Induction of protein tyrosine phosphorylation in human natural killer cells by triggering via alpha 4 beta 1 or alpha 5 beta 1 integrins

Recent studies have shown that cell-surface integrins expressed on platelets, fibroblasts, or carcinoma cell lines serve not only as adhesion receptors that connect the extracellular matrix to the cytoskeleton, but also as signal-transducing molecules involved in altering cellular patterns of tyrosine phosphorylation. In this present report we provide evidence that adhesion of freshly purified human natural killer (NK) cells to fibronectin (FN) induces tyrosine phosphorylation of intracellular proteins of approximate molecular mass of 60, 70, and 120 kD. Increases in phosphorylation induced by NK cell binding to immobilized FN were partially blocked by EILDV- (CS-1) or RGD-containing peptides, which compete specifically for a distinct binding site for either alpha 4 beta 1 or alpha 5 beta 1 integrins, respectively, within the FN molecule. The presence of either one of the inhibitory peptides alone inhibited tyrosine phosphorylation primarily during short-term (30 minutes) and, to a lesser extent, during long- term (2 to 3 hours) periods of adhesion. These observations indicate that triggering either via alpha 4 beta 1 or alpha 5 beta 1 integrins, which are constitutively expressed on NK cells, induces protein tyrosine phosphorylation. Moreover, FN fragments of 40 or 120 kD, known to contain the binding sites for alpha 4 beta 1 or alpha 5 beta 1 integrins, respectively, used as immobilized substrates for NK cell adhesion, were able to initiate tyrosine kinase activity. The induced tyrosine phosphorylation was observed mainly on intracellular proteins of greater than 50 kD molecular weight. We have identified a 70-kD tyrosine phosphoprotein as paxillin, a cytoskeletal-associated tyrosine kinase substrate previously identified in fibroblasts and shown to localize to focal adhesions. Thus, interaction of NK cells with immobilized extracellular matrix glycoproteins required for migration and extravasation of these cells involves activation of intracellular protein tyrosine kinases and tyrosine phosphorylation of cytoskeleton- associated protein, paxillin, which may play a role in signaling between beta 1 integrins and the underlying cytoskeleton.     

Detection of chromosome 20q deletions in bone marrow metaphases but not peripheral blood granulocytes in patients with myeloproliferative disorders or myelodysplastic syndromes

Myeloproliferative disorders and myelodysplastic syndromes arise in multipotent progenitors and may be associated with chromosomal deletions that can be detected in peripheral blood granulocytes. We present here seven patients with myeloproliferative disorders or myelodysplastic syndromes in whom a deletion of the long arm of chromosome 20 was detectable by G-banding and/or fluorescence in situ hybridization in most or all bone marrow metaphases. However, in each case, microsatellite polymerase chain reaction (PCR) using 15 primer pairs spanning the common deleted region on 20q showed that the deletion was absent from most peripheral blood granulocytes. The human androgen receptor clonality assay was used to show that the vast majority of peripheral blood granulocytes were clonal in all four female patients. This represents the first demonstration that the 20q deletion can arise as a second event in patients with pre-existing clonal granulopoiesis. Microsatellite PCR analysis of whole bone marrow from two patients was consistent with cytogenetic studies, a result that suggests that cytogenetic analysis was not merely selecting for a minor subclone of cells carrying the deletion. Furthermore, in one patient, the deletion was present in both erythroid and granulocyte/monocyte colonies. This implies that the absence of the deletion in most peripheral blood granulocytes did not reflect lineage restriction of the progenitors carrying the deletion but may instead result from other selective influences such as preferential retention/destruction within the bone marrow of granulocytes carrying the deletion.     

Characterization of Charge States in Conducting Organic Nanoparticles by X-ray Photoemission Spectroscopy

The metallic and semiconducting character of a large family of organic materials based on the electron donor molecule tetrathiafulvalene (TTF) is rooted in the partial oxidation (charge transfer or mixed valency) of TTF derivatives leading to partially filled molecular orbital-based electronic bands. The intrinsic structure of such complexes, with segregated donor and acceptor molecular chains or planes, leads to anisotropic electronic properties (quasi one-dimensional or two-dimensional) and morphology (needle-like or platelet-like crystals). Recently, such materials have been synthesized as nanoparticles by intentionally frustrating the intrinsic anisotropic growth. X-ray photoemission spectroscopy (XPS) has emerged as a valuable technique to characterize the transfer of charge due to its ability to discriminate the different chemical environments or electronic configurations manifested by chemical shifts of core level lines in high-resolution spectra. Since the photoemission process is inherently fast (well below the femtosecond time scale), dynamic processes can be efficiently explored. We determine here the fingerprint of partial oxidation on the photoemission lines of nanoparticles of selected TTF-based conductors.     

自身免疫性肝炎大鼠外周血CD4+细胞、肝细胞特异性脂蛋白抗体及Th细胞因子水平之间的相关性

目的:建立自身免疫性肝炎大鼠模型,分析其外周血CD4 细胞、肝细胞特异性脂蛋白抗体及Th细胞因子水平之间的相关性。方法:实验于2004-10/2006-10在南京军区肝病中心实验室完成。①实验动物:雄性3月龄Wistar大鼠80只,随机数字表法分为4组:正常对照组、模型对照组、褪黑素治疗组、猪促肝细胞生长素治疗组,20只/组。②实验方法:除正常对照组外,其余各组大鼠均给予弗氏完全佐剂 肝细胞特异性脂蛋白建立自身免疫性肝炎模型。褪黑素用含0.01%乙醇的生理盐水配制成1g/L的溶液,褪黑素治疗组腹腔注射2mg/kg;猪促肝细胞生长素用含0.01%乙醇的生理盐水配制成2g/L的溶液,猪促肝细胞生长素治疗组腹腔注射2mg/kg;正常对照组和模型对照组均腹腔注射含0.01%乙醇的生理盐水,1次/d,连续60d。③实验评估:检测各组大鼠外周血淋巴细胞亚群浓度、肝细胞特异性脂蛋白抗体水平及Th细胞因子的浓度,并分析三者之间的关系。结果:80只大鼠均进入结果分析。模型对照组CD4 细胞百分比、肝细胞特异性脂蛋白抗体水平、白细胞介素4,6水平均显著高于其余3组(P<0.01),白细胞介素2和干扰素γ水平均显著低于其余3组(P<0.01)。与正常对照组比较,褪黑素治疗组、猪促肝细胞生长素治疗组CD4 细胞百分比无明显变化(P>0.05),肝细胞特异性脂蛋白抗体水平显著升高(P<0.001)。与猪促肝细胞生长素治疗组比较,正常对照组、褪黑素治疗组白细胞介素2和干扰素γ水平显著升高(P<0.01),白细胞介素4,6水平均显著降低(P<0.01)。结论:①CD4 细胞百分比与肝细胞特异性脂蛋白抗体水平有关,当CD4 细胞百分比达到正常水平时肝细胞特异性脂蛋白抗体表达虽受到明显抑制却并未停止表达。②褪黑素调节Th细胞因子水平的作用强于猪促肝细胞生长素。③Th细胞因子水平变化与肝细胞特异性脂蛋白抗体水平有一定关系。     

Beta-trace protein, cystatin C, beta(2)-microglobulin, and creatinine compared for detecting impaired glomerular filtration rates in children

BACKGROUND: Because of the limitations of serum creatinine as a marker of glomerular filtration rate (GFR) in children, we assessed the diagnostic accuracy of the novel marker beta-trace protein (BTP) in comparison with cystatin C (Cys-C), beta(2)-microglobulin (beta(2)-MG), and creatinine as conventional indicators of reduced GFR. METHODS: We obtained serum samples from 225 children (age range, 0.2-18 years) with various renal pathologies who were referred for nuclear medicine clearance investigations (technetium-diethylenetriamine pentaacetic acid or chromium-EDTA). We measured Cys-C, BTP (nephelometric tests; Dade Behring), beta(2)-MG (Tinaquant; Roche), and creatinine (enzymatic assay; Creatinine-PAP; Roche). RESULTS: Seventy-five children had reduced GFR (<90 mL x min(-1) x 1.73 m(-2)). One hundred fifty children (independent of gender and age) with values >90 mL x min(-1) x 1.73 m(-2) comprised the control group with gaussian distributions of BTP and Cys-C concentrations. The upper reference limits (97.5 percentile) were 1.01 mg/L for BTP and 1.20 mg/L for Cys-C. The correlations of nuclear medicine clearance with the reciprocals of BTP, Cys-C, and the Schwartz GFR estimate were significantly higher (r = 0.653, 0.765, and 0.706, respectively; P <0.05) than with the reciprocal of creatinine or beta(2)-MG (r = 0.500 and 0.557, respectively). ROC analysis showed a significantly higher diagnostic accuracy of BTP, Cys-C, and the GFR estimate for the detection of impaired GFR than serum creatinine (P <0.05). Compared to creatinine, BTP increased the diagnostic sensitivity by approximately 30%, but it was not more sensitive than Cys-C or the Schwartz GFR estimate. CONCLUSIONS: BTP is superior to serum creatinine and an alternative for Cys-C to detect mildly reduced GFR in children, but it is not better than the Schwartz GFR estimate.     

Surgical Palliation for Pancreatic Malignancy: Practice Patterns and Predictors of Morbidity and Mortality

Introduction

Most patients with pancreatic cancer present with, or develop, biliary or duodenal obstruction. We sought to characterize palliative surgery utilization in a contemporary cohort and identify patients at high risk of morbidity and mortality.

Methods

The ACS NSQIP database (2005–2011) was queried for patients with a pancreatic malignancy undergoing gastrojejunostomy, biliary bypass, or laparotomy without resection. Univariate analysis and multivariate logistic regression identified factors associated with increased risk of 30-day morbidity or mortality.

Results

Operations for the 1,126 patients undergoing palliative bypass were gastrojejunostomy alone (33 %), bile duct bypass alone (27 %), both (31 %), or cholecystojejunostomy (9 %). A major complication occurred in 20 % and mortality in 6.5 % at 30 days. Risk factors for morbidity and mortality were defined in multivariate models. The number of identified risk factors stratified morbidity from 14.8–50 % and mortality from 1.6–50 % (p?<?0.0001 for each). Laparotomy alone (n?=?622) was associated with lower morbidity than bypass (12 vs. 20 %, p?<?0.0001), but equivalent mortality (5 vs. 6.5 %, p?=?0.21).

Conclusion

Palliative bypass for pancreatic cancer is associated with a high rate of morbidity and mortality. In select patients, this risk may be prohibitive. Patient selection reflecting predictors of morbidity and mortality may allow for improved outcomes.     

Quality Assessment in Pancreatic Surgery: What Might Tomorrow Require?

Introduction

The Institute of Medicine (IOM) defines healthcare quality across six domains: safety, timeliness, effectiveness, patient centeredness, efficiency, and equitability. We asked experts in pancreatic surgery (PS) whether improved quality metrics are needed, and how they could align to contemporary IOM healthcare quality domains.

Methods

We created and distributed a web-based survey to pancreatic surgeons. Respondents ranked 62 proposed PS quality metrics on level of importance (LoI) and aligned each metric to one or more IOM quality domains (multi-domain alignment (MDA)). LoI and MDA scores for a given quality metric were averaged together to render a total quality score (TQS) normalized to a 100-point scale.

Results

One hundred six surgeons (21 %) completed the survey. Ninety percent of respondents indicated a definite or probable need for improved quality metrics in PS. Metrics related to mortality, to rates and severity of complications, and to access to multidisciplinary services had the highest TQS. Metrics related to patient satisfaction, costs, and patient demographics had the lowest TQS. The least represented IOM domains were equitability, efficiency, and patient-centeredness.

Conclusions

Experts in pancreatic surgery have significant consensus on 12 proposed metrics of quality that they view as both highly important and aligned with more than one IOM healthcare quality domain.     

Induction of tumor stem cell differentiation—novel strategy to overcome therapy resistance in gastric cancer

Purpose

Metastases are a frequent finding in gastric cancer and are associated with poor prognosis. A recently discovered link between metabolic changes, differentiation, and therapy resistance due to tumor stem cells could depict a novel approach in cancer research and therapy. Phosphoglycerate kinase 1 (PGK1) is a metabolic enzyme and is known to be involved in enabling gastric cancer cells to be invasive and to disseminate. In this study, we investigated if PGK1 is a promising candidate in inducing stem cell differentiation in gastric cancer.

Materials and methods

MKN45 gastric cancer cells were used due to their known cancer stem cell population, which is defined by the surface marker CD44. MKN45 cells were separated between CD44+ and CD44? cells and, in equal parts, incubated with shRNA anti-PGK1 using fluorescence-activated cell sorting (FACS) analysis; they were then injected into nude mice to evaluate their tumor growth behavior in vivo. Further, the invasive potential of gastric cancer cells was evaluated in vitro using the xCelligence analyzing system.

Results

CD44+ gastric cancer cells treated with and without shRNA anti-PGK1 were capable to cause tumor growth in vivo, whereas tumor growth in CD44+ cells treated with shRNA anti-PGK1 was considerably smaller in comparison with that in CD44+ cells without treatment. CD44? cells did not show any noticeable tumor growth in vivo. By targeting PGK1, the invasive potential of gastric cancer cells was impressively reduced in vitro. In all our cells, which were targeted with shRNA anti-PGK1, we did not find any change that is in accordance with the phenotype of the cells using FACS analysis.

Conclusions

Our findings suggest that targeting the key metabolic enzyme PGK1 in gastric cancer cells may open a new chapter in cancer treatment, which is well worth for further exploration in combination with recent chemotherapy, and might be a promising possibility to overcome therapy resistance in gastric cancer.