BACKGROUND

Nutritional status plays an important role in the incidence of postoperative complications and the prognosis of various tumours. The prognostic value of preoperative nutritional factors in patients with pancreatic cancer is not known.

METHODS

This retrospective study included 268 patients who underwent resection for adenocarcinoma of the pancreas. The predictive value of preoperative nutritional status for postoperative outcome (survival, complications) was assessed. Nutritional factors included the three constitutional indices, serum albumin and Onodera's prognostic nutrition index (PNI), calculated as 10 × serum albumin (g/dl) + 0·005× total lymphocyte count (per mm(3)).

RESULTS

In multivariable analysis preoperative low PNI (but not low albumin) was an independent prognostic factor for poor survival: hazard ratio (HR) 1·73 (95 per cent confidence interval (c.i.) 1·21 to 2·47). The accuracy of a PNI value of less than 45 as cut-off for clinically significant preoperative malnutrition in predicting 1- or 2-year survival after surgery was, however, limited (66·4 and 56·3 per cent respectively). Low preoperative albumin concentration and PNI were significantly associated with postoperative complications: odds ratio 1·98 (95 per cent c.i. 1·18 to 3·32) and 2·14 (1·23 to 3·73) respectively. Low PNI and low body mass index were independently associated with pancreatic fistula: HR 2·52 (1·37 to 4·63) and 0·40 (0·17 to 0·93) respectively.

CONCLUSIONS

The PNI is associated with overall survival and postoperative complications, in particular pancreatic fistula, in patients with pancreatic cancer. The moderate accuracy of PNI as a predictor of survival limits its clinical use.

A simplified procedure for filter paper electrophoresis is described in which disturbing factors such as evaporation, heating, buffer concentration gradients, and pH changes in the electrode vessels were reduced to a minimum. Artificial mixtures of highly purified proteins could be separated and the components isolated. The application of the method to a variety of studies on serum proteins is demonstrated. Protein concentration in paper segments was determined by two different methods of protein estimation. Curves were obtained showing the same five major peaks for normal serum as found by the classical methods of free electrophoresis. Comparisons were made of the areas of the various components under the curves obtained with the different methods. Two dimensional electrophoresis was applied to serum and serum components. It proved of value in demonstrating the heterogeneity of fractions such as the gamma-globulin of serum. The polysaccharide dextran was used as an index of the extent of electro-osmotic flow during the course of the various experiments. The ratio of the distance of electroosmotic flow and the distance of protein migration was shown to be constant for a given type of paper. For serum albumin on Munktell 20 paper this ratio was 0.35. A formula for mobilities applicable to liquid in a highly porous supporting medium is presented. Mobility values for human serum albumin at various pH levels on paper showed approximate agreement with those obtained in free solution giving a similar isoelectric point.

Formaldehyde (HCHO) produces DNA-protein crosslinks both in vitro and in vivo. Simian virus 40 (SV40) chromosomes that have been fixed by prolonged incubation with HCHO either in vitro or in vivo (within SV40-infected cells) can be converted to nearly protein-free DNA by limit-digestion with Pronase in the presence of NaDodSO4. The remaining Pronase-resistant DNA-peptide adducts retard the DNA upon gel electrophoresis, allowing resolution of free and crosslink-containing DNA. Though efficiently crosslinking histones to DNA within nucleosomes both in vitro and in vivo, HCHO does not crosslink either purified lac repressor to lac operator-containing DNA or an (A + T)-DNA-binding protein (alpha-protein) to its cognate DNA in vitro. Furthermore, a protein that does not bind to DNA, such as serum albumin, is not crosslinked to DNA by HCHO even at extremely high protein concentrations. These properties of HCHO as a DNA-protein crosslinker are used to probe the distribution of nucleosomes in vivo. We show that there are no HCHO-crosslinkable DNA-protein contacts in a subset of SV40 chromosomes in vivo within a 325-base-pair stretch that spans the "exposed" (nuclease-hypersensitive) region of the SV40 chromosome. This replication origin-proximal region has been found previously to lack nucleosomes in a subset of isolated SV40 chromosomes. We discuss other applications of the HCHO technique, including the possibility of obtaining base-resolution in vivo nucleosome "footprints."

BACKGROUND

End-stage liver disease is a medical problem with high morbidity and mortality. We have investigated the feasibility, safety, and efficacy of using autologous mesenchymal stem cells (MSCs) as a treatment.

METHODS

Eight patients (four hepatitis B, one hepatitis C, one alcoholic, and two cryptogenic) with end-stage liver disease having Model for End-Stage Liver Disease score>> or =10 were included. Autologous MSCs were taken from iliac crest. Approximately, 30-50 million MSCs were proliferated and injected into peripheral or the portal vein. Liver function and clinical features were evaluated at baseline and 1, 2, 4, 8, and 24 weeks after injection.

RESULTS

Treatment was well tolerated by all patients. Liver function improved as verified by the Model for End-Stage Liver Disease score, which decreased from 17.9+/-5.6 to 10.7+/-6.3 (P<0.05) and prothrombin complex from international normalized ratio 1.9+/-0.4 to 1.4+/-0.5 (P<0.05). Serum creatinine decreased from 114+/-35 to 80+/-18 micromol/l (P<0.05). Serum albumin changed from 30+/-5 to 33+/-5 g/l and bilirubin from 46+/-29 to 41+/-31 micromol/l. No adverse effects were noted.

CONCLUSIONS

Our data show that MSCs injection can be used for the treatment of end-stage liver disease with satisfactory tolerability. Furthermore, this treatment may improve clinical indices of liver function in end-stage liver disease.

Studies examining the relationship between total circulating 25-hydroxyvitamin D [25(OH)D] levels and bone mineral density (BMD) have yielded mixed results. Vitamin D-binding protein (DBP), the major carrier protein for 25(OH)D, may alter the biologic activity of circulating vitamin D. We hypothesized that free and bioavailable 25(OH)D, calculated from total 25(OH)D, DBP, and serum albumin levels, would be more strongly associated with BMD than levels of total 25(OH)D. We measured total 25(OH)D, DBP, and serum albumin levels in 49 healthy young adults enrolled in the Metabolic Abnormalities in College-Aged Students (MACS) study. Lumbar spine BMD was measured in all subjects using dual-energy X-ray absorptiometry. Clinical, diet, and laboratory information also was gathered at this time. We determined free and bioavailable (free + albumin-bound) 25(OH)D using previously validated formulas and examined their associations with BMD. BMD was not associated with total 25(OH)D levels (r = 0.172, p = .236). In contrast, free and bioavailable 25(OH)D levels were positively correlated with BMD (r = 0.413, p = .003 for free, r = 0.441, p = .002 for bioavailable). Bioavailable 25(OH)D levels remained independently associated with BMD in multivariate regression models adjusting for age, sex, body mass index, and race (p = .03). It is concluded that free and bioavailable 25(OH)D are more strongly correlated with BMD than total 25(OH)D. These findings have important implications for vitamin D supplementation in vitamin D-deficient states. Future studies should continue to explore the relationship between free and bioavailable 25(OH)D and health outcomes.

BACKGROUND

Because more than 90 percent of circulating cortisol in human serum is protein-bound, changes in the binding proteins can alter measured serum total cortisol concentrations without influencing free concentrations of this hormone. We investigated the effect of decreased amounts of cortisol-binding proteins on serum total and free cortisol concentrations during critical illness, when glucocorticoid secretion is maximally stimulated.

METHODS

Base-line serum total cortisol, cosyntropin-stimulated serum total cortisol, aldosterone, and free cortisol concentrations were measured in 66 critically ill patients and 33 healthy volunteers in groups that were similar with regard to sex and age. Of the 66 patients, 36 had hypoproteinemia (albumin concentration, 2.5 g per deciliter or less), and 30 had near-normal serum albumin concentrations (above 2.5 g per deciliter).

RESULTS

Base-line and cosyntropin-stimulated serum total cortisol concentrations were lower in the patients with hypoproteinemia than in those with near-normal serum albumin concentrations (P<0.001). However, the mean (+/-SD) base-line serum free cortisol concentrations were similar in the two groups of patients (5.1+/-4.1 and 5.2+/-3.5 microg per deciliter [140.7+/-113.1 and 143.5+/-96.6 nmol per liter]) and were several times higher than the values in controls (0.6+/-0.3 microg per deciliter [16.6+/-8.3 nmol per liter], P<0.001 for both comparisons). Cosyntropin-stimulated serum total cortisol concentrations were subnormal (18.5 microg per deciliter [510.4 nmol per liter] or less) in 14 of the patients, all of whom had hypoproteinemia. In all 66 patients, including these 14 who had hypoproteinemia, the base-line and cosyntropin-stimulated serum free cortisol concentrations were high-normal or elevated.

CONCLUSIONS

During critical illness, glucocorticoid secretion markedly increases, but the increase is not discernible when only the serum total cortisol concentration is measured. In this study, nearly 40 percent of critically ill patients with hypoproteinemia had subnormal serum total cortisol concentrations, even though their adrenal function was normal. Measuring serum free cortisol concentrations in critically ill patients with hypoproteinemia may help prevent the unnecessary use of glucocorticoid therapy.

OBJECTIVE

In phase III trials, the safety profile of triple therapy (pegylated interferon/ribavirin with boceprevir or telaprevir) seems to be similar in HCV treatment-experienced cirrhotic and non-cirrhotic patients, but few cirrhotics were included. We report the week 16 safety and efficacy analysis in a cohort of compensated cirrhotics treated in the French Early Access Programme.

METHODS

674 genotype 1 patients, prospectively included, received 48 weeks of triple therapy. The analysis is restricted to 497 patients reaching week 16.

RESULTS

A high incidence of serious adverse events (40.0%), and of death and severe complications (severe infection or hepatic decompensation) (6.4%), and a difficult management of anaemia (erythropoietin and transfusion use in 50.7% and 12.1%) were observed. Independent predictors of anaemia < 8 g/dl or blood transfusion were: female gender (OR 2.19, 95% CI 1.11-4.33, p=0.024), no lead-in phase (OR 2.25, 95% CI 1.15-4.39, p=0.018), age ≥ 65 years (OR 3.04, 95% CI 1.54-6.02, p=0.0014), haemoglobin level (≤ 12 g/dl for females, ≤ 13 g/dl for males) (OR 5.30, 95% CI 2.49-11.5, p=0.0001). Death or severe complications were related to platelets count ≤ 100,000/mm(3) (OR 3.11, 95% CI 1.30-7.41, p=0.0105) and albumin <35 g/dl (OR 6.33, 95% CI 2.66-15.07, p=0.0001), with a risk of 44.1% in patients with both. However, the on-treatment virological response was high.

CONCLUSIONS

The safety profile was poor and patients with platelet count ≤ 100,000/mm(3) and serum albumin <35 g/L should not be treated with the triple therapy.

Human neutrophils contain a neutral metalloproteinase which degrades denatured collagens and potentiates the action of interstitial collagenase. This gelatinase is rapidly secreted from neutrophils stimulated with phorbol myristate acetate. The secreted enzyme has been purified by a combination of chromatography on DEAE-cellulose and gelatin-Sepharose. The purified enzyme was latent and had a specific activity of 24,000 units. Estimated molecular weight obtained by gel filtration was 150,000-180,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed three bands with relative molecular weights of 225,000, 130,000, and 92,000. Electrophoresis in the presence of a reducing agent revealed a single band of Mr = 92,000. All the proteins seen on the unreduced gel were found to contain proteolytic activity against gelatin and native type V collagen. Polyclonal antibodies were prepared against the Mr = 130,000 and 92,000 proteins. When analyzed by immunoblotting, both antibodies recognized all three proteins. Furthermore, the identical three proteins were identified by the antibodies when crude culture medium was immunoblotted. The purified enzyme was inhibited by EDTA and 1,10-phenanthroline but not by serine or thiol proteinase inhibitors, suggesting that the enzyme is a metalloendoproteinase. The enzyme had little or no activity against common protein substrates such as bovine serum albumin or casein. Native type I collagen was not cleaved under conditions where native type V collagen was extensively degraded.

Adoptive immunotherapy with lymphokine-activated killer cells and recombinant interleukin 2 (IL 2) can produce significant reduction of visceral metastases in tumor-bearing mice and, as shown recently, in humans with disseminated cancer. Because further dose escalations of IL 2 have been prevented by the development of a vascular leak syndrome (VLS) in both mice and humans, we investigated this VLS in mice undergoing the systemic administration of high-dose IL 2. A model for quantitating capillary permeability was used in which 125I-bovine serum albumin was injected i.v., and 2 hr later, tissues were counted in a gamma analyzer. A permeability index (PI) was calculated by dividing the mean counts per minute (cpm) of tissues from IL 2-treated mice by those from control animals. The injection of IL 2 produced increases in vascular permeability that were most pronounced in the thymus, spleen, lungs, liver, and kidneys (PI = 18.0, 10.0, 9.7, 6.7, and 6.3, respectively, on day 6). The development of the VLS was highly dependent on the number of days of IL 2 treatment (for example, the lungs contained 638, 1382, 3350, and 6187 cpm after 0, 1, 3, and 6 days of IL 2, respectively). Moreover, the degree of the VLS was directly related to the dose of IL 2 administered. Measurement of the wet and dry weights of lungs from IL 2-treated mice demonstrated that IL 2 produced a dramatic increase in their water weight (from 0.10 g at base line to 0.22 g after 200,000 U of IL 2 for 6 days). The injection of the IL 2 excipient failed to induce capillary leakage in tissues. Immunosuppression of mice by pretreatment irradiation (500 rad) or by injection of cyclophosphamide or by concurrent use of cortisone acetate markedly reduced or eliminated the development of the VLS. Similarly, the VLS was not observed in nude mice receiving IL 2. Thus, the administration of IL 2 produces a dose-limiting VLS that may be mediated, directly or indirectly, by host lymphoid elements.

In vitro culturing of primary neurons is a mainstay of neurobiological research. Many of these culture paradigms have taken advantage of defined culture media rather than serum additives that contain undefined survival factors to facilitate experimental manipulations and interpretation of the results. To culture neurons in the absence of serum, defined supplements such as B27 are now widely used. However, commercially available supplements exhibit large variability in their capabilities to support neurons in culture. We re-optimized and modified earlier published formulations of B27 using 21 different ingredients (NS21). NS21 supports neuronal cultures of high quality as manifested by their morphological characteristics, formation of synapses, and postsynaptic responses. Much of the variability in the quality of B27/NS21 was due to variability in the quality of different sources of bovine serum albumin. Furthermore, we found that holo-transferrin used in NS21 is preferable over apo-transferrin used in B27 for the quality of neuronal cultures.

A neoglycoprotein (a heptasaccharide of (1-->5)-alpha-L-linked-arabinosyl residues linked to bovine serum albumin) has been used to generate a rat monoclonal antibody specific to a linear chain of (1-->5)-alpha-L-arabinan which is a structural feature of the side chains of pectins. The antibody, designated LM6, detected 100 ng of debranched sugar beet arabinan in an immunodot binding assay and 1 microgram of commercial citrus pectin in a similar assay. Hapten inhibition studies indicated that the antibody recognized 5-6 Ara residues and 50% inhibition of antibody binding in a competitive inhibition ELISA was achieved with ca. 2ng (21 nM) of (1-->5)-alpha-L-Arabinohexaose. The antibody will be useful for the localization of arabinans in plant tissue and will have uses in the analyses of pectin structure. We report here on the localization of the arabinan epitope in lemon fruits using tissue printing.

BACKGROUND

We tested the hypothesis that maternal infections during pregnancy are associated with the subsequent development of schizophrenia and other psychoses in adulthood.

METHODS

We conducted a nested case-control study of 27 adults with schizophrenia and other psychotic illnesses and 54 matched unaffected control subjects (matched for sex, ethnicity, and date of birth) from the Providence, RI, cohort of the Collaborative Perinatal Project. We retrieved stored blood samples that had been obtained from these mothers at the end of pregnancy. These samples were analyzed for total class-specific immunoglobulins and for specific antibodies directed at recognized perinatal pathogens capable of affecting brain development.

RESULTS

Maternal levels of IgG and IgM class immunoglobulins before the mothers were delivered of their neonates were significantly elevated among the case series (t = 3.06, P =.003; t = 2.93, P =.004, respectively, for IgG and IgM immunoglobulin-albumin ratios). Secondary analyses indicated a significant association between maternal antibodies to herpes simplex virus type 2 glycoprotein gG2 and subsequent psychotic illness (matched t test = 2.43, P =.02). We did not find significant differences between case and control mothers in the serum levels of IgA class immunoglobulins, or in specific IgG antibodies to herpes simplex virus type 1, cytomegalovirus, Toxoplasma gondii, rubella virus, human parvovirus B19, Chlamydia trachomatis, or human papillomavirus type 16.

CONCLUSIONS

The offspring of mothers with elevated levels of total IgG and IgM immunoglobulins and antibodies to herpes simplex virus type 2 are at increased risk for the development of schizophrenia and other psychotic illnesses in adulthood.

OBJECTIVE

Umbilical cord (UC)-derived mesenchymal stem cells (MSCs) have shown marked therapeutic effects in a number of diseases in animal studies, based on their potential for self-renewal and differentiation. No data are available on the effectiveness of UC MSC transplantation (MSCT) in human autoimmune disease. This study was undertaken to assess the efficacy and safety of allogeneic UC MSCT in patients with severe and treatment-refractory systemic lupus erythematosus (SLE).

METHODS

We conducted a single-arm trial that involved 16 SLE patients whose disease was refractory to standard treatment or who had life-threatening visceral involvement. All of the patients gave consent and underwent UC MSCT. Clinical changes were evaluated before and after transplantation using the SLE Disease Activity Index (SLEDAI), measurement of serum antinuclear antibody (ANA), anti-double-stranded DNA (anti-dsDNA) antibody, serum complement C3 and C4, and albumin levels, and assessment of and renal function. Evaluation of potential mechanisms of MSCT effects focused on the percentage of peripheral blood Treg cells and serum levels of cytokines.

RESULTS

From April 2007 to July 2009, a total of 16 patients with active SLE were enrolled and underwent UC MSCT. The median followup time after MSCT was 8.25 months (range 3-28 months). Significant improvements in the SLEDAI score, levels of serum ANA, anti-dsDNA antibody, serum albumin, and complement C3, and renal function were observed. Clinical remission was accompanied by an increase in peripheral Treg cells and a re-established balance between Th1- and Th2-related cytokines. Significant reduction in disease activity was achieved in all patients, and there has been no recurrence to date and no treatment-related deaths.

CONCLUSIONS

Our findings indicate that UC MSCT results in amelioration of disease activity, serologic changes, and stabilization of proinflammatory cytokines. These data provide a foundation for conducting a randomized controlled trial of this new therapy for severe and treatment-refractory SLE.

Uric acid may mediate aspects of the relationship between hypertension and kidney disease via renal vasoconstriction and systemic hypertension. To investigate the relationship between uric acid and subsequent reduced kidney function, limited-access data of 13,338 participants with intact kidney function in two community-based cohorts, the Atherosclerosis Risks in Communities and the Cardiovascular Health Study, were pooled. Mean baseline serum uric acid was 5.9 +/- 1.5 mg/dl, mean baseline serum creatinine was 0.9 +/- 0.2 mg/dl, and mean baseline estimated GFR was 90.4 +/- 19.4 ml/min/1.73 m(2). During 8.5 +/- 0.9 yr of follow-up, 712 (5.6%) had incident kidney disease defined by GFR decrease >>or=15 ml/min/1.73 m(2) with final GFR <60 ml/min/1.73 m(2)), while 302 (2.3%) individuals had incident kidney disease defined by creatinine increase >>or=0.4 mg/dl with final serum creatinine >1.4 mg/dl in men and 1.2 mg/dl in women). In GFR- and creatinine-based logistic regression models, baseline uric acid level was associated with increased risk for incident kidney disease (odds ratio 1.07 [95% confidence interval 1.01 to 1.14] and 1.11 [95% confidence interval 1.02 to 1.21] per 1-mg/dl increase in uric acid, respectively), after adjustment for age, gender, race, diabetes, systolic BP, hypertension, cardiovascular disease, left ventricular hypertrophy, smoking, alcohol use, education, lipids, albumin, hematocrit, baseline kidney function and cohort; therefore, elevated serum uric acid level is a modest, independent risk factor for incident kidney disease in the general population.

Antisera were raised against gamma-aminobutyric acid (GABA) or glutamate (Glu) conjugated to bovine serum albumin with glutaraldehyde. After purification, these antisera reacted strongly with fixed GABA or Glu, but not significantly with other amino acids fixed with glutaraldehyde to brain macromolecules. The antisera were used to demonstrate the distributions of Glu-like and GABA-like immunoreactivities (Glu-LI and GABA-LI) in parts of the perfusion-fixed mouse and rat brain, including the olfactory bulb, cerebral neocortex, thalamus, basal ganglia, lower brain stem, and cerebellum. The level of GABA-LI varied widely among brain regions, thus it was very high in the globus pallidus and substantia nigra and low in the bulk of the thalamus. The GABA antisera labeled nonpyramidal neurons of the neocortex, most cells of the reticular nucleus of the thalamus, medium-sized cells of the caudatoputamen, and stellate, basket, Golgi, and Purkinje cells of the cerebellum. The distribution of GABA-LI closely matched that of the GABA-synthesizing enzyme, glutamic acid decarboxylase (GAD), as revealed in immunocytochemical studies by others. However, the GABA antisera seem to be better suited than GAD antisera for demonstrating putative GABA-ergic axons. The results suggest that GABA-LI, as displayed by the present method, is a good marker of neurons thought to use GABA as a transmitter. Glutamate-like immunoreactivity was much more evenly distributed among regions than GABA-LI, but was particularly low in globus pallidus and substantia nigra and high in the cerebral cortex. Mitral cells of the olfactory bulb, pyramidal neocortical cells, and other cells assumed to use Glu or aspartate as transmitter were stained for Glu-LI, but so also were neurons that are thought to use other transmitters, such as cells in the substantia nigra pars compacta, in the dorsal raphe nucleus, and in the brain stem motor nuclei. The Glu antisera seem to reveal the "transmitter pool" as well as the "metabolic pool" of Glu in perfusion-fixed material. This report shows that it is possible by means of immunocytochemistry to display reliably the tissue contents of GABA and Glu in material that has been fixed by perfusion with glutaraldehyde.

OBJECTIVE

Adefovir dipivoxil effectively inhibits both hepatitis B virus (HBV) replication and disease activity in patients with chronic hepatitis B. Resistance to treatment was not observed in 2 recent large placebo-controlled 48-week studies with this drug. The aim of this study was to characterize adefovir resistance in a patient who developed clinical and virologic evidence of breakthrough during a 96-week course of treatment.

METHODS

HBV DNA was PCR amplified and sequenced. Phenotypic studies used patient-derived HBV as well as specific mutations created by site-directed mutagenesis of a HBV/baculovirus recombinant.

RESULTS

Following the commencement of treatment with adefovir dipivoxil, the patient initially responded with a 2.4 log(10) decrease in serum HBV DNA and normalization of alanine aminotransaminase levels by week 16. During the second year of treatment, however, serum HBV DNA rose progressively, eventually returning to near-pretreatment levels. This increase in viral replication was associated with a marked increase in alanine aminotransferase and mild changes in bilirubin, albumin, and prothrombin time. Comparison of pretreatment and posttreatment HBV DNA by polymerase chain reaction sequencing identified a novel asparagine to threonine mutation at residue rt236 in domain D of the HBV polymerase. In vitro testing of a laboratory strain encoding the rtN236T mutation and testing of patient-derived virus confirmed that the rtN236T substitution caused a marked reduction in susceptibility to adefovir.

CONCLUSIONS

The development of this novel mutation in the HBV polymerase confers resistance to adefovir dipivoxil. The patient responded to subsequent lamivudine therapy, achieving normalization of alanine aminotransferase and a significant decrease in serum HBV DNA.

A study of complex I (NADH:ubiquinone oxidoreductase) activity in Parkinson's disease (PD) brain has identified loss of activity only in substantia nigra although loss of activity of this enzyme has been identified in a number of non-brain tissues. We investigated this paradox by studying complex I and other complexes of the mitochondrial electron transport chain in frontal cortex from PD and aged control brain using a variety of assay conditions and tissue preparations. We found increasingly significant losses of complex I activity in PD frontal cortex as increasingly pure mitochondria were studied. Complexes II, III, and IV were comparable in PD and controls. Inclusion of bovine serum albumin in the assay increased enzyme activity but lessened discrimination between PD and controls. Complex I deficiency in PD brain is not confined to substantia nigra. Methodological issues are critical in demonstrating this loss of activity.

The intravenous administration of superoxide dismutase (superoxide:superoxide oxidoreductase, EC 1.15.1.1) to animals with induced inflammation suppresses the inflammatory response and inhibits leukocyte infiltration into the challenged site, suggesting that neutrophil-generated superoxide reacts with an extracellular precursor to generate a substance chemotactic for neutrophils. Plasma exposed to superoxide in vitro becomes potently chemotactic. The appearance of chemotactic activity is inhibited by superoxide dismutase but not by catalase. The chemotactic factor does not stimulate superoxide production or degranulation in neurtrophils. Intradermal injection of superoxide-treated plasma or of a superoxide-generating system causes heavy infiltration of neutrophils to the injection site but does not cause overt signs of inflammation. The chemotactic factor consists of a chloroform-extractable component bound to serum albumin. The superoxide-dependent chemotactic factor appears to play a major role in communication in neutrophil-mediated inflammatory events. Prevention of production of this factor appears to be the major anti-inflammatory action of superoxide dismutase.

The binding of Shiga toxin isolated from the bacterium Shigella dysenteriae type 1 to a series of glycolipids and to cells or cell homogenates has been studied. Bound toxin was detected using either 125I-labeled toxin or specific monoclonal antibody and 125I-labeled anti-antibody. Overlay of toxin on thin-layer chromatograms with separated glycolipids and binding to glycolipids coated in microtiter wells established that the toxin specifically bound to Gal alpha 1-4Gal beta (galabiose) placed terminally or internally in the oligosaccharide chain. No glycolipid shown to lack this sequence binds the toxin. Most of the glycolipids with internally placed galabiose were not active, indicating a sterical hindrance for toxin access to the binding epitope. Binding of toxin to HeLa cells in monolayers could be inhibited by preincubation of the toxin with galabiose covalently linked to bovine serum albumin (BSA), but not with free oligosaccharides containing galabiose or with lactose coupled to BSA. This demonstrated that the inhibition is specifically dependent on galabiose and requires multivalency of the disaccharide to be efficient. The inhibitory effect was successively enhanced by increasing the substitution on BSA (7, 18, and 25 mol of galabiose/mol of BSA). The BSA-coupled galabiose could also prevent the cytotoxic effect on HeLa cells (detachment of killed cells). There are cell lines with a dense number of receptor sites, but which are resistant to toxin action (uptake and inhibition of protein synthesis) which may suggest two types of receptor substances which are functionally different and unevenly expressed. In analogy with the mechanism earlier formulated for cholera toxin, we propose glycolipid-bound, bilayer-close galabiose as the functional receptor for membrane penetration of the toxin, while galabiose bound in glycoproteins affords binding sites but is not able to mediate penetration.

Coated microneedles have been shown to deliver proteins and DNA into the skin in a minimally invasive manner. However, detailed studies examining coating methods and their breadth of applicability are lacking. This study's goal was to develop a simple, versatile and controlled microneedle coating process to make uniform coatings on microneedles and establish the breadth of molecules and particles that can be coated onto microneedles. First, microneedles were fabricated from stainless steel sheets as single microneedles or arrays of microneedles. Next, a novel micron-scale dip-coating process and a GRAS coating formulation were designed to reliably produce uniform coatings on both individual and arrays of microneedles. This process was used to coat compounds including calcein, vitamin B, bovine serum albumin and plasmid DNA. Modified vaccinia virus and microparticles of 1 to 20 micro m diameter were also coated. Coatings could be localized just to the needle shafts and formulated to dissolve within 20 s in porcine cadaver skin. Histological examination validated that microneedle coatings were delivered into the skin and did not wipe off during insertion. In conclusion, this study presents a simple, versatile, and controllable method to coat microneedles with proteins, DNA, viruses and microparticles for rapid delivery into the skin.

Quinolinic acid is an "excitotoxic" metabolite and an agonist of N-methyl-D-aspartate receptors. Of patients infected with human immunodeficiency virus type 1 (HIV-1) who were neurologically normal or exhibited only equivocal and subclinical signs of the acquired immunodeficiency syndrome (AIDS) dementia complex, concentrations of quinolinic acid in cerebrospinal fluid (CSF) were increased twofold in patients in the early stages of disease (Walter Reed stages 1 and 2) and averaged 3.8 times above normal in later-stage patients (Walter Reed stages 4 through 6). However, in patients with either clinically overt AIDS dementia complex, aseptic meningitis, opportunistic infections, or neoplasms, CSF levels were elevated over 20-fold and generally paralleled the severity of cognitive and motor dysfunction. CSF concentrations of quinolinic acid were significantly correlated to the severity of the neuropsychological deficits. After treatment of AIDS dementia complex with zidovudine and treatment of the opportunistic infections with specific antimicrobial therapies, CSF levels of quinolinic acid decreased in parallel with clinical neurological improvement. By analysis of the relationship between levels of quinolinic acid in the CSF and serum and integrity of the blood-brain barrier, as measured by the CSF:serum albumin ratio, it appears that CSF levels of quinolinic acid may be derived predominantly from intracerebral sources and perhaps from the serum. While quinolinic acid may be another "marker" of host- and virus-mediated events in the brain, the established excitotoxic effects of quinolinic acid and the magnitude of the increases in CSF levels of the acid raise the possibility that quinolinic acid plays a direct role in the pathogenesis of brain dysfunction associated with HIV-1 infection.

OBJECTIVE

This study aimed to evaluate the relationship between anemia and heart failure (HF) prognosis.

BACKGROUND

Although it is known that chronic diseases, including HF, may be associated with anemia, the impact of hemoglobin (Hb) level on symptoms and survival in HF has not been fully defined. We analyzed a cohort of 1,061 patients with advanced HF (New York Heart Association [NYHA] functional class III or IV and left ventricular ejection fraction [LVEF] <40%) referred to a single center for evaluation and management. The Hb level was drawn at time of initial evaluation. Patients were divided into quartiles of Hb: Hb <12.3; Hb 12.3 to 13.6; Hb 13.7 to 14.8; Hb >14.8 g/dl.

RESULTS

Mean Hb was 13.6, and values ranged from 7.1 to 19.0 g/dl. The Hb groups were similar in age, medication profile, LVEF, hypertension, diabetes, smoking status and serum sodium. Lower Hb was associated with an impaired hemodynamic profile, higher blood urea nitrogen and creatinine, and lower albumin, total cholesterol and body mass index. Patients in the lower Hb quartiles were more likely to be NYHA functional class IV (p < 0.0001) and have lower peak oxygen consumption (PKVO(2)) (p < 0.0001). Survival at one year was higher with increased Hb quartile (55.6%, 63.9%, 71.4% and 74.4% for quartiles 1, 2, 3 and 4, respectively). On multivariate analysis adjusting for known HF prognostic factors, low Hb proved to be an independent predictor of mortality (relative risk 1.131, confidence interval 1.045 to 1.224 for each decrease of 1 g/dl).

CONCLUSIONS

In chronic HF, relatively mild degrees of anemia are associated with worsened symptoms, functional status and survival.

A Shiga-like toxin type II variant (SLT-IIv) is produced by strains of Escherichia coli responsible for edema disease of swine and is antigenically related to Shiga-like toxin type II (SLT-II) of enterohemorrhagic E. coli. However, SLT-IIv is only active against Vero cells, whereas SLT-II is active against both Vero and HeLa cells. The structural genes for SLT-IIv were cloned from E. coli S1191, and the nucleotide sequence was determined and compared with those of other members of the Shiga toxin family. The A subunit genes for SLT-IIv and SLT-II were highly homologous (94%), whereas the B subunit genes were less homologous (79%). The SLT-IIv genes were more distantly related (55 to 60% overall homology) to the genes for Shiga toxin of Shigella dysenteriae type 1 and the nearly identical Shiga-like toxin type I (SLT-I) of enterohemorrhagic E. coli. (These toxins are referred to together as Shiga toxin/SLT-I.) The A subunit of SLT-IIv, like those of other members of this toxin family, had regions of homology with the plant lectin ricin. SLT-IIv did not bind to galactose-alpha 1-4-galactose conjugated to bovine serum albumin, which is an analog of the eucaryotic cell receptor for Shiga toxin/SLT-I and SLT-II. These findings support the hypothesis that SLT-IIv binds to a different cellular receptor than do other members of the Shiga toxin family but has a similar mode of intracellular action. The organization of the SLT-IIv operon was similar to that of other members of the Shiga toxin family. Iron did not suppress SLT-IIv or SLT-II production, in contrast with its effect on Shiga toxin/SLT-I. Therefore, the regulation of synthesis of SLT-IIv and SLT-II differs from that of Shiga toxin/SLT-I.

Albumin, transferrin, and lipids can replace serum entirely for support of LPS-stimulated murine B lymphocytes in culture. In the presence of these compounds, growth and maturation to IgM and IgG secretion, induced by lipopolysaccharide (LPS), occurs at the same or higher efficiency in serum-free conditions as in conventional serum-containing medium, even at relatively low cell concentrations. In contrast to the rapid disappearance of LPS reactivity in conventional serum-containing medium, responsiveness remains at initial levels in serum-free conditions for 2 days before slowly declining. Overall lymphocyte survival is also markedly prolonged. In the presence of thymus "filler" cells, the serum-free conditions permit growth of every LPS-responsive cell to a clone of Ig-secreting cells at dilutions as low as a single reactive B cell per culture. The results have several important implications. These include the establishment for the first time of transferrin as a requirement for B lymphocyte responses in culture, and the availability now of conditions for the assay isolation of cell products regulating lymphocyte function, free of interference from undefined serum components.