药物注射组胰高血糖素较安静对照组、训练对照组要高,且有显著性差异(P<0.01)。结论:穴位注射黄芪、生脉使大强度耐力训练大鼠体内糖储备显著增加,同时可以提高胰岛激素水平,从而提高了大鼠的运动能力。     

Influence of hydroxyethyl starch (6% HES 130/0.4) administration on hematology and clinical chemistry parameters

BACKGROUND: The chemical inertness of hydroxyethyl starch (HES) might cause interferences of the colloid with a variety of laboratory tests. We aimed to evaluate potential influences of HES 130/0.4, the newest HES type, on several common hematology and clinical chemistry parameters. METHODS AND RESULTS: A convenient sample of 25 patients scheduled for rheological therapy with 500 mL 6% HES 130/0.4 was evaluated. Blood samples were drawn before and after colloid application. Comparing pre- and post-infusion values of a battery of laboratory tests (i.e., hematology and hemostasis parameters, electrolytes, enzymes, kidney and metabolic parameters, lipids, etc.) in time course, a median difference greater than the reference change value for a specific parameter was considered clinically relevant. Among all parameters tested, only serum amylase activity displayed a clinically relevant difference between pre- and post-infusion values (median increase of 85% due to HES administration). By applying in vitro experiments, we demonstrated that serum amylase values obtained in the samples diluted in a 1:1 ratio with HES 130/0.4 and in samples diluted in a 1:1 ratio with 0.9% NaCl displayed a negligible median difference of 3%. CONCLUSIONS: The in vivo effect of HES 130/0.4 administration on serum amylase activity observed in our study was pharmacological (real) in nature. With the exception of the influence of HES 130/0.4 on amylase activity, the effects of HES 130/0.4 on other parameters tested in this study can be interpreted as having no clinical relevance.     

Signal recognition particle (SRP) positive myositis in a patient with cryptogenic organizing pneumonia (COP)

We report of a 46-year-old female patient with cryptogen organizing pneumonia preceeding the rare SRP positive necrotising myositis without cardiac involvement and no sign of dysphagia. Myositis showed full regression without oral immune suppression but with extracorporeal treatment, performed as a combined therapy of plasmaexchange and immunoadsorption. After 33-month of treatment, anti-SRP antibodies were not detectable any more.     

Flavocoxid,a dual inhibitor of cyclooxygenase and 5-lipoxygenase,blunts pro-inflammatory phenotype activation in endotoxin-stimulated macrophages

Background and purpose:

The flavonoids, baicalin and catechin, from Scutellaria baicalensis and Acacia catechu, respectively, have been used for various clinical applications. Flavocoxid is a mixed extract containing baicalin and catechin, and acts as a dual inhibitor of cyclooxygenase (COX) and 5-lipoxygenase (LOX) enzymes. The anti-inflammatory activity, measured by protein and gene expression of inflammatory markers, of flavocoxid in rat peritoneal macrophages stimulated with Salmonella enteritidis lipopolysaccharide (LPS) was investigated.

Experimental approach:

LPS-stimulated (1 µg·mL⁻¹) peritoneal rat macrophages were co-incubated with different concentrations of flavocoxid (32–128 µg·mL⁻¹) or RPMI medium for different incubation times. Inducible COX-2, 5-LOX, inducible nitric oxide synthase (iNOS) and inhibitory protein κB-α (IκB-α) levels were evaluated by Western blot analysis. Nuclear factor κB (NF-κB) binding activity was investigated by electrophoretic mobility shift assay. Tumour necrosis factor-α (TNF-α) gene and protein expression were measured by real-time polymerase chain reaction and enzyme-linked immunosorbent assay respectively. Finally, malondialdehyde (MDA) and nitrite levels in macrophage supernatants were evaluated.

Key results:

LPS stimulation induced a pro-inflammatory phenotype in rat peritoneal macrophages. Flavocoxid (128 µg·mL⁻¹) significantly inhibited COX-2 (LPS = 18 ± 2.1; flavocoxid = 3.8 ± 0.9 integrated intensity), 5-LOX (LPS = 20 ± 3.8; flavocoxid = 3.1 ± 0.8 integrated intensity) and iNOS expression (LPS = 15 ± 1.1; flavocoxid = 4.1 ± 0.4 integrated intensity), but did not modify COX-1 expression. PGE₂ and LTB₄ levels in culture supernatants were consequently decreased. Flavocoxid also prevented the loss of IκB-α protein (LPS = 1.9 ± 0.2; flavocoxid = 7.2 ± 1.6 integrated intensity), blunted increased NF-κB binding activity (LPS = 9.2 ± 2; flavocoxid = 2.4 ± 0.7 integrated intensity) and the enhanced TNF-α mRNA levels (LPS = 8 ± 0.9; flavocoxid = 1.9 ± 0.8 n-fold/β-actin) induced by LPS. Finally, flavocoxid decreased MDA, TNF and nitrite levels from LPS-stimulated macrophages.

Conclusion and implications:

Flavocoxid might be useful as a potential anti-inflammatory agent, acting at the level of gene and protein expression.     

The successful short-term treatment of flexion-distraction injuries of the thoracic spine using posterior-only pedicle screw instrumentation

STUDY DESIGN: In this retrospective study, the results of treating unstable flexion-distraction injuries (FDI) of the thoracic spine with posterior-only thoracic pedicle screw (P/TPS) instrumentation were investigated. OBJECTIVE: The objective was to determine the ability of P/TPS to correct and maintain the focal kyphosis of the injured spine. Clinical outcome and complications of the surgical procedure were also a focus of the study. SUMMARY OF BACKGROUND DATA: The treatment of FDI of the thoracic spine remains controversial. There continues to be difficulty in maintaining the corrected kyphosis angle regardless of the surgical approach used. METHODS: Eighteen patients with FDI of thoracic spine who underwent P/TPS were identified. The initial and corrected focal kyphosis was evaluated radiographically. Failure of treatment was defined as a >5-degree progression of corrected kyphosis from initial to latest follow-up. Clinical evaluation included complication rate, Injury Severity Score, and Frankel grade. RESULTS: Of the 18 eligible patients, 15 (83%) had adequate follow-up for evaluation. The mean length of follow-up care was 16.1 months. The average Injury Severity Score was 23. The average number of instrumented levels was 6.8. The focal kyphosis of the injury was reduced from a mean of 19.60 to 5.73 degrees (P<0.001), with an average progression of only 2.27 degrees (P=0.128) at final follow-up. Complications were limited to wound infections and occurred in 2 study participants. Neurologic status returned to normal in all patients with incomplete cord injuries. CONCLUSIONS: FDI of the thoracic spine may be amendable to a uniform surgical approach with P/TPS. This procedure carried a relatively low complication rate and allowed for reduction and restoration of the posterior tension band with a biomechanically rigid construct.