BACKGROUND

The targeting of the immune system through immunotherapies to prevent tumor tolerance and immune suppression are at the front lines of breast cancer treatment and research. Human and laboratory studies have attributed breast cancer progression and metastasis to secondary organs such as the bone, to a number of factors, including elevated levels of prostaglandin E2 (PGE2) and the enzyme responsible for its production, cyclooxygenase 2 (COX2). Due to the strong connection of COX2 with immune function, we focused on understanding how variance in COX2 expression manipulates the immune profile in a syngeneic, and immune-competent, mouse model of breast cancer. Though there have been correlative findings linking elevated levels of COX2 and Tregs in other cancer models, we sought to elucidate the mechanisms by which these immuno-suppressive cells are recruited to breast tumor and the means by which they promote tumor tolerance.

RESULTS

To elucidate the mechanisms by which exacerbated COX2 expression potentiates metastasis we genetically manipulated non-metastatic mammary tumor cells (TM40D) to over-express COX2 (TM40D-COX2). Over-expression of COX2 in this mouse breast cancer model resulted in an increase in bone metastasis (an observation that was ablated following suppression of COX2 expression) in addition to an exacerbated Treg recruitment in the primary tumor. Interestingly, other immune-suppressive leukocytes, such as myeloid derived suppressor cells, were not altered in the primary tumor or the circulation. Elevated levels of PGE2 by tumor cells can directly recruit CD4+CD25+ cells through interactions with their <em>EP</em>2 and/or <em>EP</em>4 receptors, an effect that was blocked using anti-PGE2 antibody. Furthermore, increased Treg recruitment to the primary tumor contributed to the greater levels of apoptotic CD8+ T cells in the TM40D-COX2 tumors.

CONCLUSIONS

Due to the systemic effects of COX2 inhibitors, we propose targeting specific EP receptors as therapeutic interventions to breast cancer progression.

Parameters of semen quality, seminal plasma indicators of secretory function of the prostate and seminal vesicles, sex hormones in serum, and biomarkers of lead, cadmium, copper, zinc, and selenium body burden were measured in 240 Croatian men 19-52 years of age. The subjects had no occupational exposure to metals and no known other reasons suspected of influencing male reproductive function or metal metabolism. After adjusting for age, smoking, alcohol, blood cadmium, and serum copper, zinc, and selenium by multiple regression, significant (P<0.05) associations of blood lead (BPb), delta-aminolevulinic acid dehydratase (ALAD), and/or erythrocyte protoporphyrin (EP) with reproductive parameters indicated a lead-related increase in immature sperm concentration, in percentages of pathologic sperm, wide sperm, round sperm, and short sperm, in serum levels of testosterone and estradiol, and a decrease in seminal plasma zinc and in serum prolactin. These reproductive effects were observed at low-level lead exposure (BPb median 49 microg/L, range 11-149 microg/L in the 240 subjects) common for general populations worldwide. The observed significant synergistic effect of BPb and blood cadmium on increasing serum testosterone, and additive effect of a decrease in serum selenium on increasing serum testosterone, may have implications on the initiation and development of prostate cancer because testosterone augments the progress of prostate cancer in its early stages.

Abnormal levels of brain-derived neurotrophic factor (BDNF) are associated with major depression, a disorder with a higher incidence in women than men. Stress affects BDNF levels in various brain regions and thus, a heightened stress response in females could contribute to the development of depression. As well, ovarian hormones directly affect brain levels of BDNF mRNA and protein. Two experiments were performed to investigate the effects of stress and sex and gonadal hormones on BDNF protein levels in CA1, CA3, and dentate gyrus (DG) subregions of the hippocampus. In the first experiment, male and female Sprague-Dawley rats were subjected to one hour of restraint stress or control handling prior to sacrifice. In the second experiment, fifty-one female rats were ovariectomized and separated into stress and control conditions, as described for the first experiment. Stressed and handled groups received a single injection of estrogen (E; 53h prior to stress), estrogen and progesterone (EP; E given at 53h and P given 5h prior to stress), or vehicle (OVX). In both experiments BDNF protein was quantified using an enzyme-linked immunosorbent enzyme assay (ELISA) in micropunches of hippocampus. Gonadally intact females had significantly higher levels of BDNF in CA3, but significantly lower levels in DG, relative to males. In CA3, stress significantly decreased BDNF in both males and females. In DG of ovariectomized female rats, the effects of stress were significantly different following EP vs. vehicle treatment. Thus, stress increased BDNF levels in EP-treated rats but decreased BDNF levels in vehicle-treated rats. Reduced trophic support in DG in the presence of estrogen and progesterone could jeopardize neurogenesis and under certain conditions could be a contributing factor to the hippocampal atrophy associated with stress-induced affective disorders. These results emphasize the need to consider sex, gonadal steroids, and hippocampal subregion when examining the effects of stress on the brain.

Experimental studies implicate late systolic load as a determinant of impaired left-ventricular relaxation. We aimed to assess the relationship between the myocardial loading sequence and left-ventricular contraction and relaxation. Time-resolved central pressure and time-resolved left-ventricular geometry were measured with carotid tonometry and speckle-tracking echocardiography, respectively, for computation of time-resolved ejection-phase myocardial wall stress (EP-MWS) among 1214 middle-aged adults without manifest cardiovascular disease from the general population. Early diastolic annular velocity and systolic annular velocities were measured with tissue Doppler imaging, and segment-averaged longitudinal strain was measured with speckle-tracking echocardiography. After adjustment for age, sex, and potential confounders, late EP-MWS was negatively associated with early diastolic mitral annular velocity (standardized β=-0.25; P<0.0001) and mitral inflow propagation velocity (standardized β=-0.13; P=0.02). In contrast, early EP-MWS was positively associated with early diastolic mitral annular velocity (standardized β=0.18; P<0.0001) and mitral inflow propagation velocity (standardized β=0.22; P<0.0001). A higher late EP-MWS predicted a lower systolic mitral annular velocity (standardized β=-0.31; P<0.0001) and lesser myocardial longitudinal strain (standardized β=0.32; P<0.0001), whereas a higher early EP-MWS was associated with a higher systolic mitral annular velocity (standardized β=0.16; P=0.002) and greater longitudinal strain (standardized β=-0.24; P=0.002). The loading sequence remained independently associated with early diastolic mitral annular velocity after adjustment for systolic mitral annular velocity or systolic longitudinal strain. In the context of available experimental data, our findings support the role of the myocardial loading sequence as a determinant of left-ventricular systolic and diastolic function. A loading sequence characterized by prominent late systolic wall stress was associated with lower longitudinal systolic function and diastolic relaxation.

Aerobic exercise training increases arterial compliance and reduces systolic blood pressure, but the effects of muscular strength training on arterial mechanical properties are unknown. We compared blood pressure, whole body arterial compliance, aortic impedance, aortic stiffness (measured by beta-index and carotid pulse pressure divided by normalized systolic expansion [Ep]), pulse wave velocity, and left ventricular parameters in 19 muscular strength-trained athletes (mean+/-SD age, 26+/-4 years) and 19 sedentary controls (26+/-5 years). Subjects were healthy, non-steroid-using, nonsmoking males, and athletes had been engaged in a strength-training program with no aerobic component for a minimum of 12 months. There was no difference in maximum oxygen consumption between groups, but handgrip strength (mean+/-SEM, 44+/-2 versus 56+/-2 kg; P<0.01) and left ventricular mass (168+/-8 versus 190+/-8 g; P<0.05) were greater in athletes. Arterial stiffness was higher in athletes, as evidenced by lower whole body arterial compliance (0.40+/-0.04 versus 0.54+/-0.04 arbitrary compliance units; P=0.01), higher aortic characteristic impedance (1.55+/-0.13 versus 1.18+/-0.08 mm Hg. s. cm-1; P<0.05), beta-index (4.6+/-0.2 versus 3.8+/-0.4; P<0. 05), and ln Ep (10.86+/-0.06 versus 10.60+/-0.08; P<0.01). Femoral-dorsalis pedis pulse wave velocity was also higher in the athletes, but carotid-femoral pulse wave velocity was not different. Furthermore, both carotid (56+/-3 versus 44+/-2 mm Hg; P<0.001) and brachial (60+/-3 versus 50+/-2 mm Hg; P<0.01) pulse pressures were higher in the athletes, but mean arterial pressure and resting heart rate did not differ between groups. These data indicate that both the proximal aorta and the leg arteries are stiffer in strength-trained individuals and contribute to a higher cardiac afterload.

The cochlear stria vascularis produces the positive endocochlear potential (EP) and the endolymph. Both the EP and the endolymph are essential for the physiological function of hair cells. The intermediate cell is one of several cell types constituting the stria vascularis. It is known that inward rectifier K+ channels can play a constitutive role in the determination of the resting membrane potential. Localization of a member of the inward rectifier K+ channel family, Kir4.1, in the stria vascularis of gerbils and rats was investigated by immunological methods. A polyclonal antibody specific to the C-terminus of the rat Kir4.1 channel was raised in rabbits. Immunostaining of dissociated cells revealed that the Kir4.1 channel was localized to the intermediate cell, but not to the epithelial marginal cell. Subcellular localization of the Kir4.1 channel to the plasma membrane of the intermediate cell was confirmed by immunoelectron microscopy. Immunostaining of whole-tissue preparations revealed a network-like structure composed of intermediate cells. It seems likely that the Kir4.1 channel mediates the inwardly rectifying K+ current in the intermediate cell as shown previously by electrophysiological methods, and that this channel plays key roles in the production of the EP and K+ transport in the stria vascularis.

Within the first few years after chlorpromazine began to be used to treat psychosis, it was observed that it could cause many kinds of neurologic reactions that resembled those seen in idiopathic Parkinson's disease. These reactions were termed "extrapyramidal side effects" (EPS) because of their resemblance to the signs of Parkinson's disease, which were associated with degeneration of the dopamine nerve tracks located in the extrapyramidal region of the central nervous system. Eventually this association of dopamine loss, antipsychotics, and parkinsonism became a central part of the dopamine hypothesis of schizophrenia. Unfortunately, this association was also used to support the hypothesis that EPS were absolutely necessary for antipsychotic efficacy--hence the term "neuroleptic" rather than "antipsychotic." This theory, now discredited, was used to justify the practice of inducing EPS as a means to gauge whether an antipsychotic would be effective. The demonstration that clozapine, an antipsychotic virtually devoid of EPS, has better efficacy for psychosis than any other "neuroleptic" disproved the theory that EPS were fundamentally linked to efficacy. Because the idea of a relationship between EPS and efficacy was so ingrained in clinical practice, clozapine was called "atypical." Our understanding of the relationship between EPS and antipsychotic response has come full circle. With the introduction of clozapine and other newer antipsychotics, it has become clear that EPS are harmful and serve no beneficial purpose. The availability of newer antipsychotics with a lower EPS burden means that, at least in theory, it is now possible to treat psychosis without EPS in the vast majority of patients. In practice, however, EPS remain a significant problem even in the era of atypical or second generation antipsychotics (SGAs). One limitation is that the concept of "atypicality," when used to denote antipsychotic efficacy without EPS, is a relative not an absolute concept. Because all of the post-clozapine SGAs still affect the dopamine D2 receptor, it may be more accurate to say these medications have lower EPS liabilities that the earlier "neuroleptic" antipsychotics; i.e., relatively fewer patients will get EPS at therapeutic doses of one of the newer medications and, when EPS do occur, they tend to be less severe. Nonetheless, reduced EPS are not the same as no EPS, and most of the newer antipsychotics can still cause EPS in some patients. The incidence of EPS differs among the SGAs, with risperidone associated with the most and clozapine and quetiapine with the fewest EPS. The likelihood of developing EPS with a first-line SGA depends not only on the specific agent, but also on the rapidity of dose escalation, the target dose, and the patient's intrinsic vulnerability to EPS. Even with the SGAs, clinicians should not be lulled into believing EPS cannot happen, but need to be able to recognize and manage both overt and subtle manifestations of EPS. This review discusses differences among the SGAs in EPS liability, relationships between dosing and type of EPS, and situations in which differences in EPS liability among the SGAs are clinically relevant.

OBJECTIVE

To determine whether diffusion-weighted echo-planar (EP) MR images with very small, small, and large gradient b-factors are useful in evaluating hepatic lesions and hepatic parenchyma.

METHODS

Approximate values of the apparent diffusion coefficients for diffusion (D) and for flowing spins (D*) for 96 hepatic lesions (26 hepatocellular carcinomas [HCCs], 28 metastases, 26 hemangiomas, and 16 cysts) and the non-lesion-bearing regions of parenchyma in 78 livers (50 noncirrhotic and 28 cirrhotic) were calculated from EP images (modified for gradient b-factors of 3, 50, and 300 second/mm(2)).

RESULTS

Liver cysts and noncirrhotic livers showed statistically higher mean D* values than HCCs, hemangiomas, metastases, and cirrhotic livers (P < 0.05 on Scheffé post hoc analysis). Liver cysts showed statistically higher mean D values than HCCs, metastases, noncirrhotic livers, and cirrhotic livers (P < 0.05). Liver hemangiomas showed statistically higher mean D values than HCCs, noncirrhotic livers, and cirrhotic livers (P < 0.05).

CONCLUSIONS

The D* value in addition to the D value may be useful for evaluating the nature of diffusion and flowing spins in hepatic lesions and hepatic parenchyma.

Brief pulses of Laser emitted radiant heat were used to induce cutaneous painful sensations in human volunteers. Accurate timing of the stimuli permitted recording of scalp averaged evoked potentials. A late negative-positive component of the EP which correlated in amplitude with the subjective sensation was observed in four subjects. The latency of this component (130-160 msec) correlated with stimulus intensity.

OBJECTIVE

Emergency department (ED) bedside echocardiography may offer useful information on cardiac function and volume status. The authors evaluated the accuracy of emergency physician (EP) performance of echocardiography in the assessment of left ventricular ejection fraction (LVEF) and central venous pressure (CVP).

METHODS

The authors conducted a cross-sectional observational study at an urban teaching ED, involving a convenience sample of patients presenting to the ED between September 2000 and February 2001. Level III-credentialed EP sonographers who had undergone a three-hour training session in limited echocardiography, focusing on LVEF and CVP measurement, performed echocardiograms. Vital signs and indication for echocardiography were documented on a study data sheet. LVEF was rated as poor (<30%), moderate (30%-55%), or normal (>55%) and an absolute percentage. Central venous pressure categories included low (<5 cm), moderate (5-10 cm), and high (>10 cm). Formal echocardiograms were obtained within a four-hour window on all patients and interpreted by a staff cardiologist. Correlation analysis was performed using the kappa correlation coefficient for LVEF and CVP categories and a Pearson correlation coefficient for LVEF measurement.

RESULTS

A total of 115 patients were assessed for LVEF, and 94 patients had complete information for CVP. Indications for echocardiography included chest pain (45.1%), congestive heart failure (38.1%), dyspnea (5.7%), and endocarditis (10.6%). Results showed a LVEF correlation of r(2) = 0.712 with 86.1% overall agreement. Subgroup analysis revealed the highest agreement (92.3%) between EP and formal echocardiograms within the normal LVEF category, followed by 70.4% agreement in the poor LVEF category and 47.8% in the moderate LVEF category. Central venous pressure measurements resulted in 70.2% overall raw agreement between EP and formal echocardiograms. Subgroup analysis revealed the highest agreement (83.3%) within the high CVP category followed by 66.6% in the moderate and 20% in the low categories.

CONCLUSIONS

Experienced EP sonographers with a small amount of focused additional training in limited bedside echocardiography can assess LVEF accurately in the ED.

Apolipoprotein E (apoE) has been implicated in modulating the central nervous system (CNS) inflammatory response. However, the molecular mechanisms involved in apoE-dependent immunomodulation are poorly understood. We hypothesize that apoE alters the CNS inflammatory response by signaling via low-density lipoprotein (LDL) receptors in glia. To address this hypothesis, we used a small bioactive peptide formed from the receptor-binding domain of apoE, apoE peptide (EP), to study LDL receptor signaling in microglia. To model glial activation, we treated primary mouse microglia and the microglial cell line BV2 with lipopolysaccharide (LPS) and studied two inflammatory responses: an increase in nitric oxide production (NO) and a decrease in apoE production. We found that treatment of primary microglia and BV2 cells with EP attenuated LPS-induced NO accumulation and apoE reduction in a dose-dependent manner. Using the receptor-associated protein to block ligand binding to members of the LDL receptor family, we found that EP attenuated both of these LPS-induced inflammatory responses via LDL receptors. We studied two intracellular signaling cascades associated with apoE: c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK). LPS induced both ERK and JNK activation, whereas EP induced ERK activation, but drastically reduced JNK activation. Inhibition of JNK with SP600125 reduced LPS-induced NO production and apoE reduction in a dose-dependent manner. Treatment of microglia with suboptimal EP in combination with JNK inhibitor enhanced attenuation of LPS-induced NO production. These data suggest that microglial LDL receptors regulate JNK activation, which is necessary for apoE modulation of the inflammatory response.

The exopolysaccharide (EPS) biosynthesis gene clusters of four Lactobacillus rhamnosus strains consist of chromosomal DNA regions of 18.5 kb encoding 17 ORFs that are highly similar among the strains. However, under identical conditions, EPS production varies considerably among these strains, from 61 to 1611 mg l(-1). Fifteen genes are co-transcribed starting from the first promoter upstream of wzd. Nevertheless, five transcription start sites were identified by 5'-RACE PCR analysis, and these were associated with promoter sequences upstream of wzd, rmlA, welE, wzr and wzb. Six potential glycosyltransferase genes were identified that account for the assembly of the heptasaccharide repeat unit containing an unusually high proportion of rhamnose. Four genes involved in the biosynthesis of the sugar nucleotide precursor dTDP-L-rhamnose were identified in the EPS biosynthesis locus, which is unusual for lactic acid bacteria. These four genes are expressed from their own promoter (P2), as well as co-transcribed with the upstream EPS genes, resulting in coordinated production of the rhamnose precursor with the enzymes involved in EPS biosynthesis. This is believed to be the first report demonstrating that the sequence, original organization and transcription of genes encoding EPS production are highly similar among four strains of Lb. rhamnosus, and do not vary with the amount of EPS produced.

OBJECTIVE

To investigate the value of using changes in three parameters (tumor size, transfer constant (K(trans)), and rate constant (k(ep))) obtained after the first treatment-cycle in predicting the final clinical response after two to four cycles of neoadjuvant anthracycline and cyclophosphamide (AC) chemotherapy.

METHODS

Early changes in the three parameters were measured in 29 patients with invasive breast cancer by MRI after one cycle of treatment. Changes were then assessed for their predictive value of final clinical response and compared among patients with four different response patterns, Group 1 = responder (R) after one cycle and also R after four cycles, Group 2 = nonresponder (NR) after one cycle, but eventual R after four cycles, Group 3 = NR after one cycle and still NR after four cycles, and Group 4 = NR after one cycle and determined as NR after two cycles, being switched to the taxane regimen.

RESULTS

Pearson's correlation analysis revealed significant correlation between early changes in tumor size and both pharmacokinetic parameters (r = 0.49 and P < 0.01 for K(trans), r = 0.66 and P < 0.001 for k(ep)). The areas under the receiver operating characteristic (ROC) curve differentiating between R (Groups 1+2) and NR (Groups 3+4) groups using changes in tumor size, K(trans), and k(ep) were 0.88 (standard error [SE] = 0.06, P < 0.0001), 0.63 (SE = 0.11, P = 0.11), and 0.77 (SE = 0.09, P = 0.001), respectively.

CONCLUSIONS

Early tumor size change in MRI after one cycle is better response predictor than that of either K(trans) or k(ep) in breast cancer undergoing neoadjuvant chemotherapy using an AC regimen.

Risperidone (Risperdal) is a novel antipsychotic drug, with beneficial effects on both positive and negative symptoms of schizophrenia, and with a low incidence of extrapyramidal side effects (EPS). These particular properties have been attributed to the predominant and very potent serotonin 5-HT2 receptor antagonism of the drug combined with less potent dopamine D2 antagonism. In order to provide data on the degree to which various central neurotransmitter receptors are occupied in vivo, we performed ex vivo receptor occupancy studies with risperidone in comparison with clozapine and haloperidol in rats and guinea pigs. Various types of receptors, to which the compounds were known to bind to in vitro, were investigated precisely using receptor autoradiography in sections of the same rat brain except for histamine H1 receptors that were measured in the guinea-pig cerebellum. Risperidone (2 h after s.c. treatment) occupied 5-HT2 receptors at very low doses (ED50 = 0.067 mg/kg). Nearly full occupancy >> 80%) was achieved before H1, D2, alpha 1 and alpha 2 receptors became occupied (ED50 = 0.45, 0.66, 0.75 and 3.7 mg/kg, respectively). Clozapine displayed occupancy of H1 and alpha 1 receptors at low doses (ED50 = 0.15 and 0.58 mg/kg, respectively) and of 5-HT2, 5-HT1C, D2, alpha 2, cholinergic muscarinic and 5-HT1A receptors at higher doses (ED50 = 1.3, 1.8, 9.0, 9.5, 11 and 15 mg/kg, respectively). Haloperidol occupied D2 and alpha 1 receptors at low doses (ED50 = 0.13 and 0.42 mg/kg, respectively) and 5-HT2 receptors at a higher dose (ED50 = 2.6 mg/kg). Occupancy of receptor types occurred with similar ED50-values in various brain areas, e.g. D2 receptors in striatum and mesolimbic areas. The ED50-values for the ex vivo measured occupancy of 5-HT2 and D2 receptors were in good agreement with ED50-values for functional effects putatively mediated by these central receptors. The dose-dependent occupancy of D2 receptors proceeded more gradually with risperidone (slope in the caudate-putamen: 0.85) than with clozapine (slope: 1.44) or haloperidol (slope: 1.51). It has previously been suggested that partial D2 receptor occupancy may suffice to control the positive symptoms of schizophrenia, whereas higher D2 receptor occupancy would induce extrapyramidal symptoms (EPS). The dose ratio for high (75%) vs. low (25%) D2 receptor occupancy in the caudate-putamen, was 37.3 for risperidone, 8.4 for clozapine, and 7.9 for haloperidol.(ABSTRACT TRUNCATED AT 400 WORDS)

Atypical antipsychotics are expected to be better tolerated than older antipsychotics because of their lower propensity to cause certain adverse effects. All atypical drugs have been shown to cause fewer acute extrapyramidal symptoms (EPS) than a standard typical agent (usually haloperidol) and some (clozapine, sertindole and quetiapine) appear to cause these effects no more often than placebo. In the longer term, clozapine, olanzapine and (less robustly) other atypical antipsychotics are thought to cause less tardive dyskinesia than typical antipsychotics. Problems caused by hyperprolactinaemia occur less often with some atypical antipsychotics than with typical drugs although risperidone and amisulpride appear to have no advantages in this respect. Other adverse effects may occur as frequently with some atypical antipsychotics as with some typical drugs. Clozapine, risperidone and quetiapine are known to cause postural hypotension; clozapine, olanzapine and quetiapine are clearly sedative; and anticholinergic effects are commonly seen with clozapine, and, much less frequently, with olanzapine. Some adverse effects are more frequent with atypical drugs. Idiosyncratic effects seem particularly troublesome with clozapine and, to a lesser extent, sertindole, olanzapine and zotepine. Bodyweight gain is probably more problematic with atypical antipsychotics than with typical drugs. Overall tolerability, as judged by withdrawals from therapy, is not clearly proven to be better with atypical drugs, although some individual trials do indicate an advantage with atypical agents. Differences in tolerability between individual atypical antipsychotics have not been clearly shown. The tolerability profile of atypical drugs certainly benefits from a lower incidence of acute EPS effects, along with less certain or less uniform benefits in symptomatic hyperprolactinaemia or tardive dyskinesia. Other, perhaps more trivial, adverse effects militate against their good tolerability, and effects such as bodyweight gain may severely reduce tolerability. Without clear advantages in tolerability in patient groups used in trials, drug choice in regard to adverse effects should continue to be on a patient to patient basis.

R. meliloti Rm1021 normally produces an acidic Calcofluor-binding exopolysaccharide, called succinoglycan or EPS I, which is required for successful nodulation of alfalfa by this strain. At least 13 loci affecting production of EPS I were previously mapped to a cluster on the second of two symbiotic megaplasmids in Rm1021, pRmeSU47b. A putative regulatory region was originally defined by the exoG and exoJ mutations. exoG and exoJ mutants produced less exopolysaccharide than wild-type strains and induced nitrogen-fixing nodules on alfalfa with reduced efficiency compared with the wild type. These mutants appeared to produce only a low-molecular-weight form of EPS I. Mutations called exoX cause an increase in exopolysaccharide production and map in the same region as the exoG and exoJ mutations. The DNA sequence of this region reveals that it contains two open reading frames, called exoX and exoY, which have homologs in other Rhizobium species. Interestingly, the exoG insertion mutations fall in an intergenic region and may affect the expression of exoX or exoY. The exoJ mutation falls in the 3' portion of the exoX open reading frame and is probably an allele of exoX that results in altered function. exoG and exoJ mutations limit EPS I production in the presence of exoR95 or exoS96 mutations, which cause overproduction of EPS I. Gene regulation studies suggest that ExoX and ExoY constitute a system that modulates exopolysaccharide synthesis at a posttranslational level. The deduced sequence of ExoY is homologous to a protein required for an early step in xanthan gum biosynthesis, further suggesting that the modulatory system may affect the exopolysaccharide biosynthetic apparatus.

OBJECTIVE

Over the past 5 years, several clinical studies on a total of approximately 2500 patients have shown that the immunocytochemical detection of occult metastatic tumor cells in bone marrow (BM) at primary surgery provides important prognostic information in breast cancer (e.g., Ref 13 ). Here, we evaluated whether these cells can survive first-line chemotherapy and express epithelial cell adhesion molecule (Ep-CAM), recently suggested as promising target for immunotherapeutic interventions in breast cancer.

METHODS

A total of 62 patients with node-negative and -positive breast cancer but without distant metastases (Tumor-Node-Metastasis stage M(0)) was treated with two or more courses of various forms of adjuvant chemotherapy (e.g., cyclophosphamide-methotrexate-5-fluorouracil, anthracyclines). After chemotherapy, BM was aspirated from the upper iliac crest and analyzed for the presence of tumor cells. A first cohort of 34 BM aspirates was enriched for tumor cells by Ficoll density gradient centrifugation, and 2-4 x 10(6) mononuclear cells were analyzed per patient. The tumor cells were detected by anticytokeratin monoclonal antibody (Mab) A45-B/B3 and double labeled with Mab 3B10 against an Ep-CAM-epitope. The subsequent 27 BM aspirates were specifically enriched for Ep-CAM(+) cells using magnetic beads coupled to Mab 3B10, and tumor cells were identified by Fab fragments of Mab A45-B/B3 directly conjugated with alkaline phosphatase.

RESULTS

After chemotherapy, 10 of 35 (28.6%) Ficoll-enriched BM samples contained cytokeratin-positive tumor cells. In total, 26 cytokeratin-positive cells were detected, but none of these cells coexpressed Ep-CAM. Even within the second cohort of 27 Ep-CAM-enriched BM samples, only 2 specimens (7.4%) harbored cytokeratin-positive cells costaining with the Ep-CAM antibody.

CONCLUSIONS

Our results indicate that disseminated breast cancer cells in BM can survive first-line adjuvant chemotherapy. Ep-CAM expression is, however, restricted to a subset of these cells, which may limit the broad applicability of Ep-CAM as target for second-line adjuvant therapy in breast cancer.

Naked DNA injection with electropermeabilization (EP) is a promising method for nucleic acid vaccination (NAV) and in vivo gene therapy. Skin is an ideal target for NAV due to ease of administration and the accessibility of large numbers of antigen-presenting cells within the tissue. This study demonstrates that in vivo skin EP may be used to increase transgene expression up to an average of 83-fold relative to naked DNA injection (50 microg DNA per dose, P < 0.005). Transfected cells were principally located in dermis and included adipocytes, fibroblasts, endothelial cells, and numerous mononuclear cells with dendritic processes in a porcine model. Transfected cells were also observed in lymph nodes draining electropermeabilized sites. A HBV sAg-coding plasmid was used to test skin EP-mediated NAV in a murine model. Analysis of humoral immune responses including immunoglobulin subclass profiles revealed strong enhancement of EP-mediated NAV relative to naked DNA injection, with a Th1-dominant, mixed-response pattern compared to immunization with HBV sAg protein that was exclusively Th2 (P = 0.02). Applications for these findings include NAV-based modulation of immune responses to pathogens, allergens, and tumor-associated antigens and the modification of tolerance.

Our goal is to model the coupling between neuronal activity, cerebral metabolic rates of glucose and oxygen consumption, cerebral blood flow (CBF), electroencephalography (EEG) and blood oxygenation level-dependent (BOLD) responses. In order to accomplish this, two previous models are coupled: a metabolic/hemodynamic model (MHM) for a voxel, linking BOLD signals and neuronal activity, and a neural mass model describing the neuronal dynamics within a voxel and its interactions with voxels of the same area (short-range interactions) and other areas (long-range interactions). For coupling both models, we take as the input to the BOLD model, the number of active synapses within the voxel, that is, the average number of synapses that will receive an action potential within the time unit. This is obtained by considering the action potentials transmitted between neuronal populations within the voxel, as well as those arriving from other voxels. Simulations are carried out for testing the integrated model. Results show that realistic evoked potentials (EP) at electrodes on the scalp surface and the corresponding BOLD signals for each voxel are produced by the model. In another simulation, the alpha rhythm was reproduced and reasonable similarities with experimental data were obtained when calculating correlations between BOLD signals and the alpha power curve. The origin of negative BOLD responses and the characteristics of EEG, PET and BOLD signals in Alzheimer's disease were also studied.

Human papillomavirus (HPV) has recently been associated with oral cancers. To prepare for a study of the natural history of oral HPV infection, the effect of the DNA purification method on HPV genomic DNA detection in Scope mouthwash oral rinse samples and the reproducibility of HPV detection in rinse samples collected 7 days apart were investigated. The study was conducted with a population at high risk for oral HPV infection: human immunodeficiency virus-infected men with CD4-cell counts <200. Five DNA purification methods were compared among equal aliquots of oral rinse samples collected from a subset of individuals. The purification methods included (i) proteinase K digestion (PKD) and heat inactivation; (ii) PKD and ethanol precipitation (EP); (iii) PKD, phenol-chloroform extraction, and EP; (iv) use of the Puregene DNA purification kit; and (v) use of the QIAamp DNA Blood Midi kit. HPV was detected by PCR amplification with PGMY09 and PGMY11 L1 primer pools and by use of a Roche linear array. Puregene-purified samples had higher human DNA yields and purities, and Puregene purification detected the greatest number of HPV-positive subjects and total HPV infections in comparison to the numbers detected by all other methods. The total number of HPV infections and HPV prevalence estimates were also higher for Puregene-processed oral rinse samples when a fixed volume (10 mul) rather than a fixed cell number ( approximately 50,000 cells) was used for PCR amplification. A good concordance was observed for oral HPV infection status (agreement, 80%; kappa value, = 0.60) and type-specific infection (agreement, 98%; kappa value, 0.57) in matched oral rinse samples. The method of DNA purification significantly affects the detection of HPV genomic DNA from oral rinse samples and may result in exposure misclassification that could contribute to the inconsistent associations reported in the literature.

OBJECTIVE

Gastroesophageal reflux disease (GERD) is increasing in Asian countries. Functional dyspepsia (FD) or irritable bowel syndrome (IBS) are also prevalent and commonly overlapped with GERD. This study was conducted to compare the proportion and risk factors for overlapping reflux esophagitis (RE) and non-erosive reflux disease (NERD) with functional gastrointestinal disorders (FGIDs).

METHODS

A total of 2,388 [male, 55.9%; mean age (+/- SD), 43.2 years (+/- 8.4)] Korean subjects who underwent the upper endoscopy for health screening were prospectively included. The subjects were asked about demographic, medical and social history by using a structured questionnaire, and FD and IBS were assessed according to the Rome III criteria.

RESULTS

The subjects with RE were 286 (12.0%, male 88.5%, 42.8 years) and 74 subjects had NERD (3.1%) while the prevalence of FD and IBS were 8.1% and 10.1%, respectively. The proportion of FD and IBS in NERD was higher than that of RE (74.3% vs. 10.5%, p = 0.000; 41.9% vs. 11.2%, p = 0.000, respectively). The epigastric pain syndrome (EPS) was more prevalent than postprandial distress syndrome in NERD. According to multiple regression analysis, high somatization score and the presence of FD increased the odd ratio for NERD. However, male gender and current smoker were significant risk factors for RE.

CONCLUSIONS

Compared to RE, NERD is more frequently overlapped with FD, especially EPS, and also are associated with significantly increased frequency of IBS. Our data draws attention to the possibility of subgrouping FGIDs and GERD to be important in understanding the pathophysiology of these conditions.

The present experiments were designed to test the hypothesis that prostaglandin (PG) E(2) causes vasodilatation through activation of endothelial NO synthase (eNOS). Aortic rings from mice with targeted deletion of eNOS and E-prostanoid (<em>EP</em>) receptors were used for contraction studies. Blood pressure changes in response to PGE(2) were measured in conscious mice. Single doses of PGE(2) caused concentration-dependent relaxations during contractions to phenylephrine (EC(50)=5*10(-8) mol/L). Relaxation after PGE(2) was absent in rings without endothelium and in rings from eNOS(-/-) mice and was abolished by N(G)-nitro-l-arginine methyl ester and the soluble guanylate cyclase inhibitor 1H(1,2,4)-oxadiazolo-[4,3-a]quinoxalin-1-one. In PGE(2)-relaxed aortic rings, the cGMP content increased significantly. PGE(2)-induced relaxations were abolished by the <em>EP</em>4 receptor antagonist AE3-208 (10(-8) mol/L) and mimicked by an <em>EP</em>4 agonist (AE1-329, 10(-7) mol/L) in the presence of endothelium and eNOS only. Relaxations were attenuated significantly in rings from <em>EP</em>4(-/-) mice but normal in <em>EP</em>2(-/-). Inhibitors of the cAMP-protein kinase A pathway attenuated, whereas the inhibitor of protein phosphatase 1C, calyculin (10(-8) mol/L), abolished the PGE(2)-mediated relaxation. In aortic rings, PGE(2) dephosphorylated eNOS at Thr(495). Chronically catheterized eNOS(-/-) mice were hypertensive (137+/-3.6 mm Hg, n=13, versus 101+/-3.9 mm Hg, n=9) and exhibited a lower sensitivity of blood pressure reduction in response to PGE(2) compared with wild-type mice. There was no difference in the blood pressure response to nifedipine. These findings show that PGE(2) elicits <em>EP</em>4 receptor-mediated, endothelium-dependent stimulation of eNOS activity by dephosphorylation at Thr(495) resulting in guanylyl cyclase-dependent vasorelaxation and accumulation of cGMP in aortic rings.