OBJECTIVE

The aim of this study was to identify the independent effect of very preterm gestation on breast milk content of biologically active proteins (secretory immunoglobulin A (sIgA), lysozyme, lactoferrin, osteoprotegerin (OPG), leptin, adiponectin and β-endorphin (b-EP)) during the first month of lactation.

METHODS

We collected samples of transitional (6 to 8 and 13 to 15 days) and mature (20 to 22 and 27 to 29 days) milk from mothers after term (38 to 41 weeks) or very preterm (24 to 31 weeks) delivery. The levels of sIgA, lysozyme, lactoferrin, OPG, leptin, adiponectin and b-EP in the breast milk were quantified using enzyme-linked immunosorbent assay or enzyme immunoassay kits. Statistical analysis included descriptive statistics and regression analysis.

RESULTS

Sixty breast milk samples were collected from 15 mothers after very preterm (preterm breast milk, PBM) and 20 samples from 5 mothers after term (term breast milk, TBM) deliveries. Decrease in lysozyme, lactoferrin, OPG, leptin, adiponectin and b-EP but no change in sIgA was recorded during the first month of lactation in both TBM and PBM. The IgA, lysozyme and adiponectin were higher in PBM than in TBM, whereas concentrations of lactoferrin, OPG and leptin were higher in TBM than in PBM (P<0.05 to 0.0001). A similar pattern was seen in the lysozyme, leptin and adiponectin concentration in mature milk. Increased b-EP levels in breast milk were associated with the vaginal mode of delivery but not gestational age.

CONCLUSIONS

Although a similar pattern of change was observed in the breast milk bioactive proteins during the first month of lactation after term and very preterm gestation, PBM is a better source of factors with antibacterial/anti-inflammatory activities but is constantly deficient in leptin, which is involved in neuroendocrine regulation.

The mechanism by which ethanol induces <em>beta</em>-endorphin (<em>beta</em>-<em>EP</em>) neuronal death during the developmental period was determined using fetal rat hypothalamic cells in primary cultures. The addition of ethanol to hypothalamic cell cultures stimulated apoptotic cell death of <em>beta</em>-<em>EP</em> neurons by increasing caspase-3 activity. Ethanol lowered the levels of adenylyl cyclase (AC)7 mRNA, AC8 mRNA, and/or cAMP in hypothalamic cells, whereas a cAMP analog blocked the apoptotic action of ethanol on <em>beta</em>-<em>EP</em> neurons. The AC inhibitor dideoxyadenosine (DDA) increased cell apoptosis and reduced the number of <em>beta</em>-<em>EP</em> neurons, and it potentiated the apoptotic action of ethanol on these neurons. <em>beta</em>-<em>EP</em> neurons in hypothalamic cultures showed immunoreactivity to transforming growth factor-<em>beta</em>1 (TGF-<em>beta</em>1) protein. Ethanol and DDA increased TGF-<em>beta</em>1 production and/or release from hypothalamic cells. A cAMP analog blocked the activation by ethanol of TGF-<em>beta</em>1 in these cells. TGF-<em>beta</em>1 increased apoptosis of <em>beta</em>-<em>EP</em> neurons, but it did not potentiate the action of ethanol or DDA actions on these neurons. TGF-<em>beta</em>1 neutralizing antibody blocked the apoptotic action of ethanol on <em>beta</em>-<em>EP</em> neurons. Determination of TGF-<em>beta</em>1-controlled cell apoptosis regulatory gene levels in hypothalamic cell cultures and in isolated <em>beta</em>-<em>EP</em> neurons indicated that ethanol, TGF-<em>beta</em>1, and DDA similarly alter the expression of these genes in these cells. These data suggest that ethanol increases <em>beta</em>-<em>EP</em> neuronal death during the developmental period by cellular mechanisms involving, at least partly, the suppression of cAMP production and activation of TGF-<em>beta</em>1-linked apoptotic signaling.

OBJECTIVE

Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) with high temporal resolution enables the detection of microcirculation variables amplitude A and exchange rate constant k(ep). In this study, the prognostic value of the DCE-MRI variables for overall survival and event-free survival in patients with progressive multiple myeloma was investigated.

METHODS

Between 1999 and 2001, 65 patients with progressive or relapse of multiple myeloma requiring therapy were investigated with DCE-MRI of the lumbar spine before start of therapy. The contrast uptake was quantified using a two-compartment model with the output variables amplitude A and exchange rate constant k(ep) reflecting bone marrow microcirculation. The estimated median follow-up was 56 months. Event-free survival and overall survival were investigated for DCE-MRI variables and for established prognosis variables (beta(2)-microglobulin, lactate dehydrogenase, albumin, and age).

RESULTS

Using a multivariate Cox regression model, beta(2)-microglobulin and amplitude A of DCE-MRI were identified as statistically significant prognostic variable of event-free survival with Ps of 0.01 and 0.02, respectively. A statistical correlation of DCE-MRI variables with overall survival could not be found. The multivariate analysis of beta(2)-microglobulin, age, lactate dehydrogenase, and albumin revealed beta(2)-microglobulin as statistically significant prognostic factor for overall survival in this group of patients (P < 0.001).

CONCLUSIONS

This analysis identifies contrast-enhanced DCE-MRI variable amplitude A reflecting increased bone marrow microcirculation and angiogenesis as a novel and possibly useful prognostic factor in patients with multiple myeloma. Prospective studies are currently done to further investigate this functional variable for prognosis and stratification of myeloma patients.

OBJECTIVE

The aim of this prospective study was to observe immunophenotypic patterns in patients with noninflammatory chronic pelvic pain syndrome (Cat IIIB CPPS) for further description and as possible surrogate markers for diagnosis and treatment.

METHODS

Eighty-eight patients with a referral diagnosis of chronic prostatitis underwent fractionated urinary cultures including expressed prostate secretion (EPS) and ejaculate analysis twice on two occasions. Monthly serum analyses included C3c, C4, IL-1alpha, sIL-2R, and IL-6. One hundred samples from healthy individuals were used as the control group for serum analysis. Monthly ejaculate testing was done for IgG, IgA, IgM, IL-1alpha, sIL-2R, and IL-6. The control group for ejaculate analysis was composed of 96 normal ejaculates (according to the WHO criteria). Immunohistochemical detection of CD3 cells (T lymphocytes) and CD20 cells (B lymphocytes) was performed in 71 biopsy cylinders of Cat IIIB CPPS patients and in 25 prostate biopsy cylinders of men without symptoms or obstruction.

RESULTS

Complete sampling of urinary, serum and ejaculate specimens was achieved in 50/88 (57%) patients. Cat IIIB CPPS was observed in 44/50 (88%) patients. Intra-acinar T-lymphocytic infiltrates were dominated by T cytotoxic cells (p = 0.05). Immunohistochemical studies showed inflammatory expression in serum complement, serum interleukin, and ejaculate interleukin concentrations in relation to the presence of large numbers of T cells (all p values < or =0.01). No difference was found in the proportion of B lymphocytes in patients with Cat IIIB CPPS compared to the control group. Serum and ejaculate IL-6 and ejaculate IgA increased significantly and dropped again, correlating with a release of clinical symptoms.

CONCLUSIONS

Interleukin, complement and immunoglobulin determinations in serum and ejaculate reveal an inflammatory process even in Cat IIIB CPPS. The findings of intra-acinar T-cell-rich infiltrates and the associated inflammatory reaction may be a significant advance in defining Cat IIIB CPPS caused by a possible autoimmune component. Serum and ejaculate IL-6 and ejaculate IgA are possible surrogate markers for the diagnosis and treatment of Cat IIIB CPPS.

The effects of ovarian hormones on glucose and fatty acid oxidation during exercise were investigated in adult female ovariectomized rats. Rats subdivided into 3 groups received intraperitoneal injections of hormones or sesame oil for 8 days. Estrogen (E) treated rats received 17-beta estradiol in daily doses of 2 micrograms. Estrogen and progesterone treated rats (EP) received 17-beta estradiol in daily doses of 2 micrograms and 2 mg, respectively. Control rats (S) received sesame oil alone. After an overnight fast, rats ran at the speed of 25 m.min-1 for 60 min. [U-14C]glucose or [1-14C]palmitate was injected into rats at 5 min of exercise and before 10 min of exercise, respectively. Expired 14CO2 was collected using bottomless chamber on a treadmill belt. No significant differences were found in mean blood glucose, lactate and plasma free fatty acid concentrations after the exercise. Until the end of the exercise 34.7 +/- 2.6 (E, n = 5), 40.8 +/- 2.9 (EP, n = 5) and 43.7 +/- 3.5% (S, n = 6) (mean +/- SE) of 14C which was injected as 14C-glucose was recovered as 14CO2. During 60 min of the exercise 27.5 +/- 1.0 (E, n = 7), 19.8 +/- 2.7 (EP, n = 6) and 25.0 +/- 1.9% (S, n = 6) of 14C which was injected as 14C-palmitate was recovered as 14CO2. A significant difference was found in this rate between E and EP (P less than 0.05). It was concluded that estrogen treatment stimulated fatty acid oxidation compared with the estrogen plus progesterone treatment and tended to inhibit glucose oxidation during prolonged exercise.

BACKGROUND

Chronic inflammation is commonly observed in benign prostate hyperplasia (BPH), and prostate tissue often contains increased inflammatory infiltrates, including T cells and macrophages. Cytokines are not only key mediators of inflammation but may also play important roles in the initiation and progression of BPH.

METHODS

In order to determine what cytokines might be involved in prostatic enlargement, expressed prostatic secretions (EPS) from ex vivo prostates were analyzed by human cytokine antibody microarray and ELISA. Prostate epithelial cells (PrEC) and prostate stromal cells (PrSC) were used for ELISA, proliferation, and Western blot assays.

RESULTS

Monocyte chemotactic protein-1 (MCP-1/CCL2) was one of the most elevated proteins in secretions from large prostate glands. PrSC were found to secrete MCP-1; Western blotting showed that both PrSC and PrEC express the MCP-1 receptor CCR2 which by RT-PCR was the CCR2b isoform. Proliferation assays showed that MCP-1 stimulates the proliferation of PrEC, but not PrSC, and that a specific MCP-1 antagonist (RS102895) suppressed this effect. Conditioned medium from PrSC stimulated the proliferation of PrEC as well, an effect completely inhibited by both RS102895 and a neutralizing anti-MCP-1 monoclonal antibody. The inflammatory cytokines interleukin (IL)-1 beta, interferon-gamma, and IL-2 enhanced the secretion of MCP-1 from PrEC and PrSC. In addition, MCP-1 levels in EPS correlated with mRNA levels of the macrophage marker CD68 in the same secretions.

CONCLUSIONS

The cytokine MCP-1, of apparent prostatic stromal cell origin, may play an important role in prostatic enlargement and BPH, and is a candidate biomarker for these pathologic processes.

Type II collagen-induced arthritis (CIA) in mice is an autoimmune experimental model for rheumatoid arthritis. Susceptibility to CIA is associated with certain major histocompatibility complex class II haplotypes. The two very closely related haplotypes H-2q and H-2p differ in susceptibility to CIA. Only mice of H-2q (DBA/1, B10G strains) but not mice of H-2p-expressing strains (like strain B10P) develop CIA and an autoimmune response to type II collagen (CII) after immunization with CII. In contrast to H-2p, the H-2q haplotype does not express I-E molecules. The purpose of the present study was to identify, at the molecular level, the structures on major histocompatibility complex class II molecules determining susceptibility to CIA and CII responsiveness. We first excluded the possible suppressive involvement of Ep or Ap molecules by showing that F1 hybrids between H-2p and H-2q haplotype strains, expressing Ep and Ap, are responders to CII and fully susceptible to CIA. Secondly, because A alpha chains appear identical, we sequenced the A beta first-domain exons of p and q allotypes and found only four diverging amino acids in the predicted amino acid sequence. These variable residues were closely located at positions 85, 86, 88, and 89 at the end of the postulated alpha-helix, which is of importance for interactions with the antigenic peptide and the T-cell receptor. We suggest that this region is a critical major histocompatibility complex restriction site for CIA and CII responsiveness in H-2q mice as compared with H-2p mice. The CIA will now be an excellent autoimmune model for studies on interactions between autoantigenic peptide, autoreactive T cells, and a particular major histocompatibility complex molecule, as has been postulated to be the initial event also in rheumatoid arthritis.

The catalytic mechanism for the enzymatic hydrolysis of a series of paraoxon analogues by the phosphotriesterase from Pseudomonas diminuta has been determined. The Brønsted plots relating the pKa of the leaving group to the observed kinetic parameters, Vmax and V/Km, are both nonlinear. This observation is consistent with a change in the rate-limiting step from chemical to physical events as the pKa of the leaving group is decreased. This conclusion is confirmed by the effects of solvent viscosity on Vmax and V/Km for the same series of analogues. The data were fitted to the scheme E k1A in equilibrium k2 EA k3----EP k7----E'P k9----E + products where EA is the enzyme-substrate complex, EP is the enzyme-product complex, E'P is the enzyme-product complex after a viscosity-independent unimolecular reaction, and the values for k1, k2, k7, and k9 are 4.1 X 10(7) M-1 s-1, 2550 s-1, 3370 s-1, and 5940 s-1, respectively. The magnitude of the chemical step, represented by k3, is dependent on the pKa of the leaving group phenol as predicted by the Brønsted equation (log k3 = beta pKa + C) where beta = -1.8 and the constant (C) = 17.7. The magnitude of beta indicates that the transition state for substrate hydrolysis is very product-like.

Increased expression of multidrug resistance efflux pump (MDR-EP) genes in clinical isolates of Staphylococcus aureus occurs frequently, but its temporal and geographic variability is unknown. Such strains may contaminate the hospital environment, posing an infection control problem. Nearly 700 clinical isolates from different geographic locales as well as 91 environmental isolates recovered from two Detroit hospitals were studied. Ethidium bromide (EtBr) minimum inhibitory concentration (MIC), quantitative expression of all characterised chromosomal MDR-EP genes, and the presence of qacA/B and smr were determined for all strains. In addition, for norA- and/or mepA-overexpressing strains, the spa type was established. MDR-EP gene overexpression varied temporally and geographically, and overexpressing strains were present in the hospital environment. Increased expression of norA was associated with meticillin resistance and spa type t002, a rare type among control strains, consistent with widespread dissemination of a norA-overexpressing, meticillin-resistant S. aureus (MRSA) clone. Clonal spread also played a role for spa type t008, mepA-overexpressing, meticillin-susceptible strains. An EtBr MIC of ≤12.5 μg/mL was highly specific (>90%) in identifying strains lacking MDR-EP gene overexpression.

The response of plasma beta-endorphin (beta-EP) and adrenocorticotropin (ACTH) was studied in seven well-trained (T) young endurance athletes and seven untrained (UT) age- and weight-matched males during treadmill exercise. Subjects ran continuously for 7 min at 60% VO2max, 3 min at 100% VO2max and 2 min at 110% VO2max. Arterialized blood was obtained periodically from a cannulated heated (41 degrees C) hand vein. Plasma beta-EP was measured by radio-immunoassay (RIA) which incorporated an antibody that did not cross-react (less than 1.5%) with beta-lipotropin. Plasma beta-EP was similar between groups at rest (T = 4.3 +/- 0.8 fmol ml-1, mean +/- SE, UT = 3.3 +/- 0.6 fmol ml-1) and did not change at the 60% VO2max stage. Beta-endorphin significantly increased at 100% VO2max with both groups responding similarly. A further increase occurred at 110% VO2max (T = 10.8 + 2.0 and UT = 6.6 + 1.0 fmol ml-1, P less than 0.05 for between group differences). This between group difference persisted 1 min after exercise when the highest beta-EP levels were reached (T = 18.7 +/- 4.7 and UT = 12.8 +/- 3.1 fmol ml-1, P less than 0.05). Plasma ACTH responses were similar to beta-EP with the highest values (T = 61.5 +/- 7.2, UT = 45.7 +/- 6.8 fmol ml-1, P less than 0.05 for between group differences) occurring at 1 min post-exercise. A positive correlation, r = 0.85, P less than 0.05, was found between beta-EP and ACTH using the 1 min post-exercise values. The enhanced response of beta-EP and ACTH in T may indicate a training-induced adaptation which increases the response capacity to extreme levels of stress.

The present study examines the influence of destruction of the medio-basal arcuate hypothalamus (MBH), the primary site of synthesis of central pools of beta-endorphin (beta-EP), upon the aversive properties of naloxone in a conditioned place preference paradigm. Bilateral radiofrequency lesions of the MBH resulted in a pronounced fall in levels of immunoreactive beta-EP in the brain. Lesioned rats, in contrast to non-operated animals, showed a clear reduction in the conditioned place aversion produced by naloxone. However, they showed no loss of the conditioned preference produced by the mu-selective opioid receptor agonist, morphine, or the conditioned aversion produced by the kappa-selective agonist, U50-488. In contrast to the effect of the lesions, suppression of circulating beta-EP by dexamethasone treatment failed to influence conditioning produced by naloxone. Thus, the data indicate that the aversive properties of naloxone are attenuated by disruption of central (but not peripheral) beta-EP activity. We suggest that these properties of naloxone reflect an antagonism of beta-EP activity in the brain. In addition, the data indicate that differing mechanisms underlie the aversive actions of naloxone as compared to U50-488.

We hypothesized that estrogen administration would attenuate skeletal muscle neutrophil infiltration, indices of muscle membrane disruption, and muscle calpain activity shortly after the termination of exercise. Ovariectomized female rats were implanted with either an estogen pellet (25 mg beta-estradiol) or a placebo pellet. Two weeks postimplant, animals were killed either at rest or 1 h after running exercise (60 min at 21 m x min(-1), 12% grade). The 4 experimental groups (n = 12) used were: unexercised placebo (UP), unexercised estrogen (UE), exercised placebo (EP), and exercised estrogen (EE). Blood samples were analyzed for creatine kinase (CK) activity and estradiol content. Plantaris and gastrocnemius muscles were removed and histochemical determination of neutrophil content or biochemical determination of myeloperoxidase (MPO), glucose-6-phosphate dehydrogenase (G6PD), and calpain-like activity determined. Estrogen supplemented animals had 10-20-fold higher circulating estradiol levels than placebo animals. EP animals had significantly higher (P < 0.05) circulating CK activities than EE or unexercised animals. Muscle neutrophil concentrations were significantly (P < 0.01) elevated in EP and EE groups compared with unexercised controls, with EP muscle neutrophil levels also being over 60% greater (P < 0.05) than in EE animals. EP animals also had higher (P < 0.05) muscle MPO activities than unexercised or EE animals. Muscle G6PD activities were not significantly different between any groups. Muscle caplain-like activities were 80% higher (P < 0.01) in EP animals than EE animals with calpain-like activities in EE animals similar to unexercised groups. These results indicate that estrogen supplementation in ovariectomized rats attenuated 1-h post-exercise serum CK activities, muscle neutrophil infiltration, MPO activities, and calpain-like activities when compared with exercised, unsupplemented animals. This supports the possibility of a relationship between estrogen, calpain dependent production of neutrophil chemo-attractant peptides, and 1-h post-exercise skeletal muscle neutrophil infiltration.

BACKGROUND

Sex steroid hormone receptor (SHR) dynamics are well-documented in human endometrium but have not been comprehensively studied in Fallopian tube (FT).

OBJECTIVE

The aim of the study was to compare expression patterns and hormonal regulation of SHR in FT with that described in endometrium and to determine whether SHR expression is altered in FT of women with ectopic pregnancy (EP).

METHODS

Tissue was analyzed and cultured.

METHODS

Women undergoing surgery for benign gynecological conditions (n = 14) and EP (n = 6) participated in the study.

METHODS

Quantitative RT-PCR and immunohistochemistry were used to determine SHR mRNA expression and protein localization, respectively. SHR levels were measured in tubal explant cultures stimulated with estrogen and progestogen.

RESULTS

ERalpha and ERbeta mRNAs were constitutively expressed in FT during the menstrual cycle. PR-AB and PR-B mRNAs were decreased in midluteal phase compared to follicular phase. ERalpha, PR-AB, and PR-B mRNAs were down-regulated in human FT in vitro by treatment with progestogen. ERalpha, ERbeta1, ERbeta2, PR, and AR proteins localized to cell nuclei of epithelium, stroma, and smooth muscle of nonpregnant FT. In FT from women with EP, PR-B mRNA was decreased when compared to midluteal FT, and ERalpha protein was not detected.

CONCLUSIONS

SHR expression in FT is different from that observed in endometrium recovered at similar stages of the menstrual cycle, and expression in FT from women with EP is also altered compared with normal FT. These data are an important benchmark for furthering the understanding of normal human FT physiology, changes in expression of SHR in FT in response to progesterone, and disorders of FT function, such as EP.

OBJECTIVE

Inferior vena cava ultrasound (IVC-US) is a noninvasive bedside tool to assess intravascular volume status. This study set out to investigate the interrater reliability of IVC-US by bedside clinician sonographers and determine whether alternative methods of IVC-US such as B-mode and visual estimation are equally reliable to traditional M-mode.

METHODS

A convenience sample of adult emergency department (ED) patients was prospectively enrolled. Each patient underwent IVC-US by two different emergency physicians (EPs), each of whom first performed visual estimation of IVC percent collapse and of volume status, followed by caliper measurements in M-mode and B-mode. EPs were blinded to patient data and to the other sonographer's results. For each technique, interrater reliability was determined between the two EPs' assessments using intraclass correlation coefficients (ICC) for continuous data and Cohen's weighted kappa for categorical data. In addition, analysis was performed on M-mode diameter measurements to determine the relationship between sonographer and patient characteristics on interrater reliability.

RESULTS

Five EPs performed 92 US exams on 46 patients. Using M-mode, the ICC for maximum IVC diameter was 0.81 (95% confidence interval [CI]=0.67 to 0.89), and for minimum diameter was 0.77 (95% CI=0.62 to 0.87). There were no statistically significant differences between the caliper methods used for IVC measurements (M-mode diameter, B-mode diameter, or B-mode area). Agreement for visually estimated IVC collapse (0.60, 95% CI=0.36 to 0.76) was similar to agreement for calculated M-mode IVC collapse index (0.52, 95% CI=0.27 to 0.71). Cohen's weighted kappa for volume status based on visual estimation of IVC filling (size, shape, and collapse) was 0.64 (95% CI=0.53 to 0.73). ICC values for M-mode diameter measurements were significantly higher in studies involving patients who were noneuvolemic and studies in which sonographers had each performed at least five prior IVC-US.

CONCLUSIONS

Emergency physicians' US measurements of IVC diameter have a high degree of interrater reliability. IVC percent collapse by visual estimation or based on caliper measurements have lower, but still moderate to good reliability. The use of the visual estimation technique should be considered by clinicians who have learned to obtain measured parameters of IVC filling because it is equally reliable to traditional M-mode and can be performed more rapidly.

The cell surface polysaccharides of wild-type Bradyrhizobium japonicum USDA 110 and a nonnodulating mutant, strain HS123, were analyzed. The capsular polysaccharide (CPS) and exopolysaccharide (EPS) of the wild type and the mutant strain do not differ in their sugar composition. CPS and EPS are composed of mannose, 4-O-methylgalactose/galactose, glucose, and galacturonic acid in a ratio of 1:1:2:1, respectively. H nuclear magnetic resonance spectra of the EPS and CPS of the wild type and mutant strain are very similar, but not identical, suggesting minor structural variation in these polysaccharides. The lipopolysaccharides (LPS) of the above two strains were purified, and their compositions were determined. Gross differences in the chemical compositions of the two LPS were observed. Chemical and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses indicated that strain HS123 is a rough-type mutant lacking a complete LPS. The LPS of mutant strain HS123 is composed of mannose, glucose, glucosamine, 2-keto-3-deoxyoctulosonic acid, and lipid A. The wild-type LPS is composed of fucose, xylose, arabinose, mannose, glucose, fucosamine, quinovosamine, glucosamine, uronic acid, 2-keto-3-deoxyoctulosonic acid, and lipid A. Preliminary sugar analysis of lipid A from B. japonicum identified mannose, while traces of glucosamine were detected. 3-Hydroxydodecanoic and 3-hydroxytetradecanoic acids formed a major portion of the fatty acids in lipid A. Lesser quantities of nonhydroxylated 16:0, 18:0, 22:0, and 24:0 acids also were detected.

The structure and microbial communities of biofilms developing on cross-flow nanofiltration (NF) membranes at different temperatures (20, 25 or 34 degrees C) and operation lengths (8h-24days) were studied. Feedwater comprised tertiary quality wastewater effluent or synthetic media mimicking effluents of intermediate quality. After each run, the membranes were autopsied for bacterial enumeration, bacterial community composition and microscopy visualization (SEM, CLSM and AFM/NSOM). Community composition was analyzed by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) coupled with sequence analysis of 16S rRNA gene fragments from dominant bands. Deposition of polysaccharides and initial bacterial colonization were observed within 8h, whereas developed biofilms markedly affecting membrane permeability were evident from days 2-3 onwards. Regardless of applied conditions, the heterotrophic plate counts in the biofilm were 3-4x10(6)CFU/cm(2) and the thickness of the biofouling layer was 20-30microm. From a total of 22 sequences obtained from 14 independent experiments, most species identified were Gram negative (19 of 22 sequences). Proteobacteria were found to be a prevalent group in all cases (16 of 22 sequences) and among it, the beta-subclass was the most predominant (8 sequences), followed by the gamma-subclass (5 sequences). Pseudomonas/Burkholderia, Ralstonia, Bacteroidetes and Sphingomonas were the dominant groups found in most cases. Even though the microbial population might be important with respect to biofouling patterns, membrane permeability decline seems to be more substantially influenced by the formation and accumulation of exopolymeric substances (EPS).

BACKGROUND

Only few studies have assessed safety of in utero exposure to glatiramer acetate (GA). Following a previous study assessing the safety of interferon beta (IFNB) pregnancy exposure in multiple sclerosis (MS), we aimed to assess pregnancy and fetal outcomes after in utero exposure to GA, using the same dataset, with a specific focus on the risk of spontaneous abortion.

METHODS

We recruited MS patients, prospectively followed-up in 21 Italian MS Centres, for whom a pregnancy was recorded in the period 2002-2008. Patients were divided into 2 groups: drug-exposed pregnancies (EP: suspension of the drug less than 4 weeks from conception); non-exposed pregnancies (NEP: suspension of the drug at least 4 weeks from conception or never treated pregnancies). All the patients were administered a structured interview which gathered detailed information on pregnancy course and outcomes, as well as on possible confounders. Multivariate logistic and linear models were used for treatment comparisons.

RESULTS

Data on 423 pregnancies were collected, 17 were classified as EP to GA, 88 as EP to IFNB, 318 as NEP. Pregnancies resulted in 16 live births in the GA EP, 75 live births in the IFNB EP, 295 live births in the NEP. GA exposure was not significantly associated with an increased risk of spontaneous abortion (OR = 0.44;95% CI 0.044-4.51;p = 0.49). Mean birth weight and length were not significantly different in pregnancies exposed to GA than in non exposed pregnancies (p = 0.751). The frequency of preterm delivery, observed in 4 subjects exposed to GA (25% of full term deliveries), was not significantly higher in pregnancies exposed to GA than in those non exposed (p>> 0.735). These findings were confirmed in the multivariate analysis. There were neither major complications nor malformations after GA exposure.

CONCLUSIONS

Data in our cohort show that mother's GA exposure is not associated with a higher frequency of spontaneous abortion, neither other negative pregnancy and fetal outcomes. Our findings point to the safety of in utero GA exposure and can support neurologists in the therapeutic counselling of MS women planning a pregnancy.

OBJECTIVE

To evaluate the capability of the exopolysaccharides (EPS) produced by lactobacilli and bifidobacteria from human and dairy origin to antagonize the cytotoxic effect of bacterial toxins.

RESULTS

The cytotoxicity of Bacillus cereus extracellular factors on Caco-2 colonocytes in the presence/absence of the EPS was determined by measuring the integrity of the tissue monolayer and the damage to the cell membrane (extracellular lactate dehydrogenase activity). Additionally, the protective effect of EPS against the haemolytic activity of the streptolysin-O was evaluated on rabbit erythrocytes. The EPS produced by Bifidobacterium animalis ssp. lactis A1 and IPLA-R1, Bifidobacterium longum NB667 and Lactobacillus rhamnosus GG were able to counteract the toxic effect of bacterial toxins on the eukaryotic cells at 1mg ml(-1) EPS concentration. The EPS A1 was the most effective in counteracting the effect of B. cereus toxins on colonocytes, even at lower doses (0·5mg ml(-1) ), whereas EPS NB667 elicited the highest haemolysis reduction on erythrocytes.

CONCLUSIONS

The production of EPS by lactobacilli and bifidobacteria could antagonize the toxicity of bacterial pathogens, this effect being EPS and biological marker dependent.

CONCLUSIONS

This work allows gaining insight about the mechanisms that probiotics could exert to improve the host health.

Streptococcus thermophilus Sfi6 produces a texturizing exopolysaccharide (EPS) consisting of a -->3)[alpha-D-Galp-(1-->6)]-beta-D-Glcp-(1-->3)-alpha-D-GalpNAc-(1->> 3)-beta-D-Galp-(1->> repeating unit. We previously identified and analyzed a 14.5-kb gene cluster from S. thermophilus Sfi6 consisting of 13 genes responsible for its EPS production. Within this gene cluster, we found a central region of genes (epsE, epsF, epsG, and epsI) that showed similarity to glycosyltransferases. In this study, we investigated the sugar specificity of these enzymes. EpsE catalyzes the first step in the biosynthesis of the EPS repeating unit. It exhibits phosphogalactosyltransferase activity and transfers galactose onto the lipophilic carrier. The second step is fulfilled by EpsG, which transfers an alpha-N-acetylgalactosamine onto the first beta-galactoside. The activity of EpsF was determined by characterizing the EPS produced by an S. thermophilus epsF deletion mutant. This EPS consisted of the monosaccharides Gal, Glc, and GalNAc in an approximately equimolar ratio, thus suggesting that epsF codes for the branching galactosyltransferase. epsI probably codes for the beta-1,3-glucosyltransferase, since it is the only glycosyltransferase to which no gene has been assigned and it exhibits similarity to other beta-glycosyltransferases. EpsE shows the conserved features of phosphoglycosyltransferases, whereas EpsF and EpsG exhibit the primary structure of alpha-glycosyltransferases, belonging to glycosyltransferase family 4, whose members are conserved in all major phylogenetic lineages, including the Archaea and Eukaryota.

Enhanced prostaglandin production via upregulated cyclooxygenase-2 (COX-2) expression is a likely contributing factor in ultraviolet B (UVB)-induced non-melanoma skin cancer (NMSC), which consists primarily of squamous cell carcinoma (SCC) and basal cell carcinoma (BCC). The four E prostanoid (EP) receptors, designated EPEPB-induced SCC and BCC, as well as human NMSC from sun-exposed sites, to investigate the expression of EP receptors during UVB-induced tumorigenesis. We observed that UVB-induced murine SCC are associated with markedly altered expression patterns of the EP receptors when compared with non-irradiated skin. In contrast, expression of all EP receptors was largely absent in UVB-induced murine BCC. We also observed expression of all four EP receptors in human SCC, with altered expression of their mRNA levels as compared with adjacent tumor-free skin. Consistent with our murine studies, no EP receptor expression was detected in human BCC, and their mRNA expression levels showed no change from the adjacent non-tumor-bearing skin. These data suggest that altered EP receptor expression may play a differential role in the development of UVB-induced SCC and BCC in murine and human skin.

OBJECTIVE

Detailed analysis of phenotypic and molecular genetic aspects of Dok-7 myasthenia in 16 patients.

METHODS

We assessed our patients by clinical and electromyographic studies, by intercostal muscle biopsies for in vitro microelectrode analysis of neuromuscular transmission and quantitative electron microscopy EM of 409 end plates (EPs), and by mutation analysis, and expression studies of the mutants.

RESULTS

The clinical spectrum varied from mild static limb-girdle weakness to severe generalized progressive disease. The synaptic contacts were single or multiple, and some, but not all, were small. In vitro microelectrode studies indicated variable decreases of the number of released quanta and of the synaptic response to acetylcholine; acetylcholine receptor (AChR) channel kinetics were normal. EM analysis demonstrated widespread and previously unrecognized destruction and remodeling of the EPs. Each patient carries 2 or more heteroallelic mutations: 11 in genomic DNA, 7 of which are novel; and 6 identifiable only in complementary DNA or cloned complementary DNA, 3 of which are novel. The pathogenicity of the mutations was confirmed by expression studies. Although the functions of Dok-7 include AChR beta-subunit phosphorylation and maintaining AChR site density, patient EPs showed normal AChR beta-subunit phosphorylation, and the AChR density on the remaining junctional folds appeared normal.

CONCLUSIONS

First, the clinical features of Dok-7 myasthenia are highly variable. Second, some mutations are complex and identifiable only in cloned complementary DNA. Third, Dok-7 is essential for maintaining not only the size but also the structural integrity of the EP. Fourth, the profound structural alterations at the EPs likely contribute importantly to the reduced safety margin of neuromuscular transmission.

BACKGROUND

Congenital myasthenic syndromes (CMS) are disabling but treatable disorders. Anticholinesterase therapy is effective in most of them, but is contraindicated in endplate (EP) acetylcholinesterase (AChE) deficiency, the slow-channel syndrome, Dok-7 myasthenia, and β(2) -laminin deficiency, and is not useful in CMS due to defects in muscle-specific kinase (MuSK), agrin, and plectin. EP AChE, Dok-7, and β(2)-laminin deficiencies respond favorably to ephedrine, but ephedrine can no longer be prescribed in the USA.

METHODS

We used albuterol, another sympathomimetic agent, to treat 3 patients with EP AChE deficiency and 15 with Dok-7 myasthenia. Response to therapy was evaluated by a 9-point questionnaire pertaining to activities of daily life.

RESULTS

Comparison of the pre- and posttreatment responses indicated a beneficial response to albuterol (P < 0.001) in both patient groups. The adverse effects of therapy were like those of ephedrine.

CONCLUSIONS

Our observations should spur controlled, prospective clinical trials of albuterol in these as well as other CMS.

Six mitochondrial genome sequences, showing strong similarity to the glucocorticoid responsive element consensus sequence (GRE), four localized within the cytochrome c oxidase (COX) subunit I and II genes (GREs I-IV) and two within the D-loop region (GREs a and b) have been examined as binding sites of glucocorticoid receptor (GR) from rat liver cytosol. Purified GR from rat liver cytosol binds with high specificity to all potential mitochondrial GREs, as shown by filter retention and gel shift assays. Specific binding of protein(s), present in a mitochondrial extract from dexamethasone-induced mice, to all six putative mitochondrial GREs was also documented by the same methodology. Both purified GR and protein(s) from mitochondrial extract give the same band in the gel retardation assay. Using monospecific anti-glucocorticoid receptor polyclonal antibody (EP), a supershift of the gel retarded protein-DNA band was obtained. These results demonstrate that the mitochondrial genome sequences examined have characteristics of GREs, since they show the capacity to specifically bind the respective receptor protein. These findings support the hypothesis that the mitochondrial genome is a primary site of action of steroid and thyroid hormones (Sekeris C.E.: The mitochondrial genome: a possible primary site of action of steroid hormones, In vivo 4 (1990) 317-320).

φRSM1 and φRSM3 (φRSM phages) are filamentous phages (inoviruses) that infect Ralstonia solanacearum, the causative agent of bacterial wilt. Infection by φRSM phages causes several cultural and physiological changes to host cells, especially loss of virulence. In this study, we characterized changes related to the virulence in φRSM3-infected cells, including (i) reduced twitching motility and reduced amounts of type IV pili (Tfp), (ii) lower levels of β-1,4-endoglucanase (Egl) activity and extracellular polysaccharides (EPS) production, and (iii) reduced expression of certain genes (egl, pehC, phcA, phcB, pilT, and hrpB). The significantly lower levels of phcA and phcB expression in φRSM3-infected cells suggested that functional PhcA was insufficient to activate many virulence genes. Tomato plants injected with φRSM3-infected cells of different R. solanacearum strains did not show wilting symptoms. The virulence and virulence factors were restored when φRSM3-encoded orf15, the gene for a putative repressor-like protein, was disrupted. Expression levels of phcA as well as other virulence-related genes in φRSM3-ΔORF15-infected cells were comparable with those in wild-type cells, suggesting that orf15 of φRSM3 may repress phcA and, consequently, result in loss of virulence.