Reappraisal of the clinical and etiologic significance of immunoglobulin deviations in autoimmune hemolytic anemia (‘warm type’)

Summary 56 patients with autoimmune hemolytic anemia (warm type) (AIHA) were investigated for immunoglobulin deviations. Of these, 43 were repeatedly analyzed (mean 4 times). The mean observation time was 20 months. The immunoglobulin values were correlated with clinical (degree of hemolysis) and serological (immunoglobulin class of autoantibodies; strength of antiglobulin reaction) parameters and statistically evaluated by variance analysis. Although no significant deviations of immunoglobulins in AIHA were found as compared to a normal control group, the immuno-globulin disturbance most frequently seen was an elevation of IgM. This is inter-preted as a possible lack or functional impairment of immunoregulatory T cells in AIHA.Supported by the Deutsche Forschungsgemeinschaft (Mu 277/6).     

Coronary endothelial vasomotor function and vascular remodeling in heart transplant recipients randomized for tacrolimus or cyclosporine immunosuppression.

OBJECTIVES: This study aimed to compare changes in coronary endothelial function, systemic endothelin-1 (ET-1) levels, and vascular remodeling in heart transplant recipients randomized to cyclosporin A (CyA) or tacrolimus (Tac) immunosuppression. BACKGROUND: Functional endothelial abnormalities and intimal thickening are sensitive measures of early cardiac allograft vasculopathy (CAV). METHODS: The randomized, prospective study was performed in two groups of 22 patients, maintained on Tac or CyA and mycophenolate mofetil immunosuppression, 1 and 12 months after heart transplantation. We investigated epicardial luminal diameter, coronary blood flow velocity, and ET-1 plasma levels at 1 and 12 months after transplantation. Structural coronary alterations were determined using intravascular ultrasound. RESULTS: Epicardial vasomotor function at baseline and during follow-up was comparable between the groups. Deterioration of microvascular endothelial function during follow-up was significantly enhanced in the CyA versus Tac group (p < 0.05). Circulating ET-1 concentration increased in the CyA group but significantly decreased over time in the Tac group (CyA +17% vs. Tac -25%; p < 0.05). The time-dependent increase in mean intimal area was significantly enhanced in the CyA versus Tac group, whereas the vessel area significantly increased during follow-up in the Tac compared with the CyA group. CONCLUSIONS: Epicardial endothelial function is comparable between CyA- and Tac-treated patients. Microvascular endothelial function deteriorates more in CyA-treated patients, a finding that correlates with enhanced ET-1 concentration and an increased intimal area during follow-up. The mean vessel area in the Tac group increased over time, indicating positive vascular remodeling. Tac is superior to CyA with respect to microvascular endothelial function, intimal thickening, and vascular remodeling.     

Fatty acid composition of liver triglycerides in various stages of fat deposition in the parenchyma

Summary In 30 diabetic inpatients the fatty acid pattern of triglycerides in parenchymal liver cells was studied by gas-liquid chromatography. With increasing size of fat droplets, a significant increase in the proportion of palmitic and oleic acid was observed as well as a significant fall of arachidonic (C 20: 4) and eicosapentaenoic acid (C 20: 5).     

Quantitative proteomics and phosphoproteomics on serial tumor biopsies from a sorafenib-treated HCC patient

Compensatory signaling pathways in tumors confer resistance to targeted therapy, but the pathways and their mechanisms of activation remain largely unknown. We describe a procedure for quantitative proteomics and phosphoproteomics on snap-frozen biopsies of hepatocellular carcinoma (HCC) and matched nontumor liver tissue. We applied this procedure to monitor signaling pathways in serial biopsies taken from an HCC patient before and during treatment with the multikinase inhibitor sorafenib. At diagnosis, the patient had an advanced HCC. At the time of the second biopsy, abdominal imaging revealed progressive disease despite sorafenib treatment. Sorafenib was confirmed to inhibit MAPK signaling in the tumor, as measured by reduced ribosomal protein S6 kinase phosphorylation. Hierarchical clustering and enrichment analysis revealed pathways broadly implicated in tumor progression and resistance, such as epithelial-to-mesenchymal transition and cell adhesion pathways. Thus, we describe a protocol for quantitative analysis of oncogenic pathways in HCC biopsies and obtained first insights into the effect of sorafenib in vivo. This protocol will allow elucidation of mechanisms of resistance and enable precision medicine.Hepatocellular carcinoma (HCC) is a global health concern with an estimated 750,000 new cases per year (1). In more than 80% of cases, HCC arises in a setting of liver cirrhosis mainly of alcoholic or viral origin (2). The prognosis for HCC patients is poor, with less than 30% qualifying for curative treatments such as tumor resection or liver transplantation (2). Median survival time of patients that cannot be treated surgically is less than 1 y. Sorafenib is the only approved targeted therapy for HCC, prolonging median patient survival by ∼3 mo (3). Sorafenib is a multikinase inhibitor of Raf (B and C), vascular endothelial growth factor receptor (VEGFR), and platelet-derived growth factor receptor (PDGFR) (4), which presumably inhibits not only tumor cells but also endothelial cells responsible for tumor vascularization.Resistance to a targeted cancer drug can be intrinsic or adaptive (5). Sorafenib is largely cytostatic (6), suggesting that intrinsic resistance is more common in tumors, although some reports describe tumor shrinkage upon sorafenib treatment (7). Studies involving HCC cell lines or immunohistochemical staining of tumor sections revealed that sorafenib resistance correlates with the up-regulation of several signaling pathways, including the mammalian target of rapamycin (mTOR) pathway as assayed by S6 S235/236 (8) and Akt S473 phosphorylation (9). Other potential resistance mechanisms involve epithelial-to-mesenchymal transition (EMT) and autophagy (10, 11). However, the molecular mechanisms of sorafenib resistance in patients are largely unknown. Understanding the pathways that confer intrinsic or adaptive resistance would allow precision medicine and increase treatment efficacy.Proteomic analysis allows the identification of drug targets for cancer treatment and biomarkers for cancer classification or recurrence. In particular, MS is a powerful tool for resolving the complexity of cancer signaling pathways. With regard to HCC, qualitative proteomics has been performed on resected tumor material (12), laser-capture microdissected material from tissue sections (13, 14), and primary hepatocytes or serum derived from patients (15, 16). These studies (17, 18) identified HCC biomarkers such as glutamine synthetase and heat shock protein 70 (Hsp70) that are currently in use for diagnosis (19, 20). Quantitative proteomics has been performed on HCC resected tissue and serum (21, 22). Recently, proteomics has been performed on tumor biopsies of renal cell carcinoma patients (23). Several studies also have described phosphoproteomic analyses of resected HCC or other cancer material (24–26), in some cases quantifying up to 8,000 phosphorylated sites (hereafter referred to as “phosphosites”) starting with 2 mg of protein (18, 27–30). However, to our knowledge, quantitative proteomics and phosphoproteomics, hereafter collectively referred to as “(phospho)proteomics,” have yet to be performed on tumor biopsies, possibly because biopsy material is nonrenewable and typically provides only a very small amount of protein. Importantly, quantitative (phospho)proteomics on serial biopsies taken before and during treatment has not been described. We note that although a biopsy procedure generates less material than a resection, it has the important advantage of capturing normally dynamic properties of a tumor, such as the phosphorylation status of signaling pathways. Biopsies are immediately snap-frozen upon removal from the patient and, unlike resected tissue, are obtained without causing ischemia or hypoglycemia in the collected tissue. Needle biopsies are taken routinely to diagnose and stage the disease. Another important consideration is a method to perform quantitative (phospho)proteomics, such as super-SILAC (“SILAC” is an acronym for “stable isotope labeling of amino acids in cell culture”), that allows direct comparison of biopsies obtained at different times or from different patients (31).We describe quantitative (phospho)proteomic analyses of needle biopsies of HCC and matched nontumor tissue from a human patient. These analyses provide a global snapshot of signaling pathways in the biopsy material. Analyzing serial biopsies taken from a patient before and during therapy, we measured differences in signaling pathways between tumor and matched nontumor control tissue and the changes in these signaling pathways upon sorafenib treatment. Our findings provide insight into mechanisms of tumor progression and resistance to cancer therapy.     

Reduced CD4+,CD25- T cell sensitivity to the suppressive function of CD4+,CD25high,CD127 -/low regulatory T cells in patients with active systemic lupus erythematosus

OBJECTIVE: CD4+,CD25high regulatory T (Treg) cells play a crucial role in the maintenance of self tolerance and prevention of organ-specific autoimmunity. The presence of many in vivo-preactivated CD4+,CD25++ T cells in patients with systemic lupus erythematosus (SLE) poses a difficulty in discriminating CD25++ activated T cells from CD25high Treg cells. To overcome this problem, we analyzed the phenotype and function of CD4+,CD25high,CD127(-/low) natural Treg (nTreg) cells isolated from the peripheral blood of patients with SLE. METHODS: CD4+,CD25high,CD127(-/low) nTreg cells and CD4+,CD25- responder T (Tresp) cells from patients with SLE and normal donors were separated by fluorescence-activated cell sorting. Cell proliferation was quantified by 3H-thymidine incorporation, and immunophenotyping of the cells was done using FACScan. RESULTS: Comparable percentages of CD4+,CD25high,FoxP3+ T cells were observed in patients with SLE and normal donors. Proliferation of SLE nTreg cells sorted into the subset CD4+,CD25high,CD127(-/low) was significantly decreased compared with that of SLE nTreg cells sorted into the subset CD4+,CD25high (mean +/- SEM 2,223 +/- 351 counts per minute versus 9,104 +/- 1,720 cpm, respectively), while in normal donors, these values were 802 +/- 177 cpm and 2,028 +/- 548 cpm, respectively, confirming that effector cell contamination was reduced. Notably, the suppressive activity of nTreg cells was intact in all groups. However, CD4+,CD25- Tresp cells isolated from patients with active SLE were significantly less sensitive than those from patients with inactive SLE to the suppressive function of autologous or normal donor CD4+,CD25high,CD127(-/low) nTreg cells. Furthermore, a significant inverse correlation was observed between the extent of T cell regulation in suppressor assays and the level of lupus disease activity. CONCLUSION: This study is the first to show that, in human SLE, impaired sensitivity of Tresp cells to the suppressive effects of a comparably functional, highly purified nTreg cell population leads to a defective suppression of T cell proliferation in active SLE. Studies aiming to define the mechanisms leading to Tresp cell resistance might help in the development of highly specific, alternative immunotherapeutic tools for the control of systemic autoimmune diseases such as SLE.     

Autocrine insulin-like growth factor-II stimulation of tumor cell migration is a progression step in human hepatocarcinogenesis

The protumorigenic insulin-like growth factor (IGF)-II is highly expressed in a significant fraction of human hepatocellular carcinomas (HCC). However, a functional dissection that clarifies the contribution of IGF-II-binding receptors in tumor progression and a respective molecular characterization of IGF-II signaling has not been performed. Therefore, expression of IGF-II and its receptors IGF-receptor type I (IGF-IR) and insulin receptor (IR) was efficiently blocked using small interfering RNA (siRNA) in HCC cells. Despite functional IR-signaling, oncogenic IGF-II effects such as tumor cell viability, proliferation, and anti-apoptosis were solely transmitted by IGF-IR. Although IGF-II signaling was previously not described in the context of HCC cell migration, the IGF-II-dependent expression profile displayed a high percentage of genes involved in cell motility and adhesion. Indeed, IGF-II overexpression promoted HCC cell migration, especially in synergy with hepatocyte growth factor (HGF). The therapeutic relevance of IGF-II/IGF-IR signaling was tested in vitro and in a murine xenograft transplantation model using the IGF-IR inhibitor picropodophyllin (PPP). IGF-IR inhibition by small molecule treatment efficiently reduced IGF-II-dependent signaling and all protumorigenic properties of the IGF-II/IGF-IR pathway. CONCLUSION: In human HCC cells, IGF-IR but not IR is involved in oncogenic IGF-II signaling. Autocrine stimulation of IGF-II induces HCC motility by integration of paracrine signals for full malignant competence. Thus, activation of IGF-II/IGF-IR signaling is likely a progression switch selected by function that promotes tumor cell dissemination and aggressive tumor behavior.     

ICOS-ligand, expressed on human endothelial cells, costimulates Th1 and Th2 cytokine secretion by memory CD4+ T cells

Endothelial cells (EC) play a central role in inflammatory immune responses and efficiently induce effector functions in T cells, despite lacking the classical costimulatory ligands CD80 and CD86. By using the mAb HIL-131 we now demonstrate that human inducible costimulator-ligand (ICOS-L), a molecule related to CD80/CD86, is constitutively expressed on human EC in vivo. In vitro, ICOS-L expression was strongly enhanced on human umbilical vein EC and microvascular EC by the inflammatory cytokines tumor necrosis factor alpha and IL-1beta, and to a lower extent by stimulation of EC by CD40 or lipopolysaccharide. Coculture of MHC class II(+) EC with resting memory CD4(+) T cells in the presence of superantigen led to a marked up-regulation of ICOS on T cells and to the production of Th1 (IFN-gamma, IL-2) and Th2 cytokines (IL-4, IL-10, IL-13). When these cocultures were performed in the presence of the inhibitory mAb HIL-131, secretion of all cytokines was reduced by about 50-80%, indicating that ICOS-L is a major costimulator in EC-mediated T cell activation. Taken together, our data suggest an important physiological role of ICOS-L in the reactivation of effector/memory T cells on the endothelium controlling the entry of immune cells into inflamed tissue.     

Serum S-100B protein levels during and after successful carotid artery stenting or carotid endarterectomy.

PURPOSE: To characterize the course of S-100B serum levels, a reliable marker for cellular brain damage, in patients undergoing carotid artery stenting (CAS) or endarterectomy (CEA) for carotid artery stenosis compared to control groups undergoing hemithyroidectomy (HT) or coronary angiography (CA). METHODS: Forty-six consecutive patients scheduled for revascularization of internal carotid artery (ICA) stenosis were included in the study. Fourteen patients (11 men; median age 70 years, interquartile range [IQR] 63-74) were selected for treatment with CAS, while CEA was performed in 31 patients (24 men; median age 68 years, IQR 54-78) during the same time period. Fourteen consecutive patients (8 men; median age 60 years, IQR 48-70) undergoing CA for suspected coronary heart disease and 14 patients (10 women; median age 36 years, IQR 26-54) undergoing HT for a single thyroid nodule served as controls. RESULTS: All procedures were completed successfully. During ICA clamping in CEA patients without postoperative neurological deficits, median S-100B serum levels transiently increased from 0.04 to 0.26 ng/mL (p<0.01) and returned to baseline levels after declamping. Median S-100B serum levels of CAS patients without neurological impairment remained at baseline values. No increase in S-100B levels occurred in either control group. Three CEA patients who suffered from neurological deficits (1 transient ischemic attack and 1 major stroke) showed sustained elevation of S-100B serum levels 6 hours after extubation. CONCLUSION: In patients without neurological complications, CEA but not CAS was associated with a transient increase in the S-100B serum levels. Results indicate that the increase in S-100B does not originate from extracerebral sources, but rather appears to represent an impairment of the blood-brain barrier integrity or subtle brain cell damage probably due to hypoperfusion during clamping. Sustained elevation of S-100B serum levels corresponded to the development of postoperative neurological deficits.     

Turning Data Into Information: Opportunities to Advance Rehabilitation Quality,Research, and Policy

Attention to health care quality and safety has increased dramatically. The internal focus of an organization is not without influence from external policy and research findings. Compared with other specialties, efforts to align and advance rehabilitation research, practice, and policy using electronic health record data are in the early stages. This special communication defines quality, applies the dimensions of quality to rehabilitation, and illustrates the feasibility and utility of electronic health record data for research on rehabilitation care quality and outcomes. Using data generated at the point of care provides the greatest opportunity for improving the quality of health care, producing generalizable evidence to inform policy and practice, and ultimately benefiting the health of the populations served.     

Determination of Acoustic Cavitation Probabilities and Thresholds Using a Single Focusing Transducer to Induce and Detect Acoustic Cavitation Events: II. Systematic Investigation in an Agar Material

In the accompanying article (Part I), a method is described to determine acoustic cavitation probabilities in tissue-mimicking materials (TMMs) using a high-intensity focused ultrasound (HIFU) transducer for both inducing and detecting the acoustic cavitation events, and its suitability for different sonication modes like continuous wave, single pulses (with pulse lengths from microseconds to milliseconds) and repeated burst signals is discussed. In Part II, the use of the method for a systematic study of the dependence of the acoustic cavitation thresholds in 3% (by weight) agar phantoms on the temporal sonication parameters is discussed. The values obtained at a frequency of 1.06?MHz, ranging from (0.58?±?0.12)?MPa for a 3-s continuous wave mode sonication to (5.2?±?1.0)?MPa for single shots with a length of 10 wave cycles, are discussed and interpreted on the basis of literature values and their self-consistency.