The linker component of antibody-drug conjugates (ADC) is a key feature in developing optimized therapeutic agents that are highly active at well tolerated doses. For maximal intratumoral drug delivery, linkers are required that are highly stable in the systemic circulation, yet allow for efficient drug release at the target site. In this respect, amide bond-based technologies constitute a technological advancement, since the linker half-lives in circulation ( t 1/2 approximately 7 days) are much longer than earlier generation linkers that break down within 1-2 days. The amide linkers, some of which contain peptides, are appended to the mAb carriers through thioether/maleimide adducts. Here, we describe that use of a bromoacetamidecaproyl (bac) in place of the maleimidocaproyl (mc) increases the plasma stability of resulting thioether ADCs. One such ADC, 1F6-C4v2-bac-MMAF, which is directed against the CD70 antigen on lymphomas and renal cell carcinoma, was prepared containing a bac thioether spacer between the drug (MMAF) and the mAb carrier (1F6-C4v2). There was no measurable systemic drug release from this ADC for 2 weeks postadministration in mice. In order to assess the impact of improving linker stability beyond mc containing ADCs, a series of mc and bac-linked 1F6-MMAF conjugates were compared for tolerability, intratumoral drug delivery, and therapeutic efficacy in nude mice with renal cell carcinoma xenografts. There were no statistically significant efficacy differences between sets of mc and bac containing ADCs, although the bac linker technology led to 25% higher intratumoral drug exposure over a 7 day period compared to the corresponding mc linker. The mechanism of drug release from maleimide-adducts likely involves a retro-Michael reaction that takes place in plasma, based on in vitro studies demonstrating that some of the released drug-maleimide derivative became covalently bound to cysteine-34 of serum albumin. In summary, the data indicate that new linkers can be obtained with improved in vivo stability by replacing the maleimide with an acetamide, but the resulting ADCs had similar tolerability and activity profiles.

OBJECTIVE

Celiac disease is the most common severe food intolerance in the Western world and is due to gluten ingestion in genetically susceptible children and adults. Intestinal biopsy is the golden standard for evaluation of mucosal damage associated with celiac disease. Gluten-free diet is the key treatment for celiac disease. Data on the long-term control of celiac disease are few and limited to small series of patients. The study reports data on the control of celiac disease and on its correlates in a large cohort of celiac adults during long-term treatment with gluten-free diet.

METHODS

The study cohort comprises 91 men and 299 women having undergone treatment with a gluten-free diet for at least 2 years and with complete records for visits at the time of diagnosis of celiac disease (baseline). Data collection included gender, age, education, weight, bowel habit, blood hemoglobin, plasma albumin and cholesterol, serum antiendomysium antibodies (EMA), dietary compliance to gluten-free diet (coded as good, low, or very low), and intestinal damage at biopsy (coded as absent, mild, or severe).

RESULTS

The duration of follow-up was 6.9 +/- 7.5 years (mean +/- SD, range 2-22 years). At follow-up visit, intestinal damage was absent in 170 patients (43.6%), mild in 127 (32.6%), and severe in 93 (23.8%). At follow-up, intestinal damage was significantly associated with dietary compliance, EMA, and plasma albumin (follow-up value and change value from baseline to follow-up). Baseline education significantly predicted dietary compliance and intestinal damage at follow-up.

CONCLUSIONS

Celiac disease is often poorly controlled in the majority of patients on long-term treatment with a gluten-free diet as demonstrated by intestinal biopsy. Lack of adherence to strict gluten-free diet is the main reason of poorly controlled disease in adults. Laboratory and clinical information have a high positive predictive value and low negative predictive value for intestinal damage on long-term treatment. Dietary compliance as assessed by interview is the best marker of celiac disease control due to low cost, noninvasivity, and strong correlation with intestinal damage.

Conidia of Aspergillus fumigatus adhere in vitro to host proteins and cells via the outer cell wall layer. The rodA gene of A. fumigatus was cloned by homology with the rodA gene of Aspergillus nidulans, which is involved in the structure of the rodlets characteristic of the surface layer. The A. fumigatus RODA protein sequence has 85% similarity to that of A. nidulans RODA; the sequence codes for a hydrophobin, a low-molecular-weight protein moderately hydrophobic and rich in cysteines. The gene was disrupted with the hygromycin B resistance gene. By transformation of protoplasts with the disrupted gene, RodA- mutants were generated. These mutants are deficient in the ability to disperse their conidia; their conidia lack the rodlet layer and are hydrophilic. The adhesion of the rodletless conidia to collagen and bovine serum albumin was lower than that of the wild type; in contrast, there was no difference between RodA- and RodA+ conidia in adhesion to pneumocytes, fibrinogen, and laminin, suggesting that RODA is not the receptor for these cells and proteins. RodA- conidia were pathogenic for mice.

BACKGROUND

Dysregulated long non-coding RNAs (lncRNAs) have been found to have oncogenic and/or tumor suppressive roles in the development and progression of cancer, implying their potentials as novel independent biomarkers for cancer diagnosis and prognosis. However, the prognostic significance of expression profile-based lncRNA signature for outcome prediction in patients with multiple myeloma (MM) has not yet been investigated.

METHODS

LncRNA expression profiles of a large cohort of patients with MM were obtained and analyzed by repurposing the publically available microarray data. An lncRNA-focus risk score model was developed from the training dataset, and then validated in the testing and another two independent external datasets. The time-dependent receiver operating characteristic (ROC) curve was used to evaluate the prognostic performance for survival prediction. The biological function of prognostic lncRNAs was predicted using bioinformatics analysis.

RESULTS

Four lncRNAs were identified to be significantly associated with overall survival (OS) of patients with MM in the training dataset, and were combined to develop a four-lncRNA prognostic signature to stratify patients into high-risk and low-risk groups. Patients of training dataset in the high-risk group exhibited shorter OS than those in the low-risk group (HR = 2.718, 95 % CI = 1.937-3.815, p <0.001). The similar prognostic values of four-lncRNA signature were observed in the testing dataset, entire GSE24080 dataset and another two independent external datasets. Multivariate Cox regression and stratified analysis showed that the prognostic power of four-lncRNA signature was independent of clinical features, including serum beta 2-microglobulin (Sβ2M), serum albumin (ALB) and lactate dehydrogenase (LDH). ROC analysis also demonstrated the better performance for predicting 3-year OS. Functional enrichment analysis suggested that these four lncRNAs may be involved in known genetic and epigenetic events linked to MM.

CONCLUSIONS

Our results demonstrated potential application of lncRNAs as novel independent biomarkers for diagnosis and prognosis in MM. These lncRNA biomarkers may contribute to the understanding of underlying molecular basis of MM.

Because previous studies showed that polyunsaturated fatty acids can reduce the contraction rate of spontaneously beating heart cells and have antiarrhythmic effects, we examined the effects of the fatty acids on the electrophysiology of the cardiac cycle in isolated neonatal rat cardiac myocytes. Exposure of cardiomyocytes to 10 microM eicosapentaenoic acid for 2-5 min markedly increased the strength of the depolarizing current required to elicit an action potential (from 18.0 +/- 2.4 pA to 26.8 +/- 2.7 pA, P < 0.01) and the cycle length of excitability (from 525 ms to 1225 ms, delta = 700 +/- 212, P < 0.05). These changes were due to an increase in the threshold for action potential (from -52 mV to -43 mV, delta = 9 +/- 3, P < 0.05) and a more negative resting membrane potential (from -52 mV to -57 mV, delta = 5 +/- 1, P < 0.05). There was a progressive prolongation of intervals between spontaneous action potentials and a slowed rate of phase 4 depolarization. Other polyunsaturated fatty acids--including docosahexaenoic acid, linolenic acid, linoleic acid, arachidonic acid, and its nonmetabolizable analog eicosatetraynoic acid, but neither the monounsaturated oleic acid nor the saturated stearic acid--had similar effects. The effects of the fatty acids could be reversed by washing with fatty acid-free bovine serum albumin. These results show that free polyunsaturated fatty acids can reduce membrane electrical excitability of heart cells and provide an electrophysiological basis for the antiarrhythmic effects of these fatty acids.

The availability of disease-specific induced pluripotent stem cells (iPSCs) offers a unique opportunity for studying and modeling the effects of specific gene defects on human liver development in vitro and for testing small molecules or other potential therapies for relevant liver disorders. Here we report, for the first time, the derivation of iPSCs by the retroviral transduction of Yamanaka's factors in serum and feeder-free culture conditions from liver-specific patients with tyrosinemia, glycogen storage disease, progressive familial hereditary cholestasis, and two siblings with Crigler-Najjar syndrome. Furthermore, they were differentiated into functional hepatocyte-like cells efficiently. These iPSCs possessed properties of human embryonic stem cells (hESCs) and were successfully differentiated into three lineages that resembled hESC morphology, passaging, surface and pluripotency markers, normal karyotype, DNA methylation, and differentiation. The hepatic lineage-directed differentiation showed that the iPSC-derived hepatic cells expressed hepatocyte-specific markers. Their functionality was confirmed by glycogen and lipid storage activity, secretion of albumin, alpha-fetoprotein, and urea, CYP450 metabolic activity, as well as LDL and indocyanin green uptake. Our results provide proof of principal that human liver-disease specific iPSCs present an exciting potential venue toward cell-based therapeutics, drug metabolism, human liver development and disease models for liver failure disorders.

Between December 1979 and June 1983 the Eastern Cooperative Oncology Group (ECOG) treated 893 good-performance status patients with metastatic non-small-cell lung cancer (NSCLC) on one of seven phase III combination chemotherapies. The overall median survival was 23.5 weeks with no significant differences between treatments. One hundred sixty-eight patients (19%) survived greater than 1 year and 36 (4%) for greater than 2 years. The etoposide-platinum combination had the highest proportion of 1-year survivors (25%). Mitomycin-vinblastine-platinum (MVP), which had demonstrated the highest response rate, had significantly fewer 1-year survivors (12%) than any other regimen (P = .003). Analysis of pretreatment characteristics that distinguished patients who survived greater than 1 year from those who did not demonstrated that an initial performance status of 0, no bone metastases, female sex, no subcutaneous metastases, non-large-cell histology, less than 5% prior weight loss, no symptoms of shoulder or arm pain, and no liver metastases were predictors of longer survival. Of particular interest was the finding that response duration was significantly longer (P = .002) for those patients who experienced a longer time to best response. In addition, patients who survived greater than 1 year experienced greater degrees of nonlethal toxicity, in particular, gastrointestinal and hematologic, than patients who did not survive 1 year, (P = .006). A detailed chart review of 32 2-year survivors and 32 matched controls demonstrated that maintenance or improvement of performance status and maintenance of serum albumin levels at 3 months from the initiation of treatment were both important predictors of longer survival.

A retrospective survey of Japanese patients histologically diagnosed with chronic hepatitis B was conducted to determine the effectiveness of lamivudine in preventing hepatocellular carcinoma (HCC). Of the 2795 patients who satisfied criteria for analysis after treatment from any of 30 medical institutions, 657 had received lamivudine and the remaining 2138 had not. A Cox regression model with liver biopsy as the starting point revealed seven factors related to HCC: lamivudine therapy, gender, family clustering of hepatitis B, age at liver biopsy, hepatic fibrosis stage, serum albumin level, and platelet count. In a matched case-controlled study, 377 patients in a lamivudine-treated group and 377 matched patients in a non-treated group were selected based on their propensity scores. The mean follow-up period was 2.7 years in the lamivudine group and 5.3 years in the control group. In the lamivudine group, HCC occurred in four patients (1.1%) with an annual incidence rate of 0.4%/(patient/year), whereas in the control group HCC occurred in 50 patients (13.3%) for a rate of 2.5%/(patient/year). A comparison of the cumulative HCC incidence between the two groups by the Kaplan-Meier method showed a significantly lower incidence of HCC in the lamivudine group (p<0.001). These findings suggest that lamivudine effectively reduces the incidence of HCC in patients with chronic hepatitis B.

We present the first results from a new electrospray ionization Fourier transform ion cyclotron resonance mass spectrometer operated at a magnetic field of 9.4 T (i.e.>> or = 2.4 T higher than for any prior FTICR instrument). The 9.4 T instrument provides substantially improved performance for large molecules >> or = 50% increase in mass resolving power) and complex mixtures >> or = 100% increase in dynamic range) compared to lower-field (< or = 6 T) instruments. The higher magnetic field makes possible larger trapped-ion population without introduction of significant space--charge effects such as spectral peak shift and/or distortion, and coalescence of closely-spaced resonances. For bovine ubiquitin (8.6 kDa) we observe accurate relative isotopic abundances at a signal-to-noise ratio greater than 1000:1, whereas a complete nozzle-skimmer dissociation electrospray ionization (ESI) FTICR mass spectrum of bovine carbonic anhydrase (29 kDa) is achieved from a single scan with a signal-to-noise ratio of more than 250:1. Finally, we are able to obtain mass resolving power, m/delta m>> 200,000, routinely for porcine serum albumin (67 kDa). The present performance guides further modifications of the instrument, which should lead to significant further improvements.

Polymorphonuclear leukocytes suspended in Krebs-Ringer phosphate medium ingest paraffin oil containing Oil Red O emulsified with a variety of substances. Spectrophotometric determination of Oil Red O in the cells after uningested particles have been removed by differential centrifugation provides a quantitative measure of phagocytosis. This system has been used to investigate the effects of several drugs and hormones on the initial rate of phagocytosis and to approach the question of how the surface of a particle influences its acceptability as a substrate for phagocytosis. The rate of uptake of paraffin oil emulsified with bovine albumin was constant for 6 min and was proportional to cell concentration when saturating concentrations of paraffin oil emulsion were used. At lower concentrations of substrate, the initial rate of phagocytosis was directly proportional to paraffin oil concentration. The increment in glucose oxidation associated with phagocytosis varied directly with the initial rate of particle uptake. The rate of ingestion of the albumin emulsion was not altered by serum (2-20%, v/v), glucose (5-20 mM), or omission of potassium from the medium. The rate of phagocytosis was decreased 65% if magnesium was omitted, and was essentially zero in the absence of divalent cations. The initial rate of uptake was inhibited by inhibitors of glycolysis, by N-ethylmaleimide (0.05-1 mM), colchicine (0.001-0.1 mM), theophylline (1 and 2 mM), dibutyryl cyclic AMP (1 mM), hydrocortisone (2.1 mM), and ethanol (85 mM). Inhibitors of oxidative phosphorylation and dexamethasone (0.01 mM) were without effect, while insulin (2 mU/ml) slightly stimulated the phagocytic rate. Paraffin oil emulsified with different agents was used to approach the question of how the surface of a particle influences its acceptability as a substrate for phagocytosis. Emulsions prepared with nonionic detergents, methylated proteins, and proteins with a weak net charge at pH 7.4 were poorly ingested. On the other hand emulsions prepared with agents of strong net positive or negative charge were rapidly taken up. The effect of divalent cations on the rate of phagocytosis varied with the nature of the emulsifier, but was not related in any simple, direct fashion to the net surface charge of the particles. However, it has not been conclusively established that charge was the only variable of the emulsion particles employed.

We studied the effect of ursodeoxycholic acid on 18 women and 2 men with primary biliary cirrhosis, mainly stages I and II. After a 3-mo observation period, patients were randomized to a 9-mo treatment period with ursodeoxycholic acid, 10 mg/kg.day, or placebo. Two patients on placebo left the study. In all patients on ursodeoxycholic acid, mean values of serum glutamate dehydrogenase, aspartate and alanine aminotransferases, alkaline phosphatase, and gamma-glutamyl transpeptidase fell significantly by 48%-79% after 18-24 wk; 7 of 10 showed a mean decrease of 35% in immunoglobulin M after 24 wk. Prothrombin time, serum bilirubin, albumin, the antipyrin breath test, and plasma disappearance of indocyanine green were normal initially and did not change. Total serum bile acid concentrations increased; ursodeoxycholic acid became the predominant bile acid. No significant improvement occurred in the placebo group. Hepatic histology improved in 6 patients of the ursodeoxycholic acid group but deteriorated in 4 patients receiving placebo. In studies with erythrocyte membranes, changes in electron spin resonance revealed that ursodeoxycholic acid was less toxic than chenodeoxycholic or deoxycholic acid, and coaddition of ursodeoxycholic acid prevented their toxic effect.

The influences of insulin-like growth factor I (IGF-I) and insulin-like growth factor II (IGF-II) on biosynthesis and catabolism of proteoglycans (PG) in bovine articular cartilage explants were examined to define their potential use in a chemically defined medium. In both short- (10 days) and long-term (40 days) cultures, 10 to 20 ng/ml IGF-I maintained PG synthesis at the same or higher levels than in a medium containing 20% fetal calf serum (FCS). Catabolic rates were slower in IGF-I medium than in medium with only 0.1% albumin, but somewhat faster than for cultures in medium with 20% FCS. In long-term cultures 20 ng/ml IGF-I maintained a steady-state condition; the amounts of glycosaminoglycan and DNA per hydroxyproline content were constant throughout the culture period. The half-maximal dose response for IGF-I on PG synthesis (4.5 ng/ml) was distinctly different from that for the IGF-I effect on PG catabolism (1.5 ng/ml), indicating that these two components of PG metabolism can be experimentally uncoupled. IGF-II was less potent than IGF-I in the same batches of articular cartilage; 100 ng/ml IGF-II increased PG synthesis and decreased PG catabolism relative to 0.1% albumin alone, but the responses were only about 60% of those for 5 ng/ml IGF-I. These results suggest that the chondrocytes regulate PG synthesis primarily via the type I IGF receptor and that the IGF-II response is through the same receptor. Evidence is also provided indicating that the cartilage explants initially contain about 50 ng IGF-I per gram wet weight; this matrix-bound IGF-I diffuses into the medium during culture. The chondrocytes synthesize little or no IGF-I that is released into the medium under the culture conditions used.

BACKGROUND

Diagnosis of adrenal insufficiency in critically ill patients has relied on random or cosyntropin-stimulated cortisol levels, and has not been corroborated by a more accurate diagnostic standard.

OBJECTIVE

We used the overnight metyrapone stimulation test to investigate the diagnostic value of the standard cosyntropin stimulation test, and the prevalence of sepsis-associated adrenal insufficiency.

METHODS

This was an inception cohort study.

RESULTS

In two consecutive septic cohorts (n = 61 and n = 40), in 44 patients without sepsis and in 32 healthy volunteers, we measured (1) serum cortisol before and after cosyntropin stimulation, albumin, and corticosteroid-binding globulin levels, and (2) serum corticotropin, cortisol, and 11beta-deoxycortisol levels before and after an overnight metyrapone stimulation. Adrenal insufficiency was defined by postmetyrapone serum 11beta-deoxycortisol levels below 7 microg/dl. More patients with sepsis (31/61 [59% of original cohort with sepsis] and 24/40 [60% of validation cohort with sepsis]) met criteria for adrenal insufficiency than patients without sepsis (3/44; 7%) (p < 0.001 for both comparisons). Baseline cortisol (< 10 microg/dl), Delta cortisol (< 9 microg/dl), and free cortisol (< 2 microg/dl) had a positive likelihood ratio equal to infinity, 8.46 (95% confidence interval, 1.19-60.25), and 9.50 (95% confidence interval, 1.05-9.54), respectively. The best predictor of adrenal insufficiency (as defined by metyrapone testing) was baseline cortisol of 10 microg/dl or less or Delta cortisol of less than 9 microg/dl. The best predictors of normal adrenal response were cosyntropin-stimulated cortisol of 44 microg/dl or greater and Delta cortisol of 16.8 microg/dl or greater.

CONCLUSIONS

In sepsis, adrenal insufficiency is likely when baseline cortisol levels are less than 10 microg/dl or delta cortisol is less than 9 microg/dl, and unlikely when cosyntropin-stimulated cortisol level is 44 microg/dl or greater or Delta cortisol is 16.8 microg/dl or greater.

BACKGROUND

We determined recently that targeted treatment with calcium-based phosphate binders (calcium acetate and carbonate) led to progressive coronary artery and aortic calcification by electron beam tomography (EBT), while treatment with the non-calcium-containing phosphate binder, sevelamer, did not. Aside from the provision of calcium, we hypothesized that other factors might be related to the likelihood of progressive calcification in both or either treatment groups.

METHODS

We explored potential determinants of progressive vascular calcification in 150 randomized study subjects who underwent EBT at baseline and at least once during follow-up (week 26 or 52).

RESULTS

Among calcium-treated subjects, higher time-averaged concentrations of calcium, phosphorus and the calcium-phosphorus product were associated with more pronounced increases in EBT scores; no such associations were demonstrated in sevelamer-treated subjects. The relation between parathyroid hormone (PTH) and the progression of calcification was more complex. Lower PTH was associated with more extensive calcification in calcium-treated subjects, whereas higher PTH was associated with calcification in sevelamer-treated subjects. Serum albumin was inversely correlated with progression in aortic calcification. Sevelamer was associated with favourable effects on lipids, although the link between these effects and the observed attenuation in vascular calcification remains to be elucidated.

CONCLUSIONS

Calcium-based phosphate binders are associated with progressive coronary artery and aortic calcification, especially when mineral metabolism is not well controlled. Calcium may directly or indirectly (via PTH) adversely influence the balance of skeletal and extraskeletal calcification in haemodialysis patients.

We describe a general method for incorporating target ligands into the surface of biocompatible polyester poly(lactic-co-glycolic acid) (PLGA) 50/50 materials using fatty acids. Avidin-fatty acid conjugates were prepared and efficiently incorporated into PLGA. Avidin was chosen as an adaptor protein to facilitate the attachment of a variety of biotinylated ligands. We show that fatty acid preferentially associates with the hydrophobic PLGA matrix, rather than the external aqueous environment, facilitating a prolonged presentation of avidin over several weeks. We successfully applied this approach in both microspheres encapsulating a model protein, bovine serum albumin, and PLGA scaffolds fabricated by a salt-leaching method. Because of its ease, generality and flexibility, this strategy promises widespread utility in modifying the surface of PLGA-based materials for applications in drug delivery and tissue engineering.

BACKGROUND

Monocyte chemoattractant protein-1 (MCP-1) is a specific chemokine to recruit and activate monocytes from the circulation to inflammatory site. In diabetic nephropathy, similar to other glomerulonephropathies, infiltration and activation of monocytes/macrophages in glomerulus have been implicated in the development of glomerular injury. The aim of the present study was to examine a possible relationship of MCP-1 with diabetic nephropathy and to investigate the role of glycated albumin (Gly-Alb) as well as high concentration of glucose (HG) on MCP-1 production by cultured human mesangial cells.

METHODS

MCP-1 in serum or urine and urinary albumin (Alb) as well as several clinical parameters such as plasma glucose, serum Gly-Alb, and hemoglobin A1c (HbA1c) were measured after overnight fasting in 16 control subjects and 54 diabetic patients. The relationships between the levels of urinary Alb and urinary or serum MCP-1 and also between the values of respective clinical parameter and urinary MCP-1 levels were analyzed. Next, using cultured human mesangial cells, we investigated the role of Gly-Alb and/or HG on the gene and protein expression of MCP-1.

RESULTS

Urinary levels (ng/g creatinine), but not serum levels, of MCP-1 increased in accordance with the extent of albuminuria. In all subjects, there were significant correlations between the urinary levels of Alb and MCP-1 (r = 0.746, P < 0.0001) and between the levels of serum Gly-Alb and urinary MCP-1 (r = 0.475, P < 0.0001). In cultured human mesangial cells, the gene and protein expression of MCP-1 was dose and time dependently up-regulated by Gly-Alb. HG slightly but significantly stimulated MCP-1 expression. The combination of Gly-Alb and HG showed the greatest stimulation in more than an additive manner on MCP-1 production.

CONCLUSIONS

This study suggests that facilitated MCP-1 production by mesangial cells in diabetic milieu contributes to the initiation and progression of diabetic nephropathy.

A complex cell culture environment has been shown to maintain the differentiated state of hepatocytes, yet the mechanisms by which environmental cues selectively maintain liver-specific gene transcription have been unknown. In this paper we show that the hepatic environment regulates the activities of at least three liver-enriched transcription factors, eE-TF, eG-TF/HNF3, and eH-TF, that activate the mouse serum albumin enhancer. eE-TF is a heat-stable factor that has a DNA-binding specificity similar to that of the liver transcription factor C/EBP, but is a distinct protein. eG-TF/HNF3 contributes to the liver-specific transcription of several other serum protein genes. eH-TF binds to a TGTTTGC sequence that occurs at regulatory sites of the albumin promoter, the hepatitis B virus enhancer, and other hepatic genes. eE-TF, eG-TF/HNF3, and eH-TF are regulated by different combinations of the following cell culture conditions: a hormonally defined serum-free medium; an extracellular matrix gel; and a transformation-competent simian virus 40 large T antigen. We propose a regulatory network model to explain how cues from the cell lineage and the extracellular environment coordinately help maintain the activities of transcription factors involved in hepatocyte differentiation.

Hepatoid adenocarcinomas of the stomach are gastric carcinomas with both adenocarcinomatous and hepatocellular differentiations. They usually produce large amounts of alpha-fetoprotein (AFP) with a Concanavalin A-binding property of hepatic type. In this study, these carcinomas occurred in older persons, with the antrum being a common site. Observed grossly, growth of the tumors was nodular and massive. Prognosis was poor because of frequent liver metastases. In the cytoplasms of tumor cells, various serum proteins were identified, including AFP, alpha-1 antitrypsin (AAT), alpha-1 antichymotrypsin (ACT), albumin, and prealbumin. Localizations of ferritin, prothrombin, and transferrin were demonstrated with less frequency. Adenocarcinomatous foci were composed of well-differentiated, intestinal-type epithelial cells and often contained carcinoembryonic antigen. These adenocarcinomatous and hepatoid areas were often intermingled with each other. There were extensive venous involvements by tumor cells. The poor prognosis of the tumors may be attributed to these involvements as well as to production of AFP and presence of AAT/ACT, which have immunosuppressive and protease-inhibitory properties, respectively.

OBJECTIVE

Increased expression and activity of the lipogenic pathways in adipose tissue may contribute to the development of obesity. As a central enzyme in lipogenesis, the gene encoding fatty acid synthase (FASN) was identified as a candidate gene for determining body fat. In the present study we tested the hypothesis that increased FASN expression links metabolic alterations of excess energy intake, including hyperinsulinaemia, dyslipidaemia and altered adipokine profile to increased body fat mass.

METHODS

In paired samples of visceral and subcutaneous adipose tissue from 196 participants (lean or obese), we investigated whether FASN mRNA expression (assessed by PCR) in adipose tissue is increased in obesity and related to visceral fat accumulation, measures of insulin sensitivity (euglycaemic-hyperinsulinaemic clamp) and glucose metabolism.

RESULTS

FASN mRNA expression was increased by 1.7-fold in visceral vs subcutaneous fat. Visceral adipose tissue FASN expression was correlated with FASN protein levels, subcutaneous FASN expression, visceral fat area, fasting plasma insulin, serum concentrations of IL-6, leptin and retinol-binding protein 4 (RBP4), and inversely with measures of insulin sensitivity, independently of age, sex and BMI. Moreover, we found significant correlations between FASN expression and markers of renal function, including serum creatinine and urinary albumin excretion.

CONCLUSIONS

Increased FASN gene expression in adipose tissue is linked to visceral fat accumulation, impaired insulin sensitivity, increased circulating fasting insulin, IL-6, leptin and RBP4, suggesting an important role of lipogenic pathways in the causal relationship between consequences of excess energy intake and the development of obesity and type 2 diabetes.

A method which is capable of accurately determining low amounts of protein (i.e., 2-24 micrograms) in the presence of very high levels of lipid (i.e., 20-40 mg) has been developed. The procedure was developed from the original amido black 10B protein assay of Schaffner and Weissmann (W. Schaffner and C. Weissmann, 1973, Anal. Biochem. 56, 502-514) and incorporates several critical modifications that enable the assay to be performed with lipid-containing samples without interference. The modifications include a substantial increase in the assay volume (thereby decreasing the final lipid concentration) as well as the sodium dodecyl sulfate and trichloroacetic acid concentrations. Under these conditions, a linear standard curve is obtained with 2-24 micrograms of bovine serum albumin in both the absence and the presence of lipid (20 mg). Moreover, the assay is unaffected by as much as 40 mg of lipid in the original sample. Linearity as well as noninterference by lipid (20 mg) is also demonstrated with a sample of mitochondrial protein (i.e., a mixture of hydrophilic and hydrophobic proteins). Additionally, we show that in the presence of protein (20 micrograms) and lipid (20 mg), high concentrations of various buffers, salts, and nonionic detergents do not interfere with the assay. Finally, the enhanced ability of this new method to tolerate high lipid levels without interference relative to several existing protein estimation methods is demonstrated. This procedure should prove widely useful for measuring protein in reconstituted systems involving proteoliposomes.(ABSTRACT TRUNCATED AT 250 WORDS)

Renal impairment, as measured by reduced glomerular filtration rate (GFR) and increased urinary albumin excretion (UAE), is prevalent in patients with chronic heart failure (CHF) and is associated with reduced survival. The prevalence of structural tubular damage in CHF is unknown. We investigated 90 CHF patients and 20 age and sex matched healthy controls, and determined estimated GFR, UAE, N terminal-pro brain natriuretic peptide (NT-proBNP) and urinary neutrophil gelatinase associated lipocalin (NGAL) as a marker for tubular damage. CHF patients had significantly lower averaged estimated GFR (64+/-17 vs 90+/-12 mL/min/1.73 m(2), P<0.0001), but higher NT-proBNP and UAE levels (both P<0.0001). Median urinary NGAL levels were markedly increased in CHF patients compared to controls (175 (70-346) vs 37 (6-58) microg/gCr, P<0.0001). Both serum creatinine (r=0.26, P=0.006) and eGFR (r=-0.29, P=0.002) were significantly associated with urinary NGAL levels as were NT-proBNP and UAE but to a lesser extent. In conclusion, renal impairment in CHF patients is not only characterised by decreased eGFR and increased UAE, but also by the presence of tubular damage, as measured by increased urinary NGAL concentrations.

Thermosensitive polymer hydrogels that undergo a sol-to-gel transition in response to temperature changes are of great interest in therapeutic delivery and tissue engineering as injectable depot systems. A chitosan-based, injectable thermogel was prepared by grafting an appropriate amount of PEG onto the chitosan backbone and studied for drug release in vitro using bovine serum albumin (BSA) as a model protein. When more than approximately 40 wt.% of PEG was grafted to chitosan chains via covalent bonding, the aqueous solution of the resultant copolymer was an injectable liquid at low temperature and transformed to a semisolid hydrogel at body temperature. After an initial burst release in the first 5 h, a steady linear release of protein from the hydrogel was achieved for a period of approximately 70 h. Prolonged quasi-linear release of protein up to 40 days was achieved by crosslinking the hydrogel with genipin in situ, in a fashion suitable for protein encapsulation while maintaining the injectability of the hydrogel. The crosslinkage transformed the copolymer from a physical gel to an insoluble chemical gel and substantially reduced the initial burst release of protein. Both high performance liquid chromatography (HPLC) and gel electrophoresis indicated that the primary structure of BSA released from the hydrogels with or without genipin-crosslinking was generally conserved. The hydrogel can be prepared in solutions with a physiological pH, allowing the safe incorporation of bioactive molecules for a broad range of medical applications, particularly for sustained in vivo drug release and tissue engineering.

BACKGROUND

Markers of protein-energy wasting (PEW) and inflammation are common in chronic kidney disease (CKD) and are among the strongest predictors of mortality in dialysis patients.

OBJECTIVE

We hypothesized that markers of PEW and inflammation show similar associations in patients with non-dialysis-dependent CKD (NDD-CKD).

METHODS

We examined the associations of serum albumin, white blood cell (WBC) count, percentage of lymphocytes in WBCs (%LYM), and a combination of all 3 with all-cause mortality and with the composite of predialysis mortality or end-stage renal disease (ESRD) by using fixed-covariate and time-dependent Cox models in 1220 men with NDD-CKD.

RESULTS

Lower albumin and %LYM and a higher WBC count were significantly associated with outcomes. In time-dependent Cox models, compared with patients in whom none of these markers indicated PEW, those in whom 1, 2, or all 3 markers indicated the presence of PEW had multivariable-adjusted hazard ratios (95% CI) for all-cause mortality of 1.7 (1.2, 2.4), 2.4 (1.7, 3.4), and 3.6 (2.5, 5.1); the P for trend was <0.001. Similar associations were present in fixed-covariate models for all-cause mortality and in fixed-covariate and time-dependent models for the composite outcome.

CONCLUSIONS

Traditional and nontraditional markers of PEW display robust, strong, and independent associations with mortality in patients with NDD-CKD. Clinical trials are warranted to examine whether PEW-improving interventions can lead to better outcomes in these patients.