The full potential of diagnostic PCR is limited, in part, by the presence of inhibitors in complex biological samples that reduce the amplification efficiency. Therefore, different pre-PCR treatments are being used to reduce the effects of PCR inhibitors. The aim of the present study was to investigate the effects of 16 amplification facilitators to enhance DNA amplification in the presence of blood, feces, or meat. Different concentrations of amplification facilitators and inhibitory samples were added to PCR mixtures containing rTth or Taq DNA polymerase. The addition of 0.6% (wt/vol) bovine serum albumin to reaction mixtures containing Taq DNA polymerase reduced the inhibitory effect of blood and allowed DNA amplification in the presence of 2% instead of 0.2% (vol/vol) blood. Furthermore, the addition of bovine serum albumin (BSA) to reaction mixtures containing feces or meat enhanced the amplification capacities of both polymerases. Taq DNA polymerase was able to amplify DNA in the presence of 4% instead of 0.4% (vol/vol) feces and 4% instead of 0.2% (vol/vol) meat, and rTth was able to amplify DNA in the presence of 4% instead of 0.4% (vol/vol) feces and 20% instead of 2% (vol/vol) meat. The single-stranded DNA binding T4 gene 32 protein (gp32) had a relieving effect similar to that of BSA, except when it was added to PCR mixtures of rTth containing meat and of Taq DNA polymerase containing feces. The relieving effects of betaine and a cocktail of proteinase inhibitors were more sample specific. The addition of 11.7% (wt/vol) betaine allowed Taq DNA polymerase to amplify DNA in the presence of 2% (vol/vol) blood, while the addition of proteinase inhibitors allowed DNA amplification by both polymerases in the presence of 4% (vol/vol) feces. When various combinations of betaine, BSA, gp32, and proteinase inhibitors were tested, no synergistic or additive effects were observed. The effects of facilitators on real-time DNA synthesis instead of conventional PCR were also studied.

BACKGROUND

Dialysis access is critical for therapy delivery. Few studies have linked type of dialysis access to patient survival in the elderly population.

METHODS

We included 1995 to 1997 incidence Medicare hemodialysis patients (N = 66,595) who were 67 years and older at dialysis therapy initiation. Medicare Physician/Supplier claims were used to determine initial access type: simple fistula, autologous vein graft, synthetic graft, and hemodialysis catheter. We used International Classification of Diseases, Ninth Revision, Clinical Modification, codes to determine vascular access placement for renal failure. A Cox regression analysis assessed risk for death within 1 year, with explanatory variables of incidence year, age, sex, race, diabetes, initial access type, body mass index, days from first access placement date to initial dialysis date, and serum albumin, creatinine, and blood urea nitrogen levels.

RESULTS

One-year crude death rates were 24.9%, 27.2%, 28.1%, and 41.5% for patients with simple fistulae, autologous vein grafts, synthetic grafts, and hemodialysis catheters, respectively. Patients with simple fistulae (the reference) had the lowest (P < 0.0001) likelihood of death compared with those with synthetic grafts (hazard ratio [HR], 1.160; 95% confidence interval [CI], 1.084 to 1.241) or catheters (HR, 1.696; 95% CI, 1.593 to 1.806). No difference (P>> 0.09) in mortality risk was detected between simple fistulae and autologous vein grafts or between autologous vein grafts and synthetic grafts.

CONCLUSIONS

In the US Medicare dialysis population, type of initial hemodialysis access was associated with 1-year mortality. Mortality risks were (in ascending order) fistulae, grafts, and catheters.

OBJECTIVE

To identify novel genes involved in osteoarthritis (OA), by means of a genome-wide association study.

METHODS

We tested 500,510 single-nucleotide polymorphisms (SNPs) in 1,341 Dutch Caucasian OA cases and 3,496 Dutch Caucasian controls. SNPs associated with at least 2 OA phenotypes were analyzed in 14,938 OA cases and approximately 39,000 controls. Meta-analyses were performed using the program Comprehensive Meta-analysis, with P values <1 x 10(-7) considered genome-wide significant.

RESULTS

The C allele of rs3815148 on chromosome 7q22 (minor allele frequency 23%; intron 12 of the COG5 gene) was associated with a 1.14-fold increased risk (95% confidence interval 1.09-1.19) of knee and/or hand OA (P = 8 x 10(-8)) and also with a 30% increased risk of knee OA progression (95% confidence interval 1.03-1.64) (P = 0.03). This SNP is in almost complete linkage disequilibrium with rs3757713 (68 kb upstream of GPR22), which is associated with GPR22 expression levels in lymphoblast cell lines (P = 4 x 10(-12)). Immunohistochemistry experiments revealed that G protein-coupled receptor protein 22 (GPR22) was absent in normal mouse articular cartilage or synovium. However, GPR22-positive chondrocytes were found in the upper layers of the articular cartilage of mouse knee joints that were challenged with in vivo papain treatment or methylated bovine serum albumin treatment. GPR22-positive chondrocyte-like cells were also found in osteophytes in instability-induced OA.

CONCLUSIONS

Our findings identify a novel common variant on chromosome 7q22 that influences susceptibility to prevalence and progression of OA. Since the GPR22 gene encodes a G protein-coupled receptor, this is potentially an interesting therapeutic target.

A recombinant human TNF receptor Fc fusion protein (rhuTNFR:Fc) was assessed for antiarthritic activity using murine type II collagen-induced arthritis in mice. DBA/1 mice were immunized with bovine type II collagen and treated with rhuTNFR:Fc either from day 21 to day 28 (preventative protocol), or after disease onset for fourteen days (therapeutic protocol). Control mice received either sterile saline or human serum albumin injections. rhuT-NFR:Fc treatment significantly reduced both the incidence and the severity of collagen-induced arthritis in the preventative protocol. Mice receiving rhuTNFR:Fc therapeutically progressed to less severe disease than did control animals, and the arthritis index in rhuTNFR:Fc treated mice was significantly lower than the index in control mice from 7.5 weeks after treatment. The antibody response to collagen was significantly reduced by treatment with rhuTNFR:Fc in both the preventative and therapeutic protocols. No difference was observed in the proliferative response to type II collagen or Con A, but the response to LPS was significantly lower in rhuTNFR:Fc treated mice at the conclusion of both the preventative and therapeutic trials. The results suggest that rhuTNFR:Fc may have both immunosuppressive and antiarthritic properties in this experimental model, and may represent a useful approach to the treatment of autoimmune arthritis.

Though several risk factors for the development of hepatotoxicity due to antituberculosis drugs have been suggested, involvement of genetic factors is not fully established. We have studied the major histocompatibility complex (MHC) class II alleles and clinical risk factors for the development of hepatotoxicity in 346 North Indian patients with tuberculosis undergoing antituberculosis treatment. Of these, 56 patients developed drug-induced hepatotoxicity (DIH group), whereas the remaining 290 patients did not (non-DIH group). The DIH group was comparatively older, had lower pretreatment serum albumin, and a higher frequency of moderately/far advanced disease radiographically than the latter. Further, patients with high alcohol intake had threefold higher odds of developing hepatotoxicity. In multivariate logistic regression analysis, older age (odds ratio [OR] 1.2), moderately/far advanced disease (OR 2.0), serum albumin < 3.5 g/dl (OR 2.3), absence of HLA-DQA1*0102 (OR 4.0), and presence of HLA-DQB1*0201 (OR 1.9) were independent risk factors for DIH. Our results suggest that the risk of hepatotoxicity from antituberculosis drugs is influenced by clinical and genetic factors.

Isolated livers of normal and hypophysectomized (hypox) rats with or without GH replacement therapy were perfused in an erythrocyte-free recirculating perfusion system for 4 h in the presence of [35S]cysteine. Albumin secretion and synthesis increased in a parallel and linear fashion over 4 h. The albumin secretion rates were 0.53 and 0.21 mg/g liver h-1 in normal and hypox animals, respectively. Insulin-like growth factor (IGF) secretion, measured as insulin equivalents in the fat cell assay as well as in a competitive protein binding assay, and IGF synthesis, as determined from [35S]cysteine incorporation into immunoprecipitable IGF, likewise increased linearly and in parallel throughout the perfusion time. The IGF secretion rate was 50 microU/g liver h-1. The secreted IGF had a molecular weight of approximately 7700 daltons. Secretion and synthesis of IGF were reduced to 11% in hypox rats and were largely restored by human GH replacement therapy (to 86% of normal). A single specific binding protein with an approximate molecular weight of 35,000 was detected in the perfusate. The binding protein was measured by covalent cross-linkage to [125I]IGF I by dimethylsuberimidate. The secretion of this binding protein was 62% of normal in hypox animals and 79% in GH-treated hypox rats. The data suggest that IGF is continuously synthesized and released by the liver. Assuming a half-life for IGF of 3 h in the normal rat, a plasma volume of 8 ml, and a liver weight of 8.5 g, the rate of IGF production by the perfused normal rat liver (50 microU/g liver h-1) would be sufficient to maintain serum IGF at the concentration determined in normal rat serum (approximately 130 microU/ml). This suggests that the liver is the major site of IGF production in the rat.

Molecular recognition between biotinylated bovine serum albumin and polyclonal, biotin-directed IG antibodies has been measured directly under various buffer conditions using an atomic force microscope (AFM). It was found that even highly structured molecules such as IgG antibodies preserve their specific affinity to their antigens when probed with an AFM in the force mode. We could measure the rupture force between individual antibody-antigen complexes. The potential and limitations of this new approach for the measurement of individual antigen/antibody interactions and some possible applications are discussed.

The extracellular matrix (ECM) promotes tissue morphogenesis, cell migration, and the differentiation of a variety of cell types. However, the mechanisms by which ECM causes differentiated gene expression have been unknown. In this report, we show that culturing the hepatocyte-derived cell line H2.35 on an ECM gel changes cell morphology and selectively stimulates the transcription of a subset of liver-specific genes, including serum albumin. Transcriptional activation by ECM also occurs with transfected plasmids bearing the transcriptional enhancer of the albumin gene. ECM substrates of different composition activated the albumin enhancer only when the ECM promoted a cuboidal, differentiated cell morphology. Enhancer activation by the ECM was mediated by two liver transcription factors, HNF3 alpha and eH-TF, which appear to be regulated differently by matrix. Specifically, we found that a collagen gel substratum caused a selective increase in the factor HNF3 alpha at the levels of mRNA accumulation and DNA-binding activity in nuclear extracts, both in H2.35 cells and in the hepatoma cell line HepG2. We conclude that the ECM can stimulate cell differentiation by selectively activating transcriptional regulatory factors and that such regulation occurs coordinately with ECM-promoted changes in cell shape.

Regulation of serum anti-DNA antibody in systemic lupus erythematosus (SLE) by an antiidiotypic antibody was evaluated. Various sera from SLE patients in active and inactive states of their disease, as well as sera from normal individuals, were first completely depleted of anti-DNA and of DNA by affinity chromatography. The suppressive capacity of equimolar concentrations of the various depleted sera (blocking sera) on target lupus sera were determined. The target sera were from lupus patients with known DNA-binding capacity. Blocking sera from inactive SLE suppressed the binding of autologous anti-DNA antibody to [(3)H]DNA (n = 19,P < 0.01). Blocking sera from active SLE (n = 19), as well as human serum albumin, did not suppress. Sera from normal donors who had no contact with lupus patients or with lupus sera did not suppress (n = 14, P>> 0.5), whereas those from normal donors who had contact with lupus patients or sera did suppress the binding (n = 5,P < 0.02). The anti-anti-DNA antibody suppressive activity in the inactive lupus serum was shown to be localized within the F(ab')(2) portion of immunoglobulin (Ig)G and could not be removed upon adsorption by normal human gammaglobulin. Furthermore, immune complexes could be detected by a Clq binding assay when the inactive lupus blocking sera were incubated with the anti-DNA antibody containing target sera. The specificity of the suppressive serum factor was shown by its inability to block the binding of tetanus toxoid to antitetanus antibody and its ability to block the binding of DNA to F(ab')(2) fragments of active lupus IgG. Regulation of serum anti-DNA antibody levels by anti-antibodies could induce and maintain disease remission in lupus patients and prevent disease expression in normals.

A new apparatus is described which serves to investigate the in vitro antibacterial activity of antibiotics as a function of different concentration time curves. The apparatus can be adjusted to simulate the biexponential serum level curves observed in vivo after oral or intramuscular administration. Preliminary studies were carried out with a cephalosporin derivative, cefazolin, against Escherichia coli and Klebsiella sp. strains simulating initial concentrations of 5, 10, and 20 mug/ml that decreased exponentially with half-lives of 30, 60, and 120 min. Surviving cells were counted at 1-h intervals for 10 h. In all the situations tested there was an initial phase of rapid bactericidal activity followed by a phase of bacteriostatic activity, whose length depended on the drug elimination rate but was relatively independent of the initial concentrations. Bacterial regrowth occurred when the antibiotic concentration fell below the minimum inhibitory concentration of the drug against the strains tested. The antibacterial activity of cefazolin, cephacetrile, and cephradine against E. coli and Klebsiella strains was also investigated, in a medium containing 4% human albumin, simulating the serum level curves observed in humans after an intramuscular dose of 1 g. The results obtained suggest that, for cephalosporins, a longer half-life might be more useful than higher peak levels.

Collectins are C-type animal lectins with both collagenous and carbohydrate recognition domains and are involved in the first line host defense against pathogens. We report here a novel Ca(2+)-dependent and GlcNAc-binding lectin consisting of subunits of 35 kDa (P35) with a collagen-like sequence. When P35 is isolated from human serum, it forms a homopolymer by means of intermolecular disulfide bonding, as is the case with collectins. P35 cDNA was cloned from a human liver cDNA library, and the deduced amino acid sequence of 313 residues revealed that the mature form of P35 consists mainly of collagen- and fibrinogen-like domains. The latter contained two potential Ca(2+)-binding sites that may be involved in carbohydrate binding. The overall sequence of P35 was highly homologous to porcine ficolins alpha and beta. Northern blots of various human tissues showed that the major product of the 1.3-kilobase-long P35 transcript is expressed in liver. P35 enhanced phagocytosis of Salmonella typhimurium by neutrophils, suggesting an opsonic effect via the collagen region. P35 was found to bind to GlcNAc-conjugated bovine serum albumin, a neoglycoprotein, as well as to neoglycolipids containing complex-type oligosaccharides derived from glycoproteins, suggesting that P35 recognizes GlcNAc residues such as those found in microbial glycoconjugates and complex-type oligosaccharides. Therefore, P35 represents a new type of GlcNAc-binding lectin with structural and functional similarities to collectins involved in innate immunity.

The protein content of human serum is composed of a millieu of proteins from almost every type of cell and tissue within the body. The serum proteome has been shown to contain information that directly reflects pathophysiological states and represents an invaluable source of diagnostic information for a variety of different diseases. Unfortunately, the dynamic range of protein abundance, ranging from>>) mg/mL level to (< pg/mL level, renders complete characterization of this proteome nearly impossible with current analytical methods. To study low-abundance proteins, which have potential value for clinical diagnosis, the high-abundant species, such as immunoglobulins and albumin, are generally eliminated as the first step in many analytical protocols. This step, however, is hypothesized to concomitantly remove proteins/peptides associated with the high-abundant proteins targeted for depletion. In this study, immunoprecipitation was combined with microcapillary reversed-phase liquid chromatography (microRPLC) coupled on-line with tandem mass spectrometry (MS/MS) to investigate the low-molecular-weight proteins/peptides that associate with the most abundant species in serum. By this targeted isolation of select highly abundant serum proteins, the associated proteins/peptides can be enriched and effectively identified by microRPLC-MS/MS. Among the 210 proteins identified, 73% and 67% were not found in previous studies of the low-molecular-weight or whole-serum proteome, respectively.

Nanoscale carbon materials hold great promise for biotechnological and biomedical applications. Fluorescent nanodiamond (FND) is a recent new addition to members of the nanocarbon family. Here, we report long-term in vivo imaging of FNDs in Caenorhabditis elegans (C. elegans) and explore the nano-biointeractions between this novel nanomaterial and the model organism. FNDs are introduced into wild-type C. elegans by either feeding them with colloidal FND solution or microinjecting FND suspension into the gonads of the worms. On feeding, bare FNDs stay in the intestinal lumen, while FNDs conjugated with biomolecules (such as dextran and bovine serum albumin) are absorbed into the intestinal cells. On microinjection, FNDs are dispersed in the gonad and delivered to the embryos and eventually into the hatched larvae in the next generation. The toxicity assessments, performed by employing longevity and reproductive potential as physiological indicators and measuring stress responses with use of reporter genes, show that FNDs are stable and nontoxic and do not cause any detectable stress to the worms. The high brightness, excellent photostability, and nontoxic nature of the nanomaterial have enabled continuous imaging of the whole digestive system and tracking of the cellular and developmental processes of the living organism for several days.

A cohort of 432 ESRD (261 hemodialysis and 171 peritoneal dialysis) patients was followed prospectively for an average of 41 months. Baseline and annual demographic, clinical and echocardiographic assessments were performed, as well as serial clinical and laboratory tests measured monthly while on dialysis therapy. The average mean arterial blood pressure level during dialysis therapy was 101 +/- 11 mm Hg. After adjusting for age, diabetes and ischemic heart disease, as well as hemoglobin and serum albumin levels measured serially, each 10 mm Hg rise in mean arterial blood pressure was independently associated with: the presence of concentric LV hypertrophy (OR 1.48, P = 0.02), the change in LV mass index (beta = 5.4 g/m2, P = 0.027) and cavity volume (beta = 4.3 ml/m2, P = 0.048) on follow-up echocardiography, the development of de novo cardiac failure (RR 1.44, P = 0.007), and the development of de novo ischemic heart disease (RR 1.39, P = 0.05). The association with LV dilation was of borderline statistical significance (OR 1.48, P = 0.06). Mean arterial blood pressures greater than 106 mm Hg were associated with both echocardiographic and clinical endpoints. Paradoxically, low mean arterial blood pressure (RR 1.36 per 10 mm Hg fall, P = 0.009) was independently associated with mortality. The association of low blood pressure with mortality was a marker for having had cardiac failure prior to death. We conclude that even moderate hypertension worsens the echocardiographic and clinical outcome in ESRD patients, especially in those without previous clinical cardiac disease.

BACKGROUND

Current methods to predict survival duration of patients with pancreatic cancer are limited. The aim of this study was to determine whether certain nutritional indices and the acute-phase protein response are prognostic factors independent of disease stage for patients with unresectable pancreatic cancer.

METHODS

Variables at the time of diagnosis of 102 patients with unresectable pancreatic cancer were entered into a Cox's proportional hazards model. Included in the analysis were the serum concentration of C-reactive protein (CRP) and albumin, the extent of weight loss, age, sex, and disease stage (International Union Against Cancer criteria).

RESULTS

A multivariate analysis in which each factor was adjusted for the influence of the other factors revealed the patient age, disease stage, serum albumin, and serum CRP to be independent predictors of survival. The presence of an acute-phase protein response was the most significant independent predictors of survival duration. The median survival of those with an acute-phase protein response (CRP>> 10 mg/L, n = 45) was 66 days compared with 222 days for those with no acute-phase protein response (n = 57, P = 0.001, Mann-Whitney U test).

CONCLUSIONS

The acute-phase protein response is a useful prognostic indicator for patients with unresectable pancreatic cancer. Moreover, the metabolic disturbances associated with an acute-phase protein response of patients with pancreatic cancer may be a worthwhile therapeutic target.

OBJECTIVE

To determine the involvement of interleukin-1 (IL-1), tumor necrosis factor (TNF), and IL-6 in the cartilage pathology of murine antigen-induced arthritis (AIA) and zymosan-induced arthritis (ZIA).

METHODS

Arthritis was induced by intraarticular injection of zymosan in naive mice or by subcutaneous injection of methylated bovine serum albumin in sensitized animals. Mini-osmotic pumps releasing human recombinant IL-1 receptor antagonist (IL-1ra) protein were implanted intraperitoneally 2 days before arthritis induction, and neutralizing antibodies directed against murine IL-1 alpha, IL-1 beta, TNF alpha, or IL-6 were administered 1 day before. Proteoglycan (PG) synthesis and degradation were assessed in patellar cartilage.

RESULTS

Murine IL-1 alpha and IL-1 beta injected intraarticularly at doses of 0.1-100 ng suppressed chondrocyte PG synthesis. The highest dose of TNF tested (100 ng) decreased PG synthesis marginally. In contrast, the maximum dose of IL-6 (1 microgram) stimulated PG synthesis 2 days after injection. Treatment of AIA with neutralizing monoclonal antibodies against either TNF alpha or IL-6 did not reduce either the PG degradation or the suppression of its synthesis. However, treatment with anti-IL-1 (alpha + beta) polyclonal antibodies totally prevented PG suppression, although the initial breakdown of PG was unaffected. This effect was confirmed when IL-1ra was administered in high doses. Moreover, treatment of ZIA with anti-IL-1 (alpha + beta), but not with anti-TNF, resulted in normal PG synthesis, confirming the key role played by IL-1 in the inhibition of PG synthesis. Treatment of AIA with anti-IL-1 did not affect inflammation during the acute phase, but a significant reduction of ongoing inflammation was noted at day 7, and there was a marked reduction in the loss of cartilage PG.

CONCLUSIONS

The suppression of PG synthesis in both ZIA and AIA in mice is due to the combined local action of IL-1 (alpha + beta), and neither IL-6 nor TNF is involved. Moreover, the normalization of PG synthesis brought about by blocking of IL-1 ameliorates the cartilage damage associated with AIA.

The preferential interactions of proteins with solvent components were studied in concentrated salt by densimetric measurements. Proteins were found to be preferentially hydrated in NaCl, NaCH3COO, and Na2SO4. The resulting unfavorable free-energy change was related to the effects of these salts on solubility and stability of the proteins. This unfavorable free-energy change was correlated with the large, positive surface tension increment of these salts, i.e., their perturbation of surface free energy. On the other hand, KSCN, CaCl2, and MgCl2 showed considerable binding to bovine serum albumin, which could be related to their destabilizing and salting-in effects on macromolecules. Since the last two salts have high surface tension increments, it was concluded that this does not necessarily lead to protein preferential hydration and stabilization.

The authors measured ionic current blockages caused by protein translocation through voltage-biased silicon nitride nanopores in ionic solution. By calculating the mean amplitude, time duration, and the integral of current blockages, they estimated the relative charge and size of protein molecules at a single molecule level. The authors measured the change in protein charge of bovine serum albumin (BSA) protein induced by pH variation. They also confirmed that BSA molecules indeed traverse nanopores using an improved chemiluminescent analysis. They demonstrated that a larger protein fibrinogen could be distinguished from BSA by a solid-state nanopore measurement.

Human serum albumin (HSA), the most abundant protein in plasma, has been proposed to have an antioxidant role. The main feature responsible for this property is its only thiol, Cys34, which comprises approximately 80% of the total free thiols in plasma and reacts preferentially with reactive oxygen and nitrogen species. Herein, we show that the thiol in HSA reacted with hydrogen peroxide with a second-order rate constant of 2.26 M(-1) s(-1) at pH 7.4 and 37 degrees C and a 1:1 stoichiometry. The formation of intermolecular disulfide dimers was not observed, suggesting that the thiol was being oxidized beyond the disulfide. With the reagent 7-chloro-4-nitrobenzo-2-oxa-1,3-diazol (NBD-Cl), we were able to detect the formation of sulfenic acid (HSA-SOH) from the UV-vis spectra of its adduct. The formation of sulfenic acid in Cys34 was confirmed by mass spectrometry using 5,5-dimethyl-1,3-cyclohexanedione (dimedone). Sulfenic acid was also formed from exposure of HSA to peroxynitrite, the product of the reaction between nitric oxide and superoxide radicals, in the absence or in the presence of carbon dioxide. The latter suggests that sulfenic acid can also be formed through free radical pathways since following reaction with carbon dioxide, peroxynitrite yields carbonate radical anion and nitrogen dioxide. Sulfenic acid in HSA was remarkably stable, with approximately 15% decaying after 2 h at 37 degrees C under aerobic conditions. The formation of glutathione disulfide and mixed HSA-glutathione disulfide was determined upon reaction of hydrogen peroxide-treated HSA with glutathione. Thus, HSA-SOH is proposed to serve as an intermediate in the formation of low molecular weight disulfides, which are the predominant plasma form of low molecular weight thiols, and in the formation of mixed HSA disulfides, which are present in approximately 25% of circulating HSA.

In order to discover novel protein markers indicative of disease processes or drug effects, the proteomics technology platform most commonly used consists of high resolution protein separation by two-dimensional electrophoresis (2-DE), mass spectrometric identification of proteins from stained gel spots and a bioinformatic data analysis process supported by statistics. This approach has been more successful in profiling proteins and their disease- or treatment-related quantitative changes in tissue homogenates than in plasma samples. Plasma protein display and quantitation suffer from several disadvantages: very high abundance of a few proteins; high heterogeneity of many proteins resulting in long charge trains; crowding of 2-DE separated protein spots in the molecular mass range between 45-80 kD and in the isoelectric point range between 4.5 and 6. Therefore, proteomic technologies are needed that address these problems and particularly allow accurate quantitation of a larger number of less abundant proteins in plasma and other body fluids. The immunoaffinity-based protein subtraction chromatography (IASC) described here removes multiple proteins present in plasma and serum in high concentrations effectively and reproducibly. Applying IASC as an upfront plasma sample preparation process for 2-DE, the protein spot pattern observed in gels changes dramatically and at least 350 additional lower abundance proteins are visualized. Affinity-purified polyclonal antibodies (pAbs) are the immunoaffinity reagents used to specifically remove the abundant proteins such as albumin, immunoglobulin G, immunoglobulin A, transferrin, haptoglobin, alpha-1-antitrypsin, hemopexin, transthyretin, alpha-2-HS glycoprotein, alpha-1-acid glycoprotein, alpha-2-macroglobulin and fibrinogen from human plasma samples. To render the immunoaffinity subtraction procedure recyclable, the pAbs are immobilized and cross-linked on chromatographic matrices. Antibody-coupled matrices specific for one protein each can be pooled to form mixed-bed IASC columns. We show that up to ten affinity-bound plasma proteins with similar solubility characteristics are eluted from a mixed-bed column in one step. This facilitates automated chromatographic processing of plasma samples in high throughput, which is desirable in proteomic disease marker discovery projects.

OBJECTIVE

The advent of tumor necrosis factor (TNF)-blocking drugs has provided rheumatologists with an effective, but highly expensive, treatment for the management of established rheumatoid arthritis (RA). Our aim was to explore preclinically the application of camelid anti-TNF VHH proteins, which are single-domain antigen binding (VHH) proteins homologous to human immunoglobulin V(H) domains, as TNF antagonists in a mouse model of RA.

METHODS

Llamas were immunized with human and mouse TNF, and antagonistic anti-TNF VHH proteins were isolated and cloned for bacterial production. The resulting anti-TNF VHH proteins were recombinantly linked to yield bivalent mouse and human TNF-specific molecules. To increase the serum half-life and targeting properties, an anti-serum albumin anti-TNF VHH domain was incorporated into the bivalent molecules. The TNF-neutralizing potential was analyzed in vitro. Mouse TNF-specific molecules were tested in a therapeutic protocol in murine collagen-induced arthritis (CIA). Disease progression was evaluated by clinical scoring and histologic evaluation. Targeting properties were evaluated by 99mTc labeling and gamma camera imaging.

RESULTS

The bivalent molecules were up to 500 times more potent than the monovalent molecules. The antagonistic potency of the anti-human TNF VHH proteins exceeded even that of the anti-TNF antibodies infliximab and adalimumab that are used clinically in RA. Incorporation of binding affinity for albumin into the anti-TNF VHH protein significantly prolonged its serum half-life and promoted its targeting to inflamed joints in the murine CIA model of RA. This might explain the excellent therapeutic efficacy observed in vivo.

CONCLUSIONS

These data suggest that because of the flexibility of their format, camelid anti-TNF VHH proteins can be converted into potent therapeutic agents that can be produced and purified cost-effectively.

OBJECTIVE

Systemic inflammatory state is a hallmark of peritoneal dialysis (PD) patients, but its etiology remains obscure. Because circulating microbial products are an important cause of systemic immune activation in other conditions such as HIV infection, it was hypothesized that endotoxemia is a cause of systemic inflammatory state and atherosclerosis in PD patients.

METHODS

Plasma lipopolysaccharide (LPS) levels in 30 consecutive new PD patients were measured. The result was compared with serum C-reactive protein (CRP) level, peritoneal transport status, history of pre-existing cardiovascular diseases, and carotid intima media thickness (IMT) by Doppler ultrasound.

RESULTS

Among the 30 PD patients, there were 17 men. The average age was 53.7 +/- 15.1 yr. The average endotoxin concentration of PD patients was 0.44 +/- 0.18 EU/ml, which was significantly higher than that of patients with chronic kidney disease secondary to Ig-A nephropathy (IgAN) (0.035 +/- 0.009 EU/ml, P < 0.0001) and the controls (0.013 +/- 0.007 EU/ml, P < 0.0001). In PD patients, plasma LPS concentration had a significant correlation with serum CRP (r = 0.415, P = 0.025) and serum albumin level (r = -0.394, P = 0.034). In contrast, plasma LPS level did not correlate with Charlson's Comorbidity Index, peritoneal transport characteristics, or nutritional indices. Patients with pre-existing cardiovascular disease (CVD) had higher plasma LPS level than those without CVD (0.53 +/- 0.19 versus 0.36 +/- 0.16 EU/ml, P = 0.016). Plasma LPS level correlated with carotid IMT (r = 0.438, P = 0.016).

CONCLUSIONS

It was found that endotoxemia was probably common in PD patients, and the degree of circulating endotoxemia might be related to the severity of systemic inflammation and features of atherosclerosis. This result suggests that endotoxemia may have a contributory role to the systemic inflammatory state and accelerated atherosclerosis in PD patients.

Mortality is markedly elevated in patients with end-stage renal disease. The leading cause of death is cardiovascular disease. Lipoprotein levels are only slightly elevated in dialysis patients, and cardiovascular risk is inversely correlated with serum cholesterol, suggesting that a process other than hyperlipidemia plays a role in the incidence of cardiovascular disease. Hypoalbuminemia, ascribed to malnutrition, has been one of the most powerful risk factors that predict all-cause and cardiovascular mortality in dialysis patients. The presence of inflammation, as evidenced by increased levels of specific cytokines (interleukin-6 and tumor necrosis factor alpha) or acute-phase proteins (C-reactive protein and serum amyloid A), however, has been found to be associated with vascular disease in the general population as well as in dialysis patients. The process of inflammation, also called the acute-phase response, additionally causes loss of muscle mass and changes in plasma composition-decreases in serum albumin, prealbumin, and transferrin levels, also associated with malnutrition. Inflammation alters lipoprotein structure and function as well as endothelial structure and function to favor atherogenesis and increases the concentration of atherogenic proteins in serum, such as fibrinogen and lipoprotein (a). Inflammation in dialysis patients is episodic. The causes are likely to be multifactorial and include vascular access infection, less-than-sterile dialysate, dialysate back leak, and nonbiocompatible membranes in addition to clinically apparent infection. In addition, proinflammatory compounds, such as advanced glycation end products, accumulate in renal failure, and defense mechanisms against oxidative injury are reduced, contributing to inflammation and to its effect on the vascular endothelium.

Human embryonic stem cells (hESC) may provide a cell source for functional hepatocytes. The aim of this study is to establish a viable human hepatocyte-like cell line from hESC that can be used for cell-based therapies. The differentiated hESC were enriched by transducing with a lentivirus vector containing the green fluorescent protein (GFP) gene driven by the alpha1-antitrypsin promoter; the GFP gene is expressed in committed hepatocyte progenitors and hepatocytes. GFP+ hESC were purified by laser microdissection and pressure catapulting. In addition, differentiated hESC that were transduced with a lentivirus triple-fusion vector were transplanted into NOD-SCID mice, and the luciferase-induced bioluminescence in the livers was evaluated by a charge-coupled device camera. GFP+ hESC expressed a large series of liver-specific genes, and expression levels of these genes were significantly improved by purifying GFP+ hESC; our results demonstrated that purified differentiated hESC express nearly physiological levels of liver-specific genes and have liver-specific functions that are comparable to those of primary human hepatocytes. The differentiated hESC survived and engrafted in mouse livers, and human liver-specific mRNA and protein species were detected in the transplanted mouse liver and serum at 3 weeks after transplantation. This is the first time that human albumin generated by hESC-derived hepatocytes was detected in the serum of an animal model. This also represents the first successful transplantation of differentiated hESC in an animal liver and the first bioluminescence imaging of hESC in the liver. This study is an initial step in establishing a viable hepatocyte-like cell line from hESC. Disclosure of potential conflicts of interest is found at the end of this article.