Candida albicans is an opportunistic pathogenic fungus capable of causing infections in immunocompromised patients. Candidiasis is often associated with the formation of biofilms on the surface of inert or biological materials. Biofilms are structured microbial communities attached to a surface and encased within a matrix of exopolymeric substance (EPS). At present, very little is known about the changes in protein profiles that occur during the transition from the planktonic to the biofilm mode of growth. Here, we report the use of proteomics for the comparative analysis of subcellular fractions obtained from C. albicans biofilm and planktonic cultures, including cell surface-associated proteins and secreted components present in liquid culture supernatants (for planktonic cultures) and EPS (for biofilms). The analysis revealed a high degree of similarity between the protein profiles associated with the planktonic and biofilm extracts, and led to the identification of several differentially expressed protein spots. Among the differentially expressed proteins, there was a preponderance of metabolic enzymes that have been described as cell surface proteins and immunodominant antigens. Proteins found in the biofilm matrix included a few predicted to form part of the secretome, and also many secretion-signal-less proteins. These observations contribute to our understanding of the C. albicans biofilm lifestyle.

Mediators of inflammation, such as PGE(2), are known to sensitize the airways to inhaled irritants and circulating autacoids. Evidence from in vivo studies has shown the involvement of vagal pulmonary C-fiber afferents in the PGE(2)-elicited airway hypersensitivity. However, whether PGE(2) acts directly on these sensory nerves is unclear. The present study aimed to investigate whether PGE(2) has direct potentiating effects on nodose and jugular pulmonary C neurons cultured from adult Sprague-Dawley rats and, if so, determine whether the EP(2) prostanoid receptor is involved. Pulmonary neurons were identified by retrograde labeling with a fluorescent tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate. Using perforated patch-clamp technique, our results showed that 1) PGE(2) pretreatment (1 microM) increased the whole cell current density elicited by capsaicin and phenylbiguanide, chemical agents known to stimulate pulmonary C fibers; 2) selective activation of the EP(2) prostanoid receptor by butaprost (3-10 microM) increased the whole cell current density elicited by capsaicin; and 3) PGE(2), as well as butaprost, increased the number of action potentials evoked by current injection. Therefore, we conclude that PGE(2) directly sensitizes vagal pulmonary C neurons to chemical and electrical stimulation. Furthermore, butaprost modulates the neurons in a manner similar to that of PGE(2), suggesting that the effects of PGE(2) are mediated, at least in part, through the EP(2) prostanoid receptor.

Prostaglandin E(2) (PGE(2)) has complex effects on airway tone, and the existence of four PGE(2) [E-prostanoid (<em>EP</em>)] receptors, each with distinct signaling characteristics, has provided a possible explanation for the seemingly contradictory actions of this lipid mediator. To identify the receptors mediating the actions of PGE(2) on bronchomotor tone, we examined its effects on the airways of wild-type and <em>EP</em> receptor-deficient mice. In conscious mice the administration of PGE(2) increased airway responsiveness primarily through the <em>EP</em>1 receptor, although on certain genetic backgrounds a contribution of the <em>EP</em>3 receptor was detected. These effects of PGE(2) were eliminated by pretreatment with either atropine or bupivacaine and were undetectable in anesthetized mice or in denervated tracheal rings, where only <em>EP</em>2-mediated relaxation of airway smooth muscle was observed. Together, our findings are consistent with a model in which PGE(2) modulates airway tone by activating multiple receptors expressed on various cell populations and in which the relative contribution of these receptors might depend on the expression of modifier alleles. PGE(2)/<em>EP</em>1/<em>EP</em>3-induced airway constriction occurs indirectly through activation of neural pathways, whereas PGE(2)-induced bronchodilation results from direct activation of <em>EP</em>2 receptors on airway smooth muscle. This segregation of <em>EP</em> receptor function within the airway suggests that PGE(2) analogs that selectively activate the <em>EP</em>2 receptor without activating the <em>EP</em>1/<em>EP</em>3 receptors might prove useful in the treatment of asthma.

Rhizobia are Gram-negative bacteria that can exist either as free-living bacteria or as nitrogen-fixing symbionts inside root nodules of leguminous plants. The composition of the rhizobial outer surface, containing a variety of polysaccharides, plays a significant role in the adaptation of these bacteria in both habitats. Among rhizobial polymers, exopolysaccharide (EPS) is indispensable for the invasion of a great majority of host plants which form indeterminate-type nodules. Various functions are ascribed to this heteropolymer, including protection against environmental stress and host defense, attachment to abiotic and biotic surfaces, and in signaling. The synthesis of EPS in rhizobia is a multi-step process regulated by several proteins at both transcriptional and post-transcriptional levels. Also, some environmental factors (carbon source, nitrogen and phosphate starvation, flavonoids) and stress conditions (osmolarity, ionic strength) affect EPS production. This paper discusses the recent data concerning the function of the genes required for EPS synthesis and the regulation of this process by several environmental signals. Up till now, the synthesis of rhizobial EPS has been best studied in two species, Sinorhizobium meliloti and Rhizobium leguminosarum. The latest data indicate that EPS synthesis in rhizobia undergoes very complex hierarchical regulation, in which proteins engaged in quorum sensing and the regulation of motility genes also participate. This finding enables a better understanding of the complex processes occurring in the rhizosphere which are crucial for successful colonization and infection of host plant roots.

Cochlear endolymph has a highly positive potential of approximately +80 mV. This so-called endocochlear potential (EP) is essential for hearing. Although pivotal roles of K+ channels in the formation of EP have been suggested, the types and distribution of K+ channels in cochlea have not been characterized. Because EP was depressed by vascular perfusion of Ba2+, an inhibitor of inwardly rectifying K+ (Kir) channels, but not by either 4-aminopyridine or tetraethylammonium, we examined the expression of Kir channel subunits in cochlear stria vascularis, the tissue that is supposed to play the central role in the generation of positive EP. Of 11 members of the Kir channel family examined with reverse transcription-PCR, we could detect only expression of KAB-2 (Kir4.1) mRNA in stria vascularis. KAB-2 immunoreactivity was specifically localized at the basolateral membrane of marginal cells but not in either basal or intermediate cells. Developmental expression of KAB-2 in marginal cells paralleled formation of EP. Furthermore, deaf mutant mice (viable dominant spotting; WV/WV) expressed no KAB-2 in their marginal cells. These results suggest that KAB-2 in marginal cells may be critically involved in the generation of positive EP.

Proteins secreted by skeletal muscle, so called myokines, have been shown to affect muscle physiology and additionally exert systemic effects on other tissues and organs. Although recent profiling studies have identified numerous myokines, the amount of overlap from these studies indicates that the secretome of skeletal muscle is still incompletely characterized. One limitation of the models used is the lack of contraction, a central characteristic of muscle cells. Here we aimed to characterize the secretome of primary human myotubes by cytokine antibody arrays and to identify myokines regulated by contraction, which was induced by electrical pulse stimulation (EPS). In this study, we validated the regulation and release of two selected myokines, namely pigment epithelium derived factor (PEDF) and dipeptidyl peptidase 4 (DPP4), which were recently described as adipokines. This study reveals that both factors, DPP4 and PEDF, are secreted by primary human myotubes. PEDF is a contraction-regulated myokine, although PEDF serum levels from healthy young men decrease after 60 min cycling at VO2max of 70%. Most interestingly, we identified 52 novel myokines which have not been described before to be secreted by skeletal muscle cells. For 48 myokines we show that their release is regulated by contractile activity. This profiling study of the human skeletal muscle secretome expands the number of myokines, identifies novel contraction-regulated myokines and underlines the overlap between proteins which are adipokines as well as myokines.

This paper reviews some of our recent research on haptic exploration, perception and recognition of multidimensional objects. We begin by considering the nature of manual exploration in terms of the characteristics of various exploratory procedures (EPs) or stereotypical patterns of hand movements. Next, we explore their consequences for the sequence of EPs selected, for the relative cognitive salience of material versus geometric properties, and for dimensional integration. Finally, we discuss several applications of our research programme to the development of tangible graphics displays for the blind, autonomous and teleoperated haptic robotic systems, and food evaluation in the food industry.

Since 1990, management options available for children with paroxysmal supraventricular tachycardia (PSVT) have included radiofrequency catheter ablation (RCA). To determine the efficacy and safety of the procedure and to maintain a database for long-term follow-up, the Pediatric Electrophysiology Society began a Pediatric RCA Registry on January 1, 1991, to which 46 centers have submitted data from 4,135 total children and adolescents (patient age 0.1 to 20.9 years) who underwent 4,651 RCAs (through September 15, 1996). Of the 88% with a structurally normal heart, PSVT mechanisms (n = 4,030) included 3,110 accessory pathways and 920 atrioventricular node reentry tachycardia (AVNRT) during 3,653 procedures for 3,277 patients. During the 7 years of the Registry, analysis of indications for the procedure has shown a gradual shift. During the first year of the Registry for this PSVT group, "medically refractory tachycardia" was listed as the indication for 44% and "patient choice" was listed as 33%, compared with 29% and 58%, respectively, for the years 1995 to 1996 (p <0.005). Registry results were: 90% immediate success for accessory pathways (95% for left lateral; 87% for septal; 86% for right free wall) and 96% for AVNRT; mean fluoroscopy time 47.6 +/- 40 SD minutes; procedure time 257 +/- 157 SD minutes; major complication rate at the time of the procedure 3.2%. Procedure-related deaths included 1 immediate and 3 at 2, 12 and 68 weeks after the procedure (2 were infants). Follow-up revealed 77% and 71% freedom from recurrence at 3 years for accessory pathways AVNRT, respectively, and rare (<1%) detection of additional complications. RCA has evolved into a standard management option for PSVT in children with a structurally normal heart. RCA for children and adolescents should be recommended after consideration of the procedural risk/benefit compared with that of other management options, the natural history, and individual tolerance/symptoms related to PSVT.

OBJECTIVE

Novel therapeutic agents and strategies have been introduced into the management of myelodysplastic syndromes (MDS) in the last years. This has led to more treatment options and a better chance of long-term survival for MDS patients, but also to uncertainty regarding the optimal use and possible side effects of these treatments. The Italian Society of Hematology commissioned a project to develop guidelines for the therapy of MDS using evidence-based knowledge and consensus-formation techniques.

METHODS

An Advisory Council (AC) shaped the project around a series of key clinical questions, performed a systematic search for evidence and graded the available evidence according to the Scottish Intercollegiate Guidelines Network (SIGN). A list of clinical questions was mailed to each of 10 senior hematologists composing the Expert Panel (EP): the panelists were asked to rank the most relevant questions, and to formulate answers to the questions according to the tables of evidence. A scenario phase followed, so as to reach a consensus on the three top ranked questions. The EP was asked to score patient profiles as appropriate or not appropriate for the therapeutic strategy under scrutiny, according to the RAND technique. Finally, from September 2001 to January 2002, four Consensus Conferences conducted according to the Nominal Group Technique were held in Milan, Italy. The overall goal of the conferences was to take a final decision upon the appropriateness of the uncertain scenarios and of the uncertain responses to the clinical questions.

RESULTS

Evidence was judged sufficient for providing recommendations on the use of allogeneic stem cell transplantation, leukemia-like chemotherapy, autologous stem cell transplantation, low-dose chemotherapy, danazol, immunosuppressive therapy, hypomethylating agents and hematopoietic growth factors. Specific recommendations for supportive therapy, including iron chelation, were issued. Allogeneic stem cell transplantation was unanimously considered as the only curative treatment for MDS patients, and recommendations on its use were agreed based on patient's age, risk, clinical features and donor availability. AML-like chemotherapy was also considered a valuable therapeutic option for subsets of MDS patients. Autologous stem cell transplantation was recommended for patients who lack an HLA identical donor and have achieved complete remission with AML-like chemotherapy. Decitabine, recombinant human erythropoietin and immunosuppressive therapy were judged valuable therapeutic options for subsets of MDS patients whereas low-dose cytarabine was not. Specific therapeutic strategies for those subjects younger than 18 years or older than 75 years and the strategy of watchful waiting were decided by patient-oriented questions.

CONCLUSIONS

Using evidence and consensus, recommendations for the treatment of MDS were issued. Statements were graded according to the strength of the supporting evidence and uncertainty was explicitly declared.

Bacteria have long been thought of as little more than sacks of homogeneously distributed enzymes. However, recent cytological studies indicate that bacteria are compartmentalized with proteins involved in processes such as cell division, motility, chemotaxis, and development located at distinct sites. We have used the green fluorescent protein as a reporter to determine the cellular distribution of the extracellular protein secretion (eps)-encoded type II secretion complex responsible for extracellular secretion of cholera toxin and hemagglutinin/protease in Vibrio cholerae. Real-time monitoring of green fluorescent protein fused to EpsM in living cells indicated that, like the single polar flagellum, the Eps complex is located at the old pole after cell division. Eps-dependent protease secretion was also visualized in single cells by fluorescence microscopy by using intramolecularly quenched casein. This analysis demonstrated that active protease secretion is focused at the poles and colocalizes with the site of the polar Eps apparatus. These results suggest that the type II secretion complex is responsible for directed delivery of virulence factors during cholera pathogenesis.

In this study, we report that silver ions (Ag(+)) from the oxidative dissolution of silver engineered nanoparticles (Ag-ENs) determined the EN toxicity to the marine diatom Thalassiosira weissflogii. Most of the Ag-ENs formed non-toxic aggregates (>0.22 microm) in seawater. When the free Ag(+) concentration ([Ag(+)](F)) was greatly reduced by diafiltration or thiol complexation, no toxicity was observed, even though the Ag-ENs were better dispersed in the presence of thiols with up to 1.08 x 10(-5) M Ag-ENs found in the <0.22 microm fraction, which are orders of magnitude higher than predicted for the natural aquatic environment. The secretion of polysaccharide-rich algal exopolymeric substances (EPS) significantly increased at increasing [Ag(+)](F). Both dissolved and particulate polysaccharide concentrations were higher for nutrient-limited cells, coinciding with their higher Ag(+) tolerance, suggesting that EPS may be involved in Ag(+) detoxification.

Purpose: Pembrolizumab monotherapy has shown antitumor activity in patients with small-cell lung cancer (SCLC). The randomized, double-blind, phase III KEYNOTE-604 study compared pembrolizumab plus etoposide and platinum (EP) with placebo plus EP for patients with previously untreated extensive-stage (ES) SCLC.

Methods: Eligible patients were randomly assigned 1:1 to pembrolizumab 200 mg once every 3 weeks or saline placebo for up to 35 cycles plus 4 cycles of EP. Primary end points were progression-free survival (PFS; RECIST version 1.1, blinded central review) and overall survival (OS) in the intention-to-treat population. Objective response rate (ORR) and duration of response were secondary end points. Prespecified efficacy boundaries were one-sided P = .0048 for PFS and .0128 for OS.

Results: Of the 453 participants, 228 were randomly assigned to pembrolizumab plus EP and 225 to placebo plus EP. Pembrolizumab plus EP significantly improved PFS (hazard ratio [HR], 0.75; 95% CI, 0.61 to 0.91; P = .0023). Twelve-month PFS estimates were 13.6% with pembrolizumab plus EP and 3.1% with placebo plus EP. Although pembrolizumab plus EP prolonged OS, the significance threshold was not met (HR, 0.80; 95% CI, 0.64 to 0.98; P = .0164). Twenty-four-month OS estimates were 22.5% and 11.2%, respectively. ORR was 70.6% in the pembrolizumab plus EP group and 61.8% in the placebo plus EP group; the estimated proportion of responders remaining in response at 12 months was 19.3% and 3.3%, respectively. In the pembrolizumab plus EP and placebo plus EP groups, respectively, any-cause adverse events were grade 3-4 in 76.7% and 74.9%, grade 5 in 6.3% and 5.4%, and led to discontinuation of any drug in 14.8% and 6.3%.

Conclusion: Pembrolizumab plus EP significantly improved PFS compared with placebo plus EP as first-line therapy for patients with ES-SCLC. No unexpected toxicities were seen with pembrolizumab plus EP. These data support the benefit of pembrolizumab in ES-SCLC.

Electrochemotherapy (ECT) is a local cancer treatment that has been used over the course of more than 2 decades for the removal of cutaneous and subcutaneous tumors. Several lines of evidence support the premise that the immune system is an important factor underlying anticancer treatment efficacy, potentially including patient responses to ECT. The concept of immunogenic cell death (ICD) arose a few years ago, stating that some cancer treatments generate danger-associated molecular patterns (DAMPs) that trigger an adaptive immune response against tumors. Hence, dying cancer cells behave as a therapeutic vaccine, eliciting a cytotoxic immune response against surviving malignant cells. In our study, we sought to evaluate the ability of ECT to generate cancer cell death encompassing the immunostimulatory characteristics of ICD. To this end, we assayed CT26 murine colon cancer cells in vitro in response to either electric pulses (EPs) application only or in combination with the anticancer drug bleomycin (that is ECT) by quantification of calreticulin (CRT) membrane externalization, as well as the liberation of adenosine triphosphate (ATP) and high mobility group box 1 (HMGB1) protein. We show here that cell permeabilizing yet non-lethal electric pulses induce CRT exposure on the cell surface of EP-only treated cancer cells, as well as ATP release. However, the association of electric pulses along with the chemotherapeutic agent bleomycin was mandatory for HMGB1 release coincident with regimen-induced cell death. These data obtained in vitro were then substantiated by vaccination protocols performed in immunocompetent mice, showing that the injection of dying ECT-treated cells elicits an antitumor immune response that prevents the growth of a subsequent administration of viable cancer cells. We also confirmed previous results showing ECT treatment is much more efficient in immunocompetent animals than in immunodeficient ones, causing complete regressions in the former but not in the latter. This supports a central role for immunity in this beneficial outcome. In conclusion, we show that ECT not only possesses an intrinsic cytotoxic property toward cancer cells but also generates a systemic anticancer immune response via the activation of ICD. Hence, ECT may represent an interesting approach to treat solid tumors while preventing recurrence and metastasis, possibly in combination with immunostimulating agents.

Death ligands such as tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and certain forms of CD95L are attractive therapeutic options for metastatic melanoma. Since knowledge about the regulation of death receptor sensitivity in melanoma is sparse, we have analysed these signaling pathways in detail. The loss of CD95 or TRAIL-R1, but not of TRAIL-R2, surface expression correlated with apoptosis sensitivity in a panel of melanoma cell lines. In contrast, the expression of proteins of the apical apoptosis signaling cascade (FADD, initiator caspases-8 and cFLIP) did not predict apoptosis sensitivity. Since both TRAIL-R1 and -R2 transmit apoptotic signals, we asked whether cFLIP, highly expressed in several of the cell lines tested, is sufficient to maintain resistance to TRAIL-R2-mediated apoptosis. Downregulation of cFLIP in TRAIL-R2-positive, TRAIL-resistant IGR cells dramatically increased TRAIL sensitivity. Conversely ectopic expression of cFLIP in TRAIL-sensitive, TRAIL-R2-expressing RPM-EP melanoma cells inhibited TRAIL- and CD95L-mediated cell death. Thus, modulation of cFLIP is sufficient to sensitize TRAIL-R2-expressing cells for TRAIL. Taken together, albeit expressing all proteins necessary for death receptor-mediated apoptosis, TRAIL-R1 negative melanoma cells cannot undergo TRAIL- or CD95L-induced apoptosis due to expression of cFLIP. Hence, cFLIP represents an attractive therapeutic target for melanoma treatment, especially in combination with TRAIL receptor agonists.

Drought and high-temperature stresses have been extensively studied; however, little is known about their combined impact on plants. In the present study, we determined the photosynthetic gas exchange, chlorophyll fluorescence, nitrogen level, and lipid peroxidation of the leaves of a perennial grass (Leymus chinensis (Trin.) Tzvel.) subjected to three constant temperatures (23, 29 and 32 degrees C), and five soil-moisture levels (75-80%, 60-65%, 50-55%, 35-40% and 25-30% of field capacity, respectively). High temperature significantly decreased plant biomass, leaf green area, leaf water potential, photosynthetic rate (A), maximal efficiency of PSII photochemistry (F (v)/F (m)), actual PSII efficiency (Phi(PSII)), the activities of nitrate reductase (NR; EC 1.6.6.1) and glutamine synthetase (GS; EC 6.3.1.2), but markedly increased the ratio of leaf area to leaf weight (SLA), endopeptidase (EP; EC 3.4.24.11) activity, and malondialdehyde (MDA) content, especially under severe water stress conditions. The A and F (v)/F (m) were significantly and positively correlated with leaf-soluble protein content, and the activities of NR and GS. However, both photosynthesis parameters were significantly and negatively correlated with EP activity and MDA content (P < 0.05). It is suggested that high temperature, combined with severe soil drought, might reduce the function of PSII, weaken nitrogen anabolism, strengthen protein catabolism, and provoke lipid peroxidation. The results also indicate that severe water stress might exacerbate the adverse effects of high temperature, and their combination might reduce the plant productivity and distribution range of L. chinensis in the future.

Preoperative staging of gastric cancer is difficult and not optimal. The TNM stage is an important prognostic factor, but it can only be assessed reliably after surgery. Therefore, there is need for additional, reliable prognostic factors that can be determined preoperatively in order to select patients who might benefit from (neo) adjuvant treatment. Expression of immunohistochemical markers was demonstrated to be associated with tumour progression and metastasis. The expression of p53, CD44 (splice variants v5, v6 and v9), E-cadherin, Ep-CAM (CO17-1A antigen) and c-erB2/neu were investigated in tumour tissues of 300 patients from the Dutch Gastric Cancer Trial, investigating the value of extended lymphadenectomy compared to that of limited lymphadenectomy). The expression of tumour markers was analysed with respect to patient survival. Patients without loss of Ep-CAM-expression of tumour cells (19%) had a significantly better 10-year survival (P<0.0001) compared to patients with any loss: 42% (s.e.=7%) vs 22% (s.e.=3%). Patients with CD44v6 (VFF18) expression in more than 25% of the tumour cells (69% of the patients) also had a significantly better survival (P=0.01) compared to patients with expression in less than 25% of the tumour cells: 10 year survival rate of 29% (s.e.=3%) vs 19% (s.e.=4%). The prognostic value of both markers was stronger in stages I and II, and independent of the TNM stage. Ep-CAM and CD44v6-expression provides prognostic information additional to the TNM stage. Loss of Ep-CAM-expression identifies aggressive tumours especially in patients with stage I and II disease. This information may be helpful in selecting patients suitable for surgery or for additional treatment pre- or postoperatively.

Two anti-tumor monoclonal antibodies, L6 (anticarcinoma) and 1F5 (anti-B lymphoma), were covalently linked to alkaline phosphatase (AP), forming conjugates that could bind to the surface of antigen-positive tumor cells. The conjugates were capable of converting a relatively noncytotoxic prodrug, etoposide phosphate (EP), into etoposide--a drug with significant antitumor activity. In vitro studies with a human colon carcinoma cell line, H3347, demonstrated that while EP was less toxic than etoposide by a factor of greater than 100, it was equally toxic when the cells were pretreated with L6-AP, a conjugate that bound to the surface of H3347 cells. The L6-AP conjugate localized in H3347 tumor xenografts in nude mice and histological evaluation indicated that the targeted enzyme (AP) was distributed throughout the tumor mass. A strong antitumor response was observed in H3347-bearing mice that were treated with L6-AP followed 18-24 hr later by EP. This response, which included the rejection of established tumors, was superior to that of EP (P less than 0.005) or etoposide (P less than 0.001) given alone. The IF5-AP conjugate did not bind to H3347 cells and did not enhance the toxicity of EP on these cells in vitro. In addition, IF5-AP did not localize to H3347 tumors in nude mice and did not demonstrate enhanced antitumor activity in combination with the prodrug.

The general secretion pathway of gram-negative bacteria is responsible for extracellular secretion of a number of different proteins, including proteases and toxins. This pathway supports secretion of proteins across the cell envelope in two distinct steps, in which the second step, involving translocation through the outer membrane, is assisted by at least 13 different gene products. Two of these components, the cytoplasmic membrane proteins EpsL and EpsM of Vibrio cholerae, have been purified and characterized. Based on gel filtration analysis, both purified EpsM(His)6 and wild-type EpsL present in an Escherichia coli Triton X-100 extract are dimeric proteins. EpsL and EpsM were also found to interact directly and form a Triton X-100 stable complex that could be precipitated with either anti-EpsL or anti-EpsM antibodies. In addition, when the L and M proteins were coexpressed in E. coli, they formed a stable complex and protected each other from proteolytic degradation, indicating that these two proteins interact in vivo and that no other Eps protein is required for their association. Since EpsL is predicted to contain a large cytoplasmic domain, while EpsM is predominantly exposed on the periplasmic side, we speculate that these components might be part of a structure that is involved in bridging the inner and outer membranes. Furthermore, since EpsL has previously been shown to interact with the autophosphorylating cytoplasmic membrane protein EpsE, we hypothesize that this trimolecular complex might be involved in regulating the opening and closing of the secretion pore and/or transducing energy to the site of outer membrane translocation.

Epitope-based vaccines designed to induce CTL responses specific for HIV-1 are being developed as a means for addressing vaccine potency and viral heterogeneity. We identified a set of 21 HLA-A2, HLA-A3, and HLA-B7 restricted supertype epitopes from conserved regions of HIV-1 to develop such a vaccine. Based on peptide-binding studies and phenotypic frequencies of HLA-A2, HLA-A3, and HLA-B7 allelic variants, these epitopes are predicted to be immunogenic in greater than 85% of individuals. Immunological recognition of all but one of the vaccine candidate epitopes was demonstrated by IFN-gamma ELISPOT assays in PBMC from HIV-1-infected subjects. The HLA supertypes of the subjects was a very strong predictor of epitope-specific responses, but some subjects responded to epitopes outside of the predicted HLA type. A DNA plasmid vaccine, EP HIV-1090, was designed to express the 21 CTL epitopes as a single Ag and tested for immunogenicity using HLA transgenic mice. Immunization of HLA transgenic mice with this vaccine was sufficient to induce CTL responses to multiple HIV-1 epitopes, comparable in magnitude to those induced by immunization with peptides. The CTL induced by the vaccine recognized target cells pulsed with peptide or cells transfected with HIV-1 env or gag genes. There was no indication of immunodominance, as the vaccine induced CTL responses specific for multiple epitopes in individual mice. These data indicate that the EP HIV-1090 DNA vaccine may be suitable for inducing relevant HIV-1-specific CTL responses in humans.

Although neural circuits mediating various simple behaviors have been delineated, those generating more complex behaviors are less well described. The discrete structure of avian song control nuclei promises that circuits controlling complex behaviors, such as birdsong, can also be understood. To this end, we developed an in vitro brain slice preparation containing the robust nucleus of the archistriatum (RA), a forebrain song control nucleus, and its inputs from two other song nuclei, the caudal nucleus of the ventral hyperstriatum (HVc) and the lateral part of the magnocellular nucleus of the anterior neostriatum (L-MAN). Using intracellular recordings, we examined the pharmacological properties of the synapses made on RA neurons by L-MAN and HVc axons. Electrical stimulation of the L-MAN and the HVc fiber tracts evoked excitatory postsynaptic potentials (EPSPs) from >70% of RA neurons when slices were prepared from male birds of 40-90 days of age, suggesting that many individual RA neurons receive excitatory input from L-MAN and HVc axons. The "L-MAN" EPSPs were blocked by the N-methyl-D-aspartate (NMDA) receptor antagonist D-(-)-2-amino-5-phosphonovaleric acid (D-APV) as well as the broad-spectrum glutamate receptor antagonist kynurenic acid but were relatively unaffected by the non-NMDA receptor blocker 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). In contrast, "HVc" EP-SPs were relatively insensitive to D-APV but almost completely abolished by CNQX. These experiments suggest that L-MAN and HVc axons provide pharmacologically distinct types of excitatory input to many of the same RA neurons.

We investigated the coupling between perfusion-related brain imaging signals and evoked neuronal activity using optical imaging of intrinsic signals (OIS) at 570 and 610 nm. OIS at 570 nm reflects changes in cerebral blood volume (CBV), and the 610 nm response is related to hemoglobin oxygenation changes. We assessed the degree to which these components of the hemodynamic response were coupled to neuronal activity in rat barrel, hindpaw, and forepaw somatosensory cortex by simultaneously recording extracellular evoked field potentials (EPs) and OIS while varying stimulation frequency. In all stimulation paradigms, 10 Hz stimulation evoked the largest optical and electrophysiological responses. Across all animals, the 610 late phase and 570 responses correlated linearly with sigmaEP (P < 0.05) during both whisker deflection and electrical hindpaw stimulation, but the 610 early phase did not (whisker P = 0.27, hindpaw P = 0.28). The signal-to-noise ratio (SNR) of the 610 early phase (whisker 3.1, hindpaw 5.3) was much less than that for the late phase (whisker 14, hindpaw 51) and 570 response (whisker 11, hindpaw 46). During forepaw stimulation, however, the 610 early phase had a SNR (17) higher than that during hindpaw stimulation and correlated well with neuronal activity (P < 0.05). We conclude that the early deoxygenation change does not correlate consistently with neuronal activity, possibly because of its low SNR. The robust CBV-related response, however, has a high SNR and correlates well with evoked cortical activity.

1. A study of the effects of prostaglandin E2 (PGE2) and eleven synthetic analogues on the guinea-pig isolated ileum preparation has revealed three distinct contractile actions, each associated with a different prostaglandin E (EP-) receptor subtype. In addition, PGI2 (prostacyclin) and its stable analogues can activate prostaglandin I (IP-) receptors to elicit both contraction and relaxation of the ileum. 2. Two of the PGE actions involve direct stimulation of the smooth muscle, being unaffected by 1 microM morphine treatment. One action is blocked by AH 6809 at micromolar concentrations and ICI 80205 and 16,16-dimethyl PGE2 are particularly potent agonists. Activation of EPEPEPEPEPEPEPEPl1-receptor studies.5. In the presence of morphine and AH 6809, cicaprost inhibits histamine-induced contractions (IC25 = 22 nM). PGI2 and iloprost show mixed inhibitory/potentiating actions, whereas carbacyclin only potentiates histamine contractions. This IP-receptor-mediated inhibition may account for the bell-shaped log concentration-response curve of cicaprost (no inhibitors present) and the very marked block of iloprostinduced contractions by AH 6809.6. We have found no evidence for either IP-receptors mediating direct contraction or EP-receptors mediating inhibition of the ileum longitudinal smooth muscle, as has been suggested in the literature.7. In view of the complexity of prostanoid action on the guinea-pig ileum we feel that the preparation must be used with caution to ascertain the EPl agonist or antagonist potencies of novel compounds.

Cytochrome P450 aromatase (aromatase), a product of the CYP19 gene, catalyzes the synthesis of estrogens from androgens. Because aromatase-dependent estrogen biosynthesis has been linked to hormone-dependent breast carcinogenesis, it is important to elucidate the mechanisms that regulate CYP19 gene expression. The main objective of this study was to identify the receptors (EP) for prostaglandin E(2) (PGE(2)) that mediate the induction of CYP19 transcription in human adipocytes and breast cancer cells. Treatment with PGE(2) induced aromatase, an effect that was mimicked by either EP(2) or EP(4) agonists. Antagonists of EP(2) or EP(4) or small interference RNA-mediated down-regulation of these receptors suppressed PGE(2)-mediated induction of aromatase. PGE(2) via EP(2) and EP(4) stimulated the cAMP->>protein kinase A pathway resulting in enhanced interaction between P-CREB, p300, and the aromatase promoter I.3/II. Overexpressing a mutant form of p300 that lacks histone acetyltransferase activity suppressed PGE(2)-mediated induction of aromatase promoter activity. PGE(2) via EP(2) and EP(4) also caused a reduction in both the amounts of BRCA1 and the interaction between BRCA1 and the aromatase promoter I.3/II. Activation of the aromatase promoter by PGE(2) was suppressed by overexpressing wild-type BRCA1. Silencing of EP(2) or EP(4) also blocked PGE(2)-mediated induction of the progesterone receptor, a prototypic estrogen-response gene. In a mouse model, overexpressing COX-2 in the mammary gland, a known inducer of PGE(2) synthesis, led to increased aromatase mRNA and activity and reduced amounts of BRCA1; these effects were reversed by knocking out EP(2). Taken together, these results suggest that PGE(2) via EP(2) and EP(4) activates the cAMP->>PKA->>CREB pathway leading to enhanced CYP19 transcription and increased aromatase activity. Reciprocal changes in the interaction between BRCA1, p300, and the aromatase promoter I.3/II contributed to the inductive effects of PGE(2).

We have stressed the role of certain growth factors and cytokines in peritoneal fibrosis, including TGFbeta, TIMP-1, and inflammatory cytokines, especially IL-1beta. Recent research highlights the myofibroblast-like transformation of mesothelial cells as a central initiating event in peritoneal fibrosis. The induction, survival, and apoptosis of the myofibroblast cell population likely dictate the nature of the fibrogenic response. The accumulation of collagen occurs in a nondegradative environment, and collagenases and their inhibitors have a role in the maintenance of fibrosis. Fibrosis appears to be a ubiquitous response of peritoneal tissues to the damaging effects of uremia, bioincompatible dialysate, recurrent infection, and inflammation. Recent research has focused on the induction of angiogenesis, as this appears to correlate with increased solute transport and peritoneal membrane ultrafiltration failure. Fibrosis may play an integral part in peritoneal membrane dysfunction in several aspects. Angiogenesis may be induced as part of the fibrotic response, as many key fibrogenic cytokines are also strongly angiogenic. Fibrotic tissue may support and preserve angiogenesis. Changes in the interstitium may have a direct effect on the hydrodynamic properties of the peritoneum and may directly influence fluid movement. In its most extreme form, fibrosis manifests as the rare but devastating EPS. Peritoneal biopsy studies have identified a high prevalence of peritoneal fibrosis in PD patients. Research into peritoneal fibrosis will be enhanced by new animal models where the role of various cytokines and growth factors, cellular processes, and matrix interactions can be studied. With these models, the role of fibrosis in alteration of peritoneal membrane function can be better assessed. Clinical trials to assess the role of prevention of peritoneal injury using biocompatible solutions and treatments targeted directly at peritoneal fibrosis will be important, but challenging to design and carry out.