The liver manufactures albumin at a massive rate and decreases production in times of environmental, nutritional, toxic and trauma stress. Osmotic pressure is a basic evolutionary regulatory factor, and hormonal control over albumin production has been demonstrated. Where and why new or old albumin is degraded are questions which have not been clarified, although the vascular endothelium may well be the degradative site. Albumin is important as a transport protein, as a measure of evolution and as a model to study secretion following synthesis without the intervening steps of glycosylation. Investigations as to how this protein enters the endoplasmic membrane may well answer some of the questions concerning signal peptide insertion (288). The role of the urea cycle intermediate ornithine and its participation in polyamine synthesis, which has a positive effect on albumin synthesis, is under study. Likewise, the inverse relation between acute-phase protein synthesis and albumin synthesis regulated by interleukin 1 and other cytokines will merit further study. These are a few of the concepts which will be tested in the future.

BACKGROUND

The chemokines and cytokines CXCL13, CXCL12, CCL19, CCL21, BAFF and APRIL are believed to play a role in the recruitment of B cells to the central nervous system (CNS) compartment during neuroinflammation. To determine which chemokines/cytokines show the strongest association with a humoral immune response in the cerebrospinal fluid (CSF), we measured their concentrations in the CSF and correlated them with immune cell subsets and antibody levels.

METHODS

Cytokine/chemokine concentrations were measured in CSF and serum by ELISA in patients with non-inflammatory neurological diseases (NIND, n = 20), clinically isolated syndrome (CIS, n = 30), multiple sclerosis (MS, n = 20), Lyme neuroborreliosis (LNB, n = 8) and patients with other inflammatory neurological diseases (OIND, n = 30). Albumin, IgG, IgA and IgM were measured by nephelometry. CSF immune cell subsets were determined by seven-color flow cytometry.

RESULTS

CXCL13 was significantly elevated in the CSF of all patient groups with inflammatory diseases. BAFF levels were significantly increased in patients with LNB and OIND. CXCL12 was significantly elevated in patients with LNB. B cells and plasmablasts were significantly elevated in the CSF of all patients with inflammatory diseases. CXCL13 showed the most consistent correlation with CSF B cells, plasmablasts and intrathecal Ig synthesis.

CONCLUSIONS

CXCL13 seems to be the major determinant for B cell recruitment to the CNS compartment in different neuroinflammatory diseases. Thus, elevated CSF CXCL13 levels rather reflect a strong humoral immune response in the CNS compartment than being specific for a particular disease entity.

Cerebrospinal fluid (CSF) routine analysis for diagnosis of neurological diseases is based on the concepts for discrimination of blood-derived and brain-derived immunoglobulin fractions in CSF. The actual molecular flux/CSF flow theory of the blood/CSF barrier function, which founded the hyperbolic discrimination lines in quotient diagrams, is derived from the laws of molecular diffusion combined with CSF flow rate. It emerged from this theory that the decrease of CSF flow rate is sufficient to explain quantitatively the increase of CSF protein concentrations as observed in many neurological diseases. With this concept of CSF flow rate as the modulator of the normal and pathological blood-CSF barrier function, we got for the first time a theoretical frame work to explain also quantitatively the dynamics of brain-derived proteins and their source related (neurons and glial cells or leptomeningal cells) differences. The review of the anatomical, physiological and biophysical knowledge points to the new interpretations: The changing albumin quotient is an indicator of changing CSF flow rate and not for a morphological "leakage" of the blood-brain barrier. As an application of these concepts the dynamics of brain-derived molecules in blood are discussed with two examples: beta trace protein, flowing with CSF into venous blood, and neuron-specific enolase, passing from tissue into blood the opposite direction of serum proteins, again a gradient-dependent protein diffusion across the intact blood vessel wall.

OBJECTIVE

The liver contains large amounts of microRNA-122 (miR-122), whereas other tissues contain only marginal amounts of this miRNA. MicroRNAs have also been found to circulate in the blood in a cell-free form; their potential as readily accessible disease markers is currently evaluated. Here, we investigated if the serum levels of miR-122 might be useful as disease parameter in patients with chronic hepatitis C virus (HCV) infection.

METHODS

RNA was extracted from sera of patients with chronic HCV infection (CHC) and healthy controls and was analyzed for miR-22 content by quantitative real-time reverse-transcription polymerase chain reaction. miR-122 serum levels were correlated with standard parameters of liver function. Liver biopsies from the same patients were examined for the histologic activity index (HAI) and the degree of fibrosis.

RESULTS

Sera from patients with CHC contained higher levels of miR-122 than sera from healthy controls. Serum miR-122 levels correlated well with markers of liver inflammatory activity, that is, the serum levels of alanine leucine transaminase (ALT) and aspartate transaminase, and the HAI score. In patients with persistently normal ALT levels, serum miR-122 levels did not differ from healthy controls. There was no correlation of serum miR-122 levels with serum albumin, international normalized ratio, liver fibrosis, or serum HCV RNA.

CONCLUSIONS

The serum level of miR-122 strongly correlates with serum ALT activity and with necroinflammatory activity in patients with CHC and elevated ALT levels, but not with fibrosis stage and functional capacity of the liver.

Proteolytic degradation of oxidatively damaged [3H] bovine serum albumin [( 3H]BSA) was studied during incubation with cell-free erythrocyte extracts and a wide variety (14) of purified proteases. [3H]BSA was pretreated by exposure (60Co radiation) to the hydroxyl radical (.OH), the superoxide anion radical (O2-), or the combination of .OH + O2- + oxygen. Treated (and untreated) samples were dialyzed and then incubated with erythrocyte extract or proteases for measurements of proteolytic susceptibility (release of acid-soluble counts). Both .OH and .OH + O2- + caused severalfold increases in proteolytic susceptibility (with extract and proteases), but O2- alone had no effect. Proteolytic susceptibility reached a maximum at 15 nmol of .OH/nmol of BSA and declined thereafter. In contrast, proteolytic susceptibility was still increasing at an .OH + O2-/BSA molar ratio of 100 (50% .OH + 50% O2-). Degradation in erythrocyte extracts was conducted by a novel ATP- and Ca2+-independent pathway, with maximal activity at pH 7.8. Inhibitor profiles indicate that this pathway may involve metalloproteases and serine proteases. Comparisons of proteolytic susceptibility with multiple modifications to BSA primary, secondary, and tertiary structure revealed a high correlation (r = 0.98) with denaturation/increased hydrophobicity by low concentrations of .OH. Covalent aggregation reactions (BSA cross-linking) may explain the declining proteolytic susceptibility observed at .OH/BSA molar ratios greater than 20. Protein denaturation may also have caused the increased proteolytic susceptibility induced by .OH + O2- + O2, but no simple correlation could be obtained. Results with .OH + O2- + O2 appear to have been complicated by direct BSA fragmentation reactions involving (.OH-induced) protein radicals and oxygen. These data indicate a direct and quantitative relationship between protein damage by oxygen radicals and increased proteolytic susceptibility. Oxidative denaturation may exemplify a simple, yet effective inherent mechanism for intracellular proteolysis.

Thirty-seven strains of the genus Haemophilus and five strains of Streptococcus pneumoniae were examined for their ability to produce extracellular enzyme that cleaves immunoglobulin molecules. All strains of H. influenzae, H. aegyptius, and S. pneumoniae elaborated enzyme that selectively cleaved human immunoglobulin A1 (IgA1) myeloma proteins but was inactive against a variety of other proteins including human IgA2, IgG, and IgM, porcine and bovine secretory IgA, human and bovine serum albumins, and ovalbumin. Although susceptible, human secretory IgA remained largely undigested. Two strains of H. pleuropneumoniae isolated from fatally infected pigs cleaved porcine secretory IgA, but had no effect on human IgA proteins. None of 16 strains that belonged to nonpathogenic Haemophilus species produced IgA protease. Analyses of the cleavage products of human IgA1 and secretory IgA proteins by immunochemical methods, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and analytical ultracentrifugation revealed that Fab and Fc fragments were produced. Since the production of IgA1 protease by Neisseria meningitidis has been reported previously, our finding that H. influenzae and S. pneumoniae produce an IgA1 protease indicates that this is a property of all three major etiological agents of bacterial meningitis. This suggests that IgA1 protease production may be an important factor in the pathogenesis of this disease.

BACKGROUND

Despite the rising heart failure (HF) incidence and aging United States population, there are no validated prediction models for incident HF in the elderly. We sought to develop a new prediction model for 5-year risk of incident HF among older persons.

RESULTS

Proportional hazards models were used to assess independent predictors of incident HF, defined as hospitalization for new-onset HF, in 2935 elderly participants without baseline HF enrolled in the Health ABC study (age, 73.6 +/- 2.9 years, 47.9% males, 58.6% whites). A prediction equation was developed and internally validated by bootstrapping, allowing the development of a 5-year risk score. Incident HF developed in 258 (8.8%) participants during 6.5 +/- 1.8 years of follow-up. Independent predictors of incident HF included age, history of coronary disease and smoking, baseline systolic blood pressure and heart rate, serum glucose, creatinine, and albumin levels, and left ventricular hypertrophy. The Health ABC HF model had a c-statistic of 0.73 in the derivation dataset, 0.72 by internal validation (optimism-corrected), and good calibration (goodness-of-fit 2 6.24, P=0.621). A simple point score was created to predict incident HF risk into 4 risk groups corresponding to <5%, 5% to 10%, 10% to 20%, and >20% 5-year risk. The actual 5-year incident HF rates in these groups were 2.9%, 5.7%, 13.3%, and 36.8%, respectively.

CONCLUSIONS

The Health ABC HF prediction model uses common clinical variables to predict incident HF risk in the elderly, an approach that may be used to target and treat high-risk individuals.

The role of glyoxal and glycolaldehyde in protein cross-linking and N epsilon-(carboxymethyl)lysine (CML) formation during Maillard reaction under physiological conditions was investigated. Incubation of bovine serum albumin with these reagents lead to rapid formation of C-2-imine cross-links and CML. Initial CML formation rate from glyoxal was not dependent on oxidation, suggesting an intramolecular Cannizzaro reaction. CML formation from glucose/lysine or Amadori product of both was strongly dependent on oxidation. Blocking of Amadori product by boric acid totally suppressed CML formation from Amadori product, but only by 37% in the glucose/lysine system. Trapping of glyoxal with aminoguanidine hardly suppressed CML formation from Amadori product, whereas it blocked 50% of CML production in the glucose/lysine system. While these results would support a significant role for glucose autoxidation in CML formation, the addition of lysine to a glucose/aminoguanidine incubation system catalyzed glyoxal-triazine formation 7-fold, thereby strongly suggesting that glucose autoxidation is not a factor for glyoxal-mediated CML formation. Based on these results, it can be estimated that approximately 50% of the CML forming in a glucose/lysine system originates from oxidation of Amadori product, and 40-50% originates from a pre-Amadori stage largely independent from glucose autoxidation. This step may be related to the so-called Namiki pathway of the Maillard reaction.

BACKGROUND

Protein-energy malnutrition is a strong predictor of mortality in maintenance hemodialysis (MHD) patients. This association has generally been described for serum chemistry measures of protein-energy malnutrition. We hypothesized that body weight-for-height relationships also predict survival in MHD patients.

METHODS

During the last three months of 1993, data were obtained on 12,965 men and women concerning clinical characteristics (height, postdialysis weight, age, gender, race, and presence or absence of diabetes mellitus) and laboratory measurements (predialysis serum albumin, creatinine and cholesterol, and the urea reduction ratio). Patient survival during the next 12 months was evaluated retrospectively.

RESULTS

In comparison to values for normal Americans determined from the National Health and Nutrition Evaluation Survey II data, weight-for-height relationships tended to be slightly lower than normal in African American men and women and Caucasian men undergoing MHD and were normal or slightly greater in the taller Caucasian women. In both men and women, the mortality rate decreased progressively as the patients' weight-for-height increased. MHD patients who weighed more than normal had the lowest mortality rates. After adjustment for clinical characteristics and laboratory measurements, the inverse relationship between mortality rates and weight-for-height percentiles was still highly significant for patients within the lower 50th percentile of body weight-for-height. Serum albumin correlated directly with weight-for-height in patients in the lower 50th percentile of weight-for-height. Serum creatinine and cholesterol correlated directly with weight-for-height in the entire population of men and women. In contrast, the urea reduction ratio was inversely correlated with weight-for-height.

CONCLUSIONS

These data indicate that weight-for-height is a strong predictor of 12-month mortality in male and female MHD patients. Multivariate analyses indicate that body weight-for-height is an independent predictor of higher mortality in those patients who are in the lower 50th percentile for this measurement.

OBJECTIVE

To determine whether caloric intake is associated with risk of nosocomial bloodstream infection in critically ill medical patients.

METHODS

Prospective cohort study.

METHODS

Urban, academic medical intensive care unit.

METHODS

Patients were 138 adult patients who did not take food by mouth for>> or =96 hrs after medical intensive care unit admission.

METHODS

Daily caloric intake was recorded for each patient. Participants subsequently were grouped into one of four categories of caloric intake: <25%, 25-49%, 50-74%, and>> or =75% of average daily recommended calories based on the American College of Chest Physicians guidelines. Simplified Acute Physiology Score II and serum albumin were measured on medical intensive care unit admission. Serum glucose (average value and maximum value each day) and route of feeding (enteral, parenteral, or both) were collected daily. Nosocomial bloodstream infections were identified by infection control surveillance methods.

RESULTS

The overall mean (+/-sd) daily caloric intake for all study participants was 49.4 +/- 29.3% of American College of Chest Physicians guidelines. Nosocomial bloodstream infection occurred in 31 (22.4%) participants. Bivariate Cox analysis revealed that receiving>> or =25% of recommended calories compared with <25% was associated with significantly lower risk of bloodstream infection (relative hazard, 0.24; 95% confidence interval, 0.10-0.60). Simplified Acute Physiology Score II also was associated with risk of nosocomial bloodstream infection (relative hazard, 1.27; 95% confidence interval, 1.01-1.60). Average daily serum glucose, admission serum albumin, time to initiating nutritional support, and route of nutrition did not affect risk of bloodstream infection. After adjustment for Simplified Acute Physiology Score II in a multivariable analysis, receiving>> or =25% of recommended calories was associated with a significantly lower risk of bloodstream infection (relative hazard, 0.27; 95% confidence interval, 0.11-0.68).

CONCLUSIONS

In the context of reducing risk of nosocomial bloodstream infections, failing to provide>> or =25% of the recommended calories may be harmful. Higher caloric goals may be necessary to achieve other clinically important outcomes.

An enzyme from Treponema denticola that hydrolyzes a synthetic trypsin substrate, N-alpha-benzoyl-L-arginine-p-nitroanilide (BAPNA), was purified to near homogeneity, as judged by gel electrophoresis. The molecular weight of the enzyme was estimated to be ca. 69,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and ca. 50,000 by gel filtration on Sephadex G-100. The pH optimum for the hydrolysis of BAPNA was around 8.5. The enzyme was heat labile and irreversibly inactivated at low pH values. Enzyme activity was enhanced by Ca2+, Mg2+, and Ba2+ but inhibited by Mn2+, Hg2+, Co2+, and Zn2+. Metal chelators and sulfhydryl reagents had no effect on this activity. The enzyme was inhibited by certain protease inhibitors such as diisopropyl fluorophosphate, N-alpha-p-tosyl-L-lysine chloromethyl ketone, phenylmethylsulfonyl fluoride, L-1-tosylamide-2-phenylethylchloromethyl ketone, alpha-1-antitrypsin, and soybean trypsin inhibitor. The Km values for BAPNA and N-alpha-benzoyl-L-arginine ethyl ester were 0.05 and 0.12 mM, respectively, and the Vmax values were higher than those observed with trypsin. Although the purified enzyme hydrolyzed some low-molecular-weight synthetic trypsin substrates, it did not hydrolyze casein, hemoglobin, azocasein, azocoll, bovine serum albumin, or gelatin. Thus, this enzyme is probably not a protease but is capable of hydrolyzing ester, amide, and peptide bonds involving the carboxyl group of arginine and lysine.

Rats thymectomized at birth gained weight and otherwise developed normally, but were found to be very susceptible to intercurrent infections. Both Arthus reactivity and delayed hypersensitivity to BSA were markedly impaired in rats thymectomized during the first week of life and significantly impaired in rats thymectomized as late as 3 weeks after birth. The inhibition of Arthus reactivity in thymectomized rats was well correlated with their failure to develop significant titers of precipitating or hemagglutinating antibody. However, natural heteroagglutinin titers were not altered in these animals, and no abnormality of serum proteins, including gamma-globulin could be detected by paper electrophoresis. The loss of immunologic activity could not be corrected by injecting homogenates of spleen or thymus before and during the sensitization period. Splenectomy at birth did not influence Arthus or delayed reactivity.

BACKGROUND

Persistent pollutants, such as polychlorinated biphenyls (PCBs), affect endocrine function. Human exposure to polybrominated diphenyl ethers (PBDEs), which are similar in structure to PCBs, has increased recently, but health effects have not been well studied.

OBJECTIVE

Our goal in this study was to determine whether PBDE body burdens are related to thyroid and steroid hormone levels, thyroid antibodies, and thyroid disease in a cohort of frequent and infrequent adult male sport fish consumers.

METHODS

We tested serum from 405 adult males for PBDE congeners, PCB congeners, testosterone, sex-hormone-binding globulin (SHBG), SHBG-bound testosterone, thyroglobulin antibodies, and the thyroid hormones thyroxine (T(4)), triiodothyronine (T(3)), thyroid-stimulating hormone (TSH), and T(4)-binding globulin (TBG). We collected data on demographics, fish consumption, medical diseases, and medication use.

RESULTS

The median sum of PBDEs was 38 ng/g lipid. In 308 men without thyroid disease or diabetes, PBDEs were positively related to measures of T(4) and reverse T(3) and inversely related to total T(3) and TSH. PBDEs were positively related to the percentage of T(4) bound to albumin, and inversely related to the percentage of T(4) bound to TBG. Associations of BDE congeners with hormones varied. BDE-47 was positively associated with testosterone levels. Participants with PBDEs over the 95th percentile were more likely to have thyroglobulin antibodies, although high PBDE exposure was not associated with thyroid disease. PBDE effects were independent of PCB exposure and sport fish consumption.

CONCLUSIONS

PBDE exposure, at levels comparable with those of the general U.S. population, was associated with increased thyroglobulin antibodies and increased T(4) in adult males.

Recent studies suggest that diabetes mellitus increases the risk of developing hepatocellular carcinoma (HCC). The aim of this study is to quantify the risk of HCC among patients with both diabetes mellitus and hepatitis C in a large cohort of patients with chronic hepatitis C and advanced fibrosis. We included 541 patients of whom 85 (16%) had diabetes mellitus. The median age at inclusion was 50 years. The prevalence of diabetes mellitus was 10.5% for patients with Ishak fibrosis score 4, 12.5% for Ishak score 5, and 19.1% for Ishak score 6. Multiple logistic regression analysis showed an increased risk of diabetes mellitus for patients with an elevated body mass index (BMI) (odds ratio [OR], 1.05; 95% confidence interval [CI], 1.00-1.11; P = 0.060) and a decreased risk of diabetes mellitus for patients with higher serum albumin levels (OR, 0.81; 95% CI, 0.63-1.04; P = 0.095). During a median follow-up of 4.0 years (interquartile range, 2.0-6.7), 11 patients (13%) with diabetes mellitus versus 27 patients (5.9%) without diabetes mellitus developed HCC, the 5-year occurrence of HCC being 11.4% (95% CI, 3.0-19.8) and 5.0% (95% CI, 2.2-7.8), respectively (P = 0.013). Multivariate Cox regression analysis of patients with Ishak 6 cirrhosis showed that diabetes mellitus was independently associated with the development of HCC (hazard ratio, 3.28; 95% CI, 1.35-7.97; P = 0.009).

CONCLUSIONS

For patients with chronic hepatitis C and advanced cirrhosis, diabetes mellitus increases the risk of developing HCC.

OBJECTIVE

To show how hypoalbuminemia lowers the anion gap, which can mask a significant gap acidosis; and to derive a correction factor for it.

METHODS

Observational study.

METHODS

Intensive care unit in a university-affiliated hospital.

METHODS

Nine normal subjects and 152 critically ill patients (265 measurements).

METHODS

None.

RESULTS

Arterial blood samples analyzed for pH, PCO2, and concentrations of plasma electrolytes and proteins. Marked hypoalbuminemia was common among the critically ill patients: 49% of them had serum albumin concentration of <20 g/L. Each g/L decrease in serum albumin caused the observed anion gap to underestimate the total concentration of gap anions by 0.25 mEq/L (r2 = .94).

CONCLUSIONS

The observed anion gap can be adjusted for the effect of abnormal serum albumin concentrations as follows: adjusted anion gap = observed anion gap + 0.25 x ([normal albumin] [observed albumin]), where albumin concentrations are in g/L; if given in g/dL, the factor is 2.5. This adjustment returns the anion gap to the familiar scale of values that apply when albumin concentration is normal.

We developed a dispersal method for multiwalled carbon nanotubes (MWCNTs) that allows quantitative assessment of dispersion on profibrogenic responses in tissue culture cells and in mouse lung. We demonstrate that the dispersal of as-prepared (AP), purified (PD), and carboxylated (COOH) MWCNTs by bovine serum albumin (BSA) and dipalmitoylphosphatidylcholine (DPPC) influences TGF-β1, PDGF-AA, and IL-1β production in vitro and in vivo. These biomarkers were chosen based on their synergy in promoting fibrogenesis and cellular communication in the epithelial-mesenchymal cell trophic unit in the lung. The effect of dispersal was most noticeable in AP- and PD-MWCNTs, which are more hydrophobic and unstable in aqueous buffers than hydrophilic COOH-MWCNTs. Well-dispersed AP- and PD-MWCNTs were readily taken up by BEAS-2B, THP-1 cells, and alveolar macrophages (AM) and induced more prominent TGF-β1 and IL-1β production in vitro and TGF-β1, IL-1β, and PDGF-AA production in vivo than nondispersed tubes. Moreover, there was good agreement between the profibrogenic responses in vitro and in vivo as well as the ability of dispersed tubes to generate granulomatous inflammation and fibrosis in airways. Tube dispersal also elicited more robust IL-1β production in THP-1 cells. While COOH-MWCNTs were poorly taken up in BEAS-2B and induced little TGF-β1 production, they were bioprocessed by AM and induced less prominent collagen deposition at sites of nongranulomatous inflammation in the alveolar region. Taken together, these results indicate that the dispersal state of MWCNTs affects profibrogenic cellular responses that correlate with the extent of pulmonary fibrosis and are of potential use to predict pulmonary toxicity.

Low serum 25-hydroxy vitamin D (25OHD) predicts a higher cardiovascular risk in the general population. Because patients with chronic kidney disease are more likely to have low serum 25OHD, we determined the relationship between hypovitaminosis D and death in this group. Analysis was done using a cohort composed of 3011 patients from the Third National Health and Nutrition Examination Survey who had chronic kidney disease but were not on dialysis and who had a mean follow-up of 9 years. In analyses adjusted for demographics, cardiovascular risk factors, serum phosphorus, albumin, hemoglobin, stage of chronic kidney disease, albuminuria, and socioeconomic status, individuals with serum 25OHD levels less than 15 ng/ml had an increased risk for all-cause mortality when compared to those with levels over 30 ng/ml. This significantly higher risk for death with low serum 25OHD was evident in 15 of the 23 subgroups. The higher risk for cardiovascular and non-cardiovascular mortality became statistically nonsignificant on multivariable adjustment. The trend for higher mortality in patients with 25OHD levels 15-30 ng/ml was not statistically significant. Our results indicate there is a graded relationship between serum 25OHD and the risk for death among subjects with chronic kidney disease who are not undergoing dialysis. Randomized, controlled trials are needed to conclusively determine whether vitamin D supplementation reduces mortality.

Advanced glycation endproduct (AGE) levels are elevated in renal failure patients and may contribute to the excessive cardiovascular disease in this population. Diet-derived AGE are major contributors to the total body AGE pool. It was postulated that a reduction in dietary AGE intake might impact on the high circulating AGE levels in renal failure patients. Twenty-six nondiabetic renal failure patients on maintenance peritoneal dialysis were randomized to either a high or a low AGE diet for 4 wk. Three-day dietary records, fasting blood, 24-h urine, and dialysis fluid collections were obtained at baseline and end of study. AGE levels were determined by ELISA for N(epsilon)-carboxymethyl-lysine (CML) and methylglyoxal-derivatives (MG). Eighteen patients completed the study. Low dietary AGE intake decreased serum CML (34%; P < 0.002), serum MG (35%; P < 0.008), CML-LDL (28%; P < 0.011), CML-apoB (25%; P < 0.028), dialysate CML (39%; P < 0.03), and dialysate MG output (40%; P < 0.04). High dietary AGE intake increased serum CML (29%; P < 0.028), serum MG (26%; P < 0.09), CML-LDL (50%; P < 0.011), CML-apoB (67%; P < 0.028), and dialysate CML output (27%; P < 0.01). Serum AGE correlated with BUN (r = 0.6, P < 0.002 for CML; r = 0.4, P < 0.05 for MG), serum creatinine (r = 0.76, P < 0.05 for CML; r = 0.55, P < 0.004 for MG), total protein (r = 0.4, P < 0.05 for CML; r = 0.4, P < 0.05 for MG), albumin (r = 0.4, P < 0.02 for CML; r = 0.4, P < 0.05 for MG), and phosphorus (r = 0.5, P < 0.006 for CML; r = 0.5, P < 0.01 for MG). It is concluded that dietary glycotoxins contribute significantly to the elevated AGE levels in renal failure patients. Moreover, dietary restriction of AGE is an effective and feasible method to reduce excess toxic AGE and possibly cardiovascular associated mortality.

Virulent group A streptococci have been found to express a cell-surface factor that has the capability of inactivating complement-derived chemotactic factors. To determine the mechanism of action of this factor, we examined the interaction of purified inactivator with pure C5a chemotaxin. Ligand-receptor binding studies demonstrated that streptococcal chemotactic factor inactivator (SCFI)-treated C5a expressed a greatly reduced ability to bind to receptors of polymorphonuclear leukocytes as compared with native C5a. The inactivation of C5a occurred by a nonstoichiometric and temperature-dependent process. NaDodSO4/PAGE analysis indicated that SCFI mediated a small decrease in the molecular weight of C5adesArg, and sequencing of the carboxyl terminus of inactivated C5a demonstrated that a six-residue peptide was lost. The release of discrete peptide fragments from denatured bovine serum albumin upon prolonged incubation with SCFI was indicative of endoprotease activity. Although denatured bovine serum albumin was inefficiently cleaved, native bovine serum albumin and other native proteins were highly resistant to SCFI proteolysis; this indicated that activity was specific in nature.

The bincinchoninic acid protein assay has been adapted for use with microtiter plates and a plate reader to reduce the time needed for analyses. The total volume of the assay was reduced to 0.21 ml with various ratios of sample to reagent volume being used. The assay is sensitive to a limit of less than 1 microgram of bovine serum albumin. The assay is insensitive to the presence of the detergents sodium dodecyl sulfate, Triton X-100, and deoxycholate. In addition, the method has been adapted for determining the protein concentrations of sucrose density gradient fractions, which eliminates the need for removing the sucrose prior to assay.

We examined the role of antigenic electrical charge as a determinant of glomerular immune complex localization in the rabbit. Serum sickness nephritis was induced in groups of New Zealand white rabbits by daily 25-mg intravenous injections of bovine serum albumin (BSA) chemically modified to be cationic (pI>> 9.5) or more anionic (pI, 3.5-4.6); an additional group received unmodified native BSA (pI, 4.5-5.1). Factors known to influence immune complex localization, e.g., molecular size of the administered antigen and resulting circulating immune complexes, immunogenicity, and disappearance time from the circulation were examined and found to be similar for both anionic and cationic BSA. Charge modification did increase the nonimmune clearance of cationic and anionic BSA compared with native BSA. Injected cationic BSA was shown in paired label experiments to bind directly to glomeruli compared with native BSA. The renal lesion produced by cationic BSA was markedly different from that found in rabbits given anionic or native BSA. Animals receiving cationic BSA uniformly developed generalized diffuse granular capillary wall deposits of IgG, C3, and BSA detected after 2 wk of injections and increasing until death at 6 wk. Qualitatively similar deposits were produced by the administration of low doses of cationic BSA of only 1 or 10 mg/d. In contrast, the injection of both anionic and native BSA resulted in mesangial deposits at 2 and 4 wk with capillary wall deposits appearing by 6 wk. Ultrastructural examination of animals receiving cationic BSA revealed pure, extensive formation of dense deposits along the lamina rara externa of the glomerular basement membrane whereas such deposits were absent or rare in animals injected with the anionic or native BSA. Albuminuria was significantly greater at 6 wk in the groups receiving cationic BSA with a mean of 280 mg/24 h compared with 53 mg/24 h in the combined groups injected with anionic or native BSA. Blood urea nitrogen values were similar in all groups at 2 and 4 wk but higher in the animals receiving cationic BSA at 6 wk. These experiments describe the reproducible induction of epimembranous immune deposits by administration of an exogenous cationic antigen. They suggest that antigenic charge can play an important role in the pathogenesis of membranous nephropathy by permitting direct glomerular binding of an antigen and predisposing to in situ immune complex formation.

Several metabolites of tamoxifen, including 4-hydroxy-N-desmethyltamoxifen (metabolite BX), 4-hydroxytamoxifen (metabolite B), N-desmethyltamoxifen (metabolite X), the primary alcohol (metabolite Y), and N-desdimethyltamoxifen (metabolite Z) were identified and their concentrations determined in fluids and feces from patients receiving chronic tamoxifen treatment. The biological samples investigated were serum, pleural, pericardial and peritoneal effusions, cerebrospinal fluid, saliva, bile, feces, and urine. In serum, tamoxifen itself, and the metabolites X and Z were the prevailing species, but significant amounts of the metabolites Y, B, and BX were also detected. About 3 h after drug intake tamoxifen as well as Y, B, BX, X, and Z showed a peak in serum. This may be explained by efficient metabolism of the metabolite precursor before being distributed to peripheral compartments. Upon drug withdrawal all metabolites showed first-order elimination curves which paralleled that of tamoxifen suggesting that their rate of elimination exceeded that of tamoxifen and that the serum levels are production rate limited. The protein binding of tamoxifen and its major serum metabolites (Y, X, Z) was determined and found to be higher than 98%. Albumin was the predominant carrier for tamoxifen in human plasma. The concentrations of tamoxifen and its metabolites in pleural, pericardial, and peritoneal effusions equalled those detected in serum, corresponding to an effusion/serum ratio between 0.2 and 1. Only trace amounts of tamoxifen and metabolite X were detected in cerebrospinal fluid (CSF/serum ratio less than 0.02). In saliva, concentrations of tamoxifen and X exceeded the amounts of free drug in serum, suggesting active transport or trapping of these compounds in the salivary gland. Bile and urine were rich in the hydroxylated, conjugated metabolites (Y, B, and BX), whereas in feces unconjugated metabolite B and tamoxifen were the predominating species.

A unique and heretofore undescribed glycoprotein with unusual properties has been purified and characterized from the culture medium of endothelial cells. This protein is synthesized constitutively by bovine, porcine, and human endothelial cells, by vascular smooth muscle cells, and by fibroblasts from dermis and ligament. It is also a biosynthetic product of some murine malignant and/or transformed cell lines but was not uniformly observed in cells derived from human neoplasms. The glycoprotein exhibited an apparent molecular weight by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of approximately 39,000 before reduction, and of approximately 43,000 (43K protein) in the presence of dithiothreitol. Amino acid analysis revealed high levels of potentially acidic residues (Asx + Glx = 303 residues/1000) and of cysteine (35 residues/1000). Limited proteolysis indicated that both disulfide bonds and mannosylated sites were distributed throughout the protein chain. Neither phosphate nor sulfate was incorporated into the 43K protein during biosynthetic labeling of endothelial cells. In addition, the 43K protein did not bind to heparin, thrombin, gelatin, or fibronectin and displayed no affinity for [3H]diisopropyl fluorophosphate. In contrast, the 43K protein demonstrated a high affinity binding to bovine serum albumin which was dissociable only by sodium dodecyl sulfate. A complete lack of identity with several prominent serum and platelet proteins and with other mesenchymal cell products was shown by one- and two-dimensional peptide mapping, affinity chromatography, and immunological studies. Immunofluorescence staining of endothelial cells showed a granular distribution for the 43K protein that was typical of a secreted protein. The function of this apparently novel glycoprotein is presently not known. Its synthesis by normal mesenchymal cells and by malignant or transformed cells of both ectodermal and endodermal origin suggests a general role in cell function that is independent of transformation.

Aflatoxins are immunotoxins that frequently contaminate staple foods in The Gambia and other parts of sub-Saharan Africa, resulting in high exposure throughout life. Impaired infant immune system development may be a key predictor of mortality from infectious disease. In this study we aimed to determine the effect of dietary aflatoxin exposure on a number of immune parameters in Gambian children. A cohort of 472 Gambian children 6-9 years of age was recruited. Serum aflatoxin-albumin (AF-alb) adducts were analyzed to provide a measure of exposure. Immune parameters included secretory IgA (sIgA) in saliva, cell-mediated immunity (CMI), determined using the CMI multitest where test antigens are applied to the skin, and antibody responses to both rabies and pneumococcal polysaccharide vaccines. Birth weight, current anthropometry, and micronutrient status were also recorded. AF-alb adducts were detected in 93% of the children (geometric mean level 22.3 pg/mg; range 5-456 pg/mg). AF-alb level was strongly influenced by month of sampling. In a multivariable analysis, sIgA was markedly lower in children with detectable AF-alb compared with those with nondetectable levels [50.4 micro g/mg protein (95% confidence interval [CI] 48.0-52.8) and 70.2 micro g/mg protein (95% CI 61.1-79.2), respectively; p < 0.0001]. Antibody response to one of four pneumococcal serotypes, but not rabies vaccine, was weakly associated with higher levels of AF-alb. There was no association between CMI responses to test antigens and AF-alb. These data confirm that children in rural Gambia are frequently exposed to high levels of aflatoxin. The study provides evidence that sIgA in saliva may be reduced because of dietary levels of aflatoxin exposure. Given the high burden of infection-related mortality in West Africa, further investigation of the immune effects of aflatoxin exposure in children is merited.