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We present BVRI and unfiltered light curves of 93 Type Ia supernovae (SNe Ia) from the Lick Observatory Supernova Search (LOSS) follow-up program conducted between 2005 and 2018. Our sample consists of 78 spectroscopically normal SNe Ia, with the remainder divided between distinct subclasses (3 SN 1991bg-like, 3 SN 1991T-like, 4 SNe Iax, 2 peculiar, and 3 super-Chandrasekhar events), and has a median redshift of 0.0192. The SNe in our sample have a median coverage of 16 photometric epochs at a cadence of 5.4 d, and the median first observed epoch is ∼4.6 d before maximum B-band light. We describe how the SNe in our sample are discovered, observed, and processed, and we compare the results from our newly developed automated photometry pipeline to those from the previous processing pipeline used by LOSS. After investigating potential biases, we derive a final systematic uncertainty of 0.03 mag in BVRI for our data set. We perform an analysis of our light curves with particular focus on using template fitting to measure the parameters that are useful in standardizing SNe Ia as distance indicators. All of the data are available to the community, and we encourage future studies to incorporate our light curves in their analyses.

We combined deep U-band and optical/near-infrared imaging, in order to select Lyman Break Galaxies (LBGs) at using U-V and colors in the Subaru Deep Field. The resulting sample of 5161 LBGs gives a UV luminosity function (LF) down to , with a steep faint-end slope of . We analyze UV-To-NIR energy distributions (SEDs) from optical photometry and photometry on IR median-stacked images. In the stacks, we find a systematic background depression centered on the LBGs. This results from the difficulty of finding faint galaxies in regions with higher-Than-Average surface densities of foreground galaxies, so we corrected for this deficit. Best-fit stellar population models for the LBG SEDs indicate stellar masses and star formation rates of and M o yr-1 at , down to and yr-1 at . The faint LBGs show a ∼1 mag excess over the stellar continuum in K-band. We interpret this excess flux as redshifted [O iii] lines. The observed excesses imply equivalent widths that increase with decreasing mass, reaching A (rest-frame). Such strong [O iii] emission is seen only in a miniscule fraction of local emission-line galaxies, but is probably universal in the faint galaxies that reionized the universe. Our halo occupation distribution analysis of the angular correlation function gives a halo mass of for the full sample of LBGs, and for the brightest half of the sample.

We present optical spectra of the nearby Type Ia supernova SN 2011fe at 100, 205, 311, 349 and 578 d post-maximum light, as well as an ultraviolet (UV) spectrum obtained with the Hubble Space Telescope at 360 d post-maximum light. We compare these observations with synthetic spectra produced with the radiative transfer code PHOENIX. The day +100 spectrum can be well fitted with models that neglect collisional and radiative data for forbidden lines. Curiously, including these data and recomputing the fit yields a quite similar spectrum, but with different combinations of lines forming some of the stronger features. At day +205 and later epochs, forbidden lines dominate much of the optical spectrum formation; however, our results indicate that recombination, not collisional excitation, is the most influential physical process driving spectrum formation at these late times. Consequently, our synthetic optical and UV spectra at all epochs presented here are formed almost exclusively through recombinationdriven fluorescence. Furthermore, our models suggest that the UV spectrum even as late as day +360 is optically thick and consists of permitted lines from several iron-peak species. These results indicate that the transition to the 'nebular' phase in Type Ia supernovae is complex and highly wavelength dependent.

KA1858+4850 is a narrow-line Seyfert 1 galaxy at redshift 0.078 and is among the brightest active galaxies monitored by the Kepler mission. We have carried out a reverberation mapping campaign designed to measure the broad-line region size and estimate the mass of the black hole in this galaxy. We obtained 74 epochs of spectroscopic data using the Kast Spectrograph at the Lick 3 m telescope from 2012 February to November, and obtained complementary V-band images from five other ground-based telescopes. We measured the Hβ light curve lag with respect to the V-band continuum light curve using both cross-correlation techniques (CCF) and continuum light curve variability modeling with the JAVELIN method and found rest-frame lags of days and τJAVELINdays. The Hβ rms line profile has a width of σline = 770 ± 49 km s-1. Combining these two results and assuming a virial scale factor of f = 5.13, we obtained a virial estimate of for the mass of the central black hole and an Eddington ratio of L/L Edd 0.2. We also obtained consistent but slightly shorter emission-line lags with respect to the Kepler light curve. Thanks to the Kepler mission, the light curve of KA1858+4850 has among the highest cadences and signal-to-noise ratios ever measured for an active galactic nucleus; thus, our black hole mass measurement will serve as a reference point for relations between black hole mass and continuum variability characteristics in active galactic nuclei.

Background: Metastatic renal cell carcinoma (mRCC) prognostic models may be improved by incorporating treatment-induced toxicities., Methods: In sunitinib-treated mRCC patients (N=770), baseline prognostic factors and treatment-induced toxicities (hypertension (systolic blood pressure ⩾140 mm Hg), neutropenia (grade ⩾2), thrombocytopenia (grade ⩾2), hand-foot syndrome (grade >0), and asthenia/fatigue (grade >0)) were analysed in multivariate analyses of progression-free survival (PFS) and overall survival (OS) end points., Results: On-treatment neutropenia and hypertension were associated with longer PFS (P=0.0276 and P<0.0001, respectively) and OS (P=0.0014 and P<0.0001, respectively), independent of baseline prognostic factors, including International Metastatic Renal Cell Carcinoma Database Consortium (IMDC) criteria. By 12-week landmark analysis, neutropenia was significantly associated with longer PFS and OS (P=0.013 and P=0.0122, respectively) and hypertension or hand-foot syndrome with longer OS (P=0.0036 and P=0.0218, respectively). The concordance index was 0.65 (95% CI: 0.63-0.67) for IMDC classification alone and 0.72 (95% CI: 0.70-0.74) when combined with hypertension and neutropenia. Considering hypertension and neutropenia (developing both vs neither) changed IMDC-predicted median OS in each IMDC risk group (favourable: 45.3 vs 19.5 months; intermediate: 32.5 vs 8.0 months; poor: 21.1 vs 4.8 months)., Conclusions: On-treatment neutropenia and hypertension are independent biomarkers of sunitinib efficacy and may add prognostic accuracy to the IMDC model.

Understanding the etiology of metastasis is very important in clinical perspective, since it is estimated that metastasis accounts for 90% of cancer patient mortality. Metastasis results from a sequence of multiple steps including invasion and migration. The early stages of metastasis are tightly controlled in normal cells and can be drastically affected by malignant mutations; therefore, they might constitute the principal determinants of the overall metastatic rate even if the later stages take long to occur. To elucidate the role of individual mutations or their combinations affecting the metastatic development, a logical model has been constructed that recapitulates published experimental results of known gene perturbations on local invasion and migration processes, and predict the effect of not yet experimentally assessed mutations. The model has been validated using experimental data on transcriptome dynamics following TGF-β-dependent induction of Epithelial to Mesenchymal Transition in lung cancer cell lines. A method to associate gene expression profiles with different stable state solutions of the logical model has been developed for that purpose. In addition, we have systematically predicted alleviating (masking) and synergistic pairwise genetic interactions between the genes composing the model with respect to the probability of acquiring the metastatic phenotype. We focused on several unexpected synergistic genetic interactions leading to theoretically very high metastasis probability. Among them, the synergistic combination of Notch overexpression and p53 deletion shows one of the strongest effects, which is in agreement with a recent published experiment in a mouse model of gut cancer. The mathematical model can recapitulate experimental mutations in both cell line and mouse models. Furthermore, the model predicts new gene perturbations that affect the early steps of metastasis underlying potential intervention points for innovative therapeutic strategies in oncology.

Data visualization is an essential element of biological research, required for obtaining insights and formulating new hypotheses on mechanisms of health and disease. NaviCell Web Service is a tool for network-based visualization of 'omics' data which implements several data visual representation methods and utilities for combining them together. NaviCell Web Service uses Google Maps and semantic zooming to browse large biological network maps, represented in various formats, together with different types of the molecular data mapped on top of them. For achieving this, the tool provides standard heatmaps, barplots and glyphs as well as the novel map staining technique for grasping large-scale trends in numerical values (such as whole transcriptome) projected onto a pathway map. The web service provides a server mode, which allows automating visualization tasks and retrieving data from maps via RESTful (standard HTTP) calls. Bindings to different programming languages are provided (Python and R). We illustrate the purpose of the tool with several case studies using pathway maps created by different research groups, in which data visualization provides new insights into molecular mechanisms involved in systemic diseases such as cancer and neurodegenerative diseases., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Several decades of molecular biology research have delivered a wealth of detailed descriptions of molecular interactions in normal and tumour cells. This knowledge has been functionally organised and assembled into dedicated biological pathway resources that serve as an invaluable tool, not only for structuring the information about molecular interactions but also for making it available for biological, clinical and computational studies. With the advent of high-throughput molecular profiling of tumours, close to complete molecular catalogues of mutations, gene expression and epigenetic modifications are available and require adequate interpretation. Taking into account the information about biological signalling machinery in cells may help to better interpret molecular profiles of tumours. Making sense out of these descriptions requires biological pathway resources for functional interpretation of the data. In this review, we describe the available biological pathway resources, their characteristics in terms of construction mode, focus, aims and paradigms of biological knowledge representation. We present a new resource that is focused on cancer-related signalling, the Atlas of Cancer Signalling Networks. We briefly discuss current approaches for data integration, visualisation and analysis, using biological networks, such as pathway scoring, guilt-by-association and network propagation. Finally, we illustrate with several examples the added value of data interpretation in the context of biological networks and demonstrate that it may help in analysis of high-throughput data like mutation, gene expression or small interfering RNA screening and can guide in patients stratification. Finally, we discuss perspectives for improving precision medicine using biological network resources and tools. Taking into account the information about biological signalling machinery in cells may help to better interpret molecular patterns of tumours and enable to put precision oncology into general clinical practice., (© The Author 2015. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Crizotinib is approved to treat advanced ALK-positive non-small-cell lung cancer (NSCLC), but most patients ultimately develop progressive disease (PD). We investigated whether continuing ALK inhibition with crizotinib beyond PD (CBPD) is clinically beneficial and attempted to identify clinicopathologic characteristics associated with patients who experience clinical benefit., Patients and Methods: Patients with advanced ALK-positive NSCLC enrolled in two ongoing multicenter, single-arm trials who developed RECIST-defined PD were allowed to continue crizotinib if they were deriving ongoing clinical benefit. In the present retrospective analysis, continuation of CBPD was defined as >3 weeks of crizotinib treatment after PD documentation. Patients who had PD as best response to initial crizotinib treatment were excluded. Baseline and post-progression characteristics, sites of PD, and overall survival (OS) were compared in patients who continued CBPD versus those who did not. The impact of continuing CBPD on OS after adjusting for potential confounding factors was assessed., Results: Among 194 crizotinib-treated patients with RECIST-defined PD, 120 (62%) continued CBPD. A significantly higher proportion of patients who continued CBPD than patients who did not had an ECOG performance status (PS) of 0/1 at PD (96% versus 82%; P=0.02). CBPD patients had significantly longer OS from the time of PD [median 16.4 versus 3.9 months; hazards ratio (HR) 0.27, 95% confidence interval (CI): 0.17-0.42; P<0.0001] and from the time of initial crizotinib treatment (median 29.6 versus 10.8 months; HR 0.30, 95% CI: 0.19-0.46; P<0.0001). The multiple-covariate Cox regression analysis revealed that CBPD remained significantly associated with improved OS after adjusting for relevant factors., Conclusions: Patients who continued CBPD were more likely to have good ECOG PS (0/1) at the time of PD. Continuing ALK inhibition with crizotinib after PD may provide survival benefit to patients with advanced ALK-positive NSCLC.

Background: Molecular biology knowledge can be formalized and systematically represented in a computer-readable form as a comprehensive map of molecular interactions. There exist an increasing number of maps of molecular interactions containing detailed and step-wise description of various cell mechanisms. It is difficult to explore these large maps, to organize discussion of their content and to maintain them. Several efforts were recently made to combine these capabilities together in one environment, and NaviCell is one of them., Results: NaviCell is a web-based environment for exploiting large maps of molecular interactions, created in CellDesigner, allowing their easy exploration, curation and maintenance. It is characterized by a combination of three essential features: (1) efficient map browsing based on Google Maps; (2) semantic zooming for viewing different levels of details or of abstraction of the map and (3) integrated web-based blog for collecting community feedback. NaviCell can be easily used by experts in the field of molecular biology for studying molecular entities of interest in the context of signaling pathways and crosstalk between pathways within a global signaling network. NaviCell allows both exploration of detailed molecular mechanisms represented on the map and a more abstract view of the map up to a top-level modular representation. NaviCell greatly facilitates curation, maintenance and updating the comprehensive maps of molecular interactions in an interactive and user-friendly fashion due to an imbedded blogging system., Conclusions: NaviCell provides user-friendly exploration of large-scale maps of molecular interactions, thanks to Google Maps and WordPress interfaces, with which many users are already familiar. Semantic zooming which is used for navigating geographical maps is adopted for molecular maps in NaviCell, making any level of visualization readable. In addition, NaviCell provides a framework for community-based curation of maps.

Background: Reliable biomarkers of sunitinib response in gastrointestinal stromal tumor (GIST) are lacking. Hypertension (HTN), an on-target class effect of vascular endothelial growth factor signaling-pathway inhibitors, has been shown to correlate with clinical outcome in advanced renal cell carcinoma treated with sunitinib., Patients and Methods: This retrospective analysis examined correlations between sunitinib-associated HTN and antitumor efficacy (N = 319) and safety (N = 1565) across three advanced GIST studies. Blood pressure (BP) was measured on days 1 and 28 of each treatment cycle at a minimum. Time-to-event endpoints were estimated using Kaplan-Meier methods, and patient subgroups with and without HTN (maximum systolic BP ≥ 140 mmHg and/or diastolic BP ≥ 90 mmHg) were compared using Cox proportional hazards models. Landmark analyses evaluated associations between early HTN and efficacy endpoints. Adverse events (AEs) were compared between groups., Results: Sunitinib-associated HTN correlated with improved objective response rates, time to tumor progression, progression-free survival, and overall survival. Almost all benefits remained significant in multivariate and landmark analyses. Overall incidences of HTN-related AEs were low and similar between groups; incidences of cardiovascular AEs were somewhat higher in patients with HTN., Conclusion: Sunitinib-associated HTN appeared to correlate with improved clinical outcomes in GIST, while incidences of HTN-associated AEs were generally low and manageable.

Purpose: To analyze final long-term survival and clinical outcomes from the randomized phase III study of sunitinib in gastrointestinal stromal tumor patients after imatinib failure; to assess correlative angiogenesis biomarkers with patient outcomes., Experimental Design: Blinded sunitinib or placebo was given daily on a 4-week-on/2-week-off treatment schedule. Placebo-assigned patients could cross over to sunitinib at disease progression/study unblinding. Overall survival (OS) was analyzed using conventional statistical methods and the rank-preserving structural failure time (RPSFT) method to explore cross-over impact. Circulating levels of angiogenesis biomarkers were analyzed., Results: In total, 243 patients were randomized to receive sunitinib and 118 to placebo, 103 of whom crossed over to open-label sunitinib. Conventional statistical analysis showed that OS converged in the sunitinib and placebo arms (median 72.7 vs. 64.9 weeks; HR, 0.876; P = 0.306) as expected, given the cross-over design. RPSFT analysis estimated median OS for placebo of 39.0 weeks (HR, 0.505, 95% CI, 0.262-1.134; P = 0.306). No new safety concerns emerged with extended sunitinib treatment. No consistent associations were found between the pharmacodynamics of angiogenesis-related plasma proteins during sunitinib treatment and clinical outcome., Conclusions: The cross-over design provided evidence of sunitinib clinical benefit based on prolonged time to tumor progression during the double-blind phase of this trial. As expected, following cross-over, there was no statistical difference in OS. RPSFT analysis modeled the absence of cross-over, estimating a substantial sunitinib OS benefit relative to placebo. Long-term sunitinib treatment was tolerated without new adverse events.

Background: Malaria Rapid Diagnostic Tests (RDTs) are widely used to diagnose malaria. The present study evaluated a new RDT, the Clearview® Malaria pLDH test targeting the pan-Plasmodium antigen lactate dehydrogenase (pLDH)., Methods: The Clearview® Malaria pLDH test was evaluated on fresh samples obtained in returned international travellers using microscopy corrected by PCR as the reference method. Included samples were Plasmodium falciparum (139), Plasmodium vivax (22), Plasmodium ovale (20), Plasmodium malariae (7), and 102 negative., Results: Overall sensitivity for the detection of Plasmodium spp was 93.2%. For P. falciparum, the sensitivity was 98.6%; for P. vivax, P. ovale and P. malariae, overall sensitivities were 90.9%, 60.0% and 85.7% respectively. For P. falciparum and for P. vivax, the sensitivities increased to 100% at parasite densities above 100/μl. The specificity was 100%. The test was easily to perform and the result was stable for at least 1 hour., Conclusion: The Clearview® Malaria pLDH was efficient for the diagnosis of malaria. The test was very sensitive for P. falciparum and P. vivax detection. The sensitivities for P. ovale and P. malariae were better than other RDTs.

Background: Hypertension (HTN) is an on-target effect of the vascular endothelial growth factor pathway inhibitor, sunitinib. We evaluated the association of sunitinib-induced HTN with antitumor efficacy and HTN-associated adverse events in patients with metastatic renal cell carcinoma., Methods: This retrospective analysis included pooled efficacy (n = 544) and safety (n = 4917) data from four studies of patients with metastatic renal cell carcinoma who were treated with sunitinib 50 mg/d administered on a 4-week-on 2-week-off schedule (schedule 4/2). Blood pressure (BP) was measured in the clinic on days 1 and 28 of each 6-week cycle. Progression-free survival (PFS) and overall survival (OS) were estimated using Kaplan-Meier methods; hazard ratios (HRs) for survival were also estimated by a Cox proportional hazards models using HTN as a time-dependent covariate. Efficacy outcomes were compared between patients with and without HTN (maximum systolic BP [SBP] ≥140 mm Hg or diastolic BP [DBP] ≥90 mm Hg). Adverse events were also compared between patients with and without HTN (mean SBP ≥140 mm Hg or mean DBP ≥90 mm Hg). All P values were two-sided., Results: Patients with metastatic renal cell carcinoma and sunitinib-induced HTN defined by maximum SBP had better outcomes than those without treatment-induced HTN (objective response rate: 54.8% vs 8.7%; median PFS: 12.5 months, 95% confidence interval [CI] = 10.9 to 13.7 vs 2.5 months, 95% CI = 2.3 to 3.8 months; and OS: 30.9 months, 95% CI = 27.9 to 33.7 vs 7.2 months, 95% CI = 5.6 to 10.7 months; P < .001 for all). Similar results were obtained when comparing patients with vs without sunitinib-induced HTN defined by maximum DBP. In a Cox proportional hazards model using HTN as a time-dependent covariate, PFS (HR of disease progression or death = .603, 95% CI = .451 to .805; P < .001) and OS (HR of death = .332, 95% CI = .252 to .436; P < .001) were improved in patients with treatment-induced HTN defined by maximum SBP; OS (HR of death = .585, 95% CI = .463 to .740; P < .001) was improved in patients with treatment-induced HTN defined by maximum DBP, but PFS was not. Few any-cause cardiovascular, cerebrovascular, ocular, and renal adverse events were observed. Rates of adverse events were similar between patients with and without HTN defined by mean SBP; however, hypertensive patients had somewhat more renal adverse events (5% vs 3%; P = .013)., Conclusions: In patients with metastatic renal cell carcinoma, sunitinib-associated HTN is associated with improved clinical outcomes without clinically significant increases in HTN-associated adverse events, supporting its viability as an efficacy biomarker.

This study examined the expenditure of compensation received from legal claims by service users attending an outpatient methadone programme in Dublin. Most claims (n = 62) were as a result of road traffic accidents (74%) or personal injury (15%). There were 28 reports of claims resulting in payment of compensation totalling €912,871. Of the compensation not placed in trust (€477,871), almost 40% was spent on drugs and 8% on alcohol. Of those who reported no drug misuse at the time of the compensation being paid, seven out of 11 (64%) reported subsequently spending a significant amount on substance misuse. The risks of receiving large amounts of money in this population are substantial and include initiation and exacerbation of substance misuse, and risk of overdose. Alternative ways of managing the payment of compensation should be considered for this vulnerable population.

Purpose: To evaluate sunitinib activity and potential cellular and molecular correlates in gastrointestinal stromal tumor (GIST) patients after imatinib failure, in addition to assessing the safety and pharmacokinetics (PK) of different dose schedules., Experimental Design: In this open-label, dose-ranging, phase I/II study, 97 patients with metastatic imatinib-resistant/intolerant GIST received sunitinib at doses of 25, 50, or 75 mg/d on one of three schedules. Serial tumor imaging was done using computed tomography and [18F]fluoro-2-deoxy-d-glucose positron emission tomography scanning. PK and cell proliferation and KIT phosphorylation status in tumor biopsies were also analyzed., Results: Clinical benefit was observed in 52 patients (54%: 7 objective partial responses, 45 stable disease > or =6 months). Decreased tumor glycolytic activity was shown in most patients within 7 days of starting sunitinib using [18F]fluoro-2-deoxy-d-glucose positron emission tomography. Sunitinib treatment was associated with reduced tumor cell proliferation by >25% in 52% of cases analyzed and reduced levels of phospho-KIT in tumor biopsies (indicating target modulation). The recommended dose schedule was 50 mg/d for 4 weeks followed by 2 weeks off treatment. On the 50-mg dose across all schedules, 79% of PK-evaluable patients achieved total drug trough concentrations above the target concentration (50 ng/mL) within 14 days of dosing. In addition, adverse events were generally mild to moderate in severity., Conclusion: Cellular and molecular analyses showed that sunitinib clinical activity is associated with inhibition of KIT in GIST following imatinib failure, illustrating the rational approach used to develop a therapy aimed at the underlying oncogenic signaling pathway aberrancy.

Aims: To assess the antitumour activity, safety, pharmacokinetics and pharmacodynamics of continuous daily sunitinib dosing in patients with imatinib-resistant/intolerant gastrointestinal stromal tumour (GIST) and to assess morning dosing versus evening dosing., Patients and Methods: In this open-label phase II study, patients were randomised to receive morning or evening dosing of sunitinib 37.5mg/day. The primary end-point was clinical benefit rate (CBR; percent complete responses+partial responses [PRs]+stable disease [SD] 24 weeks). Secondary end-points included progression-free survival (PFS), overall survival (OS), safety, pharmacokinetic parameters and plasma biomarker levels., Results: Sixty of 61 planned patients received treatment (30 per dosing group); 26 completed the study. Overall, the CBR was 53% (95% exact CI, 40-66): eight patients (13%) achieved objective PRs; 24 (40%) achieved SD 24 weeks. Median PFS was 34 weeks (95% CI, 24-49); median OS was 107 weeks (95% CI, 72 - not yet calculable). Most adverse events (AEs) were of grade 1 or 2 in severity, and were manageable through dose modification or standard interventions. No new AEs were apparent compared with the approved intermittent dosing schedule. Antitumour activity and safety were generally similar with morning and evening dosing. Continuous daily sunitinib dosing achieved and sustained effective drug concentrations without additional accumulation across cycles. Decreases from baseline in plasma levels of soluble KIT after 20 and 24 weeks of dosing correlated with longer OS., Conclusion: For patients with imatinib-resistant/intolerant GIST, continuous daily sunitinib dosing appears to be an active alternative dosing strategy with acceptable safety.

Experimental Design: To investigate the antitumor effect of sunitinib and FAK/Pyk2 tyrosine kinase inhibitor (PF-562,271)combination therapy in vivo, utilizing human hepatocellular carcinoma (HCC) cells Huh7.5. Nude rats were inoculated subcutaneously with Huh7.5 hepatoma cells. Dosing for Phase 1 was initiated on day 5 post tumor inoculations with Vehicle(Group 1), sunitinib (25 mg/kg/day; Group 2) and sunitinib plus PF-562,271 combination (15 mg/kg/day; Group 3). Phase 2 of the study started on day 26, and each of the three original groups was divided in two subgroups; half of the rats remained on original therapy (Groups 1A and 2A) with the exception of Group 3A that was euthanized after Phase 1. The other half of the rats were switched to sunitinib and PF-562,271 combination (Group 1B) or vehicle (Groups 2B and 3B). Tumor volume and weight, serum alpha feto-protein (AFP), contrast-enhanced ultrasound imaging (CEUS) and tumor histology were used to evaluate effects of treatment on tumor growth., Results: The results from this study indicate that the combination of sunitinib and PF-562,271 TKI has the potential to target different aspects of angiogenesis and tumor aggressiveness and may have significantly greater effect than relevant single agent, blocking not only tumor growth, but also impacting the ability of the tumor to recover upon withdrawal of the therapy.

Purpose: Concurrent chemotherapy and radiation therapy (RT) are used to treat patients with esophageal cancer. The optimal combination of chemotherapeutic agents with RT is not well established. We evaluated the safety and preliminary efficacy of a combination of UFT/leucovorin, carboplatin, and paclitaxel with RT in a Phase I study of patients with advanced esophageal cancer., Methods and Materials: Patients with squamous cell carcinoma or adenocarcinoma of the esophagus initially received UFT/leucovorin, carboplatin, and paclitaxel with RT (1.8 Gy daily to 45 Gy). After completion, the disease was restaged and patients were evaluated for surgery. Primary end points included determination of dose-limiting toxicities (DLTs) and a recommended Phase II dose. Secondary objectives included determination of non-DLTs, as well as preliminary radiographic and pathologic response rates., Results: Twelve patients were enrolled (11 men, 1 woman). All were assessable for toxicity and efficacy. One of 6 patients at Dose Level 1 (UFT/leucovorin, 200/30 mg twice daily on RT days; carboplatin, area under the curve [AUC] 5, Weeks 1 and 4; paclitaxel, 175 mg/m2 Weeks 1 and 4) had a DLT (febrile neutropenia). Of these 6 patients, 4 underwent esophagectomy and none achieved a pathologic complete response. Six patients were then enrolled at Dose Level 2 (UFT/leucovorin, 300/30 mg in the morning and 200/30 mg in the evening on RT days; carboplatin, AUC 5, Weeks 1 and 4; paclitaxel, 175 mg/m2 Weeks 1 and 4). Two of 6 patients at Dose Level 2 developed DLTs (febrile neutropenia in both). Esophagectomy was performed in 3 patients, with 2 achieving a pathologic complete response., Conclusions: Maximum tolerated doses in this study were UFT/leucovorin, 200/30 mg twice daily on RT days; carboplatin, AUC 5, Weeks 1 and 4; and paclitaxel, 175 mg/m2 Weeks 1 and 4 when delivered with external RT. In this small study, this regimen appears active, but toxic.

Lactobacillus plantarum is part of the natural microbiota of many food fermentations as well as the human gastro-intestinal tract. The cytosolic fraction of the proteome of L. plantarum WCFS1, whose genome has been sequenced, was studied. 2-DE was used to investigate the proteins from the cytosolic fraction isolated from mid- and late-log, early- and late-stationary phase cells to generate reference maps of different growth conditions offering more knowledge of the metabolic behavior of this bacterium. From this fraction, a total of 200 protein spots were identified by MALDI-MS and a proteome production map was constructed to facilitate further studies such as detection of suitable biomarkers for specific growth conditions. More than half (57%) of the identified proteins were predicted to be involved in metabolic pathways of the bacterium. The protein profile changed during the growth of the bacteria such that 29% of the identified proteins involved in anabolic pathways were at least twofold up-regulated throughout the mid- and late-exponential and early-stationary phases. In the late-stationary phase, six proteins involved in stress or with a potential role for survival during starvation were up-regulated significantly.

Purpose: Eniluracil is an effective inactivator of dihydropyrimidine dehydrogenase (DPD). It allows for oral dosing of 5-fluorouracil (5-FU), which may potentially improve the antitumor activity of 5-FU when delivered concurrently with radiotherapy while avoiding the inconvenience and morbidity of continuous infusion (CI) 5-FU. We addressed the safety of oral eniluracil/5-FU combined with radiation therapy and determined the profile of dose-limiting toxicities and recommended Phase II dose (RPTD) in patients with pancreatic and hepatobiliary cancers., Methods and Materials: Patients with resectable or locally advanced pancreatic and biliary cancer received eniluracil (starting at 6.0 mg/m(2) q12h)/5-FU (starting at 0.6 mg/m(2) q12h). Eniluracil/5-FU were given concurrently with preoperative radiation to 4500 cGy followed by 540 cGy by reduced fields. Surgery was considered 4 weeks after completion of therapy., Results: Thirteen patients were enrolled. Chemoradiotherapy was completed in all patients. The MTD was not reached and, thus, the RPTD of eniluracil/5-FU was determined to be 10 mg/m(2) q12h/1 mg/m(2) q12h. Two patients with locally advanced disease had a 30-45 percent cross-sectional tumor reduction, one of which underwent margin-negative resection. Two of 5 patients with pancreatic cancer, and 1 of 3 patients with cholangiocarcinoma, with underwent exploratory surgery had margin-negative resections. One patient had a pathologic complete response (pCR). Patient 5-FU plasma exposure increased slightly from Day 8 to Day 31., Conclusion: Preoperative chemoradiation with oral eniluracil/5-FU is feasible, well tolerated, and potentially effective in the neoadjuvant setting. Further investigation of oral fluoropyrimidines as radiosensitizers for pancreaticobiliary malignancies is warranted.

Purpose: Eniluracil, an effective inactivator of dihydropyrimidine dehydrogenase, allows for oral dosing of 5-fluorouracil (5-FU), which avoids the morbidity of continuous infusion 5-FU. We addressed the safety of oral eniluracil and 5-FU combined with preoperative radiotherapy and determined the recommended Phase II dose and dose-limiting toxicity in patients with locally advanced rectal and colon cancer., Methods and Materials: Patients with TNM Stage II or III rectal cancer and residual or recurrent colon cancer received eniluracil (starting at 6.0 mg/m(2) every 12 h) and 5-FU (starting at 0.6 mg/m(2) every 12 h). Eniluracil and 5-FU were given with a 5-week course of preoperative radiotherapy of 4500 cGy, with a possible 540-cGy boost. Surgery was performed approximately 4 weeks after completion of chemoradiotherapy., Results: Twenty-two patients were enrolled; 1 patient was withdrawn owing to noncompliance. Chemotherapy was completed in all patients; radiotherapy was completed in 20 patients. The recommended Phase II dose of eniluracil and 5-FU was 8 mg/m(2) every 12 h and 0.8 mg/m(2) every 12 h, respectively. Diarrhea was the dose-limiting toxicity. Eleven of the 17 patients with primary rectal cancer underwent a sphincter-sparing procedure. One patient had a pathologic complete response., Conclusion: Preoperative chemoradiotherapy with oral eniluracil and 5-FU is feasible and well tolerated. Additional investigation is warranted.

Objective: To characterize the genotype and phenotype of a man with idiopathic hypogonadism with infertility., Design: Molecular analysis and clinical description., Setting: Medical school laboratory and reproductive endocrine clinic.A 40-year-old male with idiopathic hypogonadotropic hypogonadism., Intervention(s): Denaturing gradient gel electrophoresis analysis and DNA sequencing of the gonadotropin-releasing hormone receptor (GNRHR) gene were performed. The patient was treated with hCG and FSH., Main Outcome Measure(s): GNRHR mutation detection, genotype/phenotype correlation, and testicular response to exogenous gonadotropin therapy., Result(s): The proband demonstrated compound heterozygosity for Ala129Asp/Arg262Gln GNRHR mutations. He had a complete form of idiopathic hypogonadotropic hypogonadism, with descended testes and severe oligospermia but little response to exogenous gonadotropins., Conclusion(s): The phenotype of this patient differs from the one other family described with the same mutations. Exogenous gonadotropin therapy may not be as beneficial for increasing sperm concentration in older men with idiopathic hypogonadotropic hypogonadism.

Alzheimer's disease affects as many as 40% of Americans over the age of 80 and, as such, is a major public health issue. Interestingly, there is a two- to threefold greater prevalence in women than in men. It has been estimated that the prevalence of Alzheimer's disease will quadruple over the next half century. There have been implications of an effect of estrogen on neurological function for many years. As long as 50 years ago a study published in the gerontology literature suggested that the administration of i.m. estrogen in a nursing home population was associated with improvement in memory and a delay in progression of memory loss. Most recently there has been great interest in the effect of estrogen on both neurons and the CNS vasculature. A study evaluating verbal memory and abstract reasoning in over 700 women without dementia demonstrated that women who had used estrogen for as little as 1 year had significant improvements in baseline cognitive testing. The pathogenesis of Alzheimer's disease and neurodementia is better understood today but remains incompletely elucidated. It has been suggested that inflammation exists both within the neurovasculature and the stroma and that beta-amyloid creates an inflammatory reaction. In Alzheimer's patients there are abnormal deposits of proteins such as beta-amyloid, presenelin, and apolipoprotein E-4. Estrogen may act as a protectant against these inflammatory mediating proteins. While a recent trial demonstrated no impact of estrogen in patients diagnosed with mild to moderate Alzheimer's, other studies have suggested that estrogen use significantly delays disease onset. One study followed over 1,100 subjects who were free of disease at trial initiation over a period of 1 to 5 years. Even short-term use of estrogen imparted protection, although longer-term estrogen use was associated with greater protection. Unfortunately, most women are unaware of the potential beneficial effect of estrogen on cognitive function. Prospective studies are under way to try to delineate how estrogen impacts Alzheimer's disease.

Objective: To characterize the phenotype of idiopathic hypogonadotropic hypogonadism due to compound heterozygous GnRHR gene mutations (Arg262Gln/Tyr284Cys)., Design: Retrospective review., Setting: Tertiary medical center., Patient(s): Family containing four siblings (three female and one male) with complete idiopathic hypogonadotropic hypogonadism., Intervention(s): Baseline and stimulated laboratory studies. One patient received GnRH treatment and one received human menopausal gonadotropins., Main Outcome Measure(s): Clinical phenotype vs. genotype is assessed by endocrine studies, karyotype, pedigree, and review of pathology slides of ovarian neoplasm., Result(s): With GnRH stimulation, two patients with idiopathic hypogonadotropic hypogonadism had maximum LH < 10 mIU/mL, and two others had peak LH > 10 mIU/mL. With repeated GnRH stimulation 24 hours later, gonadotropin levels in all patients were increased. Stimulation of thyroid-releasing hormone and tests for insulin-induced hypoglycemia were normal. One affected patient did not ovulate after GnRH treatment, but her sister ovulated with gonadotropin treatment. Another affected sibling had bilateral oophorectomy for seromucinous cystadenomas, and her hypogonadotropic state remained after castration. The man with idiopathic hypogonadotropic hypogonadism and his unaffected brother had a ring chromosome 21., Conclusion(s): All patients with complete idiopathic hypogonadotropic hypogonadism had the same GnRHR mutations, but clinical presentations and endocrinologic responses were heterogeneous. Gonadotropin levels remained low in patients with idiopathic hypogonadotropic hypogonadism after castration, and ring chromosome 21 was present, suggesting that sequences from this chromosome could affect the idiopathic hypogonadotropic hypogonadism phenotype.

The gonadotropin-releasing hormone receptor (GnRHR) is a guanine nucleotide-binding protein-coupled receptor with a characteristic seven transmembrane domain motif. It transduces the hypothalamic message carried by the decapeptide gonadotropin-releasing hormone. At the gonadotrope cell surface the hormone binds to the receptor, leading to pituitary synthesis and secretion of gonadotropins. These glycoprotein hormones, in turn, modulate folliculogenesis and ovulation in the ovary. Failure of the GnRHR to respond appropriately to its cognate ligand has been demonstrated in humans. These patients have genomic mutations in the gene for the GnRHR and represent the first individuals with hypogonadotropic hypogonadism of autosomal inheritance. The mutant receptors are poorly expressed at the cell surface and have a diminished capacity to transduce the hypothalamic message efficiently. To date, no mutations have been identified that lead to constitutively active receptors and autonomous gonadotropin function. This article identifies the mutations in the GnRHR reported to date and reviews how these abnormal receptors help us to better understand the biology of this interesting molecule.

The topoisomerase I inhibitor camptothecin and its analogs have potent activity against a wide range of solid tumors and several hematologic malignancies. Previous studies with these compounds using the MTT metabolic inhibition assay have shown significant cytotoxicity against lymphocytes from patients with chronic B-cell lymphocytic leukemia (B-CLL). Yet the water soluble analogue, topotecan, which was inhibitory at > 1 microM in vitro, had no clinical activity in vivo. In the present study, we evaluated the in vitro cytotoxicities of SN-38, the active form of irinotecan, and two newer water soluble camptothecin derivatives 10,11-methylenedioxy-20(S)-camptothecin glycinate (MDCG) and 7-chloromethyl-10,11-methylenedioxy-20(S)-camptothecin glycinate (CMMDCG). These two glycinate esters are prodrugs for 10,11-methylenedioxy-20(S)-camptothecin (MDC) and 7-chloromethyl-10,11-methylenedioxy-20(S)-camptothecin (CMMDC), respectively. Effects on cellular metabolism, induction of apoptosis, and overall cell survival were used to evaluate chemosensitivity. We report that the relative cytotoxic potency for these compounds is MDC > or = CMMDC > or = SN-38 >> TPT > CPT-11, where MDC, CMMDC, and SN-38 were over an order of magnitude more cytotoxic than TPT and CPT-11. We also investigated potential mechanisms underlying the unexpected cytotoxicity of these camptothecin derivatives in B-CLL cells that are known to be arrested in G0/G1 of the cell cycle, and found that this class of compounds inhibited [3H]uridine incorporation. We therefore postulate that the inhibition of RNA rather than DNA synthesis may be responsible for the observed cytotoxicity in non-cycling B-CLL cells.

Objective: To determine whether a mutation in the GnRH receptor gene is responsible for polycystic ovary syndrome (PCOS)., Design: Molecular analysis of human genomic DNA., Setting: Academic research environment., Patient(s): Eighty patients with PCOS., Intervention(s): Extraction and polymerase chain reaction (PCR) analysis of genomic DNA, confirmation of PCR products by ethidium bromide staining of agarose gels after electrophoresis, denaturing gradient gel electrophoresis of PCR products, and photography., Main Outcome Measure(s): Mutations in the GnRH receptor of women with PCOS., Result(s): Denaturing gradient gel electrophoresis revealed no mutations in the exonic sequence encoding the open reading frame of the GnRH receptor., Conclusion(s): A mutation in the GnRH receptor gene is unlikely to be the underlying cause of PCOS in most patients. The molecular basis of this disorder remains unknown.

Objective: To determine whether FSH receptor gene missense mutation in Finnish women with premature ovarian failure (POF) is present in North American women with POF., Design: Analysis of DNA from patients and controls., Patient(s): Thirty-five women with POF and ten normal controls., Intervention(s): Extraction of DNA with subsequent digestion by the enzyme BsmI, polyacrylamide gel electrophoresis, ethidium bromide staining, and photography., Main Outcome Measure(s): After restriction enzyme digestion, the frequencies of the normal allele (two fragments of 51 and 27 base pairs) and the mutant allele (a single 78-base pair fragment) were determined., Result(s): BsmI digestion was noted for all 35 affected individuals and 10 controls, thus demonstrating homozygosity for the normal FSH receptor allele. No patient or control was heterozygous or homozygous for the mutant allele., Conclusion(s): The missense mutation in the human FSH receptor gene in Finnish women with POF is uncommon in North American women with POF. The molecular basis of ovarian failure for most patients remains unknown.

The human CT receptor (hCTR), which is found as three isoforms, belongs to a small, recently described subfamily of G protein-coupled receptors (GPCRs). Several mutant GPCRs have been shown to exhibit constitutive (or agonist-independent) signaling activity and cause disease in humans, but only a few GPCRs have been identified with agonist-independent activity in the wild-type (or native) form. In the hCTR subfamily, no wild-type receptor has been shown to exhibit constitutive activity and only one, a mutated receptor for PTH/PTH-related peptide, has been found with constitutive activity to cause disease in humans. We demonstrate that two wild-type isoforms of hCTR, hCTR-1 and hCTR-2, exhibit constitutive activity by showing that they cause elevation of cAMP and induction of a cAMP-responsive gene in two cell types in culture in the absence of agonist. The identical mutation that caused the PTH/PTH-related peptide receptor to be constitutively active was made in hCTR-2 and shown to have no effect on signaling. We suggest that constitutive activity of wild-type hCTR-1 and hCTR-2 may reflect an adaptation of their signaling properties to exert their regulatory function in the absence of agonist in some cell types.

Understanding molecular biology can improve the clinical acumen of the practicing obstetrician/gynecologist. An area of basic research now becoming clinically relevant involves the G proteins and G protein-coupled receptors. Clinicians already manipulate G protein-coupled receptors in their daily practice. Examples include the administration of oxytocin (oxytocin receptors), beta-2 tocolytic agents (beta 2-adrenergic receptors), GnRH agonists (GnRH receptors), exogenous gonadotropins (FSH and LH receptors), and bromocriptine (dopamine receptor). Clinically important disorders presenting to the obstetrician/gynecologist include some forms of precocious puberty, delayed puberty, premature ovarian failure, and pituitary adenomas which are due to mutations of G proteins and G protein-coupled receptors. The importance of these proteins is demonstrated by the fact that G protein-related genes comprise about 1 percent of the human genome. Additionally, the knowledge that some G protein gene mutations are present in the germ line, and others are somatic cell in origin (and not heritable), aids in more accurate genetic counseling to patients.

Overexpression of native and epitope-tagged human calcitonin (CT) receptors (hCTR-2) in COS-1 cells was performed to permit identification of the receptor protein and begin studies of receptor turnover. Data obtained with immunological techniques and cross-linking of radiolabeled salmon CT ([125I]sCT) revealed two forms of hCTR-2 in transfected cells: a larger, mature cell surface receptor (apparent size, 81 kDa) and a smaller, intracellular form (apparent size, 66 kDa). These conclusions are based on the following observations. 1) Only the larger hCTR-2 was visualized by cell surface [125I]sCT binding, whereas both species were identified by [125I]sCT binding to cell lysates. 2) Immunofluorescence studies with antibodies directed against the epitope confirmed the presence of cell surface and intracellular hCTR-2s; there were apparently many more receptors intracellularly than on the cell surface. 3) Both hCTR-2 forms were changed to a similar size of approximately 57-60 kDa by deglycosylation with endoglycosidase F; this size is consistent with that predicted by the amino acid sequence. Metabolic studies with radioactive amino acids labeled only the intracellular form. This immature form exhibited a rapid half-life of 30 min. We conclude that overexpression of native and epitope-tagged hCTR-2s in COS-1 cells leads to their intracellular retention and rapid degradation.

Objective: To assess the predominant subtype of calcium channel present in neurons of the paracervical ganglia (PG) of the female rat and the ability of neuroactive peptide to modulate total calcium current., Methods: Whole-cell patch clamp techniques were used on isolated PG neurons to assess calcium current modulation in the presence and absence of selective calcium channel subtype inhibitors and neuropeptides. Digital imaging analysis of cells was used to determine neuronal cell size distributions within the ganglia., Results: Average PG cell diameter was 28.1 microns. Most PG neurons (64%) had an N-type calcium current that contributed 41% of the total calcium current. The low voltage-activated or T-type current was not present. Very few neurons (22%) were sensitive to the P-type calcium channel blocker omega-agatoxin IVA, and in only 10% of neurons was the calcium current sensitive to the L-type channel blocker nimodipine. Neuropeptide Y (NPY) inhibited the calcium current by 41% in 79% of the neurons, but vasoactive intestinal peptide (VIP) had no effect. Calcitonin gene-related peptide (CGRP) both increased and decreased the calcium current in separate cell populations., Conclusions: Calcium currents in female rat PG neurons are carried primarily through N-type calcium channels with a small contribution from L- and P-type channels. An unidentified calcium channel type is also present. Modulation of the calcium current by NPY is demonstrated, and support for the presence of a local, CGRP-mediated circuit is presented.

The reported binding affinities of cloned human calcitonin receptors (hCTRs) for salmon calcitonin (sCT) vary over a wide range. Because the greatest differences were between values estimated in association binding versus competition binding experiments, we considered the possibility that this variation was due to differences in the affinities of hCTRs for moniodinated sCT (I-sCT) and sCT. We found that I-sCT competed with 125I-sCT for binding with 9.3 +/- 0.58 times the apparent affinity of sCT in COS-1 and Madin Darby canine kidney (MDCK) cells expressing cloned hCTRs. I-sCT was 25 times more potent than sCT in stimulating cAMP formation in MDCK cells expressing hCTRs. These data demonstrate that monoiodinated sCT interacts more avidly with hCTRs than sCT with a consequent enhanced potency in stimulating second messenger formation. Therefore, as I-sCT (or 125I-sCT) binds with different affinity to hCTRs than sCT, affinity measurements must be performed with I-sCT (or 125I-sCT) alone.

The signals required to induce S phase entry in murine splenic B cells were found to be altered by prolonged treatment with low doses of anti-Ig antibody. Whereas fresh splenic B cells are stimulated by the combination of a phorbol ester protein kinase C agonist plus a calcium ionophore, anti-Ig-treated splenic B cells were stimulated by phorbol ester alone, in the absence of a comitogen. The majority of these phorbol ester responsive B cells expressed CD5. The phorbol ester responses of anti-Ig-treated splenic B cells paralleled those previously reported for untreated peritoneal CD5+ B cells in a number of respects: responses were not idiosyncratic to phorbol esters but occurred with nonphorbol protein kinase C agonists; phorbol ester responses were enhanced by IL-4; and, phorbol ester responses occurred rapidly and were greater at 24 than at 48 h. However, the effect of agents that act to raise intracellular levels of cAMP distinguished between anti-Ig-treated splenic B cells and untreated peritoneal B cells in that the phorbol ester responses of the former were enhanced whereas the responses of the latter were inhibited. The present results add a functional dimension to the phenotypic similarity between splenic B cells treated with anti-Ig and resident peritoneal B cells that constitutively express CD5; however, some differences in behavior were noted.

Conventional murine splenic B cells are stimulated to initiate DNA synthesis by the combination of a phorbol ester protein kinase C (PKC) agonist, and a calcium ionophore; in contrast, recent work from this laboratory has shown that peritoneal B cells, enriched for the Ly-1+ B cell subset, differ in that they proliferate in response to the single signal provided by phorbol ester, acting alone. To elucidate the mechanism responsible for the abbreviated signaling requirement of peritoneal B cells, studies of intracellular Ca2+ and PKC were carried out. Measurements using the calcium sensitive dye, Indo-1, showed that base line levels of intracellular Ca2+ in peritoneal B cells were similar to those of splenic B cells, and that there was no change as a result of phorbol ester treatment. However, measurements of PKC based on the phosphorylation of histone showed enzymatic activity in peritoneal B cells to be about 60% greater than that of splenic B cells on a per microgram protein basis. Furthermore, this difference was accentuated by phorbol ester treatment, so that after 4 h, membrane and cytosol fractions from peritoneal B cells contained more than 5 times the PKC activity of the corresponding splenic B cell fractions because the down-regulation of PKC was relatively delayed in peritoneal B cells. This could not be accounted for by the onset of new PKC synthesis, but may relate to the finding that peritoneal B cells express more of the alpha-isoenzyme of PKC than splenic B cells, as shown by immunoblot analysis. Together with data from experiments using the PKC inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride(H7), these results suggest that PKC activity remaining hours after phorbol ester treatment may contribute to the unusual phorbol ester responsiveness of peritoneal B cells, and indicate that B cells from separate anatomic locations differ in terms of several parameters relating to the activity and behavior of PKC.

Women who use the oral contraceptive pill are at risk for a variety of thromboembolic complications, but the precise mechanism is unclear. Since 2-hydroxyestradiol is a major metabolite of estradiol and possesses some of the structural and functional properties of catecholamines, we examined whether this compound might influence thrombogenesis, in particular whether it would exert an epinephrine-like effect on the platelet. In vitro, 2-hydroxyestradiol does not cause or potentiate aggregation or thromboxane release, nor does it prevent the rise in cyclic AMP caused by anti-aggregatory prostaglandins. While 2-hydroxyestradiol does not exert a direct effect on the platelet in vitro, it may play a role in thrombosis in women who take oral contraceptives through another mechanism.

Adenosine 3',5'-cyclic monophosphate (cAMP) acts to inhibit a number of lymphocyte activities. The extent of this inhibition was tested by evaluating the effects of two cAMP-raising agents on B cell S phase entry induced by several different mitogenic regimens. It was found that both dibutyryl cAMP (dbcAMP) and isobutylmethylxanthine (IBMX) enhanced S phase entry induced by some regimens but inhibited S phase entry induced by others. The observed enhancing activity stands in contrast to the general notion of cAMP as being a "negative regulator," and it confirms that the observed inhibiting activity does not simply reflect cytotoxicity. Mitogenic regimens that appear to mimic each other, such as F(ab')2 fragments of goat anti-mouse immunoglobulin and the combination of a calcium ionophore and a phorbol ester, were distinguished by their responses to the addition of the two cAMP-raising agents. B cell responses were enhanced or inhibited even when dbcAMP was added 18-24 hr after the establishment of cultures. Cyclic AMP may regulate in a complex fashion S phase entry in cells of the immune system.

By using paired sera the IgM and IgG host responses were analyzed in dogs with ELISA and Western blot techniques. Antibodies in clinical seropositive dogs bound to 4-25 IgM and up to 40 or more IgG antigenic determinants. Early IgM response to the 41-kDa flagellin persisted for at least 9 months and involved as many as seven other peptides. IgG response expanded later in the disease and involved more immunogens than are currently recognized in late human disease. A percentage of asymptomatic dogs that later developed clinical symptoms were seropositive. Immunoblot studies suggested that B. burgdorferi is persistent in both asymptomatic and weakly reactive animals and if untreated could lead to disease expression. Clinical seropositive, asymptomatic seronegative, and experimentally infected horses were similarly studied. In experimentally inoculated animals IgG antibodies were initially bound to flagellin and later to the 34- and 31-kDa polypeptides, even though ELISA values were considered only slightly reactive.