A major limitation in developing applications for the use of human embryonic stem cells (HESCs) is our lack of knowledge of their responses to specific cues that control self-renewal, differentiation, and lineage selection. HESCs are most commonly maintained on inactivated mouse embryonic fibroblast feeders in medium supplemented with FCS, or proprietary replacements such as knockout serum-replacement together with FGF-2. These undefined culture conditions hamper analysis of the mechanisms that control HESC behavior. We have now developed a defined serum-free medium, hESF9, for the culture of HESCs on a type I-collagen substrate without feeders. In contrast to other reported media for the culture of HESCs, this medium has a lower osmolarity (292 mosmol/liter), l-ascorbic acid-2-phosphate (0.1 microg/ml), and heparin. Insulin, transferrin, albumin conjugated with oleic acid, and FGF-2 (10 ng/ml) were the only protein components. Further, we found that HESCs would proliferate in the absence of exogenous FGF-2 if heparin was also present. However, their growth was enhanced by the addition of FGF-2 up to 10 ng/ml although higher concentrations were deleterious in the presence of heparin.

BACKGROUND

Endothelial dysfunction and oxidative stress are matters of concern in patients with chronic renal failure (CRF). Uremic solutes retained in these patients could be involved in these processes. Notably, the protein-bound uremic solute indoxyl sulfate induces endothelial dysfunction in vitro, and has shown pro-oxidant effects.

OBJECTIVE

To demonstrate that indoxyl sulfate is a potential mediator of oxidative stress in endothelial cells in vitro.

METHODS

Indoxyl sulfate-induced oxidative stress in human umbilical vein endothelial cells (HUVEC) was studied by measuring reactive oxygen specie (ROS) production by cytofluorimetry, by analyzing the involvement of the pro-oxidative enzymes NAD(P)H oxidase, xanthine oxidase, and NO synthase, and by measuring the levels of the non-enzymatic antioxidant glutathione.

RESULTS

We showed that indoxyl sulfate induced a significant production of ROS in HUVEC, with or without human serum albumin. We then investigated the role of pro-oxidative enzymes and measured the levels of the antioxidant glutathione. The NAD(P)H oxidase inhibitors, DPI, and apocynin, inhibited ROS production, whereas inhibitors of xanthine oxidase, NO synthase, and mitochondrial ROS had no effect. Interestingly, indoxyl sulfate strongly decreased the levels of glutathione, one of the most active antioxidant systems of the cell. In addition, the ROS production mediated by indoxyl sulfate was inhibited by the antioxidants vitamin C, vitamin E, and NAC.

CONCLUSIONS

The uremic solute indoxyl sulfate enhances ROS production, increases NAD(P)H oxidase activity, and decreases glutathione levels in endothelial cells. Thus, indoxyl sulfate induces oxidative stress by modifying the balance between pro- and antioxidant mechanisms in endothelial cells.

We investigated the effect of human umbilical mesenchymal stem cells (HUMSCs) from Wharton's jelly on carbon tetrachloride (CCl4)-induced liver fibrosis in rats. Rats were treated with CCl4 for 4 weeks, and this was followed by a direct injection of HUMSCs into their livers. After 4 more weeks of CCl4 treatment (8 weeks in all), rats with HUMSC transplants [CCl4 (8W)+HUMSC liver] exhibited a significant reduction in liver fibrosis, as evidenced by Sirius red staining and a collagen content assay, in comparison with rats treated with CCl4 for 8 weeks without HUMSC transplants [CCl4 (8W)]. Moreover, rats in the CCl4 (8W)+HUMSC (liver) group had significantly lower levels of serum glutamic oxaloacetic transaminase, glutamic pyruvate transaminase, alpha-smooth muscle actin, and transforming growth factor-beta1 in the liver, whereas the expression of hepatic mesenchymal epithelial transition factor-phosphorylated type (Met-P) and hepatocyte growth factor was up-regulated, in comparison with the CCl4 (8W) group. Notably, engrafted HUMSCs scattered mostly in the hepatic connective tissue but did not differentiate into hepatocytes expressing human albumin or alpha-fetoprotein. Instead, these engrafted, undifferentiated HUMSCs secreted a variety of bioactive cytokines that may restore liver function and promote regeneration. Human cytokine assay revealed that the amounts of human cutaneous T cell-attracting chemokine, leukemia inhibitory factor, and prolactin were substantially greater in the livers of the CCl4 (8W)+HUMSC (liver) group, with considerably reduced hepatic inflammation manifested by a micro positron emission tomography scan. Our findings suggest that xenogeneic transplantation of HUMSCs is a novel approach for treating liver fibrosis and may be a promising therapeutic intervention in the future.

The ability of Escherichia coli heat-labile enterotoxin (LT) to influence the induction and maintenance of tolerance was examined in animals primed orally with a soluble protein antigen, ovalbumin (OVA), or in animals primed orally with two unrelated protein antigens administered simultaneously, OVA and bovine serum albumin (BSA). LT is immunologically and structurally related to the cholera enterotoxin (CT), which has been shown to be capable of abrogating oral tolerance to protein antigens when delivered simultaneously with the antigens. In this study, simultaneous administration of LT with OVA was shown to prevent the induction of tolerance to OVA and to increase the serum anti-OVA IgG response 30- to 90-fold over OVA-primed and PBS-primed animals, respectively. This effect was determined to be a function of the enzymatically active A subunit of the toxin since the B (binding) subunit alone was unable to influence tolerance induction. Animals fed LT with OVA after the initial OVA prime developed a significantly lower serum IgG and mucosal IgA anti-OVA response than those fed LT with OVA in the initial immunization, indicating that prior exposure to the antigen reduces the effectiveness of LT to influence tolerance and its ability to act as an adjuvant. LT was not able to abrogate tolerance once it had been established. Serum IgG and mucosal IgA responses in animals receiving LT on only a single occasion, that being upon first exposure to antigen, were equivalent to responses after three OVA/LT primes, indicating that commitment to responsiveness occurs early and upon first exposure to antigen.(ABSTRACT TRUNCATED AT 250 WORDS)

BACKGROUND

Nutritional status is strongly associated with outcomes among hemodialysis patients. We analyzed the independent predictive value of several readily measured nutritional indicators, including a modified subjective global assessment (mSGA), body mass index (BMI), serum albumin, serum creatinine, normalized protein catabolic rate (nPCR), serum bicarbonate, lymphocyte count, and neutrophil count, using baseline and six-month follow-up measurements.

METHODS

The study sample consisted of 7719 U.S. adult hemodialysis patients enrolled in the international Dialysis Outcomes and Practice Patterns Study (DOPPS), a prospective observational study that includes a random sample of hemodialysis patients from 145 dialysis facilities in the United States. Cox regression was used to estimate the relative risk of mortality associated with differences in measurements at baseline and six months later. Each analysis was adjusted for age, race, sex, and 15 summary comorbid conditions.

RESULTS

Lower baseline measurements of mSGA, BMI, serum albumin, serum creatinine, and lymphocyte count were independently associated with significantly higher risk of mortality. During six-month follow-up, decreases in BMI, serum albumin, and serum creatinine were also associated with significantly higher mortality risk. The risk of mortality increased with higher baseline and six-month increases in neutrophil count.

CONCLUSIONS

This study confirms that several readily-measured nutritional indicators predict mortality among hemodialysis patients and that changes in indicator values over six months provide additional important prognostic information. Interventions that modify these indicators of nutritional status may have an important impact on the survival of hemodialysis patients.

OBJECTIVE

Laparoscopy-assisted gastrectomy with lymph node dissection for gastric cancer is considered technically more complicated than the open method. Moreover, the safety and efficacy of laparoscopy-assisted distal gastrectomy (LADG) with extraperigastric lymph node dissection in patients with gastric cancer have not been established yet. To evaluate short-term surgical validity, surgical outcome of the laparoscopy-assisted distal gastrectomy (LADG) with extraperigastric lymph node dissection was compared with that of the conventional open distal gastrectomy (CODG) in patients with early gastric cancer.

METHODS

One hundred and forty-seven patients with early gastric cancer received radical distal gastrectomy during 2002 and 2003, where LADG was undergone in 71 patients. The clinicopathologic characteristics, postoperative outcomes and courses, and postoperative morbidities and mortalities were compared between the two groups. Data were retrieved from the stomach cancer database at Dong-A University Medical center.

RESULTS

Baseline characteristics, including sex, age, body mass index (BMI), American Society of Anesthesiology (ASA) class, tumor size, T stage, and lymph node metastasis were similar between the two groups. No significant differences were found between these groups in terms of the number of retrieved lymph nodes with respect to D1 + alpha (D1 + no. 7) and D1 + beta (D1 + no. 7, 8a, and 9) lymphadenectomy. In the LADG group, wound size was smaller (P < 0.0001), but operation time was longer (P = 0.0001) than in the CODG group. Perioperative recovery was faster in the LADG group than in the CODG group, as reflected by a shorter hospital stay (P = 0.0176) and less times of additional analgesics (P = 0.0370). Serum albumin level in LADG was higher (P = 0.0002) on day 7 than that in CODG, and the leukocyte count in LADG lower (P = 0.0445) on day 1 than that in CODG. Postoperative morbidities and mortalities were not significantly different between the two groups.

CONCLUSIONS

Our data confirmed that LADG with extraperigastric (no. 7, 8, and 9) lymph node dissection proved to be feasible and acceptable surgical technique for early gastric cancer. At least taking a surgical point of view, LADG with extraperigastric lymph node dissection is suggested to be a preferred surgical option for patients with early gastric cancer. Its oncologic validity awaits larger and prospective multicenter trials.

BACKGROUND

Measles kills about 2 million children annually, and there is no specific therapy for the disease. It has been suggested that vitamin A may be of benefit in the treatment of measles.

METHODS

We conducted a randomized, double-blind trial involving 189 children who were hospitalized at a regional center in South Africa because of measles complicated by pneumonia, diarrhea, or croup. The children (median age, 10 months) were assigned to receive either vitamin A (total dose, 400,000 IU of retinyl palmitate, given orally; n = 92) or placebo (n = 97), beginning within five days of the onset of the rash. At base line, the characteristics of the two groups were similar.

RESULTS

Although clinically apparent vitamin A deficiency is rare in this population, the children's serum retinol levels were markedly depressed (mean [+/- SEM], 0.405 +/- 0.021 mumols per liter [11.6 +/- 0.6 micrograms per deciliter]), and 92 percent of them had hyporetinemia (serum retinol level less than 0.7 mumols per liter [20 micrograms per deciliter]). Serum concentrations of retinol-binding protein (mean, 30.1 +/- 2.0 mg per liter) and albumin (mean, 33.4 +/- 0.5 g per liter) were also low. As compared with the placebo group, the children who received vitamin A recovered more rapidly from pneumonia (mean, 6.3 vs. 12.4 days, respectively; P less than 0.001) and diarrhea (mean, 5.6 vs. 8.5 days; P less than 0.001), had less croup (13 vs. 27 cases; P = 0.03), and spent fewer days in the hospital (mean, 10.6 vs. 14.8 days; P = 0.01). Of the 12 children who died, 10 were among those given placebo (P = 0.05). For the group treated with vitamin A, the risk of death or a major complication during the hospital stay was half that of the control group (relative risk, 0.51; 95 percent confidence interval, 0.35 to 0.74).

CONCLUSIONS

Treatment with vitamin A reduces morbidity and mortality in measles, and all children with severe measles should be given vitamin A supplements, whether or not they are thought to have a nutritional deficiency.

OBJECTIVE

This study sought to evaluate the impact of frailty in older adults undergoing transcatheter aortic valve replacement (TAVR) for symptomatic aortic stenosis.

BACKGROUND

Frailty status impacts prognosis in older adults with heart disease; however, the impact of frailty on prognosis after TAVR is unknown.

METHODS

Gait speed, grip strength, serum albumin, and activities of daily living status were collected at baseline and used to derive a frailty score among patients who underwent TAVR procedures at a single large-volume institution. The cohort was dichotomized on the basis of median frailty score into frail and not frail groups. The impact of frailty on procedural outcomes (stroke, bleeding, vascular complications, acute kidney injury, and mortality at 30 days) and 1-year mortality was evaluated.

RESULTS

Frailty status was assessed in 159 subjects who underwent TAVR (age 86 ± 8 years, Society of Thoracic Surgery Risk Score 12 ± 4). Baseline frailty score was not associated with conventionally ascertained clinical variables or Society of Thoracic Surgery score. Although high frailty score was associated with a longer post-TAVR hospital stay when compared with lower frailty score (9 ± 6 days vs. 6 ± 5 days, respectively, p = 0.004), there were no significant crude associations between frailty status and procedural outcomes, suggesting adequacy of the standard selection process for identifying patients at risk for periprocedural complications after TAVR. Frailty status was independently associated with increased 1-year mortality (hazard ratio: 3.5, 95% confidence interval: 1.4 to 8.5, p = 0.007) after TAVR.

CONCLUSIONS

Frailty was not associated with increased periprocedural complications in patients selected as candidates to undergo TAVR but was associated with increased 1-year mortality after TAVR. Further studies will evaluate the independent value of this frailty composite in older adults with aortic stenosis.

Freshly isolated adult rat hepatocytes, when cultured on type I collagen (commercially available as Vitrogen), assume a polygonal shape, form a stable monolayer within 24 hours, but lose the capacity to express some liver-specific functions over time in culture. We incubated hepatocytes in a serum-free medium on a reconstituted basement membrane gel, "matrigel" (prepared from an extract of extracellular matrix of the murine Engelbreth-Holm-Swarm sarcoma), and observed that the cells adhered firmly, remained rounded as single cells or clusters, and maintained liver-specific gene expression for more than 1 week in vitro. Hepatocytes on matrigel secreted substantially higher amounts of albumin, transferrin, haptoglobin, and hemopexin, Northern blot analyses of extracted cellular RNA, expressed increased amounts of mRNA for the liver-specific protein albumin (as compared with cells on vitrogen). In cultures treated with phenobarbital, cytochrome P-450b, and cytochrome P-450e, mRNAs and proteins were barely detectable in cells on Vitrogen but were induced to levels similar to those in the liver in vivo in matrigel cultures. Likewise, the use of matrigel greatly enhanced the induction of mRNA and protein for P-450c by 3-methylcholanthrene and for P-450p by steroidal and nonsteroidal inducers. However, neither substratum permitted induction of P-450d by 3-methylcholanthrene, suggesting that the effects of matrigel are selective even for expression in liver of members of the superfamily of cytochrome P-450 genes. Within 5 days in cultures on Vitrogen, hepatocytes expressed detectable amounts of fetal liver aldolase activity and also mRNA for vimentin and type I collagen, each considered a phenotypic change reflecting hepatocyte "dedifferentiation." None of these was present in cells on matrigel. Responsiveness to mitogenic stimuli, as judged by incorporation of 3H-thymidine into DNA, was also decreased in hepatocytes cultured on matrigel. Finally, there was a remarkable increase in the levels of both matrices during the first 2 days in culture. However, the continuously cytoskeleton mRNA over time in culture than did the rounded cells on matrigel. We conclude that hepatocytes cultured on matrigel, as opposed to the standard collagen, exhibit remarkably enhanced expression of many liver-specific functions.

1,25(OH)(2)D(3) has antiproliferative effects and promotes cell differentiation. This consideration has provided the rationale for studies in subtotally nephrectomized rats showing that 1,25(OH)(2)D(3) interfered with glomerulosclerosis. The cellular mechanisms involved have remained obscure, however. It was the purpose of the present study to assess glomerular structure and cellular composition in subtotally nephrectomized (SNX) rats treated with nonpharmacological doses of 1,25(OH)(2)D(3). Male Sprague-Dawley rats were sham operated (sham) or underwent SNX under general anesthesia and received either solvent or 1,25(OH)(2)D(3) (3 ng.100 g body wt(-1).day(-1) sc). Blood pressure (BP) and albuminuria were measured. After 16 wk, the remnant renal tissue was perfusion fixed and morphometric and stereological measurements were carried out. The expression of proliferating cellular antigen (PCNA), cyclin-dependent kinase inhibitor p27, Wilms tumor gene (WT1), and desmin, a marker of early podocyte damage, was investigated by immunohistology. BP, serum creatinine, and urinary albumin excretion were significantly higher in SNX than in sham rats. Albuminuria was significantly lower in SNX+1,25(OH)(2)D(3) compared with SNX+solvent rats. Mean glomerular tuft volume was significantly higher in SNX+solvent (2.69 +/- 0.21 gx 10(6) microm(3)) than in sham rats (1.44 +/- 0.17 and 1.28 +/- 0.14 x 10(6) microm(3)); it was significantly (P < 0.05) lower in SNX+1,25(OH)(2)D(3) rats (1.81 +/- 0.16 x 10(6) microm(3)). The main finding was a significantly higher number of podocytes in SNX+1,25(OH)(2)D(3) (88 +/- 9) and sham (98 +/- 17) compared with SNX+solvent rats (81 +/- 8.7). In parallel, the increase in podocyte volume in SNX+solvent rats was abrogated by treatment with 1,25(OH)(2)D(3), and immunohistochemistry revealed less expression of desmin, PCNA, and p27, suggesting less podocyte injury and activation of the cyclin cascade. This study identifies the podocyte as an important target cell for the renoprotective action of 1,25(OH)(2)D(3). This notion is suggested by less evidence of podocyte injury, decreased podocytes loss, and abrogation of podocyte hypertrophy, findings that may also explain less pronounced albuminuria and glomerulosclerosis.

BACKGROUND

The National Veterans Affairs Surgical Risk Study was designed to collect reliable, valid data on patient risk and outcomes for major surgery in the Veterans Health Administration and to report comparative risk-adjusted postoperative mortality and morbidity rates for surgical services in the Veterans Health Administration.

METHODS

This was a cohort study conducted at 44 Veterans Affairs Medical Centers closely affiliated with university medical centers. Included were 87,078 major noncardiac operations performed under general, spinal, or epidural anesthesia between October 1, 1991, and December 31, 1993. The main outcomes measures in this report are 21 postoperative adverse events (morbidities) occurring within 30 days after the index procedure. Multivariable logistic regression risk-adjustment models for all operations and for eight surgical subspecialties were developed.

RESULTS

Patient risk factors predictive of postoperative morbidity included serum albumin level, American Society of Anesthesia class, the complexity of the operation, and 17 other preoperative risk variables. Wide variation in the unadjusted rates of one or more postoperative morbidities for all operations was observed across the 44 hospitals (7.4-28.4%). Risk-adjusted observed-to-expected ratios ranged from 0.49 to 1.46. The Spearman rank order correlation between the ranking of the hospitals based on unadjusted morbidity rates and risk-adjusted observed-to-expected ratios for all operations was 0.87. There was little or no correlation between the rank order of the hospitals by risk-adjusted morbidity and risk-adjusted mortality.

CONCLUSIONS

The Department of Veterans Affairs has successfully implemented a system for the prospective collection and comparative reporting of postoperative mortality and morbidity rates after major noncardiac operations. Risk adjustment had only a modest effect on the rank order of the hospitals.

BACKGROUND

The aim of this study was to establish and validate a practical method to disperse nanoparticles in physiological solutions for biological in vitro and in vivo studies.

RESULTS

TiO2 (rutile) dispersions were prepared in distilled water, PBS, or RPMI 1640 cell culture medium. Different ultrasound energies, various dispersion stabilizers (human, bovine, and mouse serum albumin, Tween 80, and mouse serum), various concentrations of stabilizers, and different sequences of preparation steps were applied. The size distribution of dispersed nanoparticles was analyzed by dynamic light scattering and zeta potential was measured using phase analysis light scattering. Nanoparticle size was also verified by transmission electron microscopy. A specific ultrasound energy of 4.2 x 105 kJ/m3 was sufficient to disaggregate TiO2 (rutile) nanoparticles, whereas higher energy input did not further improve size reduction. The optimal sequence was first to sonicate the nanoparticles in water, then to add dispersion stabilizers, and finally to add buffered salt solution to the dispersion. The formation of coarse TiO2 (rutile) agglomerates in PBS or RPMI was prevented by addition of 1.5 mg/ml of human, bovine or mouse serum albumin, or mouse serum. The required concentration of albumin to stabilize the nanoparticle dispersion depended on the concentration of the nanoparticles in the dispersion. TiO2 (rutile) particle dispersions at a concentration lower than 0.2 mg/ml could be stabilized by the addition of 1.5 mg/ml albumin. TiO2 (rutile) particle dispersions prepared by this method were stable for up to at least 1 week. This method was suitable for preparing dispersions without coarse agglomerates (average diameter < 290 nm) from nanosized TiO2 (rutile), ZnO, Ag, SiOx, SWNT, MWNT, and diesel SRM2975 particulate matter.

CONCLUSIONS

The optimized dispersion method presented here appears to be effective and practicable for preparing dispersions of nanoparticles in physiological solutions without creating coarse agglomerates.

Mammalian preimplantation embryos normally develop within the protected environment of the female reproductive tract, which virtually precludes studies on embryogenesis in situ. Information must therefore be derived from experiments on cultured embryos. Consequently, studies on the epigenetic regulation of embryogenesis have long been interwoven with efforts to formulate culture media capable of sustaining normal development. In this review, comparative information on epigenetic regulation of embryo development is discussed, including information on energy substrate and amino acid preferences of embryos. Advantages of simple versus complex culture media, and of substituting serum albumin or synthetic macromolecules for serum, are discussed. Some potential pitfalls of co-culture are described. Culture appears to induce anomalies in embryo metabolism, which may derive from disturbed intracellular pH. Rationales for selecting endpoints to evaluate the outcome of experiments are considered, including incorporation of timing of embryo development into the analysis. Poor experimental design and/or data analysis can detract from or even negate the value of data obtained from embryo culture; examples are examined to help correct this problem. All of these points are discussed with a view to using data on the needs of embryos for making improvements in the design of culture media, so that higher yields and increased viability of embryos are achieved.

The peritoneum is one of the most common extrapulmonary sites of tuberculous infection. Peritoneal tuberculosis remains a significant problem in parts of the world where tuberculosis is prevalent. Increasing population migration, usage of more potent immunosuppressant therapy and the acquired immunodeficiency syndrome epidemic has contributed to a resurgence of this disease in regions where it had previously been largely controlled. Tuberculous peritonitis frequently complicates patients with underlying end-stage renal or liver disease that further adds to the diagnostic difficulty. The diagnosis of this disease, however, remains a challenge because of its insidious nature, the variability of its presentation and the limitations of available diagnostic tests. A high index of suspicion is needed whenever confronted with unexplained ascites, particularly in high-risk patients. Based on a systematic review of the literature, we recommend: tuberculous peritonitis should be considered in the differential diagnosis of all patients presenting with unexplained lymphocytic ascites and those with a serum-ascites albumin gradient (SAAG) of <11 g/L; culture growth of Mycobacterium of the ascitic fluid or peritoneal biopsy as the gold standard test; further studies to determine the role of polymerase chain reaction, ascitic adenosine deaminase and the BACTEC radiometric system for acceleration of mycobacterial identification as means of improving the diagnostic yield; increasing utilization of ultrasound and computerized tomographic scan for the diagnosis and as a guidance to obtain peritoneal biopsies; low threshold for diagnostic laparoscopy; treatment for 6 months with the first-line antituberculous drugs (isoniazid, rifampicin, ethambutol and pyrazinamide) in uncomplicated cases.

An enzyme isolated from rat liver cytosol (native molecular mass 78. 3 kDa; polypeptide molecular mass 42.5 kDa) is capable of catalysing the NADH/NADPH-dependent degradation of S-nitrosoglutathione (GSNO). The activity utilizes 1 mol of coenzyme per mol of GSNO processed. The isolated enzyme has, as well, several characteristics that are unique to alcohol dehydrogenase (ADH) class III isoenzyme: it is capable of catalysing the NAD+-dependent oxidations of octanol (insensitive to inhibition by 4-methylpyrazole), methylcrotyl alcohol (stimulated by added pentanoate) and 12-hydroxydodecanoic acid, and also the NADH/NADPH-dependent reduction of octanal. Methanol and ethanol oxidation activity is minimal. The enzyme has formaldehyde dehydrogenase activity in that it is capable of catalysing the NAD+/NADP+-dependent oxidation of S-hydroxymethylglutathione. Treatment with the arginine-specific reagent phenylglyoxal prevents the pentanoate stimulation of methylcrotyl alcohol oxidation and markedly diminishes the enzymic activity towards octanol, 12-hydroxydodecanoic acid and S-hydroxymethylglutathione; the capacity to catalyse GSNO degradation is also checked. Additionally, limited peptide sequencing indicates 100% correspondence with known ADH class III isoenzyme sequences. Kinetic studies demonstrate that GSNO is an exceptionally active substrate for this enzyme. S-Nitroso-N-acetylpenicillamine and S-nitrosated human serum albumin are not substrates; the activity towards S-nitrosated glutathione mono- and di-ethyl esters is minimal. Product analysis suggests that glutathione sulphinamide is the major stable product of enzymic GSNO processing, with minor yields of GSSG and NH3; GSH, hydroxylamine, nitrite, nitrate and nitric oxide accumulations are minimal. Inclusion of GSH in the reaction mix decreases the yield of the supposed glutathione sulphinamide in favor of GSSG and hydroxylamine.

Two or more oligoclonal IgG bands (OB) detected by separation of cerebrospinal fluid (CSF) proteins while not demonstrable in corresponding serum reflect a local B-cell response accompanying central nervous system (CNS) inflammation. Using optimized, standardized methodology, preferentially protein separation by isoelectric focusing followed by immunoblotting, more than 95% of patients with multiple sclerosis (MS) have CSF OB of IgG class not detectable in serum, thereby providing powerful evidence for the diagnosis of MS. Once present, CSF OB persists in the individual patient irrespective of MS course or therapy. Because of the high sensitivity of CSF OB in MS as well as its high specificity in the appropriate clinical setting, examination of CSF for OB of IgG class can be strongly recommended to obtain support for the diagnosis of MS and identify patients with clinically isolated syndrome (CIS) at increased risk of developing MS. The IgG index equal to CSF/serum IgG:CSF/serum albumin is elevated in about 70% of MS patients, but rarely in CSF OB-negative MS. Because of lower diagnostic sensitivity, IgG index cannot be recommended as replacement of CSF OB in the diagnosis of MS but, when elevated, as additional evidence for an augmented B-cell response within the CNS that is compatible with MS. Although the clinical picture as well as findings from magnetic resonance imaging of the brain and spinal cord are essential for an MS diagnosis, this should be re-evaluated in CSF OB-negative patients, keeping in mind the many disease entities imitating MS. Recommended diagnostic criteria for MS must include definitions of the role of lumbar puncture and of clearly specified, optimized and standardized routine CSF investigations including for the presence of CSF IgG OB. There is a need for concerted long-term follow-up studies of the subgroup of MS patients without CSF OB regarding e.g. prognostic and immunologic features. For inclusion in trials of disease-modulating drugs, it is recommended that patients with MS or CIS are selected regarding presence vs. absence of CSF OB. Development and evaluation of new technologies to define local vs. systemic B-cell responses in patients with MS or CIS vs. patients with other inflammatory neurological diseases should shed new light on the role of CSF OB, which remains enigmatic.

OBJECTIVE

To examine the relationship of systemic markers of inflammation, endothelial dysfunction, and serum folate level to retinal vessel diameter.

METHODS

Cross-sectional analyses were completed for data from a random sample of 396 persons aged 50 to 86 years who underwent a baseline examination from 1988 to 1990. Standardized protocols for blood collection and measurement of markers were used. Diameters of arterioles and venules were measured from digitized photographs. Standard univariate and multivariate analyses were performed.

RESULTS

While controlling for age, smoking status, diabetes status, serum high-density lipoprotein cholesterol, and hematocrit, wider retinal venular diameters were associated with higher serum high-sensitivity C-reactive protein, interleukin 6, and amyloid A levels. While controlling for age, systolic and diastolic blood pressure, smoking, serum high-density lipoprotein cholesterol level, and gout, smaller arteriolar diameters were associated with higher serum amyloid A and lower serum albumin and folate levels but not high-sensitivity C-reactive protein or interleukin 6 levels. Levels of serum soluble intercellular adhesion molecule-1 and serum soluble E-selectin, markers of endothelial dysfunction, were not associated with retinal arteriolar or venular diameters.

CONCLUSIONS

These data show an association of inflammatory markers with larger retinal venular diameter, suggesting that retinal venular caliber may be a marker of systemic inflammation.

BACKGROUND

The cardiovascular risk implications of a combined assessment of reduced kidney function and microalbuminuria are unknown. In elderly persons, traditional cardiovascular risk factors are less predictive, and measures of end organ damage, such as kidney disease, may be needed for improved cardiovascular mortality risk stratification.

METHODS

The glomerular filtration rate was estimated from calibrated serum creatinine, and the urine albumin-creatinine ratio (ACR) was measured in 3 urine samples in 9,709 participants of the second Nord-Trøndelag Health Study (HUNT II), a Norwegian community-based health study, followed for 8.3 years with a 71% participation rate.

RESULTS

An estimated glomerular filtration rate (EGFR) at levels of less than 75 mL/min/1.73 m(2) was associated with higher cardiovascular mortality risk, whereas a higher ACR was associated with higher risk with no lower limit. Low EGFR and albuminuria were synergistic cardiovascular mortality risk factors. Compared with subjects with an EGFR greater than 75 mL/min/1.73 m(2) and ACR below the sex-specific median who were at the lowest risk, subjects with an EGFR of less than 45 mL/min/1.73 m(2) and microalbuminuria had an adjusted incidence rate ratio of 6.7 (95% confidence interval, 3.0-15.1; P < .001). The addition of ACR and EGFR improved traditional risk models: 39% of subjects with intermediate risk were reclassified to low- or high-risk categories with corresponding observed risks that were 3-fold different than the original category. Age-stratified analyses showed that EGFR and ACR were particularly strong risk factors for persons 70 years or older.

CONCLUSIONS

Reduced kidney function and microalbuminuria are risk factors for cardiovascular death, independent of each other and traditional risk factors. The combined variable improved cardiovascular risk stratification at all age levels, but particularly in elderly persons where the predictive power of traditional risk factors is attenuated.

There is a direct correlation between protein levels and disease states in human serum, which makes it an attractive target for sensors and diagnostics. However, this is challenging because serum features more than 20,000 proteins, with an overall protein content greater than 1 mM. Here we report a sensor based on a hybrid synthetic-biomolecule that uses arrays of green fluorescent protein and nanoparticles to detect proteins at biorelevant concentrations in both buffer and human serum. Distinct and reproducible fluorescence-response patterns were obtained from five serum proteins (human serum albumin, immunoglobulin G, transferrin, fibrinogen and a-antitrypsin), both in buffer and when spiked into human serum. Using linear discriminant analysis we identified these proteins with an identification accuracy of 100% in buffer and 97% in human serum. The arrays were also able to discriminate between different concentrations of the same protein, as well as a mixture of different proteins in human serum.

Using the technique of centrifugal ultrafiltration isodialysis to measure the free concentration of 1,25-dihydroxyvitamin D [1,25-(OH)2D], we determined the affinity of serum proteins for 1,25-(OH)2D both by Scatchard analysis (increasing ligand concentration at fixed binding site concentrations) and by a novel analysis in which the binding site concentrations were varied (serial dilution) at fixed ligand concentrations. The high affinity binding constant in serum for 1,25-(OH)2D was 3.7 X 10(7) M-1 by Scatchard analysis and 4.2 X 10(7) M-1 by serial dilution analysis. Human serum albumin had a much lower affinity for 1,25-(OH)2D (5.4 X 10(4) M-1). When vitamin D-binding protein (DBP) was selectively removed from serum by an actin affinity column, the affinity of the remaining serum proteins for 1,25-(OH)2D was that of albumin. Postulating a two-site model (DBP and albumin) for transport of 1,25-(OH)2D in serum and incorporating the estimated affinity constants of DBP and albumin for this metabolite, we calculated that 85% of total circulating 1,25-(OH)2D is transported in blood bound to DBP in normal individuals (0.4% is free and 14.6% is bound to albumin). In patients with liver disease, 73% is bound to DBP (1.1% is free and 25.9% is bound to albumin). Using this same two site model, we found a reasonable correlation (r = 0.612; P less than 0.001) between the measured free 1,25-(OH)2D level and the calculated free 1,25-(OH)2D level in serum based on albumin and DBP concentrations in 16 normal subjects and 16 patients with liver disease. These results confirm the concept that although DBP is the principal protein carrier of 1,25-(OH)2D in serum, albumin is a major secondary carrier, especially in patients with low DBP levels.

The availability of allergen molecules ('components') from several protein families has advanced our understanding of immunoglobulin E (IgE)-mediated responses and enabled 'component-resolved diagnosis' (CRD). The European Academy of Allergy and Clinical Immunology (EAACI) Molecular Allergology User's Guide (MAUG) provides comprehensive information on important allergens and describes the diagnostic options using CRD. Part A of the EAACI MAUG introduces allergen molecules, families, composition of extracts, databases, and diagnostic IgE, skin, and basophil tests. Singleplex and multiplex IgE assays with components improve both sensitivity for low-abundance allergens and analytical specificity; IgE to individual allergens can yield information on clinical risks and distinguish cross-reactivity from true primary sensitization. Part B discusses the clinical and molecular aspects of IgE-mediated allergies to foods (including nuts, seeds, legumes, fruits, vegetables, cereal grains, milk, egg, meat, fish, and shellfish), inhalants (pollen, mold spores, mites, and animal dander), and Hymenoptera venom. Diagnostic algorithms and short case histories provide useful information for the clinical workup of allergic individuals targeted for CRD. Part C covers protein families containing ubiquitous, highly cross-reactive panallergens from plant (lipid transfer proteins, polcalcins, PR-10, profilins) and animal sources (lipocalins, parvalbumins, serum albumins, tropomyosins) and explains their diagnostic and clinical utility. Part D lists 100 important allergen molecules. In conclusion, IgE-mediated reactions and allergic diseases, including allergic rhinoconjunctivitis, asthma, food reactions, and insect sting reactions, are discussed from a novel molecular perspective. The EAACI MAUG documents the rapid progression of molecular allergology from basic research to its integration into clinical practice, a quantum leap in the management of allergic patients.

An objective and quantitative standard of inflammatory activity for patients with Crohn's disease was developed. This Activity Index (AI) was derived from data of 63 patients with Crohn's disease who had been submitted to a total of 85 clinical examinations. On the basis of 18 predictor variables three physicians gave an overall evaluation of the severity of inflammatory activity in each patient. Stepwise multiple regression analysis was used to investigate which combination of variables contributed most to the overall evaluation. The combination of the following nine variables gave a very good correlation (r = 0.95) with the overall evaluation: serum albumin, ESR, body weight related to length, abdominal mass, sex, temperature, stool consistency, bowel resection, and extraintestinal symptoms related to Crohn's disease. This combination of variables expressed in a score that was used as an activity index proved to be very useful in the assessment of disease activity and of the effect of therapy. Index values below 100 are associated with inactive disease, values between 100 and 150 can be regarded as indicating slight inflammatory activity, values between 150 and 210 as indicating moderate, and values above 210 as indicating severe-to-very-severe inflammatory activity.

Three, single-day nutritional surveys at weekly intervals were conducted in the general medical wards of an urban municipal teaching hospital. The techniques of nutritional assessment included anthropometric measures (weight/height, triceps skin fold, arm-muscle circumference, serum albumin, and hematocrit). The prevalence of protein-calorie malnutrition was 44% or greater by these criteria (weight/height, 45%; triceps skin fold, 76%; arm-muscle circumference, 55%; serum albumin, 44%; and hematocrit, 48%). These results were reproducible without significant variation between surveys. In 34% of patients, a lymphopenia of 1,200 cells/cu mm or less was found, a level likely to be associated with diminished cell-mediated immunity. Compared with a similar survey among surgical patients, the medical patients were more depleted calorically (weight/height, triceps skin fold) but had better protein status (arm-muscle circumference, serum albumin). Significant protein-calorie malnutrition occurs commonly in municipal hospitals in both medical and surgical services.

BACKGROUND

Data about the relationship of inflammation to nephropathy in type 2 diabetes mellitus are scarce. In the present study, we test the hypothesis that inflammatory parameters are independently related to urinary albumin excretion (UAE) at early stages of nephropathy.

METHODS

Sixty-five patients with type 2 diabetes with microalbuminuria (MAB) or mild proteinuria (protein < 1 g/d) were included. We analyzed serum concentrations of high-sensitivity C-reactive protein (hs-CRP) and tumor necrosis factor-alpha (TNF-alpha), as well as urinary level of this cytokine.

RESULTS

Inflammatory parameters were significantly greater in patients with diabetes than controls; furthermore, urinary TNF-alpha levels increased significantly as nephropathy progressed. Median urinary TNF-alpha level was 7 pg/mg in normoalbuminurics, 13 pg/mg in microalbuminurics (P < 0.001), and 18 pg/mg in proteinurics (P < 0.001 versus normoalbuminuria and P < 0.01 versus MAB). Albuminuria was related to hs-CRP (r= 0.68; P < 0.001) and serum (r = 0.45; P < 0.01) and urinary TNF-alpha levels (r = 0.71; P < 0.001), but there was no association between serum and urinary TNF-alpha levels. Partial correlation analysis showed that hs-CRP level, urinary TNF-alpha level, duration of diabetes, and glycated hemoglobin level remained significantly associated with UAE. A stepwise multiple regression analysis showed that UAE was significantly associated with hs-CRP level (P < 0.001), duration of diabetes (P < 0.001), urinary TNF-alpha level (P < 0.01), and glycated hemoglobin level (P < 0.05; adjusted R2 = 0.73; P < 0.001).

CONCLUSIONS

Inflammatory parameters in patients with type 2 diabetes at an early stage of nephropathy are independently associated with UAE. In addition to traditional metabolic and hemodynamic factors, it is possible to hypothesize on the participation of inflammation in the pathogenesis of diabetic nephropathy.