Safety assessment of a modified acetolactate synthase protein (GM-HRA) used as a selectable marker in genetically modified soybeans.

Acetolactate synthase (ALS) enzymes have been isolated from numerous organisms including soybeans (Glycine max; GM-ALS) and catalyze the first common step in biosynthesis of branched chain amino acids. Expression of an ALS protein (GM-HRA) with two amino acid changes relative to native GM-ALS protein in genetically modified soybeans confers tolerance to herbicidal active ingredients and can be used as a selectable transformation marker. The safety assessment of the GM-HRA protein is discussed. Bioinformatics comparison of the amino acid sequence did not identify similarities to known allergenic or toxic proteins. In vitro studies demonstrated rapid degradation in simulated gastric fluid (<30s) and intestinal fluid (<1min). The enzymatic activity was completely inactivated at 50 degrees C for 15 min demonstrating heat lability. The protein expressed in planta is not glycosylated and genetically modified soybeans expressing the GM-HRA protein produced similar protein/allergen profiles as its non-transgenic parental isoline. No adverse effects were observed in mice following acute oral exposure at a dose of at least 436 mg/kg of body weight or in a 28-day repeated dose dietary toxicity study at doses up to 1247 mg/kg of body weight/day. The results demonstrate GM-HRA protein safety when used in agricultural biotechnology.

Emergency department utilisation for inflammatory bowel disease in the United States from 2006 to 2014.

BACKGROUND: Despite advances in treatment, patients with inflammatory bowel disease (IBD) frequently require emergency department (ED) visits and hospitalisations. AIMS: To analyse trends in ED visits and subsequent hospitalisations for IBD in the United States (US). METHODS: Data were analysed from the Nationwide Emergency Department Sample (NEDS) years 2006-2014. The NEDS is the largest all-payer ED database in the US, weighted to represent 135 million visits/year. IBD was identified using ICD-9 codes for Crohn's disease (CD) or ulcerative colitis (UC). Surgeries were identified using procedure codes. RESULTS: The frequency of IBD-ED visits increased 51.8%, from 90 846 visits in 2006 to 137 946 in 2014, which was statistically significant in linear regression. For comparison, all-case ED use between 2006 and 2014 increased 14.8%. In-patient hospitalisations from the ED decreased 12.1% for IBD (from 64.7% rate of hospitalisation from the ED in 2006 to 52.6% in 2014), with a UC:CD ratio of 1.2:1 in 2006 and 1.3:1 in 2014. Chi-square analysis revealed that this was a significant decrease. Surgery rates also showed a statistically significant decrease. The mean ED charge per patient rose 102.5% and the aggregate national cost of IBD-ED visits increased 207.5%. CD accounted for over twice as many visits as UC in both years. UC, age, male gender, highest income quartile, private insurance, Medicaid/Medicare, and tobacco use were associated with in-patient admissions. CONCLUSIONS: The number of ED visits due to IBD and associated charges have continued to rise, while the rates of in-patient hospitalisations referred from the ED and surgeries have decreased.

The impact of irritable bowel syndrome on daily functioning: Characterizing and understanding daily consequences of IBS.

BACKGROUND: Despite the well-documented economic and psychosocial burden of irritable bowel syndrome (IBS), few studies have focused on the impact of IBS on daily activities. This study aims to quantitate impairment in daily activities among IBS patients and to evaluate the relationship between impairment, IBS, quality of life, and psychiatric symptoms. METHODS: A total of 179 participants meeting ROME-III criteria for IBS completed an online research survey evaluating the following variables: (i) the impact of IBS on daily activities, (ii) comorbid psychiatric diagnoses, (iii) symptom severity, (iv) quality of life, and (v) symptom-specific cognitive affective factors related to IBS. KEY RESULTS: This sample reported a high degree of impairment due to IBS, with 76% of the sample reporting some degree of IBS-related impairment in at least five different domains of daily life. Rates of impairment were significantly higher for participants who met criteria for anxiety, depression, and/or panic disorder. CONCLUSIONS & INFERENCES: This study contributes to existing literature by demonstrating a high level of daily impairment among patients with IBS, particularly those who meet criteria for anxiety, depression, and panic disorder. These findings support the importance of integrated psychosocial and medical care for IBS patients, and highlight the utility of evaluation and intervention for behavioral avoidance/impairment especially among those who exhibit signs or symptoms of psychiatric diagnoses.

Case report: comparative response of fever to corticosteroids in tuberculosis and in connective tissue disease.

A young woman with fever and pleural effusion eventually was found to have both primary tuberculosis and systemic lupus erythematosus. Before either diagnosis was confirmed, the patient had a dramatic defervescence in response to prednisone; her condition had not improved initially after anti-tuberculous therapy. Uncertainty regarding the clinical significance of this remarkable temperature response led us to review our experience with febrile tuberculous patients who had received corticosteroids. Of 14 such patients, nine had rapid declines of body temperature to below 36.5C. In a comparable group of 14 patients with a variety of connective tissue disease, 12 showed an equally dramatic defervescence after corticosteroid administration. The data suggest that a prompt temperature decline following corticosteroid therapy is a nonspecific response that cannot be used to differentiate between fever of infectious or noninfectious origin.

Chronic inflammation in older people: recognition, consequences, and potential intervention.

In elderly individuals, some chronic inflammatory diseases appear to occur with increased frequency, and recent evidence suggests that some very common chronic, age-related disorders may be propagated and perpetuated by inflammatory processes, perhaps giving rise to the abnormal acute phase protein levels that are seen with increased frequency with aging. The consequences of chronic inflammation in the elderly undoubtedly contribute to excessive morbidity in this population. Treatment of chronic inflammation in the elderly is often difficult, requiring utmost care and close follow-up by a knowledgeable and dedicated physician.

Clinical aspects of antibodies to DNA.

The detection of antibodies to DNA is one of the most important laboratory tests in rheumatology and immunology from both scientific and clinical points of view. The most useful methods for detection of anti-DNA are the liquid phase radioimmunoassay (Farr assay), solid phase enzyme-linked assays (ELISA) and immunofluorescence (Crithidia Luciliae). The clinical value of detection of anti-DNA can be summarized as follows: (a) Antibodies to DNA (particularly those reactive primarily with double-stranded DNA determinants) are highly specific for the disease SLE; (b) Levels of anti-DNA bear a close relation to disease activity in many patients. Rapidly rising levels are frequently associated with a subsequent exacerbation and clinical improvement is often accompanied by declining levels of anti-DNA. However, a minority of patients may have persistent elevations of anti-DNA for extended periods in the absence of overt clinical disease activity; (c) Although a single determination of anti-DNA has little prognostic value, the persistent presence of high levels or the absence of anti-DNA may define patient subsets with poor and good prognoses respectively; (d) Antibodies to single stranded DNA although present in some patients with discoid lupus and "ANA-negative" lupus, have little diagnostic specificity and are less valuable for disease follow-up as compared with antibodies to double-stranded DNA.

SRM 2460/2461 Standard Bullets and Casings Project.

The National Institute of Standards and Technology Standard Reference Material (SRM) 2460/2461 standard bullets and casings project will provide support to firearms examiners and to the National Integrated Ballistics Information Network (NIBIN) in the United States. The SRM bullet is designed as both a virtual and a physical bullet profile signature standard. The virtual standard is a set of six digitized bullet profile signatures originally traced from six master bullets fired at the Bureau of Alcohol, Tobacco and Firearms (ATF) and the Federal Bureau of Investigation (FBI). By using the virtual signature standard to control the tool path on a numerically controlled diamond turning machine, 40 SRM bullets were produced. A profile signature measurement system was established for the SRM bullets. The profile signature differences are quantified by the maximum of the cross correlation function and by the signature difference between pairs of compared profile signatures measured on different SRM bullets. Initial measurement results showed high reproducibility for both the measurement system and production process of the SRM bullets. A traceability scheme has been proposed to establish the measurement traceability for nationwide bullet signature measurements to NIST, ATF and FBI. Prototype SRM casings have also been developed.

Moves of murder.

A missing person report was filed with the local Police Department. In the ensuing days it became apparent this was not just a missing person case when a bloody pillow and pillowcase were recovered from a wooded area not far from the missing woman's home. Careful examination of the pillowcase revealed fragments of a bloody fingerprint and bloody patterns that seemed to indicate the type of weapon used. A thorough search of the woman's bedroom resulted in the recovery of three small blood stains, a wig fiber and a head hair. Although the body of the victim was not recovered during the investigation, the evidence collected and techniques used by the forensic investigators resulted in a guilty plea to second degree murder.

Systemic lupus erythematosus. Controversies in management.

At the core of the controversy surrounding the management of systemic lupus erythematosus are the two issues of when to treat and what treatment to use. On the basis of a review of the recent medical literature, the following conclusions can be drawn: Patients with isolated serologic or histologic renal abnormalities in the absence of clinical disease activity probably should not be treated. Such abnormalities primarily serve to indicate the need for close follow-up and to heighten the physician's concern about the possible development of clinical symptoms. For those patients with systemic manifestations who require corticosteroids, a regimen of single daily doses is appropriate. The dose should be tapered as rapidly as the degree of symptomatic control allows; a switch to alternate-day therapy can be considered as symptoms become quiescent. Intravenous methylprednisolone therapy may be used for patients with very severe systemic disease, particularly acute nephritis. In addition, use of immunosuppressive agents should be considered for all patients with clinically serious renal disease.

The National Ballistics Imaging Comparison (NBIC) project.

In response to the guidelines issued by the American Society of Crime Laboratory Directors/Laboratory Accreditation Board (ASCLD/LAB-International) to establish traceability and quality assurance in U.S. crime laboratories, a NIST/ATF joint project entitled National Ballistics Imaging Comparison (NBIC) was initialized in 2008. The NBIC project aims to establish a National Traceability and Quality System for ballistics identifications in crime laboratories within the National Integrated Ballistics Information Network (NIBIN) of the U.S. NIST Standard Reference Material (SRM) 2460 bullets and 2461 cartridge cases are used as reference standards. 19 ballistics examiners from 13 U.S. crime laboratories participated in this project. They each performed 24 periodic image acquisitions and correlations of the SRM bullets and cartridge cases over the course of a year, but one examiner only participated in Phase 1 tests of SRM cartridge case. The correlation scores were collected by NIST for statistical analyses, from which control charts and control limits were developed for the proposed Quality System and for promoting future assessments and accreditations for firearm evidence in U.S. forensic laboratories in accordance with the ISO 17025 Standard.

Binding of human C-reactive protein to monocytes: analysis by flow cytometry.

An opsonic role has been proposed as a major function of C-reactive protein (CRP) in humans. In support of this hypothesis, recent radiolabelled ligand binding studies have provided evidence for the presence of specific receptors for soluble human CRP on human phagocytic cells, including neutrophils and monocytes. In order to confirm specific binding of CRP to monocytes and to quantify the percentage of such cells capable of expressing binding sites, we employed a sensitive biotin-avidin fluorescence assay to study the CRP-monocyte interaction. It was observed that 67% of monocytes bound biotinylated CRP in a dose-dependent manner, that the binding was calcium dependent, and that it could be inhibited by 60% in the presence of a greater than 20-fold excess of competing native CRP. In other experiments, neither IgG nor heat-aggregated IgG inhibited the binding of CRP to monocytes; and no significant binding to lymphocyte population could be detected. These studies confirm the ability of human CRP to bind to a majority of human monocytes in a calcium-dependent and specific manner, and provide further support for a biologically important interaction of this acute-phase protein with phagocytic cells.

Induction of inflammatory cytokine release from cultured human monocytes by C-reactive protein.

The human acute phase protein, C-reactive protein (CRP), is capable of specifically binding to and modulating the function of mononuclear phagocytes. To investigate whether CRP can also affect the capacity of these cells to produce inflammatory cytokines, enzyme immunoassays and Western blot techniques were used to quantitate interleukin 1 beta (IL-1 beta), interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-alpha) produced by freshly-isolated normal human monocytes. CRP induced the rapid release of each cytokine, with significantly elevated levels in culture supernatants at 4 hours and maximal levels of TNF-alpha at 8 hours, and of IL-1 beta and IL-6 at 16 hours of culture. The effects of CRP were dose-dependent; greater than 10-fold increases of each cytokine were observed following culture with greater than or equal to 50 micrograms/ml CRP, concentrations which are often found in the presence of moderate to severe inflammation or tissue injury. The induction of cytokine release by CRP was unaffected by inclusion of 25 micrograms/ml polymyxin-B in culture media, but was completely abrogated by prior boiling of the CRP, a procedure which had no effect on induction of monocyte cytokine release by lipopolysaccharide. The dose-dependent induction of inflammatory cytokines by CRP provides further support for the hypothesis that interaction with mononuclear phagocytes constitutes an important biological role for this acute phase protein.

Anti-native DNA detection by the Crithidia luciliae method: an improved guide to the diagnosis and clinical management of systemic lupus erythematosus.

The clinical value of the Crithidia luciliae (CL) method for detection of antibodies to native DNA (nDNA) was assessed. Significant titers were limited almost exclusively to patients with active systemic lupus erythematosus (SLE). Evaluation of sera from patients at the onset of active lupus demonstrated elevated anti-nDNA levels in 80% of subjects with active disease and in 94% of patients with clinically evident lupus nephritis. In longitudinal studies, rising titers of anti-nDNA were invariably accompanied by exacerbation of lupus activity. These findings suggest that the CL method correlates more closely with active SLE than do other anti-DNA methods in common use and indicate that it will prove highly useful in the diagnosis and management of SLE.

Immunochemical characteristics of antibodies to DNA in patients with active systemic lupus erythematosus.

To investigate the suggestion that qualitative immunochemical characteristics of antibodies to DNA (anti-DNA) may be of importance in the pathogenesis of nephritis in systemic lupus erythematosus (SLE), we used the Crithidia luciliae (CL) immunofluorescence test to determine the titre, immunoglobulin (Ig) class and complement-fixing activity of anti-DNA in thirty-five patients with active SLE. Eighteen of these patients had active lupus nephritis (Group I) and the remaining seventeen had no clinical evidence of renal involvement (Group II). Anti-DNA was detected in twenty-eight patients, and was present more frequently and in higher titre (P less than 0.01) in Group I than in Group II. Anti-DNA of all three Ig classes studied (IgG, IgM and IgA) was present in twenty-three out of twenty-eight cases. The ratio of IgG to IgM anti-DNA did not differ in the two groups of patients. Complement-fixing antibodies were detected in thirteen patients in Group I and five patients in Group II. The titre of complement-fixing activity was strongly correlated with titre of anti-DNA. DNA-binding capacity was also determined in these by a millipore filter (MF) assay. A highly significant correlation between DNA binding by MF and CL was found in Group I patients, while no correlation was found in Group II patients. These findings suggest that (1) anti-DNA with specificity for determinants found in CL, presumably native DNA, are more highly correlated with the presence of active renal lupus than are antibodies directed toward other DNA determinants, and (2) the major characteristic of anti-DNA found to be associated with nephritis was quantity of antibody. Most patients had anti-DNA of all Ig classes regardless of the presence of renal disease. Complement-fixing activity of anti-DNA could not be related to the occurrence of renal disease independently of anti-DNA titre.

Tendon rupture in systemic lupus erythematosus.

A patient with systemic lupus erythematosus (SLE) and spontaneous rupture of the Achilles tendon is reported. Tendon rupture in SLE is extremely uncommon; review of the reported cases suggests that it may be more common in males, and is a late complication of SLE, more likely to occur in older patients. The possible role of corticosteroid therapy is discussed.

Absence of a binding reactivity of human C-reactive protein for immunoglobulin or immune complexes.

Because C-reactive protein (CRP) has been identified as a component of circulating immune complexes from patients with inflammatory diseases, we sought to evaluate a potentially clinically important interaction of this acute-phase protein with immunoglobulin or experimentally-prepared immune complexes in vitro. Highly purified human CRP was incubated with a variety of immunoglobulin substrates, including monomeric immunoglobulin G1 (IgG1), a polyclonal IgG, heat-aggregated IgG, and human serum albumin/anti-serum albumin complexes. We were unable to detect a significant binding interaction of radioiodinated CRP with any of these materials, using either polyethylene glycol (PEG) precipitation or sucrose density gradient ultracentrifugation. In contrast, binding of radioiodinated human C1q to both aggregated immunoglobulin and immune complexes was readily detected by these techniques. Incubation of radiolabeled CRP with serum samples from 22 patients with active inflammatory diseases and high levels of circulating immune complexes disclosed no difference in the amount of PEG-precipitable CRP when compared with serum samples from healthy individuals. However, a radiolabeled commercial preparation of CRP did result in some PEG-precipitable radioactivity after incubation with aggregated IgG. These findings provide no support for a biologically important binding interaction of CRP with immunoglobulin or immune complexes, and they suggest that highly purified preparations of CRP should be used in functional studies of this acute-phase protein.

Specific binding of human C-reactive protein to human monocytes in vitro.

The precise biologic function of C-reactive protein (CRP), a major acute phase protein in man, is unknown. The abilities of CRP to bind biologic substrates and to activate the C pathway, and its localization at sites of inflammation argue for an opsonic role for this protein. Such a role has been supported by recent reports of specific binding of CRP to neutrophils. Using highly purified radioiodinated human CRP, we have observed specific binding of this protein to human monocytes in vitro. The binding was reversible and rapid, with a t1/2 for the dissociation reaction of approximately 3 min. Binding was saturable at a CRP concentration of approximately 0.2 microM, with an estimated K from Scatchard analysis of 1.1 x 10(-7) M. Specific binding was calcium-dependent, with optimal binding occurring at calcium concentrations of more than 1.0 mM. No specific binding could be demonstrated to a non-adherent population of mononuclear cells (more than 80% lymphocytes). In other experiments, a 100-fold excess of human IgG failed to inhibit binding, although rabbit CRP produced competitive inhibition of binding which was quantitatively similar to human CRP. The binding was maximal at pH 7.4 and was sensitive to prior trypsin treatment of cells. These studies provide direct evidence for specific binding of soluble human CRP to human monocytes in vitro and thus provide further support for an important functional interaction of this acute phase protein with phagocytic cells in man.

Effect of flurbiprofen on cytokine production by human monocytes and U-937 and THP-1 cell lines.

Possible effects of nonsteroidal antiinflammatory drugs (NSAID) on inflammatory mediators other than arachidonic acid metabolites which might contribute to the antiinflammatory effects of these drugs have not been fully explored. We investigated the effects of an NSAID, flurbiprofen, on production of the cytokines tumor necrosis factor alpha (TNF alpha), interleukin 1 beta (IL-1 beta) and interleukin 6 (IL-6) by human peripheral blood monocytes and by the human cell lines U-937 and THP-1. Cytokine production was induced by 1 microgram/ml bacterial lipopolysaccharide (LPS) in both monocytes and cell lines, and cytokine levels in supernatants were measured by enzyme immunoassay. In monocytes, IL-6 was the major product while in both cell lines, TNF alpha was the major product. Flurbiprofen caused moderate inhibition of IL-1 beta and TNF alpha production by stimulated monocytes, but did not affect IL-6 production. In contrast, flurbiprofen completely abolished IL-6 production by both cell lines and substantially inhibited IL-1 beta and TNF alpha production. These observations raise the possibility that inhibition of cytokine production by flurbiprofen may contribute to the antiinflammatory properties of this drug.