OBJECTIVE

Diminished cholesterol efflux activity of apolipoprotein B (apoB)-depleted serum is associated with prevalent coronary artery disease, but its prognostic value for incident cardiovascular events is unclear. We investigated the relationship of cholesterol efflux activity with both prevalent coronary artery disease and incident development of major adverse cardiovascular events (death, myocardial infarction, or stroke).

RESULTS

Cholesterol efflux activity from free cholesterol-enriched macrophages was measured in 2 case-control cohorts: (1) an angiographic cohort (n=1150) comprising stable subjects undergoing elective diagnostic coronary angiography and (2) an outpatient cohort (n=577). Analysis of media from cholesterol efflux assays revealed that the high-density lipoprotein fraction (1.063<d<1.21) contained only a minority (≈ 40%) of [(14)C]cholesterol released, with the majority found within the lipoprotein particle-depleted fraction, where ≈ 60% was recovered after apolipoprotein A1 immunoprecipitation. Albumin immunoprecipitation recovered another ≈ 30% of radiolabeled cholesterol within this fraction. Enhanced cholesterol efflux activity from ATP-binding cassette transporter A1-stimulated macrophages was associated with reduced risk of prevalent coronary artery disease in unadjusted models within both cohorts; however, the inverse risk relationship remained significant after adjustment for traditional coronary artery disease risk factors only within the outpatient cohort. Surprisingly, higher cholesterol efflux activity was associated with increase in prospective (3 years) risk of myocardial infarction/stroke (adjusted hazard ratio, 2.19; 95% confidence interval, 1.02-4.74) and major adverse cardiovascular events (adjusted hazard ratio, 1.85; 95% confidence interval, 1.11-3.06).

CONCLUSIONS

Heightened cholesterol efflux to apoB-depleted serum was paradoxically associated with increased prospective risk for myocardial infarction, stroke, and death. The majority of released radiolabeled cholesterol from macrophages in cholesterol efflux activity assays does not reside within a high-density lipoprotein particle.

The acute phase response is a nonspecific inflammatory reaction of the host that occurs shortly after any tissue injury. The response includes changes in the concentration of plasma proteins called acute phase proteins (APPs), some of which decrease in concentration (negative APPs), such as albumin or transferrin, and others of which increase in concentration (positive APPs), such as C-reactive protein, serum amyloid A, haptoglobin, alpha-1-acid glycoprotein, and ceruloplasmin. Most positive APPs are glycoproteins synthesized mainly by hepatocytes upon stimulation by proinflammatory cytokines and released into the bloodstream. The acute phase response and clinical application of monitoring APPs in dogs and cats are reviewed in this article, including biochemical characteristics, assays developed for each individual APP, and preanalytic and analytic factors influencing APP results that should be taken into account for proper and adequate clinical interpretation. In addition, the diagnostic use of APPs and their possible application in monitoring treatment, which can be considered one of the most interesting and promising practical applications of these proteins, will be discussed. Finally, challenges and future developments of APPs in dogs and cats will be considered, because it is expected that new and cheaper automated assays for determination of the main APPs in small animals will contribute to a wider use of these proteins as biomarkers of infection and inflammatory lesions.

BACKGROUND

Transcatheter arterial chemoembolisation (TACE) is the treatment of choice for intermediate stage hepatocellular carcinoma (HCC). Doxorubicin-loaded drug-eluting beads (DEB)-TACE is expected to improve the performance of conventional TACE (cTACE). The aim of this study was to compare DEB-TACE with cTACE in terms of time-to-tumour progression (TTP), adverse events (AEs), and 2-year survival.

METHODS

Patients were randomised one-to-one to undergo cTACE or DEB-TACE and followed-up for at least 2 years or until death. Transcatheter arterial chemoembolisation was repeated 'on-demand'.

RESULTS

We enrolled 177 patients: 89 underwent DEB-TACE and 88 cTACE. The median number of procedures was 2 in each arm, and the in-hospital stay was 3 and 4 days, respectively (P=0.323). No differences were found in local and overall tumour response. The median TTP was 9 months in both arms. The AE incidence and severity did not differ between the arms, except for post-procedural pain, more frequent and severe after cTACE (P<0.001). The 1- and 2-year survival rates were 86.2% and 56.8% after DEB-TACE and 83.5% and 55.4% after cTACE (P=0.949). Eastern Cooperative Oncology Group (ECOG), serum albumin, and tumour number independently predicted survival (P<0.05).

CONCLUSIONS

The DEB-TACE and the cTACE are equally effective and safe, with the only advantage of DEB-TACE being less post-procedural abdominal pain.

We have identified and purified a protein complex from human red blood cells that activates the multicatalytic protease (MCP). The complex, which we call the regulator, sediments at 11 S and is composed of 30-kDa subunits. The regulator does not hydrolyze fluorogenic peptides, but when multicatalytic protease and regulator are combined, MCP cleaves succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin and Leu-Leu-Glu-p-nitroanilide as much as 60-fold faster. Hydrolysis of several other fluorogenic peptides is stimulated to a lesser extent, and activated MCP does not degrade ubiquitin-lysozyme conjugates, bovine serum albumin, or lysozyme. Latent and activated forms of MCP display similar sensitivity to protease inhibitors, suggesting that activation does not generate new kinds of catalytic sites. In addition, ATP suppresses peptide hydrolysis by activated and latent MCPs to the same extent. Activation involves binding of regulator to MCP, and activated MCP migrates slower on native acrylamide gels. Dissociation of the MCP regulator complex during prolonged sedimentation on glycerol gradients releases active regulator and MCP molecules capable of being reactivated. Moreover, two-dimensional electrophoresis does not reveal changes in MCP or regulator subunits following activation. Thus, activation appears to result from reversible association of regulator subunits with MCP.

Evidence has been reported that the proximal small intestine of the neonatal rat selectively transports antibodies into the circulation. This study describes the morphology of the absorptive epithelial cells in this region of the intestine and their transport of several immunoglobulin tracers: ferritin-conjugated immunoglobulins (IgG-Ft) and antiperoxidase antibodies. Cells exposed to rat IgG-Ft bound the tracer on the membrane of tubular invaginations of the apical cell surface. Tubular and coated vesicles within the cell also contained the tracer, as did the intercellular spaces. Uptake of tracer was highly selective and occurred only with rat or cow IgG-Ft; when cells were exposed to chicken IgG-Ft, ferritin-conjugated bovine serum albumin, or free ferritin, tracer did not enter the cell or appear in the intercellular spaces. Experiments with rat and chicken antiperoxidase showed a similar selective uptake and transport of only the homologous antibody. When cells from the distal small intestine were exposed to the tracers, all tracers were absorbed nonselectively but none were released from the cells. Cells from the proximal small intestine of the 22-day-old rat failed to absorb even rat IgG-Ft. A model is presented for selective antibody transport in proximal cells of the neonatal rat in which antibodies are selectively absorbed at the apical cell surface by pinocytosis within tubular vesicles. The antibodies are then transferred to the intercellular space within coated vesicles. Distal cells function only to digest proteins nonselectively.

During the acute phase response to tissue injury or inflammation, the concentration of several plasma proteins change. Previous work (29-34) suggested a role for interleukin 1 (IL-1) in the acute phase response. The availability of recombinant-generated mouse IL-1 prompted a study designed to directly test the function of IL-1 and its mechanism of action on hepatic synthesis of two positive acute phase proteins (serum amyloid A [SAA] and complement factor B), and a negative acute phase reactant (albumin). Intravenous injection of purified recombinant-generated murine-IL-1 into C3H/HeJ endotoxin-resistant mice induced a dose-dependent increase in SAA-specific hepatic messenger RNA (mRNA), and an increase in SAA plasma protein concentration. In primary murine hepatocyte cultures, both the recombinant IL-1 and highly purified human IL-1 induced a dose- and time-dependent, reversible increase in expression of the SAA and factor B genes, and a decrease in albumin gene expression. This regulation is pretranslational, since the kinetics and direction of change in specific mRNA for SAA, factor B, and albumin correspond to the changes in synthesis of the respective proteins. Moreover, the effect of IL-1 was specific, since actin gene expression was unaffected, and the IL-1 response was inhibited by antibody specific for IL-1. These data provide direct evidence that a single mediator, IL-1, can effect the positive and negative changes in specific hepatic gene expression characteristic of the acute phase response.

Patients with Type 1 (insulin-dependent) diabetes without proteinuria were studied to define those patients who will later develop persistent proteinuria (more than 0.5 g protein/24 h). Two investigations were performed; 71 patients were studied longitudinally for 6 years and another 227 patients were studied cross-sectionally. All were less than 50 years of age and had developed diabetes before the age of 40 years. At entry into the study they had no proteinuria (Albustix method), had normal blood pressure and urinary albumin excretion rates less than 200 micrograms/min (normal less than or equal to 20 micrograms/min). The best predictor of persistent proteinuria or an albumin excretion rate greater than 200 micrograms/min was the initial urinary albumin excretion rate. During the longitudinal study, seven patients with an urinary albumin excretion rate of more than 70 micrograms/min at the start of the study developed persistent proteinuria or an albumin excretion rate greater than 200 micrograms/min. In contrast, only three out of the remaining 64 patients with urinary albumin excretion rate less than or equal to 70 micrograms/min developed urinary albumin excretion rate greater than 200 micrograms/min. Patients with an urinary albumin excretion rate greater than 70 micrograms/min are thus at risk of developing diabetic nephropathy. We designate this stage of renal involvement incipient nephropathy. Patients with incipient nephropathy were further characterized in the cross-sectional study. Compared with normoalbuminuric patients, patients with incipient nephropathy had increased systolic and diastolic blood pressure, but normal serum creatinine. The glomerular filtration rate was higher than normal in patients with incipient nephropathy though not different from that of normoalbuminuric patients.

OBJECTIVE

To investigate the significance of preoperative Glasgow prognostic score (GPS) for postoperative prognostication of patients with colorectal cancer.

BACKGROUND

Recent studies have revealed that the GPS, an inflammation-based prognostic score that includes only C-reactive protein (CRP) and albumin, is a useful tool for predicting postoperative outcome in cancer patients. However, few studies have investigated the GPS in the field of colorectal surgery.

METHODS

The GPS was calculated on the basis of admission data as follows: patients with an elevated level of both CRP (>10 mg/L) and hypoalbuminemia (Alb <35 g/L) were allocated a score of 2, and patients showing 1 or none of these blood chemistry abnormalities were allocated a score of 1 or 0, respectively. Prognostic significance was analyzed by univariate and multivariate analyses.

RESULTS

A total of 315 patients were evaluated. Kaplan-Meier analysis and log-rank test revealed that a higher GPS predicted a higher risk of postoperative mortality (P < 0.01). Univariate analyses revealed that postoperative TNM was the most sensitive predictor of postoperative mortality (odds ratio, 0.148; 95% confidence interval, 0.072-0.304; P < 0.0001). Multivariate analyses using factors such as age, sex, tumor site, serum carcinoembryonic antigen, CA19-9, CA72-4, CRP, albumin, and GPS revealed that GPS (odds ratio, 0.165; 95% confidence interval, 0.037-0.732; P = 0.0177) was associated with postoperative mortality.

CONCLUSIONS

Preoperative GPS is considered to be a useful predictor of postoperative mortality in patients with colorectal cancer.

Peptide hormones exert unique actions via specific G protein-coupled receptors; however, the therapeutic potential of regulatory peptides is frequently compromised by rapid enzymatic inactivation and clearance from the circulation. In contrast, recombinant or covalent coupling of smaller peptides to serum albumin represents an emerging strategy for extending the circulating t(1/2) of the target peptide. However, whether larger peptide-albumin derivatives will exhibit the full spectrum of biological activities encompassed by the native peptide remains to be demonstrated. We report that Albugon, a human glucagon-like peptide (GLP)-1-albumin recombinant protein, activates GLP-1 receptor (GLP-1R)-dependent cAMP formation in BHK-GLP-1R cells, albeit with a reduced half-maximal concentration (EC(50)) (0.2 vs. 20 nmol/l) relative to the GLP-1R agonist exendin-4. Albugon decreased glycemic excursion and stimulated insulin secretion in wild-type but not GLP-1R(-/-) mice and reduced food intake after both intracerebroventricular and intraperitoneal administration. Moreover, intraperitoneal injection of Albugon inhibited gastric emptying and activated c-FOS expression in the area postrema, the nucleus of the solitary tract, the central nucleus of the amygdala, the parabrachial, and the paraventricular nuclei. These findings illustrate that peripheral administration of a larger peptide-albumin recombinant protein mimics GLP-1R-dependent activation of central and peripheral pathways regulating energy intake and glucose homeostasis in vivo.

BACKGROUND

Emergency surgery for patients with severe acute cholangitis due to choledocholithiasis is associated with substantial morbidity and mortality. Because recent results suggested that emergency endoscopic drainage could improve the outcome of such patients, we undertook a prospective study to determine the role of this procedure as initial treatment.

METHODS

During a 43-month period, 82 patients with severe acute cholangitis due to choledocholithiasis were randomly assigned to undergo surgical decompression of the biliary tract (41 patients) or endoscopic biliary drainage (41 patients), followed by definitive treatment. Hospital mortality was analyzed with respect to the use of endoscopic biliary drainage and other clinical and laboratory findings. Prognostic determinants were studied by linear discriminant analysis.

RESULTS

Complications related to biliary tract decompression and subsequent definitive treatment developed in 14 patients treated with endoscopic biliary drainage and 27 treated with surgery (34 vs. 66 percent, P greater than 0.05). The time required for normalization of temperature and stabilization of blood pressure was similar in the two groups, but more patients in the surgery group required ventilatory support. The hospital mortality rate was significantly lower for the patients who underwent endoscopy (4 deaths) than for those treated surgically (13 deaths) (10 vs. 32 percent, P less than 0.03). The presence of concomitant medical problems, a low platelet count, a high serum urea nitrogen concentration, and a low serum albumin concentration before biliary decompression were the other independent determinants of mortality in both groups.

CONCLUSIONS

Endoscopic biliary drainage is a safe and effective measure for the initial control of severe acute cholangitis due to choledocholithiasis and to reduce the mortality associated with the condition.

OBJECTIVE

To explore the links between tumor necrosis factor alpha (TNFalpha) and leptin adipose tissue expression and low-grade systemic inflammation and to determine the relationship between inflammation and the degree of adiposity, the presence of type 2 diabetes, and other cardiovascular risk factors.

METHODS

Ninety-one women (BMI 19 to 65 kg/m(2)) were divided into tertiles of CRP. Insulin resistance was calculated using the HOMA method. Albumin, fibrinogen, C-reactive protein (CRP), interleukin-6, sTNFR1, sTNFR2, and leptin levels were measured in serum and plasma samples. TNFalpha and leptin expression were measured by reverse transcription-polymerase chain reaction in abdominal subcutaneous adipose tissue samples.

RESULTS

CRP was positively related to BMI and upper distribution of adiposity. TNFalpha and leptin adipose tissue expression were higher in the upper tertile of CRP. Also, peripheral levels of both soluble TNFRs and leptin were higher in patients with the greatest inflammation degree. Diabetes, dislipidemia, and hypertension were most prevalent in patients in the upper CRP tertile. Inflammatory markers of diabetic women were significantly different from those of nondiabetic women, even after adjusting for differences in body fat. BMI, type 2 diabetes, and adipose TNFalpha mRNA levels were significant predictors of serum CRP levels (r(2) = 0.28, p < 0.001).

CONCLUSIONS

These results are in agreement with the hypothesis that the synthesis of adipose tissue TNFalpha and leptin could induce the production of interleukin-6, CRP, and other acute-phase reactants, thus contributing to the maintenance of chronic low-grade inflammation state involved in the progression of obesity and its associated comorbidities.

Multipotent stem/progenitors are present in peribiliary glands of extrahepatic biliary trees from humans of all ages and in high numbers in hepato-pancreatic common duct, cystic duct, and hilum. They express endodermal transcription factors (e.g., Sox9, SOX17, FOXA2, PDX1, HES1, NGN3, PROX1) intranuclearly, stem/progenitor surface markers (EpCAM, NCAM, CD133, CXCR4), and sometimes weakly adult liver, bile duct, and pancreatic genes (albumin, cystic fibrosis transmembrane conductance regulator [CFTR], and insulin). They clonogenically expand on plastic and in serum-free medium, tailored for endodermal progenitors, remaining phenotypically stable as undifferentiated cells for months with a cell division initially every ≈36 hours and slowing to one every 2-3 days. Transfer into distinct culture conditions, each comprised of a specific mix of hormones and matrix components, yields either cords of hepatocytes (express albumin, CYP3A4, and transferrin), branching ducts of cholangiocytes (expressing anion exchanger-2-AE2 and CFTR), or regulatable C-peptide secreting neoislet-like clusters (expressing glucagon, insulin) and accompanied by changes in gene expression correlating with the adult fate. Transplantation into quiescent livers of immunocompromised mice results in functional human hepatocytes and cholangiocytes, whereas if into fat pads of streptozocin-induced diabetic mice, results in functional islets secreting glucose-regulatable human C-peptide.

CONCLUSIONS

The phenotypes and availability from all age donors suggest that these stem/progenitors have considerable potential for regenerative therapies of liver, bile duct, and pancreatic diseases including diabetes.

A general method for the quantification of proteins in human serum was developed using mass spectrometry (MS) and stable isotope-labeled synthetic peptides as internal standards. Using this approach, C-reactive protein (CRP), a diagnostic marker of rheumatoid arthritis (RA), was detected in serum samples taken from patients with either erosive or nonerosive RA and compared to healthy individuals. Small volumes of serum samples were enriched for low-abundance proteins through the selective removal of human serum albumin (HSA), immunoglobulin G (IgG), and haptoglobin. After depletion of abundant proteins, the complexity of the protein mixture was further simplified using size exclusion chromatography (SEC) to fractionate denatured proteins into discrete molecular weight ranges. Fractions of interest containing CRP, M(r) = 25 000, were pooled, digested with trypsin, and then fixed quantities of the synthetic peptides were added to the mixture. The mixture of tryptic peptides was subsequently analyzed by nanoflow chromatography-tandem MS (nanoLC-MS/MS) using multiple-reaction monitoring (MRM) on a triple quadrupole mass spectrometer (TQ-MS). The ratio of transition ions derived from the endogenous and isotope-labeled peptides provided a quantitative measure of CRP in the original samples as assessed by independent measurement of CRP in the same patient samples using an immunoassay. The use of isotope-labeled synthetic peptides and MRM is a powerful analytical method for the prescreening of candidate protein biomarkers in human serum prior to antibody and immunoassay development.

Advanced glycation end products (AGEs) and glycoxidation products are formed during Maillard or browning reactions between sugars and proteins and are implicated in the pathophysiology of aging and the complications of diabetes. To determine the structure of AGEs, antibodies were prepared to protein browned by incubation with glucose and used in ELISA assays to measure AGEs formed in model reactions between bovine serum albumin (BSA) or N alpha-acetyllysine and glucose, fructose, or glyoxal. AGEs were formed from glucose and fructose only under oxidative conditions, but from glyoxal under both oxidative and antioxidative conditions. Gel permeation chromatographic analysis indicated that a similar AGE was formed in reactions of N alpha-acetyllysine with glucose, fructose, and glyoxal and that this AGE co-eluted with authentic N alpha-acetyl-N epsilon-(carboxymethyl)lysine. Amino acid analysis of AGE proteins revealed a significant content of N epsilon-(carboxymethyl)lysine (CML). In ELISA assays using polyclonal antibodies against AGE proteins, CML-BSA (approximately 25 mol of CML/mol of BSA), prepared by chemical modification of BSA, was a potent inhibitor of the recognition of AGE proteins and of AGEs in human lens proteins. We conclude that AGEs are largely glycoxidation products and that CML is a major AGE recognized in tissue proteins by polyclonal antibodies to AGE proteins.

Vasopressin analogues associated with albumin improve renal function in hepatorenal syndrome (HRS). The current study was aimed at assessing the efficacy of the treatment, predictive factors of response, recurrence of HRS, and survival after therapy. Twenty-one consecutive patients with HRS (16 with type 1 HRS, 5 with type 2 HRS) received terlipressin (0.5-2 mg/4 hours intravenously) until complete response was achieved (serum creatinine level < 1.5 mg/dL) or for 15 days; 13 patients received intravenous albumin together with terlipressin. Twelve of the 21 patients (57%) showed complete response. Albumin administration was the only predictive factor of complete response (77% in patients receiving terlipressin and albumin vs. 25% in those receiving terlipressin alone, P =.03). Treatment with terlipressin and albumin was associated with a remarkable decrease in serum creatinine level, increase in arterial pressure, and suppression of the renin-aldosterone system. By contrast, no significant changes in these parameters were found in patients treated with terlipressin alone. Only 1 patient showed ischemic adverse effects. Recurrence of HRS occurred in 17% of patients with complete response. The occurrence of complete response was associated with an improved survival. In conclusion, terlipressin therapy reverses HRS in a high proportion of patients. Recurrence rate after treatment withdrawal is uncommon. Albumin appears to improve markedly the beneficial effects of terlipressin.

Controlling the extent of inflammatory responses following brain injury may be beneficial since posttraumatic intracranial inflammation has been associated with adverse outcome. In order to elucidate the potential role of anti-inflammatory mediators, the production of interleukin-10 (IL-10) was monitored in paired cerebrospinal fluid (CSF) and serum of 28 patients with severe traumatic brain injury (TBI) and compared to control samples. The pattern of IL-10 was analyzed with respect to the patterns of IL-6, tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta1 (TGF-beta1) in both fluids during a time period of up to 22 days. In parallel, the function/dysfunction of the blood-brain barrier (BBB) was monitored using the CSF-/serum-albumin quotient (Q(A)) and compared to intrathecal cytokine levels. Mean IL-10 concentration in CSF was elevated in 26 out of 28 TBI patients (range: 1.3-41.7 pg/ml) compared to controls (cut-off: 1.06 pg/ml), whereas only seven patients had elevated mean IL-10 concentration in serum (range: 5.4-23 pg/ml; cut-off: 5.14 pg/ml). The time course of IL-10 was similar in both fluids, showing a peak during the first days and a second, lower rise in the second week. Intrathecal IL-10 synthesis is hypothesized since CSF-IL-10 levels exceeded serum-IL-10 levels in most of the patients, IL-10-index (CSF/serum-IL-10/QA) was elevated in 23 individuals, and elevation of CSF-IL-10 showed to be independent from severe BBB dysfunction. Neither CSF nor serum IL-10 values correlated with the dysfunction of the BBB. IL-10, IL-6 and TGF-beta1 showed similar patterns in CSF over time, whereas rises of TNF-alpha corresponded to declines of IL-10 levels. Our results suggest that IL-10 is predominantly induced intrathecally after severe TBI where it may downregulate inflammatory events following traumatic brain damage.

The polymeric immunoglobulin receptor (pIgR) on mucosal epithelial cells binds dimeric IgA (dIgA) on the basolateral surface and mediates transport of dIgA to the apical surface. Using Madin-Darby canine kidney epithelial cells stably transfected with pIgR cDNA, we found that soluble immune complexes (ICs) of 125I-labeled rat monoclonal antidinitrophenyl (DNP) dIgA (125I-dIgA) and DNP/biotin-bovine serum albumin were transported from the basolateral to the apical surface and then released. Monomeric IgA ICs were not transported, consistent with the specificity of pIgR for polymeric immunoglobulins. Essentially all the 125I-dIgA in apical culture supernatants was streptavidin precipitable, indicating that dIgA remained bound to antigen during transcytosis. While both dIgA and dIgA ICs bound pIgR with equal affinity (Kd approximately 8 nM), the number of high-affinity binding sites per cell was 2- to 3-fold greater for dIgA than for dIgA ICs. The extent of endocytosis of dIgA and dIgA ICs was correlated with the number of high-affinity binding sites. SDS/PAGE analysis of intracellular dIgA and dIgA ICs demonstrated that in both cases IgA remained undegraded during transport. The results suggest that the pathways of epithelial transcytosis of free dIgA and dIgA ICs are the same. Given the high population density of mucosal IgA plasma cells and the enormous surface area of pIgR-expressing mucosal epithelium, it is likely that significant local transcytosis of IgA ICs occurs in vivo. Such a process would allow direct elimination of IgA ICs at the mucosal sites where they are likely to form, thus providing an important defense function for IgA.

Diabetic retinopathy is characterized by blood-retinal barrier (BRB) breakdown and neurotoxicity. These pathologies have been associated with oxidative stress and proinflammatory cytokines, which may operate by activating their downstream target p38 MAP kinase. In the present study, the protective effects of a nonpsychotropic cannabinoid, cannabidiol (CBD), were examined in streptozotocin-induced diabetic rats after 1, 2, or 4 weeks. Retinal cell death was determined by terminal dUTP nick-end labeling assay; BRB function by quantifying extravasation of bovine serum albumin-fluorescein; and oxidative stress by assays for lipid peroxidation, dichlorofluorescein fluorescence, and tyrosine nitration. Experimental diabetes induced significant increases in oxidative stress, retinal neuronal cell death, and vascular permeability. These effects were associated with increased levels of tumor necrosis factor-alpha, vascular endothelial growth factor, and intercellular adhesion molecule-1 and activation of p38 MAP kinase, as assessed by enzyme-linked immunosorbent assay, immunohistochemistry, and/or Western blot. CBD treatment significantly reduced oxidative stress; decreased the levels of tumor necrosis factor-alpha, vascular endothelial growth factor, and intercellular adhesion molecule-1; and prevented retinal cell death and vascular hyperpermeability in the diabetic retina. Consistent with these effects, CBD treatment also significantly inhibited p38 MAP kinase in the diabetic retina. These results demonstrate that CBD treatment reduces neurotoxicity, inflammation, and BRB breakdown in diabetic animals through activities that may involve inhibition of p38 MAP kinase.

The prevalence of ErbB2 amplification in breast cancer has resulted in the heavy pursuit of ErbB2 as a therapeutic target. Although both the ErbB2 monoclonal antibody trastuzumab and ErbB1/ErbB2 dual kinase inhibitor lapatinib have met with success in the clinic, many patients fail to benefit. In addition, the majority of patients who initially respond will unfortunately ultimately progress on these therapies. Activation of ErbB3, the preferred dimerization partner of ErbB2, plays a key role in driving ErbB2-amplified tumor growth, but we have found that current ErbB2-directed therapies are poor inhibitors of ligand-induced activation. By simulating ErbB3 inhibition in a computational model of ErbB2/ErbB3 receptor signaling, we predicted that a bispecific antibody that docks onto ErbB2 and subsequently binds to ErbB3 and blocks ligand-induced receptor activation would be highly effective in ErbB2-amplified tumors, with superior activity to a monospecific ErbB3 inhibitor. We have developed a bispecific antibody suitable for both large scale production and systemic therapy by generating a single polypeptide fusion protein of two human scFv antibodies linked to modified human serum albumin. The resulting molecule, MM-111, forms a trimeric complex with ErbB2 and ErbB3, effectively inhibiting ErbB3 signaling and showing antitumor activity in preclinical models that is dependent on ErbB2 overexpression. MM-111 can be rationally combined with trastuzumab or lapatinib for increased antitumor activity and may in the future complement existing ErbB2-directed therapies to treat resistant tumors or deter relapse.

OBJECTIVE

Comorbid conditions affect the risk of adverse outcomes after surgery, but the magnitude of risk has not previously been quantified using multivariate statistical methods and prospectively collected data. Identifying factors that predict results of surgical procedures would be valuable in assessing the quality of surgical care. This study was performed to define risk factors that predict adverse events after colectomy for cancer in Department of Veterans Affairs Medical Centers.

METHODS

The National Veterans Affairs Surgical Quality Improvement Program contains prospectively collected and extensively validated data on more than 415,000 surgical operations. All patients undergoing colectomy for colon cancer from 1991 to 1995 who were registered in the National Veterans Affairs Surgical Quality Improvement Program database were selected for study. Independent variables examined included 68 preoperative and 12 intraoperative clinical risk factors; dependent variables were 21 specific adverse outcomes. Stepwise logistic regression analysis was used to construct models predicting the 30-day mortality rate and 30-day morbidity rates for each of the ten most frequent complications.

RESULTS

A total of 5,853 patients were identified; 4,711 (80 percent) underwent resection and primary anastomosis. One or more complications were observed in 1,639 of 5,853 (28 percent) patients. Prolonged ileus (439/5,853; 7.5 percent), pneumonia (364/5,853; 6.2 percent), failure to wean from the ventilator (334/5,853; 5.7 percent), and urinary tract infection (292/5,853; 5 percent) were the most frequent complications. The 30-day mortality rate was 5.7 percent (335/5,853). For most complications, 30-day in-hospital mortality rates were significantly higher for patients with a complication than for those without. Thirty-day mortality rates exceeded 50 percent if postoperative coma, cardiac arrest, a pre-existing vascular graft prosthesis that failed after colectomy, renal failure, pulmonary embolism, or progressive renal insufficiency occurred. Preoperative factors that predicted a high risk of 30-day mortality included ascites, serum sodium >145 mg/dl, "do not resuscitate" status before surgery, American Society of Anesthesiologists classes III and IV OR V, and low serum albumin.

CONCLUSIONS

Mortality rates after colectomy in Veterans Affairs hospitals are comparable with those reported in other large studies. Ascites, hypernatremia, do not resuscitate status before surgery, and American Society of Anesthesiologists classes III and IV OR V were strongly predictive of perioperative death. Clinical trials to decrease the complication rate after colectomy for colon cancer should focus on these risk factors.

BACKGROUND

Although the medical determinants of mortality in patients with end-stage renal disease (ESRD) treated with hemodialysis (HD) are well appreciated, the contribution of immunologic parameters to survival in such patients is unclear, especially when variations in age, medical comorbidity and nutrition are controlled. In addition, although dysregulation of cytokine metabolism has been appreciated in patients with ESRD, the association of these parameters with outcomes has not been established. Recently, the type of dialyzer used in patients' treatment has been associated with survival, but the mechanisms underlying these findings, including their immune effects, have not been established. We conducted a prospective, cross-sectional, observational multicenter study of urban HD patients to determine the contribution of immunological factors to patient survival. We hypothesized increased proinflammatory cytokines would be associated with increased mortality, and that improved immune function would be associated with survival.

METHODS

Patients were assessed using demographic and anthropometric indices, Kt/V, protein catabolic rate (PCR) and immunologic variables including circulating cytokine [interleukin (IL)-1, IL-2, IL-4, IL-5, IL-6, IL-12, IL-13 and tumor necrosis factor (TNF)-alpha] levels, total hemolytic complement activity (CH50), and T cell number and function. A severity index, previously demonstrated to be a mortality marker, was used to grade medical comorbidity. A Cox proportional hazards model, controlling for patients' age, severity index, level of serum albumin concentration, dialyzer type and dialysis site was used to asses relative survival risk.

RESULTS

Two hundred and thirty patients entered the study. The mean (+/- SD) age of the population was 54.4 +/- 14.2 years, mean serum albumin concentration was 3.86 +/- 0.47 g/dl, mean PCR was 1.1 +/- 0.28 g/kg/day, and mean Kt/V 1.2 +/- 0.3. Patients' serum albumin concentration was correlated with levels of Kt/V and PCR, and their circulating IL-13 and TNF-alpha levels, but negatively with their circulating IL-2 levels, T-cell number and T-cell antigen recall function. T-cell antigen recall function correlated negatively with PCR, but not Kt/V. There was no correlation of any other immune parameter and medical or demographic factor. Immune parameters, were all highly intercorrelated. Mean level of circulating cytokines in HD patients were in all cases greater than those of a normal control group. There were few differences in medical risk factors or immune parameters between patients treated with different types of dialyzers. After an almost three-year mean follow-up period, increased IL-1, TNF-alpha, IL-6, and IL-13 levels were significantly associated with increased relative mortality risk, while higher levels of IL-2, IL-4, IL-5, IL-12, T-cell number and function, and CH50 were associated with improved survival. The difference in survival between patients treated with unmodified cellulose dialyzers and modified or synthetic dialyzers approached the level of statistical significance, but there were no differences in levels of circulating cytokines between these two groups.

CONCLUSIONS

Higher levels of circulating proinflammatory cytokines are associated with mortality, while immune parameters reflecting improved T-cell function are associated with survival in ESRD patients treated with HD, independent of other medical risk factors. These factors may serve as markers for outcome. The mechanism underlying the relationship of immune function and survival, and the effect of interventions to normalize immune function in HD patients should be studied.

Weight loss and nutritional deterioration are associated with adverse outcomes in terms of cancer prognosis (response rate and survival) as well as increased complications, prolonged hospitalizations, increased risk of unplanned hospitalization, increased disability, and increased overall cost of care. The nutritional oncology service at Fox Chase Cancer Center defined a proactive, standardized assessment and interventional approach from 1987-1994. In 186 consecutive patients referred to the nutrition clinic and managed solely by oral intervention and aggressive symptom management, the team demonstrated a 50%-80% success rate in getting patients to maintain or gain weight during therapy, with a similar success in maintaining or improving visceral protein status as determined by serum transferrin and/or albumin. Evaluation of the home parenteral nutrition program (n = 65, from 1987-1993) demonstrated similar success when appropriate triaging was carried out, with 58% of patients able to be tapered off parenteral nutrition (PN) entirely or with transition to enteral tube feeding. The assessment of success for a nutritional intervention (e.g., a disease-specific nutritional supplement) requires the standardization of definitions, assessment tools, criteria for nutritional intervention, and appropriate end points for the assessment of outcomes. The Patient-Generated Subjective Global Assessment of nutritional status is used in conjunction with the nutritional risk of planned cancer therapy to define a standardized interventional approach in oncology patients, which can be used in clinical practice, cooperative oncology group protocols, and clinical trials of nutritional intervention regimens.

There are two distinct subtypes of multiple sclerosis in Asians, opticospinal (OS-multiple sclerosis) and conventional (C-multiple sclerosis). In OS-multiple sclerosis, selective and severe involvement of the optic nerves and spinal cord is characteristic, though its mechanisms are unknown. The present study aimed to find out possible differences in the cytokine/chemokine profiles in CSF between OS-multiple sclerosis and C-multiple sclerosis and to delineate the relationships between these profiles and neuroimaging and pathological features. Sixteen cytokines/chemokines, namely interleukin (IL)-1beta, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12 (p70), IL-13, IL-17, interferon (IFN)-gamma, tumour necrosis factor (TNF)-alpha, granulocyte colony-stimulating factor (G-CSF), monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1beta (MIP-1beta), were measured simultaneously in CSF supernatants from 40 patients with relapsing-remitting multiple sclerosis (20 OS-multiple sclerosis and 20 C-multiple sclerosis) at relapse and 19 control patients with spinocerebellar degeneration (SCD), together with intracellular production of IFN-gamma and IL-4 in CSF CD4+ T cells. In CSF supernatants relative to controls, IL-17, MIP-1beta, IL-1beta and IL-13 were only significantly increased in OS-multiple sclerosis patients, while TNF-alpha was only significantly increased in C-multiple sclerosis patients, using a cut-off level of 1 pg/ml. IL-8 was significantly elevated in both OS-multiple sclerosis and C-multiple sclerosis patients. MCP-1 was significantly decreased in both OS-multiple sclerosis and C-multiple sclerosis patients, while IL-7 was only significantly decreased in C-multiple sclerosis patients. IL-17, IL-8 and IL-5 were significantly higher in OS-multiple sclerosis patients than in C-multiple sclerosis patients. The increases in IL-17 and IL-8 in OS-multiple sclerosis were still significant even after exclusion of the patients undergoing various immunomodulatory therapies. Assays of intracellular cytokine production revealed that both the IFN-gamma+IL-4- T-cell percentage and intracellular IFN-gamma/IL-4 ratio in CSF cells were significantly greater in C-multiple sclerosis patients than in controls. Contrarily, OS-multiple sclerosis patients showed not only a significantly greater percentage of IFN-gamma+IL-4- T cells than controls but also a significantly higher percentage of IFN-gamma-IL-4+ T cells than C-multiple sclerosis patients. Among the cytokines elevated in multiple sclerosis, only IL-8 showed a significant positive correlation with the Expanded Disability Status Scale of Kurtzke score. Both the length of the spinal cord lesions on MRI and the CSF/serum albumin ratio had a significant positive correlation with IL-8 and IL-17 in multiple sclerosis, in which the spinal cord lesions were significantly longer in OS-multiple sclerosis than in C-multiple sclerosis. Three of six spinal cord specimens from autopsied OS-multiple sclerosis cases demonstrated numerous myeloperoxidase-positive neutrophils infiltrating necrotic lesions. These findings strongly suggest that in OS-multiple sclerosis, in addition to the Th1 cell upregulation seen in C-multiple sclerosis, intrathecal activation of the IL-17/IL-8 axis inducing heavy neutrophil infiltration contributes to extensive spinal cord lesion formation.

Dendritic cells (DC) isolated from lymphoid tissues are generally thought to be nonphagocytic in culture. It has therefore been unclear how these cells could acquire particulate antigens such as microorganisms for initiation of primary immune responses. Lymphoid DC derive in part from cells that have migrated from nonlymphoid tissues, such as Langerhans cells (LC) of skin. The ability of LC to internalize a variety of particles was studied by electron, ultraviolet, phase, and differential interference contrast microscopy, and by two-color flow cytometry. Freshly isolated LC in epidermal cell suspensions phagocytosed the yeast cell wall derivative zymosan, intact Saccharomyces cerevisiae, representatives of two genera of Gram-positive bacteria, Corynebacterium parvum and Staphylococcus aureus, as well as 0.5-3.5-microns latex microspheres. During maturation in culture, the phagocytic activity of these cells was markedly reduced. Likewise, freshly isolated splenic DC were more phagocytic than cultured DC for two types of particle examined, zymosan and latex beads. Unlike macrophages, LC did not bind or internalize sheep erythrocytes before or after opsonization with immunoglobulin G or complement, and did not internalize colloidal carbon. The receptors mediating zymosan uptake by LC were examined. For this particle, C57BL/6 LC were considerably more phagocytic than BALB/c LC and exhibited a reproducible increase in phagocytic activity after 6 h of culture followed by a decline, whereas this initial rise did not occur for BALB/c LC. These differential kinetics of uptake were reflected in the pattern of zymosan binding at 4 degrees C, and endocytosis of the soluble tracer fluorescein isothiocyanate-mannose-bovine serum albumin at 37 degrees C. Zymosan uptake by LC from both strains of mice was inhibited in the presence of mannan or beta-glucan, although to different extents, but not by antibodies specific for CR3 (CD11b/CD18). These data indicate that zymosan uptake by LC can be mediated by a mannose/beta-glucan receptor(s) that is differentially expressed in the two strains of mice and that is downregulated during maturation of LC in culture.