Therapeutic DNA vaccination is an attractive adjuvant option to conventional methods in the fight against cancer, like surgery radiotherapy and chemotherapy. Despite strong antitumor effects that were observed in small animals with different antigens, DNA-based vaccines remain weakly immunogenic in large animals and primates compared to protein-based vaccines. Here, we sought to enhance the immunogenicity of a therapeutic nontransforming cervical cancer DNA vaccine (HPV-16 E7SH) by introduction of a highly optimized CpG cassette into the plasmid backbone as well as by an optimized DNA delivery using an advanced electroporation (EP) technology. By integrating the means for agent administration and EP into a single device, this technology enables a simple, one-step procedure that facilitates reproducibility. We found that highly optimized CpG motifs alone triggers an enhanced IFN-γ and granzyme B response in Elispot assays as well as stronger tumor regression. Furthermore, these effects could be dramatically enhanced when the CpG cassette containing plasmid was administered via the newly developed EP technology. These data suggest that an optimized application of CpG-enriched DNA vaccines may be an attractive strategy for the treatment of cancer. Collectively, these results provide a basis for the transfer of preclinical therapeutic DNA-based immunization studies into successful clinical cancer trials.

Variations in the essential oil composition of Salvia officinalis L. growing in Estonia and in other European countries were determined. The oils were obtained in yields of 2.2-24.8 mL kg(-1). In three samples, the content of essential oil did not conform to the EP standard (10 mL kg(-1)). Variations in the essential oil composition of sage were studied using capillary gas chromatographic methods. A total of 40 components were identified. The principal components in the sage oils were 1,8-cineole, camphor, alpha-thujone, beta-thujone, borneol, and viridiflorol. The chemotypes of sage were not determined in investigated samples. The concentration of the main compounds in the drugs cultivated in Estonia varied in about the same range as the concentrations of these compounds in the oils of drugs obtained from other countries. The comparatively high concentration of toxic thujones seem to be characteristic to sage leaves cultivated in Estonia.

Probiotic microorganisms are ingested as food or supplements and impart positive health benefits to consumers. Previous studies have indicated that probiotics transiently reside in the gastrointestinal tract and, in addition to modulating commensal species diversity, increase the expression of genes for carbohydrate metabolism in resident commensal bacterial species. In this study, it is demonstrated that the human gut commensal species Bacteroides thetaiotaomicron efficiently metabolizes fructan exopolysaccharide (EPS) synthesized by probiotic Lactobacillus reuteri strain 121 while only partially degrading reuteran and isomalto/malto-polysaccharide (IMMP) α-glucan EPS polymers. B. thetaiotaomicron metabolized these EPS molecules via the activation of enzymes and transport systems encoded by dedicated polysaccharide utilization loci specific for β-fructans and α-glucans. Reduced metabolism of reuteran and IMMP α-glucan EPS molecules may be due to reduced substrate binding by components of the starch utilization system (sus). This study reveals that microbial EPS substrates activate genes for carbohydrate metabolism in B. thetaiotaomicron and suggests that microbially derived carbohydrates provide a carbohydrate-rich reservoir for B. thetaiotaomicron nutrient acquisition in the gastrointestinal tract.

Oxidation of lipoproteins is believed to play a key role in atherogenesis. In this study, low density lipoproteins (LDL) was subjected to oxidation in the presence of either human umbilical vein endothelial cells or with Cu+2 ions and the major oxides formed were identified. While cholesterol-alpha-epoxide (C-alpha EP) was the major product of cholesterol peroxidation in the presence of endothelial cells, cholest-3,5-dien-7-one (CD) predominated in the presence of Cu+2 ion. Both steroids were identified by gas chromatography/mass spectrometry. HDL cholesterol was resistant to oxidation. When tested on human skin fibroblasts in culture C-alpha EP (10 micrograms/ml) caused marked stimulation of 14C-oleate incorporation into cholesterol esters, while CD stimulated cholesterol esterification only mildly. These studies show that a) C-alpha EP is the major peroxidation product of LDL cholesterol moiety in the presence of endothelial cells and b) it causes marked stimulation of cholesterol esterification in cells. C-alpha EP may play a key role in increasing cholesterol esterification noted in atherogenesis.

Cross-linking and two-dimensional crystallization studies have suggested that the membrane-bound gastric H,K-ATPase might be a dimeric alpha,beta-heterodimer. Effects of an oligomeric structure on the characteristics of E(1), E(2), and phosphoenzyme conformations were examined by measuring binding stoichiometries of acid-stable phosphorylation (EP) from [gamma-(32)P]ATP or (32)P(i) or of binding of [gamma-(32)P]ATP and of a K(+)-competitive imidazonaphthyridine (INT) inhibitor to an enzyme preparation containing approximately 5 nmol of ATPase/mg of protein. At <10 microM MgATP, E(1)[ATP].Mg.(H(+)):E(2) is formed at a high-affinity site, and is then converted to E(1)P.Mg.(H(+)):E(2) and then to E(2)P.Mg:E(1) with luminal proton extrusion. Maximal acid-stable phosphorylation yielded 2.65 nmol/mg of protein. Luminal K(+)-dependent dephosphorylation returns this conformation to the E(1) form. At high MgATP concentrations (>0.1 mM), the oligomer forms E(2)P.Mg:E(1)[ATP].Mg.(H(+)). The sum of the levels of maximal EP formation and ATP binding was 5.3 nmol/mg. The maximal amount of [(3)H]INT bound was 2.6 nmol/mg in the presence of MgATP, Mg(2+), Mg-P(i), or Mg-vanadate with complete inhibition of activity. K(+) displaced INT only in nigericin-treated vesicles, and thus, INT binds to the luminal surface of the E(2) form. INT-bound enzyme also formed 2.6 nmol of EP/mg at high ATP concentrations by formation of E(2).Mg.(INT)(exo):E(1)[ATP].Mg.(H(+)) which is converted to E(2).Mg.(INT)(exo):E(1)P.Mg.(H(+))(cyto), but this E(1)P form was K(+)-insensitive. Binding of the inhibitor fixes half the oligomer in the E(2) form with full inhibition of activity, while the other half of the oligomer is able to form E(1)P only when the inhibitor is bound. It appears that the catalytic subunits of the oligomer during turnover in intact gastric vesicles are restricted to a reciprocal E(1):E(2) configuration.

MSP (GP63) and PSA (GP46) are abundant 63- and 46-kDa glycolipid-anchored proteins on the surface of the promastigote form of most Leishmania species. MSP is a zinc metalloprotease that confers resistance to host complement-mediated lysis. PSA contains internal repeats of 24 amino acids, and its function is unknown. The steady state levels of mRNAs for both glycoproteins are regulated post-transcriptionally, resulting in about a 30-fold increase as Leishmania chagasi promastigotes grow in vitro from logarithmic phase to stationary phase. Previous studies showed the 3'-untranslated regions (3'-UTRs) of these mRNAs are essential for this post-transcriptional regulation. These two 3'-UTRs of 1.0 and 1.3 kilobases were cloned immediately downstream of a beta-galactosidase reporter gene in a plasmid, and segments were systematically deleted to examine which portions of the 3'-UTRs contribute to the post-transcriptional regulation. The 92-nucleotide segment of greatest similarity between the two 3'-UTRs was deleted without loss of regulation, but the segments flanking this similarity region have positive regulatory elements essential for the regulation. We propose that similar, but non-identical, molecular mechanisms regulate the parallel expression of these two L. chagasi mRNAs despite their lack of sequence identity. These post-transcriptional mechanisms resemble the mechanism recently suggested for the regulation of mRNAs encoding the dipeptide (EP) and pentapeptide (GPEET) repeat proteins in Trypanosoma brucei that involves interactions between positive and negative regulatory elements in the 3'-UTR.

Three new established human germ cell tumour lines, H 12.1, H 12.5 and H 12.7, are described. Cytogenetic and growth characteristics, morphology, histology, and tumour marker and steroid hormone production in vitro and/or in vivo revealed properties commonly found in other germ cell tumour lines and in patients with these tumours. In vitro oestrone and oestradiol were produced by the H 12 lines and four other lines (833 KE, 1156 Q, 1428 A and 2102 EP) under high and low density (differentiation inducing) conditions. AFP was produced by one line and beta-hCG by six of seven lines under low density conditions. The pattern of oestrogen production suggests that these hormones could be useful in AFP and beta-hCG negative patients. Differentiated elements of somatic and extraembryonic character, observed in tumours of H 12.1 and H 12.7, underline the value of these lines in the study of differentiation and other germ cell tumour related questions.

OBJECTIVE

The aim of this study was to investigate the short-term effect of fluid resuscitation with 4% modified fluid gelatine (GEL) versus 6% hydroxyethyl starch (HES) on haemodynamics and oxygenation in patients with septic shock and acute lung injury (ALI).

METHODS

Prospective randomised clinical trial.

METHODS

Twenty-bed intensive care unit in a university hospital.

METHODS

Thirty hypovolemic patients (intrathoracic blood volume index, ITBVI <850 ml/m(2)) in septic shock with ALI were randomised into HES (mean molecular weight: 200,000 Dalton, degree of substitution 0.6) and GEL (mean molecular weight: 30,000 Dalton) groups (15 patients each).

METHODS

For fluid resuscitation 250 ml/15 min boluses (max. 1,000 ml) were given until the end point of ITBVI >900 ml/m(2) was reached. Repeated haemodynamic measurements were done at baseline (t(b)), at the end point (t(ep)) then at 30 min and 60 min after the end point was reached (t(30), t(60)). Cardiac output, stroke volume, extravascular lung water (EVLW), and oxygen delivery was determined at each assessment point. For statistical analysis two-way ANOVA was used.

RESULTS

ITBVI, cardiac index, and oxygen delivery index increased significantly at t(ep) and remained elevated for t(30) and t(60), but there was no significant difference between the two groups. The increase in the ITBVI by 100 ml of infusion was similar in both groups (HES: 26+/-19 ml/m(2) vs GEL: 30+/-19 ml/m(2)). EVLW, remained unchanged, and there was no significant difference between the groups (HES, t(b): 8+/-6, t(60): 8+/-6; GEL, t(b): 8+/-3, t(60): 8+/-3 ml/kg). The PaO(2)/FiO(2) did not change significantly over time or between groups (HES, t(b): 207+/-114, t(60): 189+/-78; GEL, t(b): 182+/-85, t(60): 182+/-85 mmHg).

CONCLUSIONS

The results of this study indicate that both HES and GEL infusions caused similar short-term change in ITBVI in septic shock, without increasing EVLW or worsening oxygenation.

In this report, we demonstrate that gp40, a molecule previously shown to be expressed by thymic epithelial cell lines in vitro and by thymic epithelial cells in vivo, is the murine homolog of human Ep-CAM, a calcium-independent homotypic adhesion molecule. gp40 is also expressed at low levels by thymocytes and peripheral T cells. In the adult thymus, gp40 expression was inversely related to the state of thymocyte maturation, with the highest levels associated with CD4-CD8- and CD4+CD8+ thymocyte populations. Ultrastructural immunohistochemistry revealed gp40 localization to areas of thymocyte/epithelial contact and demonstrated that gp40 is also expressed by thymic dendritic cells. During fetal development, thymocytes at days 14-16 of gestation expressed high levels of gp40. At later stages, the observed decline in the frequency of gp40+ cells and levels of expression correlated with the emergence of alpha beta+ thymocytes by day 18 of gestation. In short-term cultures, stimulation of unfractionated adult thymocytes with concanavalin A increased gp40 expression, particularly among CD3hi and CD3int thymocyte populations. This demonstration that Ep-CAM, initially considered to be expressed primarily by epithelial cells, is also expressed by thymocytes, T cells and antigen-presenting cells, raises the possibility that Ep-CAM may contribute to adhesive interactions between thymocytes and epithelial cells or dendritic cells, either in the context of thymocyte development or peripheral T cell trafficking and function.

The aim of this study was to reexamine and compare the characteristics of the deficit and nondeficit schizophrenic patients. This cross-sectional study consisted of 62 in- and out-patients, 18-65 years of age, diagnosed with schizophrenia according to DSM-IV. The sociodemographic variables, premorbid adjustment, clinical course and general functioning level in the past five years were evaluated by utilizing the appropriate sections of Comprehensive Assessment of Symptoms and History (CASH). In addition, GAF, the Schedule for the Deficit Syndrome (SDS), Positive and Negative Syndrome Scale (PANSS), Montgomery Asberg Depression Scale (MADRS), the Neurological Evaluation Scale (NES) and the Simpson Angus Extrapyramidal Side Effects (EPS) Rating Scale, Trail A and B, Verbal Fluency, Stroop, Block Design and Finger Tapper tests were administered. Using the SDS, 19 patients (30.6 %) were categorized as deficit; 43 (69.4 %) were categorized as nondeficit. The deficit patients were worse on the Functioning During Past Five Years score of CASH. The PANSS and MADRS mean scores were not significantly different between the two groups, except a higher level of negative symptoms observed in the deficit group. NES scores were also significantly higher in the deficit group. However, sociodemographic and other clinical variables, neurocognitive measures and EPS symptoms did not show any significant difference between the two groups. Our findings suggest that the deficit schizophrenia is a distinct subgroup comprised of patients who have more negative symptoms, neurological impairment and poor functioning which may have a common underlying pathology.

Mitral regurgitation (MR) is associated with increased neuronal release of norepinephrine (NE) and epinephrine (<em>EP</em>) into myocardial interstitial fluid (ISF) that may be necessary in sustaining left ventricular (LV) function via activation of cardiomyocyte <em>beta</em>-adrenergic receptors (ARs). However, activation of neuronal <em>beta</em>-ARs on cardiac neurons may lead to further catecholamine release, with an attendant risk of functional deterioration. We hypothesize that a beneficial effect of <em>beta</em>-AR blockade may therefore mitigate excessive catecholamine release from cardiac adrenergic neurons in dogs with MR. We measured the effects of chronic <em>beta</em>-receptor blockade (<em>beta</em>-RB) on ISF NE and <em>EP</em> release using in vivo microdialysis in open-chest anesthetized dogs after 4 wk of MR with or without extended release of metoprolol succinate (100 mg/day) as well as in control dogs. Fractional shortening increased by 30% in both MR and MR + <em>beta</em>-RB dogs after 4 wk of MR. In MR + <em>beta</em>-RB dogs, stellate-stimulated heart rate change was attenuated compared with control and MR dogs, whereas peak change of LV pressure over time (+dP/dt) increased equally in all groups. Stellate-stimulated ISF NE increased fivefold over baseline in MR versus twofold in control dogs (< 0.05), but the NE release was significantly attenuated in MR + <em>beta</em>-RB dogs. In contrast, stellate-stimulated increases in ISF <em>EP</em> did not differ in control, MR, and MR + <em>beta</em>-RB dogs. This study demonstrates that <em>beta</em>-RB attenuates ISF NE release from cardiac neurons and that the LV functional response to MR is not dependent on an excess increase in ISF NE. Thus <em>beta</em>1-RB may exert a beneficial effect by attenuating untoward effects of excessive sympathetic efferent neural NE release while sustaining early LV functional adaptation to MR.

Intradermal inoculation of rats at the tail base with Mycobacterium butyricum led to the gradual development of an arthritic swelling of the limbs which peaked at 3 weeks and subsided thereafter. Arthritic rats displayed a loss of body weight, hypophagia, and hypodipsia in addition to a disruption of the diurnal rhythms of ingestive behavior and of core temperature. The activity of adenohypophyseal beta-endorphin-(beta-EP) secreting corticotrophs, in contrast to prolactin-(PRL) secreting lactotrophs, was increased in arthritic rats. Indeed, hypertrophy of the adrenal glands was seen. Arthritic rats also showed an elevation in spinal cord levels of immunoreactive dynorphin (DYN), an endogenous ligand of the kappa-opioid receptor. The paws and tail of arthritic rats showed lower thresholds in response to noxious pressure (hyperalgesia), higher thresholds in response to noxious heat (hypoalgesia), and no change in their response to noxious electrical stimulation. Neither naloxone nor ICI-154, 129 (a preferential delta-receptor antagonist) modified the responses of the paw or tail to pressure. However, MR 2266 (an antagonist with higher activity at kappa-receptors) decreased thresholds to pressure in arthritic, but not control, rats; that is, it potentiated the hyperalgesia. This action was stereospecific. None of the antagonists modified the response to heat. MR 2266 did not affect the response to pressure in rats with acute inflammation produced by yeast. Thus, the potentiation of pressure hyperalgesia by MR 2266 in chronic arthritic rats is highly selective. Arthritic rats showed a reduced response to the analgesic effect of a kappa-agonist (U-50,488H), whereas the response to a mu-agonist (morphine) was enhanced. These effects were specific to nociception in that their influence upon endocrine secretion (PRL and beta-EP) was otherwise changed. The secretion of beta-EP and PRL was stimulated by both morphine and U-50,488H, and the influence of U-50,488H upon the release of beta-EP (from the adenohypophysis) was enhanced in arthritic rats. It is suggested that polyarthritis is a complex condition entailing many changes, both behavioral and endocrinological. Further, arthritic rats cannot simply be described as "hyperalgesic": of critical importance is the nature of the nociceptive stimulus applied. The parallel alterations in spinal cord pools of DYN and kappa-receptors (see also Millan et al., 1986) and the changes in the influence on nociception of kappa-agonists and kappa-antagonists suggest an increased activity of spinal DYN. Thus, spinal kappa-receptors may play a role in the modulation of nociception under chronic pain.(ABSTRACT TRUNCATED AT 400 WORDS)

Aerobic conditioned muscle shows increased oxidative metabolism or glucose relative to untrained muscle at a given absolute exercise intensity. The studies of a targeted risk reduction intervention through defined exercise (STRRIDE) study is an aerobic exercise intervention in men and women with features of metabolic syndrome (Kraus WE, Torgan CE, Duscha BD, Norris J, Brown SA, Cobb FR, Bales CW, Annex BH, Samsa GP, Houmard JA, and Slentz CA, Med Sci Sports Exerc 33: 1774-1784, 2001), with four muscle biopsies taken during training and detraining time points. Here, we expanded a previous study (Hittel DS, Kraus WE, and Hoffman EP, J Physiol 548: 401-410, 2003) and used mRNA profiling to investigate gene transcripts associated with energy and substrate metabolism in STRRIDE participants. We found coordinate regulation of key metabolic enzymes with aerobic training in metabolic syndrome (aspartate aminotransferase 1, lactate dehydrogenase B, and pyruvate dehydrogenase-alpha(1)). All were also quickly downregulated by detraining, although the induction was not an acute response to activity. Protein and enzymatic assays were used to validate mRNA induction with aerobic training and loss with detraining (96 h to 2 wk) in 10 male and 10 female STRRIDE subjects. We propose that training coordinately increases the levels of aspartate aminotransferase 1, lactate dehydrogenase B, and pyruvate dehydrogenase-alpha(1) subunit, increasing glucose metabolism in muscle by liberating pyruvate for oxidative metabolism and, therefore, limiting lactate efflux. Serial measurement of fasting plasma lactate from 62 subjects from the same exercise group demonstrated a significant decrease of circulating lactate with training. We also found evidence for sex-specific molecular remodeling of muscle with ubiquinol-cytochrome c reductase core protein II, a component of mitochondrial respiratory complex III, which showed an increase after training that was specific to women. These biochemical adaptations complement existing molecular models for improved glucose tolerance with exercise intervention in prediabetic individuals.

The Na+/K(+)-ATPase couples the hydrolysis of ATP to the transport of Na+ and K+ via a phosphorylated intermediate and conformational changes. In order to identify these conformational changes, we have probed the sequence of steps from EP(3Na+ in) to EP + 3Na+ out with three fluorescent probes (IAF: 5-iodoacetamidofluorescein; BIPM: N-[p-(2-benzimidazolyl)phenyl]maleimide; and RH421) and the sensitivity of their fluorescence change to oligomycin and divalent cations (Ca2+ and Mn2+). The magnitude (% delta F) and rate constant (k(obs)) of ATP-induced fluorescence changes were measured on a fluorescence stopped-flow apparatus, and yielded the following results. (a) With RH421, k(obs) and % delta F varied with [Na+] (maximal k(obs) = 100 s-1, K1/2 = 6 mM; % delta Fmax = 6%, K1/2 = 1 mM); these values are comparable to those previously reported using IAF-labeled enzyme (Pratap, P.R., Robinson, J.D. and Steinberg, M.I. (1991) Biochim. Biophys. Acta 1069, 288-298). (b) With BIPM-labeled enzyme k(obs) did not vary with [Na+] over the range tested, and was twice as high as the maximum k(obs) for RH421. (c) Treatment with oligomycin reduced k(obs) for all three probes to about the same level (approximately 1-2 s-1) while % delta Fmax was largely unaffected. (d) Replacing Mg2+ with Ca2+ had similar effects to treatment with oligomycin. (e) RH421 fluorescence change was completely abolished in the presence of oligomycin and Ca2+. (f) Replacing Mg2+ with Mn2+ decreased IAF fluorescence, i.e., put the enzyme in an E2-like form, reduced k(obs), and rendered oligomycin less effective in reducing k(obs). From these results, we conclude: (a) the release of the second/third Na+ is the rate-limiting step for the conformational change measured by IAF and charge transfer measured with RH421; (b) BIPM indicates an earlier step, either the deocclusion of Na+ and/or the release of the first Na+; (c) oligomycin blocks Na+ deocclusion, and this step is sensitive to the divalent cation used to activate enzyme phosphorylation; and (d) Ca2+ slows the step reported by IAF as well. These experiments indicate that a simple model with two conformations (E1 and E2) is insufficient to explain transient kinetic data.

DNA vaccines require significant engineering in order to generate strong CTL responses in both non-human primates and humans. In this study, we designed a clade C env gene (EY3E1-C) to decrease the genetic distances of virus isolates within clade C and focus the induced T cell responses to conserved clade C epitopes. After generating a consensus sequence by analyzing full-length clade C env early transmitter sequences, several modifications were performed to increase the expression of the EY3E1-C, including codon/RNA optimization, addition of Kozak sequence and addition of an IgE leader sequence. We also shortened the V1 and V2 loops to approximate early transmitter isolate sequences and the cytoplasmic tail was truncated to prevent envelope recycling. When studied as a DNA vaccine in Balb/c mice, compared to a primary codon-optimized clade C envelope DNA vaccine (p96ZM651gp140-CD5), this novel construct is up to three times more potent in driving CTL responses. Importantly this construct not only induces stronger cross-reactive cellular responses within clade C, it also induces stronger immune responses against clade B and group M envelope peptide pools than p96ZM651gp140-CD5. Epitope mapping demonstrated that EY3E1-C was able to induce clade C envelope-specific immune responses against 15 peptide pools, clade B envelope-specific immune responses against 19 peptide pools and group M envelope-specific immune responses against 16 peptide pools out of 29, respectively, indicating that a significant increase in the breadth of induced immune responses. The analysis of antibody responses suggested that vaccination of pEY3E1-C could induce a clade C envelope-specific antibody response. The cellular immune responses of pEY3E1-C could be further enhanced when the DNA was delivered by using electroporation (EP). Thus, the synthetic engineered consensus EY3E1-C gene is capable of eliciting stronger and broader CTL responses than primary clade C envelopes. This finding suggests that such synthetic immunogens could be important for examination of their potential as part of an efficient HIV DNA vaccine.

Mannose-binding lectin (MBL) plays an important role in innate immunity. The effect of low MBL levels producing variants of MBL2 gene on tuberculosis (TB) has been controversial with some studies reporting it to confer protection against the disease, whereas others estimating a susceptibility relation. Other than conducting a case-control study to evaluate the role of MBL A/B polymorphism on TB, we conducted a longitudinal study to check whether this MBL variant can influence the host response to Mycobacterium tuberculosis infection. A total of 357 TB patients (286 pulmonary TB, 71 extrapulmonary (EP) TB) and 392 healthy controls belonging to same ethnicity were included in the study. We found the mutant allele 'B' allele confers a protective role against TB in our study population. This effect was absent in EP patients. On stratification on the basis of sex, the protective role of the 'B' allele was found to be limited to females only and males reported no significant difference. No effect of MBL A/B polymorphism on sputum conversion time was reported. We conclude that MBL 'B' allele is associated with protection against TB, but no influence was found on sputum conversion rate.

A new enzymatic technique for the detachment of bacteria from soil particles was developed and applied to different soil samples taken at various sampling sites and depths. Many soil microorganisms are closely associated with the organic matrix of soil particles. They produce extracellular polymeric substances (EPS), which promote the irreversible adhesion of cells to soil particulates. To characterize the EPS, a prestaining of the soil samples with different lectins was performed. Samples from a sewage field, an urban park, a farmland, a mixed forest and garden mold were stained with a set of FITC-labelled lectins from Triticum vulgaris, Ulex europaeus, Concanavalin A and Pseudomonas aeruginosa. Based on the results, a combination of alpha-glucosidase, beta-galactosidase and a lipase was chosen for degradation of the EPS structures, followed by gentle mechanical and chemical dispersion in a modified sodium pyrophosphate buffer. The samples were fixed with formaldehyde and total cell counts were determined by DAPI staining. With the exception of the wheat field sample, this technique revealed up to 22-fold higher total cell counts for all investigated soil samples compared to the conventional detachment method, a simple dispersion with sodium pyrophosphate buffer. Efficiency of the technique was assessed by scanning electron microscopy. These images showed convincingly that the enzymatic treatment followed by sonication efficiently detached the bacteria and left the soil particles almost blank.

OBJECTIVE

To examine in a nationally representative sample (a) the differential association of specific anxiety and depressive disorders defined according to DSM-IV with pain disorder (PD) and pain symptoms, and (b) whether pain-associated anxiety and depressive disorders and their comorbidity have different implications in terms of impairment, disability, health care utilization, and substance use.

METHODS

A nationally representative community study was conducted in Germany. Symptoms, syndromes and diagnoses of mental disorders, and pain were assessed in N = 4,181 participants aged 18-65 years using the DSM-IV/M-CIDI.

RESULTS

Logistic regressions revealed that pain is associated with both specific anxiety and depressive disorders, with increasing significant odds ratios (OR) for medically explained pain symptoms (EPS; OR range: 1.9-2.0), to unexplained pain symptoms (UPS; OR range: 2.4-7.3), to PD (OR range: 3.3-14.8). PD and UPS persistently showed associations after adjusting for comorbid other anxiety and depressive disorders and physical illnesses. All types of pain, particularly PD/UPS, are associated with decreased quality of life, greater impairment in role functioning, disability, health care utilization, and substance use. Depressive disorders, even more so anxiety disorders and their comorbidity account for a substantial proportion of variance in these functional correlates.

CONCLUSIONS

Pain is strongly associated with specific anxiety and depressive disorders. In light of the individual and societal burden due to pain, and the demonstrated role of comorbid anxiety or/and depression, our results call for further investigation of the underlying mechanisms for this association as well as targeted treatments for these comorbidities.

Three cases of adult pure red cell aplasia (PRCA) ARE REPORTED. All patients proved refractory to various combinations of androgens and corticosteroids. The first case, harboring a thymoma, showed a complete clinical remission following cyclophosphamide therapy. The second and third responded similarly to either a combined cyclophosphamide + antilymphocyte globulin (ALG) treatment or to ALG administration preceded by a small dosage of cyclophosphamide, which had proved ineffective when administered alone. Serum IgG inhibitors to erythropoiesis were demonstrated in all cases by means of in vivo and/or in vitro techniques. The inhibitor(s), although directed against the erythroid marrow in both the first and third patients (PRCA type A), apparently functioned as an antibody to circulating erythropoientin (Ep) in the second case (PRCA type B). The inhibitor(s) was always absent in postremission samples. Additionally, experimental models for both types of human PRCA were established in normal rodents. The present studies support the contention that adult PRCA is an autoimmune disease. The therapeutic role of cytotoxic-immunodepressive agents in PRCA patients is confirmed. It is emphasized that ALG may represent an additional therapeutic tool in cases resistant to cyclophosphamide and/or steroids. In addition, cyclophosphamide proved effective in a patient harboring a thymoma not amenable to surgery. Finally, it is postulated that IgG serum autoantibodies, directed against either an early erythroid precursor (PRCA type A) or, more rarely, circulating Ep (PRCA type B), play a major role in the pathogenesis of the disease.

Prostaglandins (PGs) interact with specific receptors on plasma membranes to regulate myometrial activity in many species. The present study examined whether the expression of relaxant prostaglandin E receptor subtype two (EP(2)) and contractile prostaglandin F receptor (FP) mRNA in the rat uterus is changed during various states of pregnancy and regulated by steroid hormones. Expression of mRNA for EP(2) and FP receptors in the full-thickness uteri was analyzed by reverse transcription-polymerase chain reaction using specific primers. Abundance of receptor mRNA was expressed relative to beta-actin mRNA. Results showed that 1) mRNA for EP(2) receptors in the rat uterus was substantially increased during pregnancy (320%) compared with the nonpregnant state (100%, P < 0.01), and declined during labor at term (36% vs. 100% in control, P < 0.01); 2) mRNA expression for FP receptors in rat uterus was increased during pregnancy (333% vs. 100% in nonpregnant rats, P < 0. 01) and reached maximal levels during labor (515% vs. 100% in control, P < 0.01); 3) upon RU-486 treatment on Day 19 of pregnancy, uterine EP(2) receptor mRNA levels were decreased (18% vs. 100% in control, P < 0.01), and FP mRNA levels were increased (357% vs. 100% in control, P < 0.01); 4) with ICI 164384 (an antiestrogen) treatment on Day 19 of gestation, uterine FP receptor mRNA levels were decreased without effects on EP(2) receptors; 5) in ovariectomized (ovx) rats, progesterone increased EP(2) (163% vs. 100% in control, P < 0.01) and had no effects on FP receptor mRNA expression in the rat uterus; 6) estradiol increased FP receptor mRNA levels (358% vs. 100% in control, P < 0.01) and had no effects on EP(2) mRNA in the ovx rat uterus. Therefore, we conclude that steroid hormones modulate the mRNA for relaxant EP(2) and contractile FP receptors for PGs in the uterus and thus regulate uterine activity during pregnancy and labor.

Sublethal anoxia/ischemia protects against subsequent damaging insults in intact brain or hippocampal slices. To help further understand mechanisms underlying anoxic/ischemic preconditioning, we tested three hypotheses which were that: (a) anoxic preconditioning (APC) improves electrical recovery in rat hippocampal slices; (b) anoxic preconditioning requires nitric oxide (NO); and (c) anoxic preconditioning blocks mitochondrial dysfunction that occurs following re-oxygenation after anoxia. Control hippocampal slices underwent a single 'test' anoxic insult. Experimental slices were preconditioned by 3 short anoxic insults prior to the 'test' insult. Evoked potentials (EPs), and NADH redox status were recorded prior to, during and after preconditioning and/or 'test' anoxic insults. To examine the role of NO, studies sought to determine whether APC could be produced by the NO donor, DEA/NO, and whether APC could be inhibited by NO synthase (NOS) inhibitor (7-nitroindazole). EP amplitudes recovered significantly better after reoxygenation in preconditioned slices and after NO-emulated preconditioning (90.0+/-17.7% and 90.0+/-21.3%, respectively, n=9, ** p<0.01, vs. 17.0+/-7.9%, n=9, in control slices). Inhibition of NOS blocked APC protection (6.8+/-6.8%, n=9). The intensity of NADH hyperoxidation was not significantly different among groups following 'test' anoxia. These data confirm that preconditioning by anoxia improves electrical recovery after anoxia in hippocampal slices. Evidence supports that NO from constitutive hippocampal NOS may be involved in the neuroprotection afforded by preconditioning by a mechanism that does not change the apparent mitochondrial hyperoxidation after anoxia.

BACKGROUND

To evaluate whether serum concentrations of the non-placental markers vascular endothelial growth factor (VEGF), glycodelin (GLY) and progesterone (P) and the novel placental markers pregnancy-associated plasmaprotein A (PAPP-A), human placental lactogen (HPL) and leukaemia inhibiting factor (LIF) differ in ectopic pregnancy (EP) when compared with abnormal intrauterine pregnancy (aIUP).

METHODS

A prospective clinical study was conducted at the University Hospital of Larissa, Greece. The study included 50 patients admitted with failed pregnancy and suspected ectopic pregnancy that were treated with curettage or laparoscopy and classified as histologically confirmed EPs (n = 27) or histologically confirmed aIUPs (n = 21) (mean gestational age of 7.15 and 7.3 weeks, respectively). Two suspected EPs proved to be normal IUPs and were excluded. VEGF, GLY, P, beta-HCG, PAPP-A, HPL and LIF were measured by enxyme-linked immunosorbent assay (ELISA) methods in a single pre-operative blood sample.

RESULTS

The median VEGF concentration was 227.2 pg/ml in the EP group versus 107.2 pg/ml in the aIUP group (P < 0.001), with a suggested threshold value of 174 pg/ml for their differential diagnosis. LIF, P, PAPP-A, HPL and GLY serum measurements did not differ significantly between EP and aIUP.

CONCLUSIONS

VEGF serum levels might be a useful marker in differentiating between EPs and aIUPs.

Epigallocatechin-3-gallate (EGCG), a polyphenol derived from green tea, exerts antioxidant effects. Oxidative stress is one of the consequences of heat stress (HS), which also depresses performance in poultry. This experiment was conducted to elucidate the action mode of EGCG in alleviation of oxidative stress in heat-stressed quail (Coturnix coturnix japonica). A total of 180 five-week-old female Japanese quails were reared either at 22°C for 24 h/d (thermoneutral, TN) or 34°C for 8 h/d (HS) for 12 wk. Birds in both environments were randomly fed 1 of 3 diets: basal diet and basal diet added with 200 or 400 mg of EGCG/kg of diet. Each of the 2×3 factorially arranged groups was replicated in 10 cages, each containing 3 quails. Performance variables [feed intake (FI) and egg production (EP)], oxidative stress biomarkers [malondialdehyde (MDA), catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px)] and hepatic transcription factors [nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) and nuclear factor (erythroid-derived 2)-like 2 (Nrf2)] were analyzed using 2-way ANOVA. Exposure to HS caused reductions in FI by 9.7% and EP by 14.4%, increased hepatic MDA level by 84.8%, and decreased hepatic SOD, CAT, and GSH-Px activities by 25.8, 52.3, and 45.5%, respectively (P<0.0001 for all). The hepatic NF-κB expression was greater (156 vs. 82%) and Nrf2 expression was lower (84 vs. 118%) for quails reared under the HS environment than for those reared under the TN environment (P<0.0001 for both). In response to increasing supplemental EGCG level, there were linear increases in FI from 29.6 to 30.9 g/d and EP from 84.3 to 90.1%/d, linear decreases in hepatic MDA level from 2.82 to 1.72 nmol/g and Nrf2 expression from 77.5 to 123.3%, and linear increases in hepatic SOD (146.4 to 182.2), CAT (36.2 to 47.1), and GSH-Px (13.5 to 18.5) activities (U/mg of protein) and NF-κB expression (149.7 to 87.3%) (P<0.0001 for all). Two-way treatment interactions revealed that the degree of restorations in all response variables was more notable under the HS environment than under the TN environment as supplemental EGCG level was increased. Moreover, levels of oxidative biomarkers were strongly correlated with expressions of hepatic nuclear transcription factors. In conclusion, supplemental EGCG alleviates oxidative stress through modulating the hepatic nuclear transcription factors in heat-stressed quails.

Common carotid arterial (CCA) stiffness can be assessed during carotid ultrasonography, but its association with aortic stiffness, a well-defined cardiovascular risk factor, has not been clarified. This study examines the relationship between CCA and aortic stiffness. CCA pressure-strain elastic modulus (Ep) and aortic pulse wave velocity (APWV) were evaluated in 110 healthy volunteers (age 56.2 +/- 14.6 y) by B-mode and Doppler ultrasonography. CCA Ep increased linearly with age and was higher in men than in women (model r2 = 0.50, p < 0.001). APWV increased quadratically with age (model r2 = 0.54, p < 0.001), similarly for women and men. Both CCA Ep and APWV were linearly associated with systolic blood pressure (BP) (r = 0.53 and 0.46, respectively) but not with diastolic BP. A linear relationship was found between CCA Ep and APWV (APWV = 194.7 + 5.67 x Ep [model r2 = 0.42, p < 0.001]). CCA Ep was associated with APWV (p < 0.001) independent of age, gender, and BP (model r2 = 0.62, p < 0.001), and the most parsimonious model to explain APWV included CCA Ep and age (APWV = 601.73 - 15.64 x age + 0.223 x age2 + 2.69 x Ep [model r2 = 0.60, p < 0.001]). Thus, CCA Ep is moderately associated with APWV. CCA stiffness as assessed by B-mode may be useful as a surrogate for aortic stiffness.