BACKGROUND

Hypoalbuminaemia is a marker of malnutrition-inflammation complex syndrome (MICS) and a strong predictor of cardiovascular (CV) death in maintenance haemodialysis (MHD) patients. However, serum albumin may change over time. Hence, its time-varying associations with outcome may be different.

METHODS

Associations between 3-month averaged serum albumin levels, measured in a single laboratory using bromocresol green, and CV mortality were studied longitudinally in a 2-year cohort of 58,058 MHD patients. Mortality predictability of fixed baseline and trimonthly-varying serum albumin concentrations were compared.

RESULTS

Hazard ratios (HRs) of CV death strictly increased across decrements of baseline serum albumin, whereas the HR for time-varying serum albumin decrements below 3.8 g/dl did not differ. A drop in serum albumin in the first 6 months was associated with increasing all-cause and CV death risks in the subsequent 18 months, while a rise in serum albumin was a predictor of better survival independent of baseline serum albumin. The multivariate adjusted population-attributable fraction of death due to baseline serum albumin <3.8 g/dl was 19%.

CONCLUSIONS

Time-varying hypoalbuminaemia predicts all-cause and CV death differently from fixed measures of serum albumin in MHD patients. An increase in serum albumin over time is associated with better survival independent of baseline serum albumin or other MICS surrogates. If this association is causal, an intervention that could increase serum albumin >3.8 g/dl might reduce the number of MHD deaths in the USA by approximately 10,000 annually. Nutritional interventions examining benefits of increasing serum albumin in MHD patients are urgently needed.

Clusterin is a highly conserved protein which is expressed at increased levels by many cell types in response to a broad variety of stress conditions. A genuine physiological function for clusterin has not yet been established. The results presented here demonstrate for the first time that clusterin has chaperone-like activity. At physiological concentrations, clusterin potently protected glutathione S-transferase and catalase from heat-induced precipitation and alpha-lactalbumin and bovine serum albumin from precipitation induced by reduction with dithiothreitol. Enzyme-linked immunosorbent assay data showed that clusterin bound preferentially to heat-stressed glutathione S-transferase and to dithiothreitol-treated bovine serum albumin and alpha-lactalbumin. Size exclusion chromatography and SDS-polyacrylamide gel electrophoresis analyses showed that clusterin formed high molecular weight complexes (HMW) with all four proteins tested. Small heat shock proteins (sHSP) also act in this way to prevent protein precipitation and protect cells from heat and other stresses. The stoichiometric subunit molar ratios of clusterin:stressed protein during formation of HMW complexes (which for the four proteins tested ranged from 1.0:1.3 to 1.0:11) is less than the reported ratios for sHSP-mediated formation of HMW complexes (1.0:1.0 or greater), indicating that clusterin is a very efficient chaperone. Our results suggest that clusterin may play a sHSP-like role in cytoprotection.

OBJECTIVE

To investigate whether bacterial lipopolysaccharide (LPS) activity in human serum is associated with the components of the metabolic syndrome (MetS) in type 1 diabetic patients with various degrees of kidney disease and patients with IgA glomerulonephritis (IgAGN).

METHODS

Serum LPS activity was determined with the Limulus Amoebocyte Lysate chromogenic end point assay in type 1 diabetic patients with a normal albumin excretion rate (n = 587), microalbuminuria (n = 144), macroalbuminuria (n = 173); patients with IgAGN (n = 98); and in nondiabetic control subjects (n = 345). The relationships of the LPS/HDL ratio and MetS-associated variables were evaluated with Pearson correlation.

RESULTS

The MetS was more prevalent in type 1 diabetic patients (48%) than in patients with IgAGN (15%). Diabetic patients with macroalbuminuria had a significantly higher serum LPS/HDL ratio than patients with IgAGN. In the normoalbuminuric type 1 diabetic group, patients in the highest LPS/HDL quartile were diagnosed as having the MetS three times more frequently than patients in the lowest quartile (69 vs. 22%; P < 0.001). High LPS activity was associated with higher serum triglyceride concentration, earlier onset of diabetes, increased diastolic blood pressure, and elevated urinary excretion of monocyte chemoattractant protein-1.

CONCLUSIONS

High serum LPS activity is strongly associated with the components of the MetS. Diabetic patients with kidney disease seem to be more susceptible to metabolic endotoxemia than patients with IgAGN. Bacterial endotoxins may thus play an important role in the development of the metabolic and vascular abnormalities commonly seen in obesity and diabetes-related diseases.

Engineered nanoparticles with theranostic functions have attracted a lot of attention for their potential role in the dawning era of personalized medicine. Iron oxide nanoparticles (IONPs), with their advantages of being non-toxic, biodegradable and inexpensive, are candidate platforms for the buildup of theranostic nanostructures; however, progress in using them has been limited largely due to inefficient drug loading and delivery. In the current study, we utilized dopamine to modify the surface of IONPs, yielding nanoconjugates that can be easily encapsulated into human serum albumin (HSA) matrices (clinically utilized drug carriers). This nanosystem is well-suited for dual encapsulation of IONPs and drug molecules, because the encapsulation is achieved in a way that is similar to common drug loading. To assess the biophysical characteristics of this novel nanosystem, the HSA coated IONPs (HSA-IONPs) were dually labeled with (64)Cu-DOTA and Cy5.5, and tested in a subcutaneous U87MG xenograft mouse model. In vivo positron emission tomography (PET)/near-infrared fluorescence (NIRF)/magnetic resonance imaging (MRI) tri-modality imaging, and ex vivo analyses and histological examinations were carefully conducted to investigate the in vivo behavior of the nanostructures. With the compact HSA coating, the HSA-IONPs manifested a prolonged circulation half-life; more impressively, they showed massive accumulation in lesions, high extravasation rate, and low uptake of the particles by macrophages at the tumor area.

The intracellular site of sphingomyelin (SM) synthesis was examined in subcellular fractions from rat liver using a radioactive ceramide analog N-([1-14C]hexanoyl)-D-erythro-sphingosine. This lipid readily transferred from a complex with bovine serum albumin to liver fractions without disrupting the membranes, and was metabolized to radioactive SM. To prevent degradation of the newly synthesized SM to ceramide, all experiments were performed in the presence of EDTA to minimize neutral sphingomyelinase activity and at neutral pH to minimize acid sphingomyelinase activity. An intact Golgi apparatus fraction gave an 85-98-fold enrichment of SM synthesis and a 58-83-fold enrichment of galactosyltransferase activity. Controlled trypsin digestion demonstrated that SM synthesis was localized to the lumen of intact Golgi apparatus vesicles. Although small amounts of SM synthesis were detected in plasma membrane and rough microsome fractions, after accounting for contamination by Golgi apparatus membranes, their combined activity contributed less than 13% of the total SM synthesis in rat liver. Subfractions of the Golgi apparatus were obtained and characterized by immunoblotting and biochemical assays using cis/medial (mannosidase II) and trans (sialyltransferase and galactosyltransferase) Golgi apparatus markers. The specific activity of SM synthesis was highest in enriched cis and medial fractions but far lower in a trans fraction. We conclude that SM synthesis in rat liver occurs predominantly in the cis and medial cisternae of the Golgi apparatus and not at the plasma membrane or endoplasmic reticulum as has been previously suggested.

BACKGROUND

Delirium is a common outcome after cardiac surgery. Delirium prediction rules identify patients at risk for delirium who may benefit from targeted prevention strategies, early identification, and treatment of underlying causes. The purpose of the present prospective study was to develop a prediction rule for delirium in a cardiac surgery cohort and to validate it in an independent cohort.

RESULTS

Prospectively, cardiac surgery patients>> or =60 years of age were enrolled in a derivation sample (n=122) and then a validation sample (n=109). Beginning on the second postoperative day, patients underwent a standardized daily delirium assessment, and delirium was diagnosed according to the confusion assessment method. Delirium occurred in 63 (52%) of the derivation cohort patients. Multivariable analysis identified 4 variables independently associated with delirium: prior stroke or transient ischemic attack, Mini Mental State Examination score, abnormal serum albumin, and the Geriatric Depression Scale. Points were assigned to each variable: Mini Mental State Examination < or =23 received 2 points, and Mini Mental State Examination score of 24 to 27 received 1 point; Geriatric Depression Scale >4, prior stroke/transient ischemic attack, and abnormal albumin received 1 point each. In the derivation sample, the cumulative incidence of delirium for point levels of 0, 1, 2, and>> or =3 was 19%, 47%, 63%, and 86%, respectively (C statistic, 0.74). The corresponding incidence of delirium in the validation sample was 18%, 43%, 60%, and 87%, respectively (C statistic, 0.75).

CONCLUSIONS

Delirium occurs frequently after cardiac surgery. Using 4 preoperative characteristics, clinicians can determine cardiac surgery patients' risk for delirium. Patients at higher delirium risk could be candidates for close postoperative monitoring and interventions to prevent delirium.

Hydrogen sulfide (H(2)S) is rapidly emerging as a biologically significant signaling molecule. Studies published before 2000 report low or undetectable H(2)S (usually as total sulfide) levels in blood or plasma, whereas recent work has reported sulfide concentrations between 10 and 300 microM, suggesting it acts as a circulating signal. In the first series of experiments, we used a recently developed polarographic sensor to measure the baseline level of endogenous H(2)S gas and turnover of exogenous H(2)S gas in real time in blood from numerous animals, including lamprey, trout, mouse, rat, pig, and cow. We found that, contrary to recent reports, H(2)S gas was essentially undetectable (<100 nM total sulfide) in all animals. Furthermore, exogenous sulfide was rapidly removed from blood, plasma, or 5% bovine serum albumin in vitro and from intact trout in vivo. To determine if blood H(2)S could transiently increase, we measured oxygen-dependent H(2)S production by trout hearts in vitro and in vivo. H(2)S has been shown to mediate ischemic preconditioning (IPC) in mammals. IPC is present in trout and, unlike mammals, the trout myocardium obtains its oxygen from relatively hypoxic systemic venous blood. In vitro, myocardial H(2)S production was inversely related to Po(2), whereas we failed to detect H(2)S in ventral aortic blood from either normoxic or hypoxic fish in vivo. These results provide an autocrine or paracrine mechanism for myocardial coupling of hypoxia to H(2)S in IPC, i.e., oxygen sensing, but they fail to provide any evidence that H(2)S signaling is mediated by the circulation.

The aim of the study was to investigate whether inhibition of gastric emptying of meals plays a role in the mechanism of the blood glucose-lowering action of glucagon-like peptide-1-(7-36) amide [GLP-1-(7-36) amide] in type 2 diabetes. Eight poorly controlled type 2 diabetic patients (age, 58 +/- 6 yr; body mass index, 30.0 +/- 5.2 kg/m2; hemoglobin A1c, 10.5 +/- 1.2%) were studied in the fasting state (plasma glucose, 11.1 +/- 1.1 mmol/L). A liquid meal of 400 mL containing 8% amino acids and 50 g sucrose (327 Kcal) was administered at time zero by a nasogastric tube. Gastric volume was determined by a dye dilution technique using phenol red. In randomized order, GLP-1-(7-36) amide (1.2 pmol/kg.min; Saxon Biochemicals) or placebo (0.9% NaCl with 1% human serum albumin) was infused between -30 and 240 min. In the control experiment, gastric emptying was completed within 120 min, and plasma glucose, insulin, C-peptide, GLP-1-(7-36) amide, and glucagon concentrations transiently increased. With exogenous GLP-1-(7-36) amide (plasma level, approximately 70 pmol/L), gastric volume remained constant over the period it was measured (120 min; P < 0.0001 vs. placebo), and plasma glucose fell to normal fasting values (5.4 +/- 0.7 mmol/L) within 3-4 h, whereas insulin was stimulated in most, but not all, patients, and glucagon remained at the basal level or was slightly suppressed. In conclusion, GLP-1-(7-36) amide inhibits gastric emptying in type 2 diabetic patients. Together with the stimulation of insulin and the inhibition of glucagon secretion, this effect probably contributes to the blood glucose-lowering action of GLP-1-(7-36) amide in type 2-diabetic patients when studied after meal ingestion. At the degree observed, inhibition of gastric emptying, however, must be overcome by tachyphylaxis, reduction in dose, or pharmacological interventions so as not to interfere with the therapeutic use of GLP-1-(7-36) amide in type 2 diabetic patients.

OBJECTIVE

Viable bacteria are often isolated from airway secretions in clinically stable patients with chronic bronchitis. We hypothesized that the number of organisms and bacterial species might be important modulators of airway inflammation.

METHODS

We performed quantitative sputum cultures in 160 stable patients [55 with chronic obstructive pulmonary disease (COPD) and normal serum alpha(1)-antitrypsin levels, 62 with COPD and severe alpha(1)-antitrypsin deficiency (PiZ), and 43 with idiopathic bronchiectasis]. The results were related to several indicators of the mechanisms and severity of airway inflammation.

RESULTS

Airway bacterial load correlated with sputum myeloperoxidase level, an indirect measure of neutrophil activation and number (r = 0.50, P<0. 001); sputum neutrophil chemoattractants [interleukin-8 level (r = 0. 68, P<0.001) and leukotriene B4 level (r = 0.53, P<0.001)]; sputum leukocyte elastase activity (r = 0.55, P<0.001); and albumin leakage from serum to sputum (r = 0.26, P<0.01). Markers of inflammation increased at bacterial loads of 10(6) to 10(7) colony-forming units per milliliter, and increased progressively with increasing bacterial load. For example, the median (interquartile range) sputum myeloperoxidase level was 0.3 U/mL (0.1 to 0.5 U/mL) for patients who were not colonized or who had mixed normal oropharyngeal flora alone; 0.5 U/mL (0.2 to 0.7 U/mL) for patients with 10(5) to 10(6) colony-forming units per milliliter (P = 0.07); 0.5 U/mL (0.3 to 1.2 U/mL) for patients with 10(6) to 10(7) colony-forming units per milliliter (P<0.01); 0.7 U/mL (0.3 to 1.2 U/mL) for patients with 10(7) to 10(8) colony-forming units per milliliter (P <0.005); and 2.4 U/mL (0.7 to 4.8 U/mL) for patients with 10(8) or greater colony-forming units per milliliter (P<0.0001). The bacterial species influenced airway inflammation; for example, sputum myeloperoxidase activity was greater (P<0.005) in patients colonized with Pseudomonas aeruginosa [median 32 U/mL (interquartile range, 20 to 65 U/mL)] than those colonized with nontypeable Hemophilus influenzae [4 U/mL (2 to 31 U/mL)], which in turn was greater (P = 0.01) than among those colonized with Moraxella catarrhalis [1.1 U/mL (0.6 to 1.8 U/mL)]. We did not find a relation between bacterial load and lung function.

CONCLUSIONS

The bacterial load and species contribute to airway inflammation in patients with stable chronic bronchitis. Further studies are required to determine the consequences of bacterial colonization on patient morbidity and decline in lung function.

BACKGROUND

Diabetic nephropathy has become the single most important cause of end-stage renal disease (ESRD) worldwide. Strategies to slow the rate of loss of renal function in these patients have been developed. We examined the risk factors that predict loss of kidney function (doubling of serum creatinine) or ESRD (dialysis or transplantation) in patients with type 2 diabetes in whom blood pressure was controlled.

METHODS

We evaluated risk factors for doubling of serum creatinine or the development of ESRD in the Reduction of End Points in NIDDM with the Angiotensin II Receptor Antagonist Losartan (RENAAL) study, which included 1513 patients with type 2 diabetes and nephropathy.

RESULTS

Univariate analyses demonstrated a group of 23 risk factors that significantly predicted doubling of serum creatinine or ESRD. From these univariate analyses, a multivariate model was developed that demonstrated four independent risk factors: proteinuria, serum creatinine, serum albumin, and hemoglobin level. Proteinuria was the strongest and most consistent risk factor. The multivariate risk model was derived from only the placebo group and was similar to that derived for the total population, suggesting that the risk predictors for progression of kidney disease were independent of therapy.

CONCLUSIONS

After control of blood pressure in type 2 diabetic patients with nephropathy, proteinuria, degree of renal failure, serum albumin, and hemoglobin level are independent risk factors that predict renal outcomes. The level of proteinuria proved to be the most important risk for progressive kidney injury in these diabetic patients.

Circulating permeability factors may be important in idiopathic nephrotic syndrome (INS) including focal segmental glomerulosclerosis (FSGS) and in recurrence after renal transplantation. Evidence for plasma factors includes posttransplant recurrence of proteinuria and its response to plasmapheresis or immunoadsorption and induction of proteinuria in experimental animals by infusion of patient plasma or its fractions. The authors and other investigators have used proteomic techniques to seek pathogenic molecules. The authors have recently proposed cardiotrophin-like cytokine-1 (CLC-1) as an active factor in FSGS. Other potential permeability factors include hemopexin and vascular permeability factor in minimal change nephrotic syndrome (MCNS) and soluble urokinase receptor in FSGS. In the authors' studies, in vitro plasma permeability activity is blocked by diverse substances that may decrease levels of active molecules or block the effects of circulating permeability factors. It has been shown that the simple sugar galactose blocks the effect of FSGS serum on albumin permeability in vitro and decreases permeability activity when administered to patients. Because identities of permeability factors and their mechanisms of action are not well defined, therapy of INS/FSGS is empiric. Corticosteroids are the mainstay of initial therapy whereas calcineurin inhibitors such as cyclosporine A (CsA) and immunosuppressive medications provide adjunctive therapy. Nonspecific therapies such as blocking the renin-angiotensin system and controlling blood pressure and plasma lipids may also diminish proteinuria and slow progression. Identification of molecules that initiate proteinuria and application of findings from in vitro studies may lead to development of new treatments to arrest progression and prevent recurrence after transplantation.

Serum hepatic protein (albumin, transferrin, and prealbumin) levels have historically been linked in clinical practice to nutritional status. This paradigm can be traced to two conventional categories of malnutrition: kwashiorkor and marasmus. Explanations for both of these conditions evolved before knowledge of the inflammatory processes of acute and chronic illness were known. Substantial literature on the inflammatory process and its effects on hepatic protein metabolism has replaced previous reports suggesting that nutritional status and protein intake are the significant correlates with serum hepatic protein levels. Compelling evidence suggests that serum hepatic protein levels correlate with morbidity and mortality. Thus, serum hepatic protein levels are useful indicators of severity of illness. They help identify those who are the most likely to develop malnutrition, even if well nourished prior to trauma or the onset of illness. Furthermore, hepatic protein levels do not accurately measure nutritional repletion. Low serum levels indicate that a patient is very ill and probably requires aggressive and closely monitored medical nutrition therapy.

BACKGROUND

The authors evaluated the significance of the preoperative serum C-reactive protein (CRP) level as a prognostic indicator in patients with hepatocellular carcinoma (HCC).

METHODS

One hundred forty-one patients who underwent curative resection for HCC were reviewed retrospectively. Clinicopathologic variables were compared between patients with serum CRP levels>>/= 1.0 mg/dL (n = 22 patients; the CRP-positive group) and patients with serum CRP levels < 1.0 mg/dL (n = 119 patients; the CRP-negative group). Univariate and multivariate analyses were conducted to identify factors that affected survival and disease recurrence.

RESULTS

There was a significant correlation between the preoperative serum CRP level and tumor size. Invasion to the portal vein in the CRP-positive group was significantly more frequent than that in the CRP-negative group. Even after they underwent curative resection, 75.3% of patients in the CRP-positive group experienced recurrence within 1 year. The overall survival and recurrence-free survival rates in the CRP-positive group were significantly lower compared with the rates in the CRP-negative group. On multivariate analysis, the preoperative serum CRP level was selected as one of the unfavorable indicators regarding survival and recurrence. When CRP levels, albumin levels, and platelet counts that were available before surgery were scored as a combined index, the total score demonstrated a good stratification value for survival after hepatic resection.

CONCLUSIONS

The current results showed that the preoperative serum CRP level is an independent and significant indicator predictive of poor prognosis and early recurrence in patients with HCC. The new CRP-based scoring system offers reliable information for predicting survival.

OBJECTIVE

To evaluate the long-term effect of angiotensin-converting enzyme inhibition on proteinuria and on the rate of decline in kidney function in patients with type II diabetes mellitus and microalbuminuria.

METHODS

Randomized, double-blind, placebo-controlled trial. Each patient was followed for 5 years.

METHODS

Six clinics for diabetes mellitus coordinated by a department of medicine in a university hospital in Israel.

METHODS

Ninety-four normotensive, type II diabetic patients with microalbuminuria and normal renal function.

METHODS

The patients were randomly assigned to receive enalapril, 10 mg per day, or placebo. Any increase in blood pressure was treated with long-acting nifedipine.

METHODS

Albuminuria, blood pressure, serum creatinine, fasting blood glucose, and glycosylated hemoglobin levels, every 3 to 4 months.

RESULTS

In the patients treated with enalapril, albuminuria decreased from 143 +/- 64 (mean +/- SD) mg/24 h to 122 +/- 67 mg/24 h during the first year. Thereafter, we observed a slow increase to 140 +/- 134 mg/24 h after 5 years. In the placebo group, albuminuria increased from 123 +/- 58 mg/24 h to 310 +/- 167 mg/24 h after 5 years. (Difference in rate of change in proteinuria [P < 0.05]). Kidney function (expressed as mean reciprocal creatinine) declined by 13% in the placebo group and remained stable (-1%) in the enalapril group (P < 0.05). Control of blood glucose levels remained stable, in both groups, throughout the study. The mean blood pressure was stable in the enalapril group (initial group mean, 99 +/- 2.1 mm Hg; fifth-year mean, 100 +/- 3.2 mm Hg) and increased in the placebo group from an initial mean value of 97 +/- 3.2 mm Hg to 102 +/- 3.4 mm Hg at the end of the study period (P = 0.082).

CONCLUSIONS

In normotensive patients with diabetes mellitus type II, the institution of angiotensin-converting enzyme inhibition during early stages of diabetic nephropathy results in long-term stabilization of plasma creatinine levels and of the degree of urinary loss of albumin. These effects are probably independent of the antihypertensive action of these agents.

The nonstructural (ns) proteins nsP1 to -4, the components of Semliki Forest virus (SFV) RNA polymerase, were localized in infected cells by confocal microscopy using double labeling with specific antisera against the individual ns proteins. All ns proteins were associated with large cytoplasmic vacuoles (CPV), the inner surfaces of which were covered by small invaginations, or spherules, typical of alphavirus infection. All ns proteins were localized by immuno-electron microscopy (EM) to the limiting membranes of CPV and to the spherules, together with newly labeled viral RNA. Along with earlier observations by EM-autoradiography (P. M. Grimley, I. K. Berezesky, and R. M. Friedman, J. Virol. 2:326-338, 1968), these results suggest that individual spherules represent template-associated RNA polymerase complexes. Immunoprecipitation of radiolabeled ns proteins showed that each antiserum precipitated the other three ns proteins, implying that they functioned as a complex. Double labeling with organelle-specific and anti-ns-protein antisera showed that CPV were derivatives of late endosomes and lysosomes. Indeed, CPV frequently contained endocytosed bovine serum albumin-coated gold particles, introduced into the medium at different times after infection. With time, increasing numbers of spherules were also observed on the cell surfaces; they were occasionally released into the medium, probably by secretory lysosomes. We suggest that the spherules arise by primary assembly of the RNA replication complexes at the plasma membrane, guided there by nsP1, which has affinity to lipids specific for the cytoplasmic leaflet of the plasma membrane. Endosomal recycling and fusion of CPV with the plasma membrane can circulate spherules between the plasma membrane and the endosomal-lysosomal compartment.

Studies of the possibility that thyroxine (T4) is converted to 3.5,3'-triiodo-L-thyronine (T3) in the extrathyroidal tissues in man have been conducted in 13 patients, all but two of whom were athyreotic or hypothyroid, and all of whom were receiving at least physiological replacement doses of synthetic sodium-L-thyroxine.T3 was found in the sera of all patients, in concentrations ranging between 243 and 680 ng/100 ml (normal range 170-270 ng/100 ml). These concentrations were far in excess of those which would have been expected on the basis of the T3 contamination of the administered T4, as measured by the same technique employed in the analysis of serum. When oral medication was enriched with (125)I-labeled T4 for 8 or more days, labeled T3 and tetraiodothyroacetic acid (Tetrac or TA(4)) were found in the serum to the extent of approximately 2-5% of total radioactivity, as assessed by unidimensional paper chromatography. The same results were obtained with a specially purified lot of radioactive T4 containing less than 0.1% T3 as a contaminant. The identities of the (125)I-labeled T3 and TA(4) were verified by two-dimensional chromatography as well as by specific patterns of binding in serum. The labeled T3 isolated was bound by albumin and by T4-binding globulin (TBG), but not by T4-binding prealbumin (TBPA): in contrast the labeled TA(4) was bound by albumin and TBPA, but not by TBG. To exclude the possibility that the conversion of T4 to T3 was a peculiarity of the oral route of administration, the sera of two additional patients were obtained 48 hr after 7-day courses of daily intravenous injections of a mixture of stable and (125)I-labeled T4. Both stable and labeled T3 were likewise found in these sera. In contrast to earlier experiments in humans in which (131)I-labeled T3 was not definitively demonstrated in serum after a single intravenous injection of (131)I-labeled T4, the present findings are taken to provide conclusive evidence of the extrathyroidal conversion of T4 to T3 in man. These results raise once again the question of the extent to which the metabolic effect of T4 is mediated through the peripheral generation of T3.

Hepatocytes are extensively used in studies of gene regulation but cannot be maintained in long-term culture as replicating, differentiated cells while remaining nontumorigenic. We have derived two hepatocyte lines from livers of transgenic mice overexpressing transforming growth factor alpha, a potent hepatocyte mitogen, which overcome these limitations. The transgenic hepatocytes were maintained for>> or = 2 months in serum-supplemented primary culture and gave rise to cell lines, of which two (AML12 and AML14) have been cultured for>> 1.5 years >> 80 passages). Both lines have typical hepatocyte features such as peroxisomes and bile canalicular-like structures, do not grow in soft agar, and are nontumorigenic in nude mice. Like normal hepatocytes, AML cells express high levels of mRNA for serum (albumin, alpha 1-antitrypsin, and transferrin) and gap junction (connexins 26 and 32) proteins, secrete albumin, and contain solely isozyme 5 of lactate dehydrogenase. After extensive passaging, AML12 cells continue to strongly coexpress hepatocyte connexin mRNAs but do not display nonparenchymal cell markers. Although mRNA levels for some serum proteins progressively fall, high expression in late AML12 cultures may be regained by passage in serum-free medium. The AML14 line loses expression of both differentiated markers and transgene mRNA with extended passaging, and hepatocytic traits are only partially restored by passage in serum-free medium. These differentiated, nontumorigenic cell lines should serve as models in which to study hepatocyte growth and differentiation.

OBJECTIVE

To identify risk factors for the development of high-risk proliferative diabetic retinopathy (PDR) and for the development of severe visual loss or vitrectomy (SVLV) in eyes assigned to deferral of photocoagulation in the Early Treatment Diabetic Retinopathy Study (ETDRS).

METHODS

Multivariable Cox models were constructed to evaluate the strength and statistical significance of baseline risk factors for development of high-risk PDR and of SVLV.

RESULTS

The baseline characteristics identified as risk factors for high-risk PDR were increased severity of retinopathy, decreased visual acuity (or increased extent of macular edema), higher glycosylated hemoglobin, history of diabetic neuropathy, lower hematocrit, elevated triglycerides, lower serum albumin, and persons with mild to moderate nonproliferative retinopathy, younger age (or type 1 diabetes). The predominant risk factor for development of SVLV was the prior development of high-risk PDR. The only other clearly significant factor was decreased visual acuity at baseline. In the eyes that developed SVLV before high-risk proliferative retinopathy was observed, baseline risk factors were decreased visual acuity (or increased extent of macular edema), older age (or type 2 diabetes), and female gender.

CONCLUSIONS

These analyses supported the view that the retinopathy-inhibiting effect of better glycemic control extends across all ages, both diabetes types, and all stages of retinopathy up to and including the severe nonproliferative and early proliferative stages and the possibility that reducing elevated blood lipids and treating anemia slow the progression of retinopathy.

BACKGROUND

Most studies of coronary artery involvement and associated risk factors in Kawasaki disease have used the Japanese Ministry of Health dichotomous criteria. Analysis of serial normalized artery measurements may reveal a broader continuous spectrum of involvement and different risk factors.

RESULTS

Clinical, laboratory, and echocardiographic measurements obtained at baseline and 1 week and 5 weeks after presentation were examined in 190 Kawasaki disease patients as part of a clinical trial of primary therapy with pulse steroids in addition to standard intravenous immunoglobulin. Maximum coronary artery z score normalized to body surface area was significantly greater than normal at all time points, decreasing significantly over time from baseline. A maximal z score>> or = 2.5 at any time was noted in 26% of patients. Japanese Ministry of Health dimensional criteria were met by 23% of patients. Significant independent factors associated with greater z score at any time included younger patient age, longer interval from disease onset to treatment with intravenous immunoglobulin, lower serum IgM level at baseline, and lower minimum serum albumin level. z scores of the proximal right coronary artery were higher than those in the left anterior descending branch.

CONCLUSIONS

Analyses of serial normalized coronary artery measurements in optimally treated Kawasaki disease patients demonstrated that for most patients, measurements are greatest at baseline and subsequently diminish; baseline measurements appear to be good predictors of involvement during early follow-up. When a more precise assessment is used, risk factors for coronary artery involvement are similar to those defined with arbitrary dichotomous criteria.

The extracellular signal-regulated kinase (ERK) pathway is critical for various forms of learning and memory, and is activated by the potent estrogen 17beta-estradiol (E(2)). Here, we asked whether E(2) modulates memory via ERK activation and putative membrane-bound estrogen receptors (ERs). Using ovariectomized mice, we first demonstrate that intraperitoneal injection of 0.2 mg/kg E(2) significantly increases dorsal hippocampal levels of phosphorylated ERK protein 1 h after injection. Second, we show that E(2) administered intraperitoneally (0.2 mg/kg) or via intrahippocampal infusion (5.0 microg/side) immediately after training in an object recognition task significantly enhances memory retention, and that the beneficial effect of intraperitoneal E(2) is blocked by dorsal hippocampal inhibition of ERK activation. Third, using bovine serum albumin-conjugated 17beta-estradiol (BSA-E(2)), we demonstrate that E(2) binding at membrane-bound ERs can increase dorsal hippocampal ERK activation and enhance object memory consolidation in an ERK-dependent manner. Fourth, we show that this effect is independent of nuclear ERs, but is dependent on the dorsal hippocampus. By demonstrating that E(2) enhances memory consolidation via dorsal hippocampal ERK activation, this study is the first to identify a specific molecular pathway by which E(2) modulates memory and to demonstrate a novel role for membrane-bound ERs in mediating E(2)-induced improvements in hippocampal memory consolidation.

Parenchymal cells from adult rat liver have been established in primary monolayer culture. Donor animals are subjected to a partial hepatectomy and, 4 days later, cells are prepared by collagenase perfusion of the regenerated liver. The hepatic parenchymal cells, separated from nonparenchymal material and suspended in serum-free medium, are placed in plastic tissue culture dishes, where they form a monolayer within 24 h. The monolayer cells exhibit minimal mitotic activity and demonstrate several major metabolic functions characteristic of liver in vivo; these include albumin synthesis and secretion, gluconeogenesis from 3-carbon precursors, responsiveness to insulin and glucagon, glycogen synthesis, and activity of two microsomal enzymes. These functions are present in the monolayer cells for several days at activities similar to those observed in the liver in vivo. The findings indicate that hepatic parenchymal cells in this monolayer system are viable and behave in many respects like normal adult rat liver.

ClpB is a heat-shock protein from Escherichia coli with an unknown function. We studied a possible molecular chaperone activity of ClpB in vitro. Firefly luciferase was denatured in urea and then diluted into the refolding buffer (in the presence of 5 mM ATP and 0.1 mg/ml bovine serum albumin). Spontaneous reactivation of luciferase was very weak (less than 0.02% of the native activity) because of extensive aggregation. Conventional chaperone systems (GroEL/GroES and DnaK/DnaJ/GrpE) or ClpB alone did not reactivate luciferase under those conditions. However, ClpB together with DnaK/DnaJ/GrpE greatly enhanced the luciferase activity regain (up to 57% of native activity) by suppressing luciferase aggregation. This coordinated function of ClpB and DnaK/DnaJ/GrpE required ATP hydrolysis, although the ClpB ATPase was not activated by native or denatured luciferase. When the chaperones were added to the luciferase refolding solutions after 5-25 min of refolding, ClpB and DnaK/DnaJ/GrpE recovered the luciferase activity from preformed aggregates. Thus, we have identified a novel multi-chaperone system from E. coli, which is analogous to the Hsp104/Ssa1/Ydj1 system from yeast. ClpB is the only known bacterial Hsp100 protein capable of cooperating with other heat-shock proteins in suppressing and reversing protein aggregation.

While aspects of cellular fatty acid uptake have been studied as early as 50 years ago, recent developments in this rapidly evolving field have yielded new functional insights on the individual mechanistic steps in this process. The extremely low aqueous solubility of long chain fatty acids (LCFA) together with the very high affinity of serum albumin and cytoplasmic fatty acid binding proteins for LCFA have challenged the limits of technology in resolving the individual steps of this process. To date no single mechanism alone accounts for regulation of cellular LCFA uptake. Key regulatory points in cellular uptake of LCFA include: the aqueous solubility of the LCFA; the driving force(s) for LCFA entry into the cell membrane; the relative roles of diffusional and protein mediated LCFA translocation across the plasma membrane; cytoplasmic LCFA binding protein-mediated uptake and/or intracellular diffusion; the activity of LCFA-CoA synthetase; and cytoplasmic protein mediated targeting of LCFA or LCFA-CoAs toward specific metabolic pathways. The emerging picture is that the cell has multiple, overlapping mechanisms that assure adequate uptake and directed intracellular movement of LCFA required for maintenance of physiological functions. The upcoming challenge is to take advantage of new advances in this field to elucidate the differential interactions between these pathways in intact cells and in tissues.

Hypovitaminosis D is associated with poor dietary intake and inadequate sunshine exposure. It is common worldwide, particularly in European elderly people. Information about vitamin D status in young adult populations from the Middle East is scarce. Furthermore, the relationship between hypovitaminosis D and some lifestyle factors such as style of clothing and dwelling location is not well defined. We assessed vitamin D intake and measured serum calcium, phosphorus, albumin, alkaline phosphatase, 25-hydroxyvitamin D [25(OH)D], parathyroid hormone (PTH), osteocalcin, and urinary-free deoxypyridinoline (DPD) in 316 Lebanese volunteers (99 men and 217 women) aged 30-50 years; 156 were recruited from rural areas and 160 from urban areas. Fifty-one women from each area were veiled. The average daily vitamin D intake was 100.3 +/- 67.9 IU and was found to be higher in men compared with women, in urban subjects compared with rural ones and in nonveiled women compared with veiled ones. The mean level of 25(OH)D was 9.71 +/- 7.07 ng/ml. Hypovitaminosis D [25(OH)D < 12 ng/ml] affected 72.8% of our population. It was more common in women than in men (83.9% vs. 48.5%). Severe hypovitaminosis D [25(OH)D < 5 ng/ml] was observed in 30.7% of our subjects and was more prevalent in women (41.5%), particularly in the veiled ones (61.8%). 25(OH)D levels were the lowest in veiled women, and in women living in rural areas. Rural men had the highest 25(OH)D levels despite their very low vitamin D intake. In a multivariate model, inadequate vitamin D intake, urban dwelling, veil wearing, and high parity in women were independent predictors of hypovitaminosis D. 25(OH)D was related inversely to PTH and free DPD whereas osteocalcin achieved only a weak positive correlation with 25(OH)D. In the absence of information regarding time spent outdoors, our results show that hypovitaminosis D is common among young Lebanese people and is related mostly to low vitamin D intake. This should emphasize the need for more vitamin D in our population.