BACKGROUND

Infection is second to cardiovascular disease as a cause of death in patients with end-stage renal disease (ESRD), and septicemia causes a majority of these infectious deaths. To identify patients at high risk and to characterize modifiable risk factors for septicemia, we examined the incidence, risk factors, and prognosis for septicemia in a large, representative group of U.S. dialysis patients.

METHODS

We conducted a longitudinal cohort study of incident ESRD patients in the case-mix study of the U.S. Renal Data System with seven years of follow-up from hospitalization and death records. Poisson regression was used to examine independent risk factors for hospital-managed septicemia. Cox proportional hazards analysis was used to assess the independent effect of septicemia on all-cause mortality and on death from septicemia. Separate analyses were performed for patients on peritoneal dialysis (PD) and hemodialysis (HD).

RESULTS

Over seven years of follow-up, 11.7% of 4005 HD patients and 9.4% of 913 PD patients had at least one episode of septicemia. Older age and diabetes were independent risk factors for septicemia in all patients. Among HD patients, low serum albumin, temporary vascular access, and dialyzer reuse were also associated with increased risk. Among PD patients, white race and having no health insurance at dialysis initiation were also risk factors. Patients with septicemia had twice the risk of death from any cause and a fivefold to ninefold increased risk of death from septicemia.

CONCLUSIONS

Septicemia, which carries a marked increased risk of death, occurs frequently in patients on PD as well as HD. Early referral to a nephrologist, improving nutrition, and avoiding temporary vascular access may decrease the incidence of septicemia. Further study of how race, insurance status, and dialyzer reuse can contribute to the risk of septicemia among ESRD patients is indicated.

The gonadal steroid estrogen exerts an important modulatory influence on the activity of multiple neuronal networks. In addition to classical genomic mechanisms of action, estrogen also exerts poorly understood rapid, nongenomic effects on neurons. To examine whether estrogen may exert rapid actions on intracellular signaling within gonadotropin-releasing hormone (GnRH) neurons in vivo,we examined the phosphorylation status of cAMP response element-binding protein (CREB) in these cells after the administration of 17-beta-estradiol to ovariectomized (OVX) mice. The percentage of GnRH neurons expressing phosphorylated CREB was increased more than sixfold (p < 0.05) in a time- and dose-dependent manner by estrogen, with the increase first observed 15 min after estrogen administration. A series of in vitro studies demonstrated that estrogen acted directly on native GnRH neurons to phosphorylate CREB, but that estrogen conjugated to bovine serum albumin was without effect. The role of classical estrogen receptors (ERs) was evaluated using ER knock-out mice in vivo. The effect of estrogen on CREB phosphorylation in GnRH neurons was normal in ERalpha knock-out mice but completely absent in ERbeta knock-out mice. Finally, studies in intact female mice revealed levels of CREB phosphorylation within GnRH neurons that were equivalent to those of estrogen-treated OVX mice. These observations demonstrate that ERbeta mediates the rapid, direct effects of estrogen on the GnRH neuronal phenotype, and that these actions persist under physiological conditions. They also provide the first evidence for a role of ERbeta in nongenomic estrogen signaling within the brain in vivo.

BACKGROUND

Optimal levels of vitamin D have been a topic of heavy debate, and the correlation between 25-hydroxyvitamin D [25(OH)D] levels and mortality still remains to be established.

OBJECTIVE

The aim of the study was to determine the association between all-cause mortality and serum levels of 25(OH)D, calcium, and PTH.

METHODS

We conducted a retrospective, observational cohort study, the CopD Study, in a single laboratory center in Copenhagen, Denmark.

METHODS

Serum 25(OH)D was analyzed from 247,574 subjects from the Copenhagen general practice sector. In addition, serum levels of calcium, albumin-adjusted calcium, PTH, and creatinine were measured in 111,536; 20,512; 34,996; and 189,496 of the subjects, respectively.

METHODS

Multivariate Cox regression analysis was used to compute hazard ratios for all-cause mortality.

RESULTS

During follow-up (median, 3.07 yr), 15,198 (6.1%) subjects died. A reverse J-shaped association between serum level of 25(OH)D and mortality was observed. A serum 25(OH)D level of 50-60 nmol/liter was associated with the lowest mortality risk. Compared to 50 nmol/liter, the hazard ratios (95% confidence intervals) of all-cause mortality at very low (10 nmol/liter) and high (140 nmol/liter) serum levels of 25(OH)D were 2.13 (2.02-2.24) and 1.42 (1.31-1.53), respectively. Similarly, both high and low levels of albumin-adjusted serum calcium and serum PTH were associated with an increased mortality, and secondary hyperparathyroidism was associated with higher mortality (P < 0.0001).

CONCLUSIONS

In this study from the general practice sector, a reverse J-shaped relation between the serum level of 25(OH)D and all-cause mortality was observed, indicating not only a lower limit but also an upper limit. The lowest mortality risk was at 50-60 nmol/liter. The study did not allow inference of causality, and further studies are needed to elucidate a possible causal relationship between 25(OH)D levels, especially higher levels, and mortality.

When [14C]oleate-bovine serum albumin complexes were incubated in vitro with rat liver plasma membranes (LPM), specific, saturable binding of oleate to the membranes was observed. Maximal heat-sensitive (i.e., specific) binding was 3.2 nmol/mg of membrane protein. Oleate-agarose affinity chromatography of Triton X-100-solubilized LPM was used to isolate a single 40-kDa protein with high affinity for oleate. On gel filtration, the protein comigrated with various fatty acids but not with [14C]bilirubin, [35S]sulfobromophthalein, [14C]taurocholate, [14C]phosphatidylcholine, or [14C]cholesteryloleate. A rabbit antibody to this membrane fatty acid-binding protein gave a single precipitin line with the antigen but no reactivity with concentrated cytosolic proteins, LPM bilirubin/sulfobromophthalein-binding protein, or rat albumin or other rat plasma proteins. The antibody selectively inhibited heat-sensitive binding of [14C]oleate to LPM. Immunofluorescence studies localized the antigen in liver-cell plasma membranes as well as in other major sites of fatty acid transport. These data are compatible with the hypothesis that this protein may act as a receptor in a hepatocellular uptake mechanism for fatty acids.

BACKGROUND

The Malnutrition-Inflammation Score (MIS), an inexpensive and easy-to-assess score of 0 to 30 to examine protein-energy wasting (PEW) and inflammation, includes 7 components of the Subjective Global Assessment, body mass index, and serum albumin and transferrin concentrations. We hypothesized that MIS risk stratification of hemodialysis (HD) patients in predicting outcomes is better than its components or laboratory markers of inflammation.

METHODS

5-Year cohort study.

METHODS

We examined 809 stable HD outpatients and followed them for up to 5 years (October 2001 to December 2006).

METHODS

MIS and other nutritional and inflammatory markers.

METHODS

Prospective all-cause mortality, health-related quality of life using the 36-Item Short Form Health Survey (SF-36), and tests of body composition.

RESULTS

The MIS correlated with logarithm of serum interleukin 6 level (r = +0.26; P < 0.001), logarithm of C-reactive protein level (r = +0.16; P < 0.001), and several measures of nutritional status. Patients with a higher MIS had lower SF-36 scores. After multivariate adjustment for case-mix and other measures of PEW, HD patients in the second (3 to 4), third (5 to 7), and fourth >>or=8) quartiles of MIS had worse survival rates than those in the first (0 to 2) quartile (P < 0.001). Each 2-unit increase in MIS was associated with a 2-fold greater death risk, ie, adjusted death hazard ratio of 2.03 (95% confidence interval, 1.76 to 2.33; P < 0.001). Cubic spline survival models confirmed linear trends. Adding MIS to the constellation of age, sex, race/ethnicity, and vintage significantly improved the area under the receiver operating characteristic curve developed for predicting mortality (0.71 versus 0.67; P < 0.001).

CONCLUSIONS

Selection bias and unknown confounders.

CONCLUSIONS

In HD patients, the MIS is associated with inflammation, nutritional status, quality of life, and 5-year prospective mortality. The mortality predictability of the MIS appears equal to serum interleukin 6 and somewhat greater than C-reactive protein levels. Controlled trials are warranted to examine whether interventions to improve the MIS can also improve clinical outcomes in HD patients.

Rat and rabbit IgG immunoglobulins conjugated to horseradiah peroxidase as a histochemical marker bind at 0 degrees C to the luminal surface of absorptive cells in isolated segments of jejunum from 10-12-day old rats. Binding is observed at pH 6.0, near the normal luminal pH of the duodenum and jejunum at this age, but not at pH 7.4. Furthermore, no binding occurs when cells are exposed at pH 6.0 to either free peroxidase or peroxidase conjugated to chicken or sheep IgG immunoglobulins or bovine serum albumin. The sensitivity of binding to pH suggests a means whereby immunoglobulins which are selectively absorbed by the cells can be released efficiently at the abluminal surface.

Crohn's disease and ulcerative colitis are chronic inflammatory disorders resulting from immune dysregulation. Patients who fail conventional medical therapy require biological treatment with monoclonal antibodies (mAbs). Although mAbs are highly effective for induction and maintenance of clinical remission, not all patients respond, and a high proportion of patients lose response over time. One factor associated with loss of response is immunogenicity, whereby the production of antidrug antibodies accelerates mAb clearance. However, other factors related to patient and disease characteristics also influence the pharmacokinetics of mAbs. These factors include gender, body size, concomitant use of immunosuppressive agents, disease type, serum albumin concentration, and the degree of systemic inflammation. Because it is important to maintain clinically effective concentrations to provide optimal clinical response and drug exposure is affected by patient factors, a better understanding of the pharmacology of mAbs will ultimately result in better patient care.

BACKGROUND

In maintenance hemodialysis (MHD) outpatients, a reverse epidemiology is described, ie, baseline obesity appears paradoxically associated with improved survival. However, the association between changes in weight over time and prospective mortality is not known.

METHODS

Using time-dependent Cox models and adjusting for changes in laboratory values over time, the relation of quarterly-varying 3-month averaged body mass index (BMI) to all-cause and cardiovascular mortality was examined in a 2-year cohort of 54,535 MHD patients from virtually all DaVita dialysis clinics in the United States.

RESULTS

Patients, aged 61.7 +/- 15.5 (SD) years, included 54% men and 45% with diabetes. Time-dependent unadjusted and multivariate-adjusted models, based on quarterly-averaged BMI controlled for case-mix and available time-varying laboratory surrogates of nutritional status, were calculated in 11 categories of BMI. Obesity, including morbid obesity, was associated with better survival and reduced cardiovascular death, even after accounting for changes in BMI and laboratory values over time. Survival advantages of obesity were maintained for dichotomized BMI cutoff values of 25, 30, and 35 kg/m2 across almost all strata of age, race, sex, dialysis dose, protein intake, and serum albumin level. Examining the regression slope of change in weight over time, progressively worsening weight loss was associated with poor survival, whereas weight gain showed a tendency toward decreased cardiovascular death.

CONCLUSIONS

Weight gain and both baseline and time-varying obesity may be associated with reduced cardiovascular mortality in MHD patients independent of laboratory surrogates of nutritional status and their changes over time. Morbidly obese patients have the lowest mortality. Clinical trials need to verify these observational findings.

BACKGROUND

Cardiovascular disease is highly prevalent in patients with chronic kidney disease. In hemodialysis patients, the protein-bound uremic retention solute p-cresol is independently associated with cardiovascular disease. The underlying mechanisms have not been elucidated.

METHODS

(1) Prospective observational study of humans and (2) in vitro study in human umbilical vein endothelial cells.

METHODS

Hemodialysis patients.

METHODS

p-Cresol and its main derivative p-cresyl sulfate.

RESULTS

Endothelial dysfunction.

METHODS

We studied: (1) the relation between p-cresol and blood markers of endothelial dysfunction, including soluble P-selectin and endothelial microparticles; and (2) direct effects of p-cresol and p-cresyl sulfate on endothelial cell cultures.

RESULTS

(1) In a cohort of 100 maintenance hemodialysis patients, free serum p-cresol concentrations (median, 11.7 micromol/L; interquartile range, 15.2) were directly associated with circulating endothelial microparticles (P = 0.007), but not with soluble P-selectin (mean, 37.7 +/- 14.4 [SD] pg/mL). Other independent determinants of the degree of circulating microparticles were greater serum phosphorus (mean, 4.8 +/- 1.5 mg/dL; P = 0.008) and serum calcium concentrations (mean, 9.3 +/- 0.8 mg/dL; P = 0.03), whereas treatment with active vitamin D (P = 0.008) and vintage (median, 25 months; P = 0.04) were inversely associated. (2) In vitro, p-cresyl sulfate induced a dose-dependent increase in the shedding of endothelial microparticles (P < 0.001) by human umbilical vein endothelial cells. Shedding was reduced, but not completely aborted, in the presence of albumin, whereas the selective Rho kinase inhibitor Y-27632 abrogated the p-cresyl sulfate-induced generation of endothelial microparticles.

CONCLUSIONS

The relationship between p-cresyl sulfate and shedding of endothelial microparticles in vivo was not mechanistically explored.

CONCLUSIONS

p-Cresyl sulfate induces shedding of endothelial microparticles in the absence of overt endothelial damage in vitro and is independently associated with the number of endothelial microparticles in hemodialysis patients. These findings suggest that p-cresyl sulfate alters endothelial function in hemodialysis patients.

BACKGROUND

Activation of the hypothalamic-pituitary-adrenal (HPA) axis represents one of several important responses to stressful events and critical illnesses. Despite a large volume of published data, several controversies continue to be debated, such as the definition of normal adrenal response, the concept of relative adrenal insufficiency, and the use of glucocorticoids in the setting of critical illness.

OBJECTIVE

The primary objective was to review some of the modulating factors and limitations of currently used methods of assessing HPA function during critical illness and provide alternative approaches in that setting.

METHODS

This was a critical review of relevant data from the literature with inclusion of previously published as well as unpublished observations by the author. Data on HPA function during three different forms of critical illnesses were reviewed: experimental endotoxemia in healthy volunteers, the response to major surgical procedures in patients with normal HPA, and the spontaneous acute to subacute critical illnesses observed in patients treated in intensive care units.

METHODS

The study was conducted at an academic medical center.

METHODS

Participants were critically ill subjects.

METHODS

There was no intervention.

METHODS

The main measure was to provide data on the superiority of measuring serum free cortisol during critical illness as contrasted to those of total cortisol measurements.

RESULTS

Serum free cortisol measurement is the most reliable method to assess adrenal function in critically ill, hypoproteinemic patients. A random serum free cortisol is expected to be 1.8 microg/dl or more in most critically ill patients, irrespective of their serum binding proteins. Because the free cortisol assay is not currently available for routine clinical use, alternative approaches to estimate serum free cortisol can be used. These include calculated free cortisol (Coolens' method) and determining the free cortisol index (ratio of serum cortisol to transcortin concentrations). Preliminary data suggest that salivary cortisol measurements might be another alternative approach to estimating the free cortisol in the circulation. When serum binding proteins (albumin, transcortin) are near normal, measurements of total serum cortisol continue to provide reliable assessment of adrenal function in critically ill patients, in whom a random serum total cortisol would be expected to be 15 microg/dl or more in most patients. In hypoproteinemic critically ill subjects, a random serum total cortisol level is expected to be 9.5 microg/dl or more in most patients. Data on Cosyntropin-stimulated serum total and free cortisol levels should be interpreted with the understanding that the responses in critically ill subjects are higher than those of healthy ambulatory volunteers. The Cosyntropin-induced increment in serum total cortisol should not be used as a criterion for defining adrenal function, especially in critically ill patients.

CONCLUSIONS

The routine use of glucocorticoids during critical illness is not justified except in patients in whom adrenal insufficiency was properly diagnosed or others who are hypotensive, septic, and unresponsive to standard therapy. When glucocorticoids are used, hydrocortisone should be the drug of choice and should be given at the lowest dose and for the shortest duration possible. The hydrocortisone dose (50 mg every 6 h) that is mistakenly labeled as low-dose hydrocortisone leads to excessive elevation in serum cortisol to values severalfold greater than those achieved in patients with documented normal adrenal function. The latter data should call into question the current practice of using such doses of hydrocortisone even in the adrenally insufficient subjects.

Several diseases and disorders are treatable with therapeutic proteins, but some of these products may induce an immune response, especially when administered as multiple doses over prolonged periods. Antibodies are created by classical immune reactions or by the breakdown of immune tolerance; the latter is characteristic of human homologue products. Many factors influence the immunogenicity of proteins, including structural features (sequence variation and glycosylation), storage conditions (denaturation, or aggregation caused by oxidation), contaminants or impurities in the preparation, dose and length of treatment, as well as the route of administration, appropriate formulation and the genetic characteristics of patients. The clinical manifestations of antibodies directed against a given protein may include loss of efficacy, neutralization of the natural counterpart and general immune system effects (including allergy, anaphylaxis or serum sickness). An upsurge in the incidence of antibody-mediated pure red cell aplasia (PRCA) among patients taking one particular formulation of recombinant human erythropoietin (epoetin-alpha, marketed as Eprex(R)/Erypo(R); Johnson & Johnson) in Europe caused widespread concern. The PRCA upsurge coincided with removal of human serum albumin from epoetin-alpha in 1998 and its replacement with glycine and polysorbate 80. Although the immunogenic potential of this particular product may have been enhanced by the way the product was stored, handled and administered, it should be noted that the subcutaneous route of administration does not confer immunogenicity per se. The possible role of micelle (polysorbate 80 plus epoetin-alpha) formation in the PRCA upsurge with Eprex is currently being investigated.

BACKGROUND

The general increase in reactive oxygen species generated from glucose-derived advanced glycation endproducts (AGEs) is among the key mechanisms implicated in tissue injury due to diabetes. AGE-rich foods could exacerbate diabetic injury, at least by raising the endogenous AGE.

METHODS

Herein, we tested whether, prior to ingestion, diet-derived AGEs contain species with cell activating (TNFalpha), chemical (cross-linking) or cell oxidative properties, similar to native AGEs. Glutathione (GSH) and GSH peroxidase (GPx) were assessed after exposure of human umbilical vein endothelial cell (HUVECs) to affinity-purified food-AGE extracts, each exposed to 250 degrees C, for 10 min, along with synthetic AGEs.

RESULTS

Animal product-derived AGE, like synthetic methylglyoxal-bovine serum albumin (MG-BSA), AGE-BSA, and AGE-low density lipoprotein (AGE-LDL), induced a dose- and time-dependent depletion of GSH (()60-75%, p, 0.01) and an increase in GPx activity (()500-600%, p < 0.01), consistent with marked TNFalpha and cross-link formation (p < 0.05); this contrasted with the low bioreactivity of starch/vegetable AGE-extracts, which was similar to that of control BSA and CML- BSA and BSA (p:NS). Anti-AGE-R1,2,3 and -RAGE IgG each inhibited cell-associated (125) I-dAGE by approximately 30-55%; GSH/GPx were effectively blocked by N-acetyl-cysteine (NAC, 800 uM, p < 0.01) and aminoguanidine-HCl (AG, 100 uM, p < 0.01).

CONCLUSIONS

Thus, food-derived AGE, prior to absorption, contain potent carbonyl species, that can induce oxidative stress and promote inflammatory signals.

We have studied the binding of long-chain free fatty acids (FFA) to crystalline bovine serum albumin (BSA) that had been extracted with charcoal to remove endogenous fatty acids. The data were analyzed in terms of a model consisting of six high-energy binding sites and a large number of weak binding sites. The high-energy sites were resolved into two distinct classes, each containing three sites. At 37 degrees C and pH 7.4, k'(1) (the apparent association constant of a class of binding sites) was about 10(6) m(-1) for binding to the three primary sites, and k'(2) was about 10(5) m(-1) for binding to the three secondary sites. The number of weak (tertiary) sites was estimated to be 63 with a k'(3) of 10(3) m(-1). In general, palmitate and palmitoleate were bound more tightly than oleate, linoleate, stearate, or myristate, and much more tightly than laurate. The association of palmitate with human and rabbit albumin also was analyzed in terms of this model. Palmitate was bound less firmly by human or rabbit albumin than by BSA. Palmitate binding to BSA was dependent upon the pH and temperature of the incubation medium. Long-chain hydrocarbons that did not contain a free carboxyl group (methyl palmitate, cetyl alcohol, and hexadecane) were bound to a limited extent and weakly. The presence of positively charged protein sites and native protein tertiary structure were required for maximal binding of palmitate to BSA. Of nine other proteins tested, only -lactoglobulin exhibited a significant capacity to bind palmitate.

Glycation of proteins leads to the formation of early glycation adducts (fructosamine derivatives) and advanced glycation endproducts (AGEs). Formation of AGEs has been linked to the development of cataract, diabetic complications, uraemia, Alzheimer's disease and other disorders. AGEs are a group of compounds of diverse molecular structure and biological function. To characterize AGE-modified proteins used in studies of structural and functional effects of glycation, an assay was developed that surveys the content of early and advanced glycation adducts in proteins. The assay procedure involved enzymic hydrolysis of protein substrate, derivatization of the hydrolysate with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) and HPLC of the resulting adducts with fluorimetric detection. Structural isomers of methylglyoxal-derived hydroimidazolone, glyoxal-derived hydroimidazolone, 3-deoxyglucosone-derived hydroimidazolone and N(delta)-(4-carboxy-4,6-dimethyl-5,6-dihydroxy-1,4,5,6-tetrahydropyrimidin-2-yl)-ornithine (THP) were determined for the first time. AGEs with intrinsic fluorescence (argpyrimidine, pentosidine) were assayed without derivatization. Limits of detection were 2-17 pmol and levels of recovery were 50-99%, depending on the analyte. The AQC assay resolved structural and epimeric isomers of methylglyoxal-derived hydroimidazolones and THP. Hydroimidazolones, THP and argpyrimidine were AGEs of short-to-intermediate stability under physiological conditions, with half-lives of 1-2 weeks. Their measurement provides further insight into the glycation process. The assay was applied to the characterization of human serum albumin minimally and highly modified by N(epsilon)-carboxymethyl-lysine and N(epsilon)-(1-carboxyethyl)-lysine.

Intraoperative near-infrared (NIR) fluorescence imaging provides the surgeon with real-time image guidance during cancer and other surgeries. We have previously reported the use of NIR fluorescent quantum dots (QDs) for sentinel lymph node (SLN) mapping. However, because of concerns over potential toxicity, organic alternatives to QDs will be required for initial clinical studies. We describe a family of 800 nm organic heptamethine indocyanine-based contrast agents for SLN mapping spanning a spectrum from 775 Da small molecules to 7 MDa nanocolloids. We provide a detailed characterization of the optical and physical properties of these contrast agents and discuss the advantages and disadvantages of each. We present robust methods for the covalent conjugation, purification, and characterization of proteins with tetra-sulfonated heptamethine indocyanines, including mass spectroscopic site mapping of highly substituted molecules. One contrast agent, NIR fluorescent human serum albumin (HSA800), emerged as the molecule with the best overall performance with respect to entry to lymphatics, flow to the SLN, retention in the SLN, fluorescence yield and reproducibility. This preclinical study, performed on large animals approaching the size of humans, should serve as a foundation for future clinical studies.

Signs of protein-energy malnutrition are common in maintenance hemodialysis (HD) patients and are associated with increased morbidity and mortality. To evaluate the nutritional status and relationship between various parameters used for assessing malnutrition, we performed a cross-sectional study in 128 unselected patients treated with hemodialysis (HD) thrice weekly for at least two weeks. Global nutritional status was evaluated by the subjective global nutritional assessment (SGNA). Body weight, skinfold thicknesses converted into % body fat mass (BFM), mid-arm muscle circumference, hand-grip strength and several laboratory values, including serum albumin (SA1b), plasma insulin-like growth factor I (p-IGF-I), serum C-reactive protein (SCRP) and plasma free amino acids, were recorded. Dose of dialysis and protein equivalence of nitrogen appearance (nPNA) were evaluated by urea kinetic modeling. The patients were subdivided into three groups based on SGNA: group I, normal nutritional status (36%); group II, mild malnutrition (51%); and group III, moderate or (in 2 cases) severe malnutrition (13%). Clinical factors associated with malnutrition were: high age, presence of cardiovascular disease and diabetes mellitus. nPNA and Kt/V(urea) were similar in the three groups. However, when normalized to desirable body wt, both were lower in groups II and III than in group I. Anthropometric factors associated with malnutrition were low body wt, skinfold thickness, mid-arm muscle circumference (MAMC), and handgrip strength. Biochemical factors associated with malnutrition were low serum levels of albumin and creatinine and low plasma levels of insulin-like growth factor 1 (IGF-1) and branched-chain amino acids (isoleucine, leucine and valine). The serum albumin (SAlb) level was not only a predictor of nutritional status, but was independently influenced by age, sex and SCRP. Plasma IGF-1 levels also reflected the presence and severity of malnutrition and appeared to be more closely associated than SAlb with anthropometric and biochemical indices of somatic protein mass. Elevated SCRP >> 20 mg/liter), which mainly reflected the presence of infection/inflammation and was associated with hypoalbuminemia, was more common in malnourished patients than in patients with normal nutritional status, and also more common in elderly than in younger patients. Plasma amino acid levels, with the possible exception of the branched-chain amino acids (isoleucine, leucine, valine), seem to be poor predictors of nutritional status in hemodialysis patients.

Advanced glycation end product (AGE) activation of the signal-transducing receptor for AGE (RAGE) has been linked to a proinflammatory phenotypic change within cells. However, the precise intracellular signaling pathways involved have not been elucidated. We demonstrate here that human serum albumin modified with N(epsilon)-(carboxymethyl)lysine (CML), a major AGE adduct that progressively accumulates with aging, diabetes, and renal failure, induced nuclear factor (NF)-kappaB-driven reporter gene expression in human monocytic THP-1 cells. The NF-kappaB response was blocked with a synthetic peptide corresponding to the putative ligand-binding domain of RAGE, with anti-RAGE antiserum, and by coexpression of truncated receptors lacking the intracellular domain. Signal transduction from RAGE to NF-kappaB involved the generation of reactive oxygen species, since reporter gene expression was blocked with the antioxidant N-acetyl-L-cysteine. CML-modified albumin produced rapid transient activation of tyrosine phosphorylation, extracellular signal-regulated kinase 1 and 2, and p38 mitogen-activated protein kinase (MAPK), but not c-Jun NH(2)-terminal kinase. RAGE-mediated NF-kappaB activation was suppressed by the selective p38 MAPK inhibitor SB203580 and by coexpression of a kinase-dead p38 dominant-negative mutant. Activation of NF-kappaB by CML-modified albumin increased secretion of proinflammatory cytokines (tumor necrosis factor-alpha, interleukin-1beta, and monocyte chemoattractant protein-1) severalfold, and inhibition of p38 MAPK blocked these increases. These results indicate that p38 MAPK activation mediates RAGE-induced NF-kappaB-dependent secretion of proinflammatory cytokines and suggest that accelerated inflammation may be a consequence of cellular activation induced by this receptor.

We tested the ability of serum-free media to support the in vitro growth of human non-small cell lung carcinoma. A medium containing insulin, transferrin, sodium selenite, hydrocortisone, epidermal growth factor, and bovine serum albumin (1 mg/ml) with serum precoating of culture dishes (modified LA medium) supported three previously established cell lines of non-small cell lung cancer and prevented fibroblast proliferation in fresh tumor specimens but did not support long term tumor cell growth from fresh specimens. We added triiodothyronine, sodium pyruvate, and additional glutamine, insulin, and epidermal growth factor to modified LA medium, precoated with fibronectin and collagen instead of serum, and deleted bovine serum albumin, defining a new medium called ACL-3. ACL-3 medium alone supported the short term growth of 10 of 12 cell lines and the soft agarose cloning of 9 of 12 cell lines tested, and ACL-3 supplemented by an optimal concentration of bovine serum albumin (5 mg/ml) supported the long term growth of 10 of 12 cell lines tested. Moreover, we have grown tumor cells for more than 6 months from 11 of 33 (33%) consecutive fresh clinical specimens of human lung adenocarcinoma in ACL-3 with bovine serum albumin. ACL-3 medium provides a defined environment for the study of growth factor requirements of human non-small cell lung cancer and enhances our ability to grow human lung cancer, particularly adenocarcinoma, in vitro.

A phase I study was performed to determine the safety and tolerability of injecting autologous CD34(+) cells into five patients with liver insufficiency. The study was based on the hypothesis that the CD34(+) cell population in granulocyte colony-stimulating factor (G-CSF)-mobilized blood contains a subpopulation of cells with the potential for regenerating damaged tissue. We separated a candidate CD34(+) stem cell population from the majority of the CD34(+) cells (99%) by adherence to tissue culture plastic. The adherent and nonadherent CD34(+) cells were distinct in morphology, immunophenotype, and gene expression profile. Reverse transcription-polymerase chain reaction-based gene expression analysis indicated that the adherent CD34(+) cells had the potential to express determinants consistent with liver, pancreas, heart, muscle, and nerve cell differentiation as well as hematopoiesis. Overall, the characteristics of the adherent CD34(+) cells identify them as a separate putative stem/progenitor cell population. In culture, they produced a population of cells exhibiting diverse morphologies and expressing genes corresponding to multiple tissue types. Encouraged by this evidence that the CD34(+) cell population contains cells with the potential to form hepatocyte-like cells, we gave G-CSF to five patients with liver insufficiency to mobilize their stem cells for collection by leukapheresis. Between 1 x 10(6) and 2 x 10(8) CD34(+) cells were injected into the portal vein (three patients) or hepatic artery (two patients). No complications or specific side effects related to the procedure were observed. Three of the five patients showed improvement in serum bilirubin and four of five in serum albumin. These observations warrant further clinical trials.

Tissue advanced glycation end products (AGE) are a measure of cumulative metabolic stress and trigger cytokines driven inflammatory reactions. AGE are thought to contribute to the chronic complications of diabetes and ESRD. Tissue autofluorescence is related to the accumulation of AGE. Therefore, skin autofluorescence (AF) may provide prognostic information on mortality in hemodialysis (HD) patients. Skin AF was measured noninvasively with an AF reader at baseline in 109 HD patients. Overall and cardiovascular mortality was monitored prospectively during a period of 3 yr. The AF reader was validated against AGE contents in skin biopsies from 29 dialysis patients. Forty-two of the 109 (38.5%) HD patients died. Cox regression analysis showed that AF was an independent predictor of overall and cardiovascular mortality (for overall mortality odds ratio [OR] 3.9), as were pre-existing cardiovascular disease (CVD; OR 3.1), C-reactive protein (OR 1.1), and serum albumin (OR 0.3). Multivariate analysis revealed that 65% of the variance in AF could be attributed to the independent effects of age, dialysis and renal failure duration, presence of diabetes, triglycerides levels, and C-reactive protein. AF was also independently linked to the presence of CVD at baseline (OR 8.8; P < 0.001). AF correlated with collagen-linked fluorescence (r = 0.71, P < 0.001), pentosidine (r = 0.75, P < 0.001), and carboxy(m)ethyllysine (both r = 0.45, P < 0.01). Skin AF is a strong and independent predictor of mortality in ESRD. This supports a role for AGE as a contributor to mortality and CVD and warrants interventions specifically aimed at AGE accumulation.

1. A quantitative theory of the precipitin reaction based on the laws of classical chemistry has now been found applicable to the crystalline egg albumin-antibody system. Equations derived from the theory permit the calculation of the behavior of an anti-egg albumin serum over most of the reaction range after a few quantitative analyses have been made for the nitrogen precipitated. Data of other workers also conform to the proposed equations. 2. The empirical relation, shown to have advantages in the dye antidye system, may also be used for the Ea-A reaction. 3. Serum from the same animal after successive courses exhibits progressive changes which have been described graphically and quantitatively. These changes are believed to consist in the formation of more and more antibody capable of reacting with a larger number of chemically different groupings in the antigen molecule. 4. Evidence is presented that anti-egg albumin is not homogeneous, and that even after prolonged immunization the antiserum contains much low-grade antibody, incapable of forming precipitates unless more reactive precipitin is present. 5. Factors affecting the equivalence point ratio are discussed.

Because of evidence that increased body iron stores are associated with an increased risk of cancer, we examined iron status and cancer risk in the first National Health and Nutrition Examination Survey, a survey of more than 14,000 adults begun in 1971, with follow-up between 1981 and 1984. Among 242 men in whom cancer developed, the mean total iron-binding capacity was significantly lower (61.4 vs. 62.9 mumol per liter; P = 0.01) and transferrin saturation was significantly higher (33.1 vs. 30.7 percent; P = 0.002) than among 3113 men who remained free of cancer. The risk of cancer in men in each quartile of transferrin-saturation level relative to the lowest quartile was 1.00, 1.01, 1.10, and 1.37 (P = 0.02 for trend). The serum albumin level was significantly lower in men in whom cancer developed than in those who remained cancer-free. Among women, those in whom cancer developed did not have significantly lower total iron-binding capacity or higher transferrin saturation than those who remained cancer-free. However, a post hoc examination of 5367 women (203 with cancer) yielded a relative risk of 1.3 (95 percent confidence interval, 0.9 to 1.9) associated with a very high transferrin saturation (greater than or equal to 36.8 percent, a value in the highest quartile among men); in 5228 women with at least six years of follow-up (149 with cancer), the relative risk associated with transferrin saturation above this level was 1.5 (1.0 to 2.2). These results are consistent with the hypothesis that high body iron stores increase the risk of cancer in men. The possibility that a similar association exists in women requires further study.

Autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly 100% in patients with rheumatoid arthritis and have been suggested to be involved in the disease pathogenesis. The targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deiminase (PAD), which converts positively charged arginine to polar but uncharged citrulline. The aim of this study was to explore the effects of citrullination on the immunogenicity of autoantigens as well as on potential arthritogenicity. Thus, immune responses to citrullinated rat serum albumin (Cit-RSA) and to unmodified rat serum albumin (RSA) were examined as well as arthritis development induced by immunisation with citrullinated rat collagen type II (Cit-CII) or unmodified CII. In addition, to correlate the presence of citrullinated proteins and the enzyme PAD4 with different stages of arthritis, synovial tissues obtained at different time points from rats with collagen-induced arthritis were examined immunohistochemically. Our results demonstrate that citrullination of the endogenous antigen RSA broke immunological tolerance, as was evident by the generation of antibodies directed against the modified protein and cross-reacting with the native protein. Furthermore we could demonstrate that Cit-CII induced arthritis with higher incidence and earlier onset than did the native counterpart. Finally, this study reveals that clinical signs of arthritis precede the presence of citrullinated proteins and the enzyme PAD4. As disease progressed into a more severe and chronic state, products of citrullination appeared specifically in the joints. Citrullinated proteins were detected mainly in extracellular deposits but could also be found in infiltrating cells and on the cartilage surface. PAD4 was detected in the cytoplasm of infiltrating mononuclear cells, from day 21 after immunisation and onwards. In conclusion, our data reveal the potency of citrullination to break tolerance against the self antigen RSA and to increase the arthritogenic properties of the cartilage antigen CII. We also show that citrullinated proteins and the enzyme PAD4 are not detectable in healthy joints, and that the appearance and amounts in arthritic joints of experimental animals are correlated with the severity of inflammation.

OBJECTIVE

Interleukin 33 (IL-33) is a new member of the IL-1 family of cytokines which signals via its receptor, ST2 (IL-33R), and has an important role in Th2 and mast cell responses. This study shows that IL-33 orchestrates neutrophil migration in arthritis.

RESULTS

Methylated bovine serum albumin (mBSA) challenge in the knee joint of mBSA-immunised mice induced local neutrophil migration accompanied by increased IL-33R and IL-33 mRNA expression. Cell migration was inhibited by systemic and local treatments with soluble (s)IL-33R, an IL-33 decoy receptor, and was not evident in IL-33R-deficient mice. IL-33 injection also induced IL-33R-dependent neutrophil migration. Antigen- and IL-33-induced neutrophil migration in the joint was dependent on CXCL1, CCL3, tumour necrosis factor alpha (TNFalpha) and IL-1beta synthesis. Synovial tissue, macrophages and activated neutrophils expressed IL-33R. IL-33 induces neutrophil migration by activating macrophages to produce chemokines and cytokines and by directly acting on neutrophils. Importantly, neutrophils from patients with rheumatoid arthritis successfully treated with anti-TNFalpha antibody (infliximab) expressed significantly lower levels of IL-33R than patients treated with methotrexate alone. Only neutrophils from patients treated with methotrexate alone or from normal donors stimulated with TNFalpha responded to IL-33 in chemotaxis.

CONCLUSIONS

These results suggest that suppression of IL-33R expression in neutrophils, preventing IL-33-induced neutrophil migration, may be an important mechanism of anti-TNFalpha therapy of inflammation.