Various studies suggest a strong association between nutrition and clinical outcome in hemodialysis (HD) patients. Several morbidity factors that per se increase the risk of a poor outcome, such as cardiovascular disease (CVD) and inflammation, may also cause malnutrition. Among laboratory parameters used to assess nutritional status, serum albumin appears to be a particularly strong predictor of morbidity and mortality. This study assessed the importance of nutritional status and inflammation and other comorbidity factors as predictors of mortality in HD patients. Nutritional status was evaluated in 128 HD patients by subjective global nutritional assessment (SGNA) and by measuring several anthropometric markers (actual body weight, percentage of actual body weight to desirable body weight, midarm muscle circumferences, triceps skinfold thickness), and serum albumin, plasma insulin such as insulin growth factor-1 and as a marker of inflammation, serum C-reactive protein (s-CRP) levels. The mortality during the next 36 mo was analyzed in relation to age, gender, CVD, SGNA, serum albumin, CRP, and several other factors by Kaplan-Meier analysis multivariate. Cox proportional hazard analysis was used to identify independent predictors of mortality. After 36 mo, 58 patients were still on HD treatment, 57 patients (45%) had died while receiving treatment, and 13 had received a kidney transplant. The main cause of death was CVD (58%), followed by infection (18%); malnutrition/cachexia was a rare direct cause of death (5%). Kaplan-Meier analysis showed that age, female gender, CVD, diabetes, SGNA, all anthropometric parameters, serum albumin, plasma insulinlike growth factor-1, and s-CRP were significant predictors of mortality. Analysis by the Cox model showed that age, gender, CVD, nutritional status (SGNA), and CRP were independent predictors of mortality at 36 mo. A low albumin level was not an independent predictor, although it was strongly associated with a reduced survival rate in the Kaplan-Meier analysis. Inflammation, malnutrition, and CVD appeared to contribute to increased mortality in a stepwise manner. The mortality at 36 mo was 0% when none of these complications was present, whereas the mortality was 75% in those patients with all three risk factors present at baseline. It is concluded that in addition to malnutrition and comorbidities (CVD, diabetes mellitus), inflammation (elevated s-CRP) is a significant independent risk factor for mortality in HD patients. Inflammation, malnutrition, and CVD appear to be interrelated, each additionally contributing to the high mortality in these patients.

OBJECTIVE; Morbidity and mortality involved in the resection of hilar cholangiocarcinoma were reviewed retrospectively. The clinicopathologic and laboratory parameters that might influence the patient's survival also were re-evaluated.

BACKGROUND

Although much progress has been made in the diagnosis and management of hilar cholangiocarcinoma, long-term outlook for most patients remains poor. Surgical resection is usually prohibited because of its local invasiveness, and most patients can only be managed by palliative drainage. Recently, many surgeons have adopted a more aggressive resection with varying degrees of success. Several prognostic factors in bile duct carcinoma have been proposed; however, no reports have specifically focused on resected hilar cholangiocarcinoma and its prognostic survival factors using multivariate analysis.

METHODS

The clinical records and pathologic slides of 49 cases with resected hilar cholangiocarcinoma were reviewed retrospectively. Twenty clinical and laboratory parameters were evaluated for their correlation with postoperative morbidity and mortality, whereas 31 variables were evaluated for their significance with postoperative survival. Variables showing statistical significance in the first univariate analysis were included in the following multivariate analysis using stepwise logistic regression test for factors affecting morbidity and mortality and Cox stepwise proportional hazard model for factors influencing survival.

RESULTS

There were 5 in-hospital deaths, and the cumulative 5-year survival rate in 44 patients who survived was 14.9%, with a median survival of 14.0 months. Multivariate analysis disclosed that co-existent hepatolithiasis and lower serum asparate aminotransferase levels (<90 U/L) had a significant low incidence of postoperative morbidity, whereas a serum albumin of less than 3 g/dL was the only significant factor affecting mortality. Regarding survival, univariate analysis identified eight significant factors: 1) total bilirubin>> or = 10 mg/dL, 2) curative resection, 3) histologic type, 4) perineural invasion, 5) liver invasion, 6) depth of cancer invasion, 7) positive proximal resected margin, and 8) positive surgical margin. However, multivariate analysis disclosed total bilirubin>> or = 10 mg/dL, curative resection, and histologic type as the three most significant independent variables.

CONCLUSIONS

Surgical resection provides the best survival for hilar cholangiocarcinoma. An adequate nutritional support to increase serum albumin over 3 g/dL is the most important factor to decrease postoperative mortality. Moreover, preoperative biliary drainage to decrease jaundice and a curative resection with adequate surgical margin are recommended if longer survival is anticipated. Patients with well-differentiated adenocarcinoma seem to survive longer compared to those with moderately or poorly differentiated tumors.

One of the major challenges of molecular allergy is to predict the allergenic potential of a protein, particularly in novel foods. Two aspects have to be distinguished: immunogenicity and cross-reactivity. Immunogenicity reflects the potential of a protein to induce IgE antibodies, whereas cross-reactivity is the reactivity of (usually preexisting) IgE antibodies with the target protein. In addition to these two issues, the relation between IgE-binding potential and clinical symptoms is of interest. This is influenced by physical properties (eg, stability and size) and immunologic properties (affinity and epitope valence). Discussions on immunogenicity and cross-reactivity of allergens rely on the establishment of structural similarities and differences among allergens and between allergens and nonallergens. For comparisons between the 3-dimensional protein folds, the representation as 2-dimensional proximity plots provides a convenient visual aid. Analysis of approximately 40 allergenic proteins (or parts of these proteins), of which the protein folds are either known or can be predicted on the basis of homology, indicates that most of these can be classified into 4 structural families: (1) antiparallel beta-strands: the immunoglobulin-fold family (grass group 2, mite group 2), serine proteases (mite group 3, 6, and 9), and soybean-type trypsin inhibitor (Ole e 1, grass group 11); (2) antiparallel beta-sheets intimately associated with one or more alpha-helices: tree group 1, lipocalin, profilin, aspartate protease (cockroach group 2); (3) (alpha+beta) structures, in which the alpha- and beta-structural elements are not intimately associated: mite group 1, lysozyme/lactalbumin, vespid group 5; and (4) alpha-helical: nonspecific lipid transfer protein, seed 2S protein, insect hemoglobin, fish parvalbumin, pollen calmodulin, mellitin from bee venom, Fel d 1 chain 1, serum albumin. Allergens with parallel beta-strands (in combination with an alpha-helix linking the two strands, a motif commonly found in, for example, nucleotide-binding proteins) seem to be underrepresented. The conclusion is that allergens have no characteristic structural features other than that they need to be able to reach (and stimulate) immune cells and mast cells. Within this constraint, any antigen may be allergenic, particularly if it avoids activation of T(H)2-suppressive mechanisms (CD8 cells and T(H)1 cells).

LC-MS-MS experiments in proteomics are usually performed with packed microcolumns employing frits or outlets smaller than the particle diameter to retain the packing material. We have developed packed microcolumns using self-assembled particles (SAPs) as frits that are smaller than the size of the outlet. A five to one ratio of outlet size to particle diameter appears to be the upper maximum. In these situations the particles assembled into an arch over the outlet like the stones in a stone bridge. When 3 microm particles were packed into a tapered column with an 8 microm outlet, two particles bridged the outlet with 0.3 pl dead volume and perfect success rate. In peptide analysis by LC-MS, the peak width at half height was normally less than 6 s, compared to 12 s without SAPs. The LC-MS-MS system provided 37% sequence coverage (21 matched peptides) for a tryptically-digested sample of 10 fmol bovine serum albumin. We also describe application of the SAP principle to make disposable pipette tip columns with short pieces of fused-silica capillary as the outlet.

In this study, we validated measurements of free testosterone (fT) and free estradiol (fE(2)) concentrations calculated from total serum concentrations of testosterone (T), estradiol (E(2)), and sex hormone-binding globulin (SHBG), measured by direct, commercial radioimmunoassays, by comparison with reference measurements obtained by dialysis plus an in-house radioimmunoassay after extraction and chromatographic purification. The study was conducted in serum samples from 19 postmenopausal women who were part of an ongoing prospective cohort study. We also performed sensitivity analyses to examine the robustness of the theoretical calculations. Sensitivity analyses showed that in this population, competitive binding of dihydrotestosterone and total T could be ignored in the calculation of fE(2), and competitive binding by dihydrotestosterone does not need to be taken into account for calculation of fT. Furthermore, variations in albumin and SHBG concentrations had negligible effects on fT and fE(2) calculations. Values of fT and fE(2), calculated from total T and E(2) concentrations obtained by the same in-house radioimmunoassay used for the dialysis method, correlated highly with the measurements by dialysis (Pearson's coefficients of correlation above 0.97). When calculating fT and fE(2) using total T and total E(2) concentrations obtained by different direct radioimmunoassays, almost all kits gave good correlations with the reference method for fT (Pearson's r>> 0.83), but only a few gave good correlations for fE(2) (Diagnostic System Laboratories and DiaSorin; r>> 0.80). The direct radioimmunoassays giving the best correlation for fT and fE(2) with the dialysis method were those that best measured total concentrations of T and E(2). Furthermore, mean values of fT and fE(2) corresponded well to mean values by the reference method if SHBG measurements were also well calibrated. We conclude that in postmenopausal women, theoretical calculations are valid for the determination of fT and fE(2) concentrations and can give reliable estimation of cancer risk in epidemiological studies when the total concentrations of T, E(2), and SHBG are measured accurately.

Hepatitis delta virus (HDV)-associated particles were purified from the serum of an experimentally infected chimpanzee by size chromatography and by density centrifugation. Hepatitis delta antigen (HDAg) was detected after mild detergent treatment at a column elution volume corresponding to 36-nm particles and banded at a density of 1.25 g/ml. The serum had an estimated titer of 10(9) to 10(10) HDV-associated particles and had only a 10-fold excess of hepatitis B surface antigen (HBsAg) not associated with HDAg. Therefore, HDV appears to be much more efficiently packed and secreted than is its helper virus, hepatitis B virus (HBV), which is usually accompanied by a 1,000-fold excess of HBsAg. The protein compositions of the HDAg-containing particles were analyzed by immunoblotting with HDAg-, HBsAg-, and hepatitis B core antigen-specific antisera and monoclonal antibodies to HBV surface gene products. The HBsAg envelope of HDAg contained approximately 95% P24/GP27s, 5% GP33/36s, and 1% P39/GP42s proteins. This protein composition was more similar to that of the 22-nm particles of HBsAg than to that of complete HBV. The significant amount of GP33/36s suggests that the HBsAg component of the HDV-associated particle carries the albumin receptor. Two proteins of 27 and 29 kilodaltons which specifically bound antibody to HDAg but not HBV-specific antibodies were detected in the interior of the 36-nm particle. Since these proteins were structural components of HDAg and were most likely coded for by HDV, they were designated P27d and P29d.

Tuberculosis can involve any part of the gastrointestinal tract and is the sixth most frequent site of extrapulmonary involvement. Both the incidence and severity of abdominal tuberculosis are expected to increase with increasing incidence of HIV infection. Tuberculosis bacteria reach the gastrointestinal tract via haematogenous spread, ingestion of infected sputum, or direct spread from infected contiguous lymph nodes and fallopian tubes. The gross pathology is characterized by transverse ulcers, fibrosis, thickening and stricturing of the bowel wall, enlarged and matted mesenteric lymph nodes, omental thickening, and peritoneal tubercles. Peritoneal tuberculosis occurs in three forms : wet type with ascitis, dry type with adhesions, and fibrotic type with omental thickening and loculated ascites. The most common site of involvement of the gastrointestinal tuberculosis is the ileocaecal region. Ileocaecal and small bowel tuberculosis presents with a palpable mass in the right lower quadrant and/or complications of obstruction, perforation or malabsorption especially in the presence of stricture. Rare clinical presentations include dysphagia, odynophagia and a mid oesophageal ulcer due to oesophageal tuberculosis, dyspepsia and gastric outlet obstruction due to gastroduodenal tuberculosis, lower abdominal pain and haematochezia due to colonic tuberculosis, and annular rectal stricture and multiple perianal fistulae due to rectal and anal involvement. Chest X-rays show evidence of concomitant pulmonary lesions in less than 25 per cent of cases. Useful modalities for investigating a suspected case include small bowel barium meal, barium enema, ultrasonography, computed tomographic scan and colonoscopy. Ascitic fluid examination reveals straw coloured fluid with high protein, serum ascitis albumin gradient less than 1.1 g/dl, predominantly lymphocytic cells, and adenosine deaminase levels above 36 U/l. Laparoscopy is a very useful investigation in doubtful cases. Management is with conventional antitubercular therapy for at least 6 months. The recommended surgical procedures today are conservative and a period of preoperative drug therapy is controversial.

A panel of members of the 2009 International Myeloma Workshop developed guidelines for standard investigative workup of patients with suspected multiple myeloma. Both serum and urine should be assessed for monoclonal protein. Measurement of monoclonal protein both by densitometer tracing and/by nephelometric quantitation is recommended, and immunofixation is required for confirmation. The serum-free light chain assay is recommended in all newly diagnosed patients with plasma cell dyscrasias. Bone marrow aspiration and/or biopsy along with demonstration of clonality of plasma cells are necessary. Serum β(2)-microglobulin, albumin, and lactate dehydrogenase are necessary for prognostic purposes. Standard metaphase cytogenetics and fluorescent in situ hybridization for 17p, t(4;14), and t(14;16) are recommended. The skeletal survey remains the standard method for imaging screening, but magnetic resonance imaging frequently provides valuable diagnostic and prognostic information. Most of these tests are repeated during follow-up or at relapse.

BACKGROUND

Long-term immunosuppressive treatment does not efficiently prevent relapses of lupus nephritis (LN). This investigator-initiated randomised trial tested whether mycophenolate mofetil (MMF) was superior to azathioprine (AZA) as maintenance treatment.

METHODS

A total of 105 patients with lupus with proliferative LN were included. All received three daily intravenous pulses of 750 mg methylprednisolone, followed by oral glucocorticoids and six fortnightly cyclophosphamide intravenous pulses of 500 mg. Based on randomisation performed at baseline, AZA (target dose: 2 mg/kg/day) or MMF (target dose: 2 g/day) was given at week 12. Analyses were by intent to treat. Time to renal flare was the primary end point. Mean (SD) follow-up of the intent-to-treat population was 48 (14) months.

RESULTS

The baseline clinical, biological and pathological characteristics of patients allocated to AZA or MMF did not differ. Renal flares were observed in 13 (25%) AZA-treated and 10 (19%) MMF-treated patients. Time to renal flare, to severe systemic flare, to benign flare and to renal remission did not statistically differ. Over a 3-year period, 24 h proteinuria, serum creatinine, serum albumin, serum C3, haemoglobin and global disease activity scores improved similarly in both groups. Doubling of serum creatinine occurred in four AZA-treated and three MMF-treated patients. Adverse events did not differ between the groups except for haematological cytopenias, which were statistically more frequent in the AZA group (p=0.03) but led only one patient to drop out.

CONCLUSIONS

Fewer renal flares were observed in patients receiving MMF but the difference did not reach statistical significance.

Fluorescence enhancement was studied on silver colloidal metal films (CMFs) using two systems: (1) Langmuir--Blodgett monolayers of fluorescein-labeled phospholipids separated from the surface of the films by spacer layers of octadecanoic acid and (2) biotin--fluorescein conjugates captured by avidin molecules adsorbed on top of a multilayer structure formed by alternating layers of bovine serum albumin--biotin conjugate (BSA--biotin) and avidin. The dependence of fluorescence intensity on the number of lipid or protein spacer layers deposited on the surface of the CMF was investigated. The results demonstrate the requirement for adsorbate location within the region between Ag particles for maximal enhancement. The density of avidin molecules on the surface of the BSA--biotin/avidin multilayers adsorbed on the CMF was also determined. A procedure for forming a rigid, uniform silica layer around the Ag particles on the CMF is described. The layer protects the particles from undesirable chemical reactions such as etching by halide ions, for example, and provides the requisite stability for bioanalytical applications. Colloidal films composed of Ag particles covered by approximately 10-nm-thick silica layers were tested for fluorescence enhancement using goat immunoglobulin and a conjugate of rabbit anti-goat immunoglobulin with 6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-amino)hexanoate. An enhancement factor of approximately 20 was obtained.

OBJECTIVE

To evaluate the prognostic significance of microalbuminuria and overt diabetic nephropathy and other putative risk factors for cardiovascular and all cause mortality in insulin dependent diabetes.

METHODS

Ten year observational follow up study.

METHODS

Outpatient diabetic clinic in a tertiary referral centre.

METHODS

All 939 adults with insulin dependent diabetes (duration of diabetes five years or more) attending the clinic in 1984; 593 had normal urinary albumin excretion (< or = 30 mg/24 h), 181 persistent microalbuminuria (31-299 mg/24 h), and 165 overt nephropathy >> or = 300 mg/24 h).

METHODS

All cause and cardiovascular mortality.

RESULTS

Fifteen per cent of patients (90/593) with normoalbuminuria, 25% (45/181) with microalbuminuria, and 44% (72/165) with overt nephropathy at baseline died during follow up. Cox multiple regression analysis identified the following significant predictors of all cause mortality: male sex (relative risk 2.03; 95% confidence interval 1.37 to 3.02), age (1.07; 1.06 to 1.08), height (0.96; 0.94 to 0.98), smoking (1.51; 1.09 to 2.08), social class V versus social class IV (1.70; 1.25 to 2.31), log10 urinary albumin excretion (1.45; 1.18 to 1.77), hypertension (1.63; 1.18 to 2.25), log10 serum creatinine concentration (8.96; 3.34 to 24.08), and haemoglobin A1c concentration (1.11; 1.03 to 1.20). Age, smoking, microalbuminuria, overt nephropathy, and hypertension were significant predictors of cardiovascular mortality. Mortality in patients with microalbuminuria was only slightly increased compared with that in patients with normoalbuminuria. Median survival time after the onset of overt diabetic nephropathy was 13.9 years (95% confidence interval 11.8 to 17.2 years).

CONCLUSIONS

Abnormally increased urinary albumin excretion and other potentially modifiable risk factors such as hypertension, smoking, poor glycaemic control, and social class predict increased mortality in insulin dependent diabetes. Microalbuminuria by itself confers only a small increase in mortality. The prognosis of patients with overt diabetic nephropathy has improved, probably owing to effective antihypertensive treatment.

We have adapted the techniques of DNA footprint analysis to an Applied Biosystems 3730 DNA Analyzer. The use of fluorescently labeled primers eliminates the need for radioactively labeled nucleotides, as well as slab gel electrophoresis, and takes advantage of commonly available automated fluorescent capillary electrophoresis instruments. With fluorescently labeled primers and dideoxynucleotide DNA sequencing, we have shown that the terminal base of each digested fragment may be accurately identified with a capillary-based instrument. Polymerase chain reaction (PCR) was performed with a 6FAM-labeled primer to amplify a typical target promoter region. This PCR product was then incubated with a transcriptional activator protein, or bovine serum albumin as a control, and then partially digested with DNase I. A clone of the promoter was sequenced with the Thermo Sequenase Dye Primer Manual Cycle Sequencing kit (USB) and the FAM-labeled primer. Through the use of Genemapper software, the Thermo sequenase and DNasei digestion products were accurately aligned, providing a ready means to assign correct nucleotides to each peak from the DNA footprint. This method was used to characterize the binding of two different transcriptional activator proteins to their respective promoter regions.

BACKGROUND

Low values of estimated glomerular filtration rate (eGFR) predispose to acute kidney injury, and proteinuria is a marker of kidney disease. We aimed to investigate how eGFR and proteinuria jointly modified the risks of acute kidney injury and subsequent adverse clinical outcomes.

METHODS

We did a cohort study of 920,985 adults residing in Alberta, Canada, between 2002 and 2007. Participants not needing chronic dialysis at baseline and with at least one outpatient measurement of both serum creatinine concentration and proteinuria (urine dipstick or albumin-creatinine ratio) were included. We assessed hospital admission with acute kidney injury with validated administrative codes; other outcomes were all-cause mortality and a composite renal outcome of end-stage renal disease or doubling of serum creatinine concentration.

RESULTS

During median follow-up of 35 months (range 0-59 months), 6520 (0·7%) participants were admitted with acute kidney injury. In those with eGFR 60 mL/min per 1·73 m(2) or greater, the adjusted risk of admission with this disorder was about 4 times higher in those with heavy proteinuria measured by dipstick (rate ratio 4·4 vs no proteinuria, 95% CI 3·7-5·2). The adjusted rates of admission with acute kidney injury and kidney injury needing dialysis remained high in participants with heavy dipstick proteinuria for all values of eGFR. The adjusted rates of death and the composite renal outcome were also high in participants admitted with acute kidney injury, although the rise associated with this injury was attenuated in those with low baseline eGFR and heavy proteinuria.

CONCLUSIONS

These findings suggest that information on proteinuria and eGFR should be used together when identifying people at risk of acute kidney injury, and that an episode of acute kidney injury provides further long-term prognostic information in addition to eGFR and proteinuria.

BACKGROUND

The study was funded by an interdisciplinary team grant from Alberta Heritage Foundation for Medical Research.

OBJECTIVE

To describe the sources, production rates, circulating concentrations, and regulatory mechanisms of the major androgen precursors and androgens in women.

METHODS

Review of the major published literature.

RESULTS

Quantitatively, women secrete greater amounts of androgen than of estrogen. The major circulating steroids generally classified as androgens include dehydroepiandrosterone sulphate (DHEAS), dehydroepiandrosterone (DHEA), androstenedione (A), testosterone (T), and dihydrotestosterone in descending order of serum concentration, though only the latter two bind the androgen receptor. The other three steroids are better considered as pro-androgens. Dehydroepiandrosterone is primarily an adrenal product, regulated by adrenocorticotropic hormone (ACTH) and acting as a precursor for the peripheral synthesis of more potent androgens. Dehydroepiandrosterone is produced by both the ovary and adrenal, as well as being derived from circulating DHEAS. Androstenedione and testosterone are products of the ovary and the adrenal. Testosterone circulates both in its free form, and bound to protein including albumin and sex steroid hormone-binding globulin (SHBG), the levels of which are an important determinant of free testosterone concentration.

CONCLUSIONS

The postmenopausal ovary is an androgen-secreting organ and the levels of testosterone are not directly influenced by the menopausal transition or the occurrence of menopause. Dihydrotestosterone (DHT) is primarily a peripheral product of testosterone metabolism. Severe androgen deficiency occurs in hypopituitarism, but other causes may lead to androgen deficiency, including Addison's disease, corticosteroid therapy, chronic illness, estrogen replacement (leads to elevated SHBG and, therefore, low free testosterone), premenopausal ovarian failure, or oophorectomy.

After oral administration on an empty stomach, the absorption of rifampicin (rifampin) is rapid and practically complete. With a single 600mg dose, peak serum concentration of the order of 10microgram/ml generally occur 2 hours after administration. The half-life of rifampicin for this dose level is of the order of 2.5 hours. The amount of rifampicin extracted by the liver during its first passage through the hepatoportal system and transferred to bile is relevance for the time course of distribution of the antibiotic in the blood compartment. With dose of the order of 300 to 450mg, the excretory capacity of the liver for the antibiotic is saturated. As a consequence, increasing the dose of antibiotic results in a more than proportional increase in serum concentrations. On repeated administration, and most likely as a consequence of self-induced (autoinduction) metabolism, the rate of disappearance of rifampicin from the blood compartment increases in the early phase of treatment, the phenomenon affecting mainly the levels following the peak, with a consequent reduction in half-life. Approximately 80% of rifampicin is transported in blood bound to plasma proteins, mainly albumin. Rifampicin is well distributed, although to a different degree, in the various tissues of the human body. Probably in the hepatocyte, rifampicin undergoes a process of desacetylation. The metabolic derivative, desacetylrifampicin, is more polar than the parent compound, and microbiologically active. This metabolite accounts for the majority of the antibacterial activity in the bile Rifampicin is almost equally excreted in the bile and urine, the recovery in the 2 fluids being of the same order of magnitude. Administration of rifampicin to newborn infants and children is followed by blood levels generally lower than those found in adults for the same dose levels. In patients with impaired liver and kidney function the elimination of the antibiotic from the blood compartment is slower than in normal subjects. Rifampicin has been found to compete with bilirubin and other cholefil substances for biliary excretion, giving rise to transient and reversible increased bilirubin and BSP retention values. A kinetic model study on the transfer constants between various body compartments has indicated that rifampicin is rapidly absorbed from the intestine and that the absorption rate increases with time. Rifampicin as such is transferred into urine at a rate 3 times higher than the rate of transfer into bile. Desacetylrifampicin, the more polar metabolic derivative of rifampicin, behaves in the opposite way since its rate of transfer into bile is 4 times higher than that into urine. The rate of biotransformation of rifampicin into desacetylrifampicin is of the same order of magnitude as than of biotransformation of the latter into a further metabolic derivative, which could be a glucuronide conjugate...

BACKGROUND

Neuromyelitis optica (NMO, Devic disease) is a severely disabling autoimmune disorder of the CNS, which was considered a subtype of multiple sclerosis (MS) for many decades. Recently, however, highly specific serum autoantibodies (termed NMO-IgG or AQP4-Ab) have been discovered in a subset (60-80%) of patients with NMO. These antibodies were subsequently shown to be directly involved in the pathogenesis of the condition. AQP4-Ab positive NMO is now considered an immunopathogenetically distinct disease in its own right. However, to date little is known about the cerebrospinal fluid (CSF) in AQP4-Ab positive NMO.

OBJECTIVE

To describe systematically the CSF profile of AQP4-Ab positive patients with NMO or its formes frustes, longitudinally extensive myelitis and optic neuritis.

METHODS

Cytological and protein biochemical results from 211 lumbar punctures in 89 AQP4-Ab positive patients of mostly Caucasian origin with neuromyelitis optica spectrum disorders (NMOSD) were analysed retrospectively.

RESULTS

CSF-restricted oligoclonal IgG bands, a hallmark of MS, were absent in most patients. If present, intrathecal IgG (and, more rarely, IgM) synthesis was low, transient, and, importantly, restricted to acute relapses. CSF pleocytosis was present in around 50% of samples, was mainly mild (median, 19 cells/μl; range 6-380), and frequently included neutrophils, eosinophils, activated lymphocytes, and/or plasma cells. Albumin CSF/serum ratios, total protein and CSF L-lactate levels correlated significantly with disease activity as well as with the length of the spinal cord lesions in patients with acute myelitis. CSF findings differed significantly between patients with acute myelitis and patients with acute optic neuritis at the time of LP. Pleocytosis and blood CSF barrier dysfunction were also present during remission in some patients, possibly indicating sustained subclinical disease activity.

CONCLUSIONS

AQP4-Ab positive NMOSD is characterized by CSF features that are distinct from those in MS. Our findings are important for the differential diagnosis of MS and NMOSD and add to our understanding of the immunopathogenesis of this devastating condition.

These studies explore the role of conformational change and exposed carbohydrate residues in the clearance of alpha 2-macroglobulin-trypsin (alpha 2M-T) complexes in the mouse. Human alpha 2-macroglobulin (alpha 2M) was purified and demonstrated to be homogeneous in the electrophoretic "slow" form. Two conformationally altered derivatives, alpha 2M-T and alpha 2-macroglobulin-methylamine (alpha 2M-MeNH2), were prepared and demonstrated to exist in the electrophoretic "fast" form. Radiolabeled alpha 2M-T and alpha 2M-MeNH2 were cleared rapidly with a half-life of 2-4 min following injection into mice. Radiolabeled native alpha 2M, however, remained in the circulation with a half-life of several hours. Both alpha 2M-T and alpha 2M-MeNH2 bound specifically to mouse peritoneal macrophages at 4 degrees C and occupancy of receptor sites increased with increasing time and radioligand concentration. Excess amounts of unlabeled alpha 2M-T or alpha 2M-MeNH2 cross-completed with trace amounts of the other in both clearance studies and binding assays, indicating that both derivatives were removed by the same receptor pathway. The clearance and binding of alpha 2M-T and alpha 2M-MeNH2 were not inhibited by excess amounts of unlabeled asialoorosomucoid, fucosyl-bovine serum albumin, mannosyl-BSA, or N-acetylglucosaminyl-BSA. Our results indicate that the clearance pathway removing alpha 2M-T complexes from the circulation recognizes a fundamental conformational change in alpha 2M secondary to protease binding, which can also be induced by exposure to methylamine. Therefore, other chemical or physical alterations that occur in alpha 2M upon binding trypsin, apart from the conformational change also present in alpha 2M-MeNH2, do not seem necessary for the recognition of alpha 2M-T by cells in the clearance pathway. In addition, this pathway appears distinct from several systems already described mediating clearance of glycoproteins through recognition of terminal galactose, fucose, N-acetylglucosamine, or mannose on oligosaccharide side chains.

Interleukin-22 (IL-22), which acts as either a proinflammatory or anti-inflammatory cytokine in various disease models, is markedly up-regulated in chronic liver diseases, including hepatitis B and C. In this report, we demonstrate a strong correlation between IL-22 expression in the liver with active, inflammatory human liver disease. To clarify the role of IL-22 up-regulation in the pathogenesis of liver diseases, liver-specific IL-22 transgenic (IL-22TG) mice, under the control of albumin promoter, were developed. Despite elevated IL-22 serum levels ranging from 4,000 to 7,000 pg/mL, IL-22TG mice developed normally without obvious adverse phenotypes or evidence of chronic inflammation (except for slightly thicker epidermis and minor inflammation of the skin) compared with wild-type mice. Interestingly, IL-22TG mice were completely resistant to concanavalin A-induced T cell hepatitis with minimal effect on liver inflammation and had accelerated liver regeneration after partial hepatectomy. Although they did not spontaneously develop liver tumors, IL-22TG mice were more susceptible to diethylnitrosamine-induced liver cancer. Microarray analyses revealed that a variety of antioxidant, mitogenic, acute phase genes were up-regulated in the livers of IL-22TG mice compared with those from wild-type mice.

CONCLUSIONS

These findings indicate that localized production of IL-22 in the liver promotes hepatocyte survival and proliferation but primes the liver to be more susceptible to tumor development without significantly affecting liver inflammation.

Equilibrium binding of long-chain fatty acids (FA) with albumin from human serum (HSA), bovine serum (BSA), and murine serum (MSA) has been studied by measuring the equilibrium levels of free fatty acids (FFA). FFA levels were measured directly, using a new fluorescent probe composed of acrylodan-derivatized intestinal fatty acid binding protein (ADIFAB). Measurements of [FFA] were done as a function of the ratio of total FA to total albumin (v) for v values between 0 and 6, at pH 7.4 and 37 degrees C. Under conditions observed in normal human physiology (v < or = 2), [FFA] values of the most abundant serum FA (palmitate, stearate, oleate) in equilibrium with human or bovine albumin are less than 15 nM. These values are considerably smaller than the generally quoted values of [FFA] in equilibrium with albumin: more than 20-fold for palmitate and more than 50-fold for oleate. FFA levels were found to increase monotonically with for all three albumins and all FA. In most cases [FFA] increased, for the same chain length, with increasing degree of acyl chain unsaturation, suggesting that FA aqueous solubility may play a significant role in the equilibrium between FA association with albumin and the aqueous phase. [The highest FFA levels (approximately 3000 nM), for example, were observed for linoleate (18:3) at the maximum v value (6).] Although aqueous-phase solubility of the FA may be important in understanding the interaction between FA and albumin, protein structure, as reflected in differences among the three albumins, also significantly affects the equilibrium.(ABSTRACT TRUNCATED AT 250 WORDS)

The transcription factor CCAAT enhancer binding protein alpha (C/EBPalpha) is expressed at high levels in liver and adipose tissue. Cell culture studies show that C/EBPalpha is sufficient to trigger differentiation of preadipocytes into mature adipocytes, suggesting a central role for C/EBPalpha in the development of adipose tissue. C/EBPalpha knockout mice die within 7-12 h after birth. Defective gluconeogenesis of the liver and subsequent hypoglycemia contribute to the early death of these animals. This short life span impairs investigation of the development of adipose tissue in these mice. To improve the survival of C/EBPalpha-/- animals, we generated a transgenic line that expresses C/EBPalpha under the control of the albumin enhancer/promoter. This line was bred into the knockout strain to generate animals that express C/EBPalpha in the liver but in no other tissue. The presence of the transgene improved survival of C/EBPalpha-/- animals almost 3-fold. Transgenic C/EBPalpha-/- animals at 7 days of age show an absence of s.c., perirenal, and epididymal white fat despite excess lipid substrate in the serum, whereas brown adipose tissue is somewhat hypertrophied and shows minimal biochemical alterations. Interestingly, mammary gland fat tissue is present and exhibits normal morphology. The absence of white adipose tissue in many depots in the presence of high serum lipid levels shows that C/EBPalpha is required for the in vivo development of this tissue. In contrast, brown adipose tissue differentiation is independent of C/EBPalpha expression. The presence of lipid in brown adipose tissue serves as an internal nutritional control, indicating that neither nutritional intake nor lipoprotein composition is likely responsible for the absence of white fat.

OBJECTIVE

Increased risk of mortality in patients with CKD has been attributed to inflammation. However, the association between kidney function, albuminuria, and biomarkers of inflammation has not been examined in a large cohort of CKD patients.

METHODS

This study measured the plasma levels of IL-1β, IL-1 receptor antagonist (IL-1RA), IL-6, TNF-α, TGF-β, high-sensitivity C-reactive protein (hs-CRP), fibrinogen, and serum albumin in 3939 participants enrolled in the Chronic Renal Insufficiency Cohort study between June 2003 and September 2008. An inflammation score was established based on plasma levels of IL-1β, IL-6, TNF-α, hs-CRP, and fibrinogen. Estimated GFR (eGFR) and serum cystatin C were used as measures of kidney function. Albuminuria was quantitated by urine albumin to creatinine ratio (UACR).

RESULTS

Plasma levels of IL-1β, IL-1RA, IL-6, TNF-α, hs-CRP, and fibrinogen were higher among participants with lower levels of eGFR. Inflammation score was higher among those with lower eGFR and higher UACR. In regression analysis adjusted for multiple covariates, eGFR, cystatin C, and UACR were strongly associated with fibrinogen, serum albumin, IL-6, and TNF-α. Each unit increase in eGFR, cystatin C, and UACR was associated with a -1.2% (95% confidence interval, -1.4, -1), 64.9% (56.8, 73.3) and 0.6% (0.4, 0.8) change in IL-6, respectively (P<0.001).

CONCLUSIONS

Biomarkers of inflammation were inversely associated with measures of kidney function and positively with albuminuria.

OBJECTIVE

Toll-like receptors (TLRs) initiate inflammatory signaling in response to conserved microbial molecules. It has been proposed that dietary saturated fatty acids (SFAs) may also serve as endogenous ligands of TLR2 or TLR4, thereby promoting diseases associated with inflammation and dyslipidemia, including atherosclerosis and insulin resistance.

RESULTS

We investigated the effects of SFAs on TLR-dependent signaling using a broad range of cell types and readouts. In HEK-293 cells transfected with TLR2, TLR4, or TLR5, SFAs complexed with fatty-acid-free bovine serum albumin (BSA)-stimulated TLR-dependent signaling. However, SFAs alone did not elicit a similar response. Further analysis showed that the effect seen with the complexed SFAs was attributable to LPS and lipopeptide contamination of fatty-acid-free BSA. Additional studies in macrophages, endothelial cells, smooth muscle cells, adipocytes, skeletal muscle cells, and human peripheral blood mononuclear cells confirmed the lack of stimulation of TLR-dependent signaling pathways or expression of TLR-target genes by SFAs.

CONCLUSIONS

SFAs do not directly stimulate TLR-dependent signaling, suggesting that alternative mechanisms link dietary fat intake with TLR-associated pathologies. LPS and lipopeptide contamination of the widely used reagent fatty-acid-free BSA explains the previously reported stimulation of TLR2 and TLR4 by SFAs.

OBJECTIVE

To evaluate putative risk factors for the development of incipient diabetic nephropathy (persistent microalbuminuria) and overt diabetic nephropathy (persistent macroalbuminuria) in patients with non-insulin dependent diabetes.

METHODS

Prospective, observational study of a cohort of white, non-insulin dependent diabetic patients followed for a median period of 5.8 years.

METHODS

Outpatient clinic in tertiary referral centre.

METHODS

191 patients aged under 66 years with non-insulin dependent diabetes and normoalbuminuria (urinary albumin excretion rate < 30 mg/24 h) who attended the clinic during 1987.

METHODS

Incipient and overt diabetic nephropathy.

RESULTS

Fifteen patients were lost to follow up. Thirty six of the 176 remaining developed persistent microalbuminuria (30-299 mg/24 h in two out of three consecutive 24 hour urine collections) and five developed persistent macroalbuminuria >> or = mg/24 h in two out of three consecutive collections) during follow up. The five year cumulative incidence of incipient diabetic nephropathy was 23% (95% confidence interval 17% to 30%). Cox's multiple stepwise regression analysis revealed the following risk factors for the development of incipient or overt diabetic nephropathy: increased baseline log urinary albumin excretion rate (relative risk 11.1 (3.4 to 35.9); P < 0.0001); male sex (2.6 (1.2 to 5.4); P < 0.02); presence of retinopathy (2.4 (1.3 to 4.7); P < 0.01); increased serum cholesterol concentration (1.4 (1.1 to 1.7); P < 0.01); haemoglobin A1c concentration (1.2 (1.0 to 1.4); P < 0.05); and age (1.07 (1.02 to 1.12); P < 0.01). Known duration of diabetes, body mass index, arterial blood pressure, serum creatinine concentration, pre-existing coronary heart disease, and history of smoking were not risk factors.

CONCLUSIONS

Several potentially modifiable risk factors predict the development of incipient and overt diabetic nephropathy in normoalbuminuric patients with non-insulin dependent diabetes.

The resolution limit of fluorescence correlation spectroscopy for two-component solutions is investigated theoretically and experimentally. The autocorrelation function for two different particles in solution were computed, statistical noise was added, and the resulting curve was fitted with a least squares fit. These simulations show that the ability to distinguish between two different molecular species in solution depends strongly on the number of photons detected from each particle, their difference in size, and the concentration of each component in solution. To distinguish two components, their diffusion times must differ by at least a factor of 1.6 for comparable quantum yields and a high fluorescence signal. Experiments were conducted with Rhodamine 6G and Rhodamine-labeled bovine serum albumin. The experimental results support the simulations. In addition, they show that even with a high fluorescence signal but significantly different quantum yields, the diffusion times must differ by a factor much bigger than 1.6 to distinguish the two components. Depending on the quantum yields and the difference in size, there exists a concentration threshold for the less abundant component below which it is not possible to determine with statistical means alone that two particles are in solution.