BACKGROUND

Invisible near-infrared (NIR) fluorescent light permits high sensitivity, real-time image-guidance during oncologic surgery without changing the look of the surgical field. In this study, we complete pre-clinical development of the technology for sentinel lymph node (SLN) mapping using a large animal model of spontaneous melanoma.

METHODS

Sinclair swine with spontaneous melanoma metastatic to regional lymph nodes were used because of their similarity to human melanoma. Organic lymphatic tracers tested included FDA-approved indocyanine green adsorbed non-covalently to human serum albumin (HSA), and NIR fluorophore CW800 conjugated covalently to HSA (HSA800). The inorganic/organic hybrid tracer tested was type II NIR quantum dots with an anionic coating. Primary tumors received four peri-tumoral injections of each tracer, with a fluorophore dose of 100 pmol to 1 nmol per injection. SLN mapping and image-guided resection were performed in real-time.

RESULTS

Each of the 3 lymphatic tracers was injected into n = 4 separate primary melanomas in a total of 6 animals. All 12 injections resulted in identification of the SLN(s) and their associated lymphatic channels within 1 minute in 100% of cases, despite highly pigmented skin and black fur. Hydrodynamic diameter had a profound impact on tracer behavior in vivo.

CONCLUSIONS

This study completes the pre-clinical development of NIR fluorescence-guided SLN mapping and provides insight into imaging system optimization and tracer choice for future human clinical trials. The technology is likely to eliminate the need for radioactive and colored tracers, permits real-time image guidance throughout the procedure, and assists the pathologist in tissue analysis.

OBJECTIVE

To investigate the effect of microparticle size on gastrointestinal tissue uptake.

METHODS

Biodegradable microparticles of various sizes using polylactic polyglycolic acid (50:50) co-polymer (100 nm, 500 nm, 1 micron, and 10 microns) and bovine serum albumin as a model protein were formulated by water-in-oil-in-water emulsion solvent evaporation technique. The uptake of microparticles was studied in rat in situ intestinal loop model and quantitatively analyzed for efficiency of uptake.

RESULTS

In general, the efficiency of uptake of 100 nm size particles by the intestinal tissue was 15-250 fold higher compared to larger size microparticles. The efficiency of uptake was dependent on the type of tissue, such as Peyer's patch and non patch as well as on the location of the tissue collected i.e. duodenum or ileum. Depending on the size of microparticles, the Peyer's patch tissue had 2-200 fold higher uptake of particles than the non-patch tissue collected from the same region of the intestine. Histological evaluation of the tissue sections demonstrated that 100 nm particles were diffused throughout the submucosal layers while the larger size nano/microparticles were predominantly localized in the epithelial lining of the tissue.

CONCLUSIONS

There is a microparticle size dependent exclusion phenomena in the gastrointestinal mucosal tissue with 100 nm size particles showing significantly greater tissue uptake. This has important implications in designing of nanoparticle-based oral drug delivery systems, such as an oral vaccine system.

In the short term, the endothelin antagonist avosentan reduces proteinuria, but whether this translates to protection from progressive loss of renal function is unknown. We examined the effects of avosentan on progression of overt diabetic nephropathy in a multicenter, multinational, double-blind, placebo-controlled trial. We randomly assigned 1392 participants with type 2 diabetes to oral avosentan (25 or 50 mg) or placebo in addition to continued angiotensin-converting enzyme inhibition and/or angiotensin receptor blockade. The composite primary outcome was the time to doubling of serum creatinine, ESRD, or death. Secondary outcomes included changes in albumin-to-creatinine ratio (ACR) and cardiovascular outcomes. We terminated the trial prematurely after a median follow-up of 4 months (maximum 16 months) because of an excess of cardiovascular events with avosentan. We did not detect a difference in the frequency of the primary outcome between groups. Avosentan significantly reduced ACR: In patients who were treated with avosentan 25 mg/d, 50 mg/d, and placebo, the median reduction in ACR was 44.3, 49.3, and 9.7%, respectively. Adverse events led to discontinuation of trial medication significantly more often for avosentan than for placebo (19.6 and 18.2 versus 11.5% for placebo), dominated by fluid overload and congestive heart failure; death occurred in 21 (4.6%; P = 0.225), 17 (3.6%; P = 0.194), and 12 (2.6%), respectively. In conclusion, avosentan reduces albuminuria when added to standard treatment in people with type 2 diabetes and overt nephropathy but induces significant fluid overload and congestive heart failure.

Porphyromonas gingivalis produces arginine-specific cysteine proteinase (Arg-gingipain, RGP) and lysine-specific cysteine proteinase (Lys-gingipain, KGP) in the extracellular and cell-associated forms. Two separate genes (rgpA and rgpB) and a single gene (kgp) have been found to encode RGP and KGP, respectively. We constructed rgpA rgpB kgp triple mutants by homologous recombination with cloned rgp and kgp DNA interrupted by drug resistance gene markers. The triple mutants showed no RGP or KGP activity in either cell extracts or culture supernatants. The culture supernatants of the triple mutants grown in a rich medium had no proteolytic activity toward bovine serum albumin or gelatin derived from human type I collagen. Moreover, the mutants did not grow in a defined medium containing bovine serum albumin as the sole carbon/energy source. These results indicate that the proteolytic activity of P. gingivalis toward bovine serum albumin and gelatin derived from human type I collagen appears to be attributable to RGP and KGP. The hemagglutinin gene hagA of P. gingivalis possesses the adhesin domain regions responsible for hemagglutination and hemoglobin binding that are also located in the C-terminal regions of rgpA and kgp. A rgpA kgp hagA triple mutant constructed in this study exhibited no hemagglutination using sheep erythrocytes or hemoglobin binding activity, as determined by a solid-phase binding assay with horseradish peroxidase-conjugated human hemoglobin, indicating that the adhesin domains seem to be particularly important for P. gingivalis cells to agglutinate erythrocytes and bind hemoglobin, leading to heme acquisition.

An optical method for measuring oxygen concentrations in aqueous solutions is described. This method is based upon the oxygen-dependent quenching of phosphorescence. Phosphorescence excitation and emission spectra and lifetimes of some of the probe molecules suitable for measurement of oxygen in aqueous solutions are given. The probes include fluorescein derivatives, 4'5'-diiodofluorescein, eosin Y, 5(and 6)-carboxyeosin, erythrosin, and 5(and 6)-carboxyerythrosin as well as the Zn(II), Y(III), Sn(IV), Lu(III), and Pd(II) derivatives of meso-tetra-(4-sulfonatophenyl)-porphine, meso-tetra-(N-methyl-4-pyridyl)-porphine and coproporphyrin. The phosphorescence lifetimes of the given probes were found to depend upon the oxygen concentration by a simple Stern-Volmer relationship with a quenching constant of approximately 10(9) M-1 S-1. Binding of the molecules to bovine serum albumin decreased the quenching constant for oxygen by approximately an order of magnitude and also inhibited probe self-quenching, indicating that at the protein binding site the probes are somewhat protected from collision with quenchers. The use of this optical method for measuring oxygen is demonstrated for reactions catalyzed by glucose oxidase and by cytochrome c oxidase. It is shown that, using this method oxygen concentrations can be measured from approximately 250 microM (air saturation) down to the nanomolar range.

Ceramides are known to play a major regulatory role in apoptosis by inducing cytochrome c release from mitochondria. We have previously reported that C(2)- and C(16)-ceramide, but not dihydroceramide, form large channels in planar membranes (Siskind, L. J., and Colombini, M. (2001) J. Biol. Chem. 275, 38640-38644). Here we show that ceramides do not trigger a cytochrome c secretion or release mechanism, but simply raise the permeability of the mitochondrial outer membrane, via ceramide channel formation, to include small proteins. Exogenously added reduced cytochrome c was able to freely permeate the mitochondrial outer membrane with entry to and exit from the intermembrane space facilitated by ceramides in a dose- and time-dependent manner. The permeability pathways were eliminated upon removal of C(2)-ceramide by bovine serum albumin, thus ruling out a detergent-like effect of C(2)-ceramide on membranes. Ceramide channels were not specific to cytochrome c, as ceramides induced release of adenylate kinase, but not fumerase from isolated mitochondria, showing some specificity of these channels for the outer mitochondrial membrane. SDS-PAGE results show that ceramides allow release of intermembrane space proteins with a molecular weight cut-off of about 60,000. These results indicate that the ceramide-induced membrane permeability increases in isolated mitochondria are via ceramide channel formation and not a release mechanism, as the channels that allow cytochrome c to freely permeate are reversible, and are not specific to cytochrome c.

BACKGROUND

The Department of Veterans Affairs National Surgical Quality Improvement Program is a unique resource to prospectively analyze surgical outcomes from a cross-section of surgical services nationally. We used this database to assess risk factors for morbidity and mortality after esophagectomy in Veterans Affairs Medical Centers from 1991 to 2001.

METHODS

A total of 1,777 patients underwent an esophagectomy at 109 Veterans Affairs hospitals with complete in-hospital and 30-day outcomes recorded. Bivariate and multivariable analyses were completed.

RESULTS

Thirty-day mortality was 9.8% (174/1,777) and the incidence of one or more of 20 predefined complications was 49.5% (880/1,777). The most frequent postoperative complications were pneumonia in 21% (380/1,777), respiratory failure in 16% (288/1,777), and ventilator support more than 48 hours in 22% (387/1,777). Preoperative predictors of mortality based on multivariable analysis included neoadjuvant therapy, blood urea nitrogen level of more than 40 mg/dL, alkaline phosphatase level of more than 125 U/L, diabetes mellitus, alcohol abuse, decreased functional status, ascites, and increasing age. Preoperative factors impacting morbidity were increasing age, dyspnea, diabetes mellitus, chronic obstructive pulmonary disease, alkaline phosphatase level of more than 125 U/L, lower serum albumin concentration, increased complexity score, and decreased functional status. Intraoperative risk factors for mortality included the need for transfusion; intraoperative risk factors for morbidity included the need for transfusion and longer operative time.

CONCLUSIONS

These data constitute the largest prospective outcomes cohort in the literature and document a near 50% morbidity rate and 10% mortality rate after esophagectomy. Data from this study can be used to better stratify patients before esophagectomy.

Infections in critically ill patients are associated with persistently poor clinical outcomes. These patients have severely altered and variable antibiotic pharmacokinetics and are infected by less susceptible pathogens. Antibiotic dosing that does not account for these features is likely to result in suboptimum outcomes. In this Review, we explore the challenges related to patients and pathogens that contribute to inadequate antibiotic dosing and discuss how to implement a process for individualised antibiotic therapy that increases the accuracy of dosing and optimises care for critically ill patients. To improve antibiotic dosing, any physiological changes in patients that could alter antibiotic concentrations should first be established; such changes include altered fluid status, changes in serum albumin concentrations and renal and hepatic function, and microvascular failure. Second, antibiotic susceptibility of pathogens should be confirmed with microbiological techniques. Data for bacterial susceptibility could then be combined with measured data for antibiotic concentrations (when available) in clinical dosing software, which uses pharmacokinetic/pharmacodynamic derived models from critically ill patients to predict accurately the dosing needs for individual patients. Individualisation of dosing could optimise antibiotic exposure and maximise effectiveness.

BACKGROUND

Serum albumin (S-Alb), C-reactive protein (CRP), and interleukin 6 (IL-6) predict malnutrition, atherosclerotic cardiovascular disease (CVD), and mortality in patients with end-stage renal disease (ESRD). Fetuin A, an inhibitor of vascular calcification, also is associated strongly with clinical outcome in patients with ESRD. In this study, multivariate analyses were performed to assess these 4 biomarkers as predictors of malnutrition, CVD, and mortality in patients with ESRD.

METHODS

One hundred seventy-six patients with ESRD (54 +/- 12 years) underwent measurements of S-Alb, high-sensitivity CRP (hs-CRP), plasma IL-6, and fetuin A close to the start of dialysis therapy and were followed up for a median of 26 months (range, 1 to 66 months). Nutritional status was evaluated by means of subjective global assessment. CVD was defined based on medical history. Associations between biomarker levels and malnutrition, CVD, and mortality were evaluated by means of receiver operating characteristic curve, logistic regression analysis, and Cox proportional hazards model.

RESULTS

All 4 biomarkers predicted malnutrition, CVD, and mortality. Multivariate analysis, according to receiver operating characteristic analysis, showed that malnutrition was predicted best by hs-CRP and IL-6 levels; CVD, by IL-6 level; and mortality, by albumin and IL-6 levels. When using the cutoff levels derived from receiver operating characteristics, logistic regression analysis showed that only hs-CRP level (odds ratio [OR], 3.6) was associated with malnutrition, and only IL-6 level (OR, 3.7) was associated with CVD. Levels of S-Alb, IL-6, and fetuin A, but not hs-CRP, were associated with increased relative risk for mortality as assessed by Cox in a model adjusting for age, sex, and diabetes mellitus.

CONCLUSIONS

Multivariate analyses show that in patients with ESRD, malnutrition is predicted best by hs-CRP and IL-6 levels; CVD, by IL-6 level; and mortality, by S-Alb, IL-6, and fetuin A levels, but not by hs-CRP level. This comparative analysis indicates that of these biomarkers, IL-6 level may be the most reliable predictor of CVD and mortality in patients with ESRD.

OBJECTIVE

To compare the quality of life (QoL) of patients receiving oxaliplatin, irinotecan, fluorouracil, and leucovorin (FOLFIRINOX) or gemcitabine as first-line chemotherapy and to assess whether pretreatment QoL predicts survival in patients with metastatic pancreatic cancer.

METHODS

Three hundred forty-two patients with performance status 0 or 1 were randomly assigned to receive FOLFIRINOX (oxaliplatin, 85 mg/m(2); irinotecan, 180 mg/m(2); leucovorin, 400 mg/m(2); and fluorouracil, 400 mg/m(2) bolus followed by 2,400 mg/m(2) 46-hour continuous infusion, once every 2 weeks) or gemcitabine 1,000 mg/m(2) weekly for 7 of 8 weeks and then weekly for 3 of 4 weeks. QoL was assessed using European Organisation for the Research and Treatment of Cancer Quality of Life Questionnaire C30 every 2 weeks.

RESULTS

Improvement in global health status (GHS; P < .001) was observed in the FOLFIRINOX arm and improvement in emotional functioning (P < .001) was observed in both arms, along with a decrease in pain, insomnia, anorexia, and constipation in both arms. A significant increase in diarrhea was observed in the FOLFIRINOX arm during the first 2 months of chemotherapy. Time until definitive deterioration ≥ 20 points was significantly longer for FOLFIRINOX compared with gemcitabine for GHS, physical, role, cognitive, and social functioning, and six symptom domains (fatigue, nausea/vomiting, pain, dyspnea, anorexia, and constipation). Physical functioning, constipation, and dyspnea were independent significant prognostic factors for survival with treatment arm, age older than 65 years, and low serum albumin.

CONCLUSIONS

FOLFIRINOX significantly reduces QoL impairment compared with gemcitabine in patients with metastatic pancreatic cancer. Furthermore, baseline QoL scores improved estimation of survival probability when added to baseline clinical and demographic variables.

The bursa of Fabricius and the thymus are "central lymphoid organs" in the chicken, essential to the ontogenetic development of adaptive immunity in that species. Surgical removal of one or both of these organs in the newly hatched chicken, followed by sublethal X-irradiation the next day, has permitted recognition of two morphologically distinct cell systems in the "peripheral lymphoid tissues" of the spleen, gut, and other organs, and clear definition of the separate functions of each cell system. The thymus-dependent development is represented morphologically by the small lymphocytes of the circulation and the white pulp type of development in the tissues. As in mammals, the thymus-dependent tissues of the chicken are basic to the ontogenesis of cellular immunity: graft versus host reactions, responses of delayed hypersensitivity and homograft rejection; and play a less clearly defined role in the antibody response to at least some antigens. Thymectomized-irradiated chickens are deficient in all these responses, and grow more slowly than any of the other experimental groups. In these animals germinal centers, plasma cells, and capacity for immunoglobulin synthesis remain intact. The bursa-dependent development is represented morphologically by the larger lymphocytes of the germinal centers and the plasma cells, and functionally by the immunoglobulins. Bursectomized-irradiated chickens are agammaglobulinemic and unable to produce detectable antibody despite intense, repeated stimulation with bovine serum albumin and Brucella abortus organisms. The thymus-dependent development in these animals seems to be normal; they have adequate numbers of lymphocytes in the circulation and tissues, are able to reject skin homografts, though more slowly than usual, and to exercise graft versus host reactions. The short life span of these chickens has precluded adequate study of responses of delayed hypersensitivity. There was no evidence of significant impairment of reticuloendothelial function in either the bursectomized-irradiated or the thymectomized-irradiated group, as judged by the clearance of colloidal gold and I(131)-tagged keyhole limpet hemocyanin.

Microalbuminuria (MA) is associated with adverse health outcomes in diabetic and hypertensive adults. The prevalence and clinical significance of MA in nondiabetic populations is less clear. The purpose of this study was to generate national estimates of the prevalence of MA in the US population. Untimed urinary albumin concentrations (UACs) and creatinine concentrations were evaluated in a nationally representative sample of 22,244 participants aged 6 years and older. Persons with hematuria and menstruating or pregnant women were excluded from analysis. The percent prevalence of clinical proteinuria (UAC>> or = 300 mg/L) was similar for males and females. However, the prevalence of MA (urinary albumin-creatinine ratio [ACR], 30 to 299 mg/g) was significantly lower in males (6.1%) compared with females (9.7%). MA prevalence was greater in children than young adults and increased continuously starting at 40 years of age. MA prevalence was greater in non-Hispanic blacks and Mexican Americans aged 40 to 79 years compared with similar-aged non-Hispanic whites. MA prevalence was 28.8% in persons with previously diagnosed diabetes, 16.0% in those with hypertension, and 5.1% in those without diabetes, hypertension, cardiovascular disease, or elevated serum creatinine levels. In adults aged 40+ years, after excluding persons with clinical proteinuria, albuminuria (defined as ACR>> or = 30 mg/g) was independently associated with older age, non-Hispanic black and Mexican American ethnicity, diabetes, hypertension, and elevated serum creatinine concentration. MA is common, even among persons without diabetes or hypertension. Age, sex, race/ethnicity, and concomitant disease contribute to the variability of MA prevalence estimates.

We initially observed a phenomenon of reduced in vitro binding of exogenous cobalt [Co(II)] to the N-terminus of human <em>serum</em> <em>albumin</em> (HSA) in emergency chest pain patients with early onset unstable angina and myocardial infarction. We then developed a colorimetric assay to measure cobalt-HSA binding and record the results in absorbance units (ABSU). In a preliminary clinical study of 139 emergency patients with acute chest pain, 99 patients with evidence of myocardial ischemia (Group 1) had elevated assay levels (mean ABSU +/- SD; 0.519 +/- 0.086) compared to 40 patients (Group 2) with no evidence of ischemia (0.316 +/- 0.092) (p < 0.00001). In Group 1, 95 of 99 (96.0%) patients had levels higher than a decision threshold of 0.400 ABSU and in Group 2, 37 of 40 (92.5%) samples had higher cobalt binding capacity (ABSU </= 0.400). Further studies are warranted to determine if an assay measuring altered cobalt-HSA binding is a clinically useful diagnostic test to rule out myocardial ischemia.

A serum-free lymphocyte culture medium is described in which serum is replaced by bovine serum albumin, transferrin, insulin, ethanolamine and a mixture of saturated and unsaturated fatty acids (linoleic acid, oleic acid and palmitic acid). In this serum-free medium proliferative and cytotoxic responses induced in mixed lymphocyte culture were comparable with those obtained in medium containing serum. Antigen-specific cytotoxic and helper T cells were isolated and could be propagated in serum-free medium without loss of function.

Antigen-induced arthritis was established in the mouse by immunizaiton with methylated bovine serum albumin (mBSA) in complete Freund's adjuvant with B pertusis vaccine. The knee joint was injected after 21 days with mBSA in saline. The arthritis was chronic, antigen-specific, and T-cell dependent in hypothmic nu/nu mice. C57BL and balb/c mice were susceptible, whereas CBA mice were relatively resistant. Susceptibility was dominant; one gene was loosely linked to the "b" allele of the H-2 complex of C57BL mice.

BACKGROUND

The importance of clinical risk factors for postoperative pulmonary complications and the value of preoperative testing to stratify risk are the subject of debate.

OBJECTIVE

To systematically review the literature on preoperative pulmonary risk stratification before noncardiothoracic surgery.

METHODS

MEDLINE search from 1 January 1980 through 30 June 2005 and hand search of the bibliographies of retrieved articles.

METHODS

English-language studies that reported the effect of patient- and procedure-related risk factors and laboratory predictors on postoperative pulmonary complication rates after noncardiothoracic surgery and that met predefined inclusion criteria.

METHODS

The authors used standardized abstraction instruments to extract data on study characteristics, hierarchy of research design, study quality, risk factors, and laboratory predictors.

RESULTS

The authors determined random-effects pooled estimate odds ratios and, when appropriate, trim-and-fill estimates for patient- and procedure-related risk factors from studies that used multivariable analyses. They assigned summary strength of evidence scores for each factor. Good evidence supports patient-related risk factors for postoperative pulmonary complications, including advanced age, American Society of Anesthesiologists class 2 or higher, functional dependence, chronic obstructive pulmonary disease, and congestive heart failure. Good evidence supports procedure-related risk factors for postoperative pulmonary complications, including aortic aneurysm repair, nonresective thoracic surgery, abdominal surgery, neurosurgery, emergency surgery, general anesthesia, head and neck surgery, vascular surgery, and prolonged surgery. Among laboratory predictors, good evidence exists only for serum albumin level less than 30 g/L. Insufficient evidence supports preoperative spirometry as a tool to stratify risk.

CONCLUSIONS

For certain risk factors and laboratory predictors, the literature provides only unadjusted estimates of risk. Prescreening, variable selection algorithms, and publication bias limited reporting of risk factors among studies using multivariable analysis.

CONCLUSIONS

Selected clinical and laboratory factors allow risk stratification for postoperative pulmonary complications after noncardiothoracic surgery.

The available data on bone fractures in hemodialysis (HD) patients are limited to results of a few studies of subgroups of patients in the United States. This study describes the prevalence of hip fractures and the incidence and risk factors associated with hip and other fractures in representative groups of HD facilities (n=320) and patients (n=12 782) from the 12 countries in the second phase of the Dialysis Outcomes and Practice Patterns Study (2002-2004). Among prevalent patients, 2.6% had a prior hip fracture. The incidence of fractures was 8.9 per 1000 patient years for new hip fractures and 25.6 per 1000 for any new fracture. Older age (relative risk (RR)(HIP)=1.91, RR(ANY)=1.33, P<0.0001), female sex (RR(HIP)=1.41, P=0.02; RR(ANY)=1.59, P<0.0001), prior kidney transplant (RR(HIP)=2.35, P=0.04; RR(ANY)=1.76, P=0.007), and low serum albumin (RR(HIP)=1.85, RR(ANY)=1.45, per 1 g/dl lower, P<0.0001) were predictive of new fractures. Elevated risk of new hip fracture was observed for selective serotonin reuptake inhibitors and combination narcotic medications (RR=1.63, RR=1.74, respectively, P<0.05). Several medications were associated with risk of any new fracture: narcotic pain medications (RR=1.67, P=0.02), benzodiazepines (RR=1.31, P=0.03), adrenal cortical steroids (RR=1.40, P<0.05), and combination narcotic medications (RR=1.72, P=0.001). Parathyroid hormone (PTH) levels >900 pg/ml were associated with an elevated risk of any new fracture (RR=1.72, P<0.05) versus PTH 150-300. The results suggest that greater selectivity in prescribing several classes of psychoactive drugs and more efficient treatment of secondary hyperparathyroidism may help reduce the burden of fractures in HD patients.

Patients on maintenance hemodialysis (MHD) often show substantial reductions in quality of life (QoL). The SF36 (Short Form with 36 questions), a well-documented, self-administered QoL scoring system that includes eight independent scales and two main dimensions, has been widely used and validated. In 65 adult outpatients on MHD, the SF36 and its scales and dimensions, scored as a number between 0 and 100, and the nutritional and inflammatory state measured by subjective global assessment, near-infrared (NIR) body fat, body mass index (BMI), and pertinent laboratory values, including hemoglobin, albumin, and C-reactive protein were assessed. Twelve-month prospective hospitalization rates and mortality were used as the clinical outcomes. Multivariate (case-mix) adjusted correlation coefficients were statistically significant between SF36 scores and serum albumin and hemoglobin concentrations. There were significant inverse correlations between SF36 scores and the BMI and NIR body fat percentage. Hypoalbuminemic, anemic, and obese patients on MHD had a worse QoL. Prospective hospitalizations correlated significantly with the SF36 total score and its two main dimensions (r between -0.28 and -0.40). The Cox proportional regression relative risk of death for each 10 unit decrease in SF36 was 2.07 (95% CI, 1.08 to 3.98; P = 0.02). Of the eight components and two dimensions of the SF36, the Mental Health dimension and the SF36 total score had the strongest predictive value for mortality. Thus, in patients on MHD the SF36 appears to have significant associations with measures of nutritional status, anemia, and clinical outcomes, including prospective hospitalization and mortality. Even though obesity, unlike undernutrition, is not generally an indicator of poor outcome in MHD, the SF36 may detect obese patients on MHD at higher risk for morbidity and mortality.

The arginine-rich fraction of calf thymus histone (histone B) exerts bactericidal activity on various coliform bacilli and micrococci under certain conditions in vitro. Final concentrations of less than 1 microg. histone per ml. kill susceptible microbes without detectable morphological alteration or lysis. Among the microorganisms highly susceptible to histone are Escherichia, Salmonella, Shigella, Pseudomonas, Klebsiella, and Micrococcus pyogenes var. albus. Less susceptible or completely resistant are Proteus, Serratia, Micrococcus pyogenes var. aureus, and various types of hemolytic streptococci. Coliforms grown on solid media are much more resistant to the lethal effect of histone than are those cultured in liquid media. This difference is apparently related to the physiological state of the bacteria; agar grown microorganisms washed with water remain resistant to histone, whereas incubation in broth rapidly renders them more susceptible. Histone is adsorbed onto heat-killed E. coli K-12 under conditions suitable for lethal action on this organism. The bactericidal activity of histone is but little affected by pH of the test system, but ionic strength of the medium exerts a marked influence, the lethal action being reduced or blocked as the salt concentration reaches levels higher than that of 0.15-0.2 M NaCl. Relatively high concentrations of rabbit serum or of bovine plasma albumin reduce the bactericidal activity of histone in a medium at pH 7; these serum preparations are, however, essentially without effect in the test system at pH 5.6. The bactericidal effect of histone is antagonized by addition to the medium of small amounts of certain basic substances (protamine, spermine), or of various acid polysaccharides (heparin, nucleic acid, bacterial lipopolysaccharides). The rate of killing of E. coli K-12 by histone increases as the temperature and the concentration of histone are raised. Within the limits studied, this rate also appears to be directly proportional to the concentration of bacteria in the system.

We propose a mechanism for oligonucleotide (ODN) release from cationic lipid complexes in cells that accounts for various observations on cationic lipid-nucleic acid-cell interactions. Fluorescent confocal microscopy of cells treated with rhodamine-labeled cationic liposome/ fluorescein-labeled ODN (F-ODN) complexes show the F-ODN separates from the lipid after internalization and enters the nucleus leaving the fluorescent lipid in cytoplasmic structures. ODN displacement from the complex was studied by fluorescent resonance energy transfer. Anionic liposome compositions (e.g., phosphatidylserine) that mimic the cytoplasmic facing monolayer of the cell membrane released ODN from the complex at about a 1:1 (-/+) charge ratio. Release was independent of ionic strength and pH. Physical separation of the F-ODN from monovalent and multivalent cationic lipids was confirmed by gel electrophoresis. Fluid but not solid phase anionic liposomes are required, whereas the physical state of the cationic lipids does not effect the release. Water soluble molecules with a high negative linear charge density, dextran sulfate, or heparin also release ODN. However, ATP, spermidine, spermine, tRNA, DNA, polyglutamic acid, polylysine, bovine serum albumin, or histone did not release ODN, even at 100-fold charge excess (-/+). Based upon these results, we propose that the complex, after internalization by endocytosis, induces flip-flop of anionic lipids from the cytoplasmic facing monolayer. Anionic lipids laterally diffuse into the complex and form a charged neutralized ion-pair with the cationic lipids. This leads to displacement of the ODN from the cationic lipid and its release into the cytoplasm.

BACKGROUND

The medical risk factors associated with increased mortality in hemodialysis (HD) patients are well known, but the psychosocial factors that may affect outcome have not been clearly defined. One key psychosocial factor, depression, has been considered a predictor of mortality, but previous studies have provided equivocal results regarding the association. We sought to determine whether depressive affect is associated with mortality in a longitudinal study of end-stage renal disease (ESRD) patients treated with HD, using multiple assessments over time.

METHODS

Two hundred ninety-five outpatients with ESRD treated with HD were recruited from three outpatient dialysis units in Washington D.C. to participate in a prospective cohort study with longitudinal follow-up. Patients were assessed every six months for up to two years using the Beck Depression Inventory (BDI), age, serum albumin concentration, Kt/V, and protein catabolic rate (PCR). A severity index, previously demonstrated to be a mortality marker, was used to grade medical comorbidity. The type of dialyzer with which the patient was treated was noted. Patient mortality status was tracked for a minimum of 20 and a maximum of 60 months after the first interview. Cox proportional hazards models, treating depression scores as time-varying covariates in a univariable analysis, and controlling for age, medical comorbidity, albumin concentration, and dialyzer type and site in multivariable models, were used to assess the relative mortality risk.

RESULTS

The mean (+/- SD) age of our population at initial interview was 54.6 +/- 14.1 years. The mean PCR was 1.06 +/- 0.27 g/kg/day, and the mean Kt/V was 1.2 +/- 0.4 at baseline, suggesting that the patients were well nourished and dialyzed comparably to contemporary U.S. patients. The patients' mean BDI at enrollment was 11.4 +/- 8.1, in the range of mild depression. Patients' baseline level of depression was not a significant predictor of mortality at 38.6 months of follow-up. In contrast, when depression was treated as a time-varying covariate based on periodic follow-up assessments, the level of depressive affect was significantly associated with mortality in both single variable and multivariable analyses.

CONCLUSIONS

Higher levels of depressive affect in ESRD patients treated with HD are associated with increased mortality. The effects of depression on patient survival are of the same order of magnitude as medical risk factors. Our findings using both controls for factors possibly confounded with depressive affect in patients with ESRD and time-varying covariate analyses may explain the inconsistent results of previous studies of depression and mortality in ESRD patients. Time-varying analyses in longitudinal studies may add power to defining and sensitivity to establishing the association of psychosocial factors and survival in ESRD patients. The mechanism underlying the relationship of depression and survival and the effect of interventions to improve depression in HD outpatients and general medical inpatients should be studied.

Aldosterone promotes glomerular and tubular sclerosis independent of angiotensin II in animal models of diabetic nephropathy. Most human studies testing the renoprotective benefit of adding an angiotensin receptor blocker or a mineralocorticoid receptor antagonist to a regimen based on inhibition of angiotensin-converting enzyme (ACE) used relatively low doses of ACE inhibitors. Furthermore, these studies did not determine whether antiproteinuric effects were independent of BP lowering. We conducted a double-blind, placebo-controlled trial in 81 patients with diabetes, hypertension, and albuminuria (urine albumin-to-creatinine ratio>> or =300 mg/g) who all received lisinopril (80 mg once daily). We randomly assigned the patients to placebo, losartan (100 mg daily), or spironolactone (25 mg daily) for 48 wk. We obtained blood and urine albumin, urea, creatinine, electrolytes, A1c, and ambulatory BP at baseline, 24, and 48 wk. Compared with placebo, the urine albumin-to-creatinine ratio decreased by 34.0% (95% CI, -51.0%, -11.2%, P = 0.007) in the group assigned to spironolactone and by 16.8% (95% CI, -37.3%, +10.5%, P = 0.20) in the group assigned to losartan. Clinic and ambulatory BP, creatinine clearance, sodium and protein intake, and glycemic control did not differ between groups. Serum potassium level was significantly higher with the addition of either spironolactone or losartan. In conclusion, the addition of spironolactone, but not losartan, to a regimen including maximal ACE inhibition affords greater renoprotection in diabetic nephropathy despite a similar effect on BP. These results support the need to conduct a long-term, large-scale, renal failure outcomes trial.

The colored complex formed between Cu+ and bicinchoninic acid is the basis of the bicinchoninic acid protein assay (P. K. Smith, R. I. Krohn, G. T. Hermanson, A. K. Mallia, F. H. Gartner, M. D. Provenzano, E. K. Fujimoto, N. M. Goeke, B.J. Olson, and D.C. Klenk (1985) Anal. Biochem. 150, 76-85). Studies show that cysteine, tryptophan, tyrosine, and the peptide bond are capable of reducing Cu2+ to Cu+. Electrochemical studies and the magnitude of the color changes observed when the reaction is carried out at 37 degrees C indicate that tryptophan, tyrosine, and the peptide bond are not completely oxidized at this temperature. When the reaction temperature is increased to 60 degrees C, significantly more color formation is observed for these three groups. Studies with di-, tri-, and tetrapeptides and with proteins indicate that the extent of color formation is not the sum of the contributions of the individual color producing functional groups. Compounds with functional groups similar to those of cysteine, cystine, tyrosine, or tryptophan are shown to react with the bicinchoninic acid reagent. The color formed by these compounds in the presence of bovine serum albumin cannot be compensated for by using a reagent blank containing an identical concentration of the interfering compound.

Metabolomics is becoming common in epidemiology due to recent developments in quantitative profiling technologies and appealing results from their applications for understanding health and disease. Our team has developed an automated high-throughput serum NMR metabolomics platform that provides quantitative molecular data on 14 lipoprotein subclasses, their lipid concentrations and composition, apolipoprotein A-I and B, multiple cholesterol and triglyceride measures, albumin, various fatty acids as well as on numerous low-molecular-weight metabolites, including amino acids, glycolysis related measures and ketone bodies. The molar concentrations of these measures are obtained from a single serum sample with costs comparable to standard lipid measurements. We have analyzed almost 250 000 samples from around 100 epidemiological cohorts and biobanks and the new international set-up of multiple platforms will allow an annual throughput of more than 250 000 samples. The molecular data have been used to study type 1 and type 2 diabetes etiology as well as to characterize the molecular reflections of the metabolic syndrome, long-term physical activity, diet and lipoprotein metabolism. The results have revealed new biomarkers for early atherosclerosis, type 2 diabetes, diabetic nephropathy, cardiovascular disease and all-cause mortality. We have also combined genomics and metabolomics in diverse studies. We envision that quantitative high-throughput NMR metabolomics will be incorporated as a routine in large biobanks; this would make perfect sense both from the biological research and cost point of view - the standard output of over 200 molecular measures would vastly extend the relevance of the sample collections and make many separate clinical chemistry assays redundant.