Multicenter study to assess presenteeism in systemic lupus erythematosus and its relationship with clinical and sociodemographic features.

Objective The aim of this study was to measure presenteeism (productivity impairment while the patient is at work) and the related risk factors in patients with systemic lupus erythematosus (SLE) from Argentina. Methods A total of 130 consecutive (1997 American College of Rheumatology (ACR) criteria) working patients with SLE were assessed using a standardized data collection form. Sociodemographic, disease and work-related variables were collected. The Work Productivity and Activity Impairment (WPAI) questionnaire was performed. Results Overall, 130 patients were included in the analysis; 91% were women, and the mean age was 39 years (range 19-77). A total of 43% were White, 43% Mestizo and 13% Amerindian. Overall, 38% were single and 38% were married. A total of 75% had more than 12 years of formal education. The median disease duration was 7 years (interquartile range 25-75 (IQR) 4-13). Median Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) score was 0 (IQR 0-2), and median Systemic Lupus International Collaborating Clinics/ACR Damage Index (SLICC-SDI) score was 0 (IQR 0-1). Lupus quality of life (LupusQoL) domains scores were: physical health 87 (IQR 70-96), emotional health 78 (IQR 54-91), burden to others 75 (IQR 50-92), intimate relationships 87 (IQR 50-100), and body image 85 (IQR 70-100). Absenteeism was 8%, presenteeism was 19%, and overall work impairment (absenteeism + presenteeism) was 26%. In the multiple regression analysis, considering presenteeism as dependent variable, (adjusting by age, disease duration, >12 years of education, Non-white race, Visual Analogue Scale (VAS) pain, VAS fatigue, SLICC-SDI, LupusQoL, physical and emotional domains), we found that SLICC-SDI (odds ratio (OR) 1.68, confidence interval (CI) 1-2.7) and Non-white race (OR 3.27, CI 1.04-10) were related to presenteeism and >12 years of education (OR 0.30, CI 0.09-0.98) and higher scores of LupusQoL emotional health domain (OR 0.95, CI 0.92-0.98) were protective. Conclusions organ damage and Non-white race were significantly associated with presenteeism while >12 years of education and higher scores of LupusQoL emotional health domain were protective.

A pilot study of a new assessment of physical activity in eating disorder patients.

BACKGROUND/AIM: In order to get more detailed information about exercise regimes and disturbances among patients with eating disorders, a new self report questionnaire was developed. The Exercise and Eating Disorders (EED) was developed to capture aspects not included in existing questionnaires. The aim of this study was to test the internal consistency and concurrent validity of the EED, and to investigate to what extent the questionnaire discriminates between inpatients and controls. METHOD: Fifty female eating disorder patients (anorexia nervosa n=25, bulimia nervosa n=10, EDNOS n=15) in a specialized inpatient unit and 51 female age-matched student controls were assessed with the EED and the Body Attitude Test (BAT). RESULTS: The results indicate satisfactory internal consistency (Cronbach's Alpha 0.92) of the sum score of the whole sample. The validity of the EED was supported by the correlation analysis between EED and Body Attitude Test (BAT) (Spearman's rho=0.84, p<0.01). There was a significant statistical difference between patients and controls in total score and subscales of the EED (p<0.001). CONCLUSION: The preliminary test of the EED questionnaire was promising. It is a short instrument, and seems to distinguish well between patients and controls. EED captures other dimensions of physical activity and exercise disturbances not captured in other questionnaires related to exercise. Further research is needed to test the psychometric properties of EED in bigger samples.

Evaluation of three scoring methods for Fluorescence Optical Imaging in erosive hand osteoarthritis and rheumatoid arthritis.

Objective: Fluorescence Optical Imaging (FOI) demonstrates indocyanine green (ICG)-enhanced microcirculation in wrist and finger joints, as a sign of inflammation. We wanted to assess the reliability of three FOI scoring methods from Berlin, Stockholm, and Copenhagen, to assess the validity of FOI with MRI as reference and to compare enhancement in hand joints in erosive hand osteoarthritis (OA) vs. rheumatoid arthritis (RA). Design: Five readers scored all finger and wrist joints of 26 patients with erosive hand OA and RA on semi-quantitative 0-3 scales using three different FOI scoring methods. To evaluate inter-reader reliability, we calculated the intraclass correlation coefficients (ICC) for sum scores and prevalence and bias adjusted kappa values for ordinal scales (Pabak-OS) on joint level. Enhancement in joint groups in erosive hand OA vs. RA was compared using Mann-Whitney test. Sensitivities and specificities of FOI was calculated with MRI as reference for hand OA patients only. Results: We found moderate to good inter-reader reliability for all FOI scoring methods (Pabak-OS: 0.50-0.78, ICC: 0.43-0.85) and different patterns of enhancement in erosive hand OA vs. RA with significantly more FOI enhancement in DIP joints in erosive hand OA across all methods. With MRI as reference the different FOI scoring methods reached similar sensitivities (63-65%) and specificities (76-91%). Conclusion: FOI enhancement can be measured reliably in erosive hand OA and RA using three different scoring methods. More DIP enhancement in erosive hand OA patients and good agreement with MRI support the diagnostic performance of FOI.

Olive oil quality classification and measurement of its organoleptic attributes by untargeted GC-MS and multivariate statistical-based approach.

The capabilities of dynamic headspace entrainment followed by thermal desorption in combination with gas chromatography (GC) coupled to single quadrupole mass spectrometry (MS) have been tested for the determination of volatile components of olive oil. This technique has shown a great potential for olive oil quality classification by using an untargeted approach. The data processing strategy consisted of three different steps: component detection from GC-MS data using novel data treatment software PARADISe, a multivariate analysis using EZ-Info, and the creation of the statistical models. The great number of compounds determined enabled not only the development of a quality classification method as a complementary tool to the official established method "PANEL TEST" but also a correlation between these compounds and different types of defect. Classification method was finally validated using blind samples. An accuracy of 85% in oil classification was obtained, with 100% of extra virgin samples correctly classified.

Down-regulation of glucocorticoid receptors in mouse lymphoma cell variants.

The mouse thymoma-derived cell line W7 is sensitive to the cytolytic action of glucocorticoids. We have isolated a novel class of cell variant that apparently overcomes its inherent sensitivity to glucocorticoids by reversibly down-regulating the level of glucocorticoid receptors. This phenotype is stable during subcloning in the presence and in the absence of glucocorticoids and is dominant in somatic cell hybrids with wild-type cells. Fusion of this variant with wild-type cells produces hybrids that down-regulate and are less sensitive to glucocorticoids than hybrids of receptor-negative and wild-type cells. This is the first demonstration of a phenotypic change which correlates with down-regulation of the glucocorticoid receptor.

Glucocorticoid-resistant lymphoma cell variants that contain functional glucocorticoid receptors.

A mouse T-lymphosarcoma cell line stably infected with mouse mammary tumor virus (MMTV) was used as the parent line for a genetic analysis of two glucocorticoid hormone responses, hormone-induced cytolysis and stimulation of viral gene expression. Variants were selected for survival and elevated expression of MMTV proteins in the presence of the steroid. The MMTV marker provided a sensitive test for glucocorticoid receptor (GR) function in the hormone-resistant variants. This strategy resulted in the isolation of two novel types of hormone-resistant variants. One type of variant with only about 25% of the level of GR found in the parent line was resistant to the cytolytic effects of glucocorticoid but produced increased levels of MMTV gene products in response to the hormone. This variant phenotype demonstrated that the MMTV response requires fewer GR than the cytolytic response. Another variant, which required approximately 100-fold higher concentrations of hormone than the wild-type cells for both responses, apparently contained GR with altered hormone-binding properties.

Immunological selection of variant mouse lymphoid cells with altered glucocorticoid responsiveness.

We have devised an immunological procedure to separate cells on the basis of expression of mouse mammary tumor virus (MMTV) gene products. Plastic petri dishes coated with specific antibodies against MMTV proteins bind cells with an efficiency that correlates with the level of MMTV gene expression. Glucocorticoid-sensitive mouse thymoma cell line W7 was infected with MMTV. Clones from the infected population retain the relatively slow cytolytic glucocorticoid response and, in addition, exhibit a rapid induction of MMTV-specific RNA and proteins. By combining our immunological selection with the selection for resistance to hormone-mediated cytolysis, we have isolated variant cells which are resistant to the cytotoxic effect of glucocorticoids but which retain the induction of viral gene products and must therefore have a functional glucocorticoid receptor protein.

Mutational analysis of the mouse glucocorticoid receptor.

We have cloned the mouse glucocorticoid receptor (GR) from both wild-type and glucocorticoid-resistant variants of the mouse lymphoma cell lines WEHI-7 and S49. Mapping of the mutations present in the variant receptors, together with deletion analysis of wild-type receptor, reveals that the receptor has three clearly defined domains. The COOH-terminal domain contains the hormone-binding site. Within this domain is a small region which is important for the suppression of receptor activity in the absence of hormone. The large NH2-terminal domain is essential for full receptor activity and contains within it a highly acidic region that potentiates receptor activity. The presence of this acidic region reduces nonspecific DNA binding and may therefore be crucial in the discrimination between specific and nonspecific DNA-binding sites by the receptor. A small centrally located domain contains all the information necessary to bind specifically to DNA and to activate transcription. Although this region is absolutely conserved in the GR of different species, many mutations introduced in vitro give rise to functional receptor. In addition, part of this region in the GR can be substituted for by the corresponding sequence in the estrogen receptor to give a GR with the DNA-binding and transcriptional specificity of an estrogen receptor. Lastly, we have succeeded in obtaining stable high-level expression of wild-type and mutant GR in transfected Chinese hamster ovary cells.

Role of the C terminus of the glucocorticoid receptor in hormone binding and agonist/antagonist discrimination.

The glucocorticoid receptor (GR) is a hormone-inducible intracellular modulator of specific gene transcription. Both glucocorticoids and progestins bind to the GR, and some progestins are able to activate the receptor. We have characterized a mutation of the mouse GR that restricts transcriptional activation, but not hormone binding, to glucocorticoids. This mutation, Y77ON, is located 13 amino acids from the C terminus of the mouse GR and helps define a region of the receptor that is important for transcriptional specificity. To further characterize this region of the GR, we have constructed a series of chimeric receptors between the glucocorticoid, progesterone, and androgen receptors. We find that the C-terminal 14 amino acids of the GR can be replaced by the equivalent region of the progesterone or androgen receptors with little alteration in either hormone-binding specificity or transcriptional response to agonists and antagonists. The region is required for hormone binding, however, since C-terminal deletions yield inactive receptors. We conclude that even though mutation of the C-terminal 14 amino acids of the GR can lead to alterations in hormone binding specificity and agonist potential, the differential hormone-binding capacities of the glucocorticoid, progesterone, and androgen receptors are not encoded in this region.

High level expression of wild type and variant mouse glucocorticoid receptors in Chinese hamster ovary cells.

We have isolated Chinese hamster ovary (CHO) cell lines expressing elevated levels of wild-type (W) and mutant forms of the glucocorticoid receptor (GR) using the technique of coamplification with a selectable dihydrofolate reductase (dhfr) cDNA. A prominent doublet at 90/92 kilodaltons was observed by Western blotting or labeling with [3H]-dexamethasone mesylate in extracts from cells transfected with W, the hormone binding mutant (NA), and the DNA binding mutant (NB). Quantification of receptor number by [3H]dexamethasone binding revealed the presence of approximately 10(6) receptors per cell in the W and NB-producing lines. This represents a 25- to 50-fold increase in receptor density over control CHO cells which were not transfected with GR. Comparative quantitation by Western blotting of extracts from cells expressing GR showed that cells producing NA contain a level approximately 500-fold over control CHO cells. Function of the amptified receptors was examined by transient transfection with the glucocorticoid-responsive reporter plasmid pMMTV-chloramphenicol acetyl transferase (CAT). Our results indicate that inducible CAT activity increases with the abundance of W receptor and no evidence of saturability was observed even at the highest levels of receptor. This supports previous suggestions that the concentration of the hormone-regulated transcription factor is definitely limiting with regard to maximal transcription efficiency. Interestingly, cells expressing even highly amplified levels of NA-GR or NB-GR showed no inducible response above that seen with control CHO cells. Thus these mutations are exceedingly nonleaky and are not dominant over the low endogenous activity of the CHO GR.(ABSTRACT TRUNCATED AT 250 WORDS)

Domains of the glucocorticoid receptor involved in specific and nonspecific deoxyribonucleic acid binding, hormone activation, and transcriptional enhancement.

We have analyzed the domain structure of the mouse glucocorticoid receptor by expression of in vitro mutated receptor in COS-7 cells. The receptor consists of a core domain rich in Cys, Lys, and Arg amino acids which can bind specific DNA sequences (glucocorticoid response elements) and activate transcription. The activity of this centrally located domain is modulated by the activity of the other two domains. The N-terminal domain of the receptor plays a role in decreasing nonspecific DNA binding and may therefore improve the ability of the protein to discriminate between specific and nonspecific DNA binding sites. This activity maps to a small, highly acidic region of the N-terminal domain. The C-terminal domain of the receptor contains the glucocorticoid binding site and in addition represses the transcriptional activity of the receptor in the absence of hormone. Hormone binding relieves the repression allowing transcription activation. The C-terminal domain contains a short sequence conserved among steroid receptors; its deletion yields a receptor that activates transcription in the absence of hormone.

Phenylalanine-780 near the C-terminus of the mouse glucocorticoid receptor is important for ligand binding affinity and specificity.

Site-directed mutagenesis was employed to make two single amino acid substitutions for highly conserved amino acid residues near the C-terminus of the 783-amino acid mouse glucocorticoid receptor. Substitution of leucine for histidine-781 caused little or no change in the concentration of dexamethasone required for half-maximal activation of a chloramphenicol acetyltransferase reporter gene expressed from a mouse mammary tumor virus promoter. However, when phenylalanine-780 was changed to alanine, the half-maximal concentrations of various agonists were increased as follows, compared with the wild-type glucocorticoid receptor: triamcinolone acetonide by 7-fold, dexamethasone by 25-fold, and hydrocortisone and deoxycorticosterone by more than 150-fold. Binding of labeled steroids by the mutant receptor in vitro and in vivo was also decreased. In contrast, this mutation caused a small decrease in the concentration of RU486 required for antagonist or partial agonist activity. Thus, the phenyl group of phenylalanine-780 of the mouse glucocorticoid receptor is an important determinant of ligand binding affinity and specificity.

Two point mutations in the hormone-binding domain of the mouse glucocorticoid receptor that dramatically reduce its function.

Mouse lymphoma cell line W7M320b, a mutant WEH17 line, requires higher than normal concentrations of glucocorticoid to elicit the hormone responses that are characteristic of this lineage. Complementary DNA clones representing the glucocorticoid receptor (GR) mRNA were derived from the mutant cells, and the sequences coding for the hormone-binding domain were substituted for the analogous wild-type sequences in a GR cDNA expression vector. The function of the resulting GR proteins was tested by transient expression in COS-7 cells along with a glucocorticoid-inducible reporter gene in the presence of varying concentrations of glucocorticoid. From these assays and DNA sequence analyses, two independent functionally significant point mutations in the GR hormone-binding domain were identified. A mutant GR protein containing the single amino acid substitution, Pro547 to Ala, was still functional as a transcriptional activator, but only at hormone concentrations 100 times higher than those required by the wild-type receptor. A second mutant GR protein with a Cys742 to Gly substitution was unstable and almost completely nonfunctional.

The frequency of mutators in populations of Escherichia coli.

Owing to occasional spontaneous mutations in genes encoding DNA repair, any population of a reasonable size is expected to harbor a sub-population of genetic mutators. Using a genetically modified strain of Escherichia coli K-12, we have estimated the frequency of mutators to be about 3x10(-5). By and large, this corresponds to a mutation rate from non-mutators to mutators of 5x10(-6) per bacterium per generation. Using a mutS∷Tn10 derivative as representative for mutators, we estimated the increase in mutation rates in mutators to be 19- to 82-fold, depending on the test-mutation under consideration. The load associated with this increase in mutation rate resulted in a growth inhibition of 1%. From these data, we estimated that the rate of detrimental mutations in the non-mutators to be 2x10(-4)-8x10(-4). The situations where adaptive mutations may result in an increase in the frequency of mutators are discussed.

The left ventricular ejection effect.

A recently developed model of the left ventricle, based on experimental data, has been shown to exhibit the main features of the heart's ability to pump. Two special cases during blood ejection, termed pressure deactivation and hyperactivation, were identified. This study proposes an 'ejection effect' correction to the model that addresses deactivation, hyperactivation and adjusts the shape of the computed ventricular ejection curve in late systole. Also, a new approach based on new animal experiments is proposed to identify the ejection effect mechanism(s).

Susceptibility of Pediococcus spp. to antimicrobial agents.

AIM: To investigate the susceptibility of Pediococcus species to antimicrobial agents. METHODS AND RESULTS: The susceptibility to 14 antimicrobial agents of 31 genotypically distinct strains of six Pediococcus species was assessed by using Etests on ISO-sensitest agar supplemented with horse blood. The species included were Pediococcus acidilactici, Pediococcus damnosus, Pediococcus dextrinicus, Pediococcus inopinatus, Pediococcus parvulus and Pediococcus pentosaceus. For several antimicrobial agents, some species were more susceptible than others. The two industrially important species, P. acidilactici and P. pentosaceus, differed with respect to erythromycin and trovafloxacin susceptibility, and in general both species had higher minimum inhibitory concentrations than the other species. In an erythromycin-resistant P. acidilactici, an erythromycin resistance methylase B [erm(B)] gene was identified by PCR. Using a plasmid preparation from strain P. acidilactici 6990, a previously erythromycin-sensitive Lactococcus lactis strain was made resistant. Transformants harboured a single plasmid, sized at 11.6 kb through sequence analysis. In addition, the erm(B) gene was identified within the plasmid sequence. CONCLUSIONS: The phenotypic test indicated the absence of acquired antimicrobial resistance genes in 30 of the strains. SIGNIFICANCE AND IMPACT OF THE STUDY: These results will help in selection of the best Pediococcus strains for use as starter cultures.

pEOC01: a plasmid from Pediococcus acidilactici which encodes an identical streptomycin resistance (aadE) gene to that found in Campylobacter jejuni.

The complete nucleotide sequence of pEOC01, a plasmid (11,661 bp) from Pediococcus acidilactici NCIMB 6990 encoding resistance to clindamycin, erythromycin, and streptomycin was determined. The plasmid, which also replicates in Lactococcus and Lactobacillus species contains 16 putative open reading frames (ORFs), including regions annotated to encode replication, plasmid maintenance and multidrug resistance functions. Based on an analysis the plasmid replicates via a theta replicating mechanism closely related to those of many larger Streptococcus and Enterococcus plasmids. Interestingly, genes homologous to a toxin/antitoxin plasmid maintenance system are present and are highly similar to the omega-epsilon-zeta operon of Streptococcus plasmids. The plasmid contains two putative antibiotic resistance homologs, an ermB gene encoding erythromycin and clindamycin resistance, and a streptomycin resistance gene, aadE. Of particular note is the aadE gene which holds 100% identity to an aadE gene found in Campylobacter jejuni plasmid but which probably originated from a Gram-positive source. This observation is significant in that it provides evidence for recent horizontal transfer of streptomycin resistance from a lactic acid bacterium to a Gram-negative intestinal pathogen and as such infers a role for such plasmids for dissemination of antibiotic resistance genes possibly in the human gut.

Differential regulation of androgen and glucocorticoid receptors by retinoblastoma protein.

The androgen receptor (AR) plays a major role in the development and maintenance of male primary and secondary sexual characteristics. The growth promoting effects of androgens are clearly seen in prostate cancer where treatment by androgen ablation usually leads to tumor regression, followed sometime later, by growth of tumor cells that are resistant to endocrine therapy. We have found that the level of pRB in cells controls AR activity. Overexpression of pRB leads to increased transcriptional activity of the AR. This is similar to the previously reported potentiation of glucocorticoid receptor activity by pRB. In contrast, loss of pRB activity inhibits AR but not glucocorticoid receptor activity. This inhibition correlates with the unique ability of the AR to form a protein-protein complex with pRB in vitro. The site of interaction with pRB lies within the N-terminal domain of the AR and co-localizes with the region of the AR that specifies a requirement for pRB. Thus, the AR has a novel requirement for pRB raising the possibility that the growth promoting activity of AR is due to its direct interaction with pRB. Furthermore, loss of pRB activity during progression of prostate cancer may directly result in a decreased response to androgens.