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Machine learning (ML) is ever more frequently used as a tool to aid decision making. The need to understand the decisions made by ML algorithms has sparked a renewed interest in explainable ML models. A number of known models are often regarded as interpretable by human decision makers with varying degrees of difficulty. The size of such models plays a crucial role in determining how easily they can be understood by a human. In this article we propose the use of binary decision diagrams (BDDs) as an interpretable ML model. BDDs can be deemed as interpretable as decision trees (DTs) while offering a often more compact representation due to node sharing. Fixed variable ordering also allows for more concise explanations. We propose a SAT-based approach for learning optimal BDDs that exhibit perfect accuracy on training data. We also explore heuristic methods for computing suboptimal BDDs, in order to improve scalability. We also investigate procedures to transform instances of known ML models into BDDs in order to provide more concise explanations.

This article explores the significance of self-care for leaders and offers a practical checklist to help them assess their well-being in various areas. It acknowledges the challenges leaders face in prioritizing self-care, including emotional demands and cultural influences. The checklist encourages leaders to adopt a mindset that values self-care, make gradual changes, and seek support from others. The article also provides specific tips for managing stress, emotions, and communication. Overall, it emphasizes the importance of leaders prioritizing their well-being to enhance their leadership abilities. [Extracted from the article]

OBJECTIVE--To develop and validate a parsimonious model for predicting short-term all-cause mortality in patients with type 2 diabetes mellitus (T2DM). RESEARCH DESIGN AND METHODS--Two cohorts of patients with T2DM were [...]

The Verifiability Approach (VA) is a verbal veracity tool that assumes that truth tellers provide more details that can be verified and obtain a higher ratio of such details (verifiable details/total details) than liars. A VA meta-analysis was conducted. Results showed that truth tellers reported more verifiable details than liars (k= 20, N= 1532, g= 0.42). The effect was moderated by the presence of the information protocol and by the nature of the statement (event-related or not). Truth tellers reported a higher ratio of verifiable details than liars (k= 18, N= 1359, g= 0.49). The effect was moderated by the medium through which the statement was provided. Unverifiable details did not discriminate truth tellers from liars (k= 15, N= 957, g= −0.25). In conclusion, results showed good potential for the use the VA, although replications and field studies are needed.

Tumor cell plasticity exhibited as an epithelial–mesenchymal transition (EMT) has been identified as a major obstacle for the effective treatment of many cancers. This process, which involves the dedifferentiation of epithelial tumor cells towards a motile, metastatic, and mesenchymal tumor phenotype, mediates resistance to conventional therapies and small-molecule targeted therapies. In this review, we highlight current research correlating the role of tumor plasticity with resistance to current immunotherapy approaches and discuss future and ongoing combination immunotherapy strategies to reduce tumor cell plasticity-driven resistance in cancer.

Background: Mathematical modeling in modified drug release is an important tool that allows predicting the release rate of drugs in their surrounding environment and elucidates the transport mechanisms involved in the process. Objective: The aim of this work was to develop a mathematical model that allows evaluating the release profile of drugs from polymeric carriers in which the swelling phenomenon is present. Methods: Swellable matrices based on ionic complexes of alginic acid or carboxymethylcellulose with ciprofloxacin were prepared and the effect of adding the polymer sodium salt on the swelling process and the drug release was evaluated. Experimental data from the ciprofloxacin release profiles were mathematically adjusted, considering the mechanisms involved in each stage of the release process. Results: A proposed model, named “Dual Release” model, was able to properly fit the experimental data of matrices presenting the swelling phenomenon, characterized by an inflection point in their release profile. This entails applying the extended model of Korsmeyer-Peppas to estimate the percentage of drug released from the first experimental point up to the inflection point and then a model called Lumped until the final time, allowing to adequately represent the complete range of the drug release profile. Different parameters of pharmaceutical relevance were calculated using the proposed model to compare the profiles of the studied matrices. Conclusion: The “Dual Release” model proposed in this article can be used to predict the behavior of complex systems in which different mechanisms are involved in the release process.

Critical limb ischemia (CLI), defined as ischemic rest pain or nonhealing ulceration due to arterial insufficiency, represents the most severe and limb-threatening manifestation of peripheral artery disease. A major challenge in the optimal treatment of CLI is that multiple specialties participate in the care of this complex patient population. As a result, the care of patients with CLI is often fragmented, and multidisciplinary societal guidelines have not focused specifically on the care of patients with CLI. Furthermore, multidisciplinary care has the potential to improve patient outcomes, as no single medical specialty addresses all the facets of care necessary to reduce cardiovascular and limb-related morbidity in this complex patient population. This review identifies current gaps in the multidisciplinary care of patients with CLI, with a goal toward increasing disease recognition and timely referral, defining important components of CLI treatment teams, establishing options for revascularization strategies, and identifying best practices for wound care post-revascularization.

Purpose: The aim of this project is to explore practice patterns of experienced vascular specialists involved in the treatment of patients with arterial femoro-popliteal in-stent occlusion (ISO) and to understand key concepts and shared thoughts, throughout an international cross-sectional survey.Materials and Methods: The web-based survey was in English and included 31 questions. One-hundred experts were invited by email to fill in the survey anonymously on Google Forms using a dedicated link. Reminders were sent after the first 2 weeks. In addition to reporting of pooled responses, subanalyses of answers were also performed, according to country of origin (European vs non-European), years of experience (≤20 years vs >20 years), and type of institution (Academic/University vs Non-academic/Private).Results: A total of 77 physicians from 22 countries completed the survey. Most responders were males (70/77, 91%). Most of the participants were vascular surgeons (58/77, 75%). Endovascular therapy was the preferred option in patients presenting with either acute limb ischemia, disabling intermittent claudication, or chronic limb-threatening ischemia. Atherectomy/thrombectomy+Drug-Coated Balloon (DCB) was the preferred endovascular modality (32/77, 42%). No differences between country of origin, years of experience, and type of institution of the participants were found.Conclusions: The international SUrvey oN TReatment Of Occluded Femoro-Popliteal Stent (SUNROOF) survey has suggested that endovascular therapy is the preferred method for the treatment of femoro-popliteal ISO. This survey could represent a good starting point for future research on comparison between open and endovascular approach and different devices, which currently lacks good-level scientific evidence and appropriate guidelines.Clinical Impact The international SUNROOF survey included 31 questions. It was performed by 77 vascular physicians from 22 countries. More than a half of respondents considered an endovascular approach as the preferred treatment modality in acute limb ischemia, disabling intermittent claudication, or chronic limb-threatening ischemia. Most responders agreed they would intensify the follow-up protocol and anti-thrombotic medications regimen after a first in-stent occlusion episode. This survey could represent a good starting point for future research on comparison between open and endovascular approach and different devices, which currently lacks of good level scientific evidence and appropriate guidelines.

Purpose:To describe a novel technique designed to safely and precisely deploy the Supera stent accurately at the ostium of the proximal superficial femoral artery (SFA) without compromising the profunda and common femoral arteries. Technique:After antegrade crossing of the chronic total occlusion (CTO) at the SFA ostium and accurate predilation of the entire SFA lesion, a retrograde arterial access is obtained. The Supera stent is navigated in retrograde fashion to position the first crown to be released just at the SFA ostium. Antegrade dilation is performed across the retrograde access site to obtain adequate hemostasis. The technique has been applied successfully in 21 patients (mean age 78.1±8.2 years; 13 men) with critical limb ischemia using retrograde Supera stenting from the proximal anterior tibial artery (n=6), the posterior tibial artery (n=2), retrograde stent puncture in the mid to distal SFA (n=2), the native distal SFA/proximal popliteal segment (n=6), and the distal anterior tibial artery (n=5). No complications were observed. Conclusion:Distal retrograde Supera stent passage and reverse deployment allow precise and safe Supera stenting at the SFA ostium.

The best treatment option for patients with type 2 diabetes in whom treatment with metformin alone fails to achieve adequate glycaemic control is debated. We aimed to compare the long-term effects of pioglitazone versus sulfonylureas, given in addition to metformin, on cardiovascular events in patients with type 2 diabetes.

Molecular factors that define stem cell identity have recently emerged as oncogenic drivers. For instance, brachyury, a key developmental transcriptional factor, is also implicated in carcinogenesis, most notably of chordoma, through mechanisms that remain elusive. Here, we show that brachyury is a crucial regulator of stemness in chordoma and in more common aggressive cancers. Furthermore, this effect of brachyury is mediated by control of synthesis and stability of Yes-associated protein (YAP), a key regulator of tissue growth and homeostasis, providing an unexpected mechanism of control of YAP expression. We further demonstrate that the brachyury-YAP regulatory pathway is associated with tumor aggressiveness. These results elucidate a mechanism of controlling both tumor stemness and aggressiveness through regulatory coupling of two developmental factors.

Purpose:To report a prospective, multicenter, observational study (ClinicalTrials.govidentifier NCT01609621) of the safety and effectiveness of tibiopedal access and retrograde crossing in the treatment of infrainguinal chronic total occlusions (CTOs). Methods:Twelve sites around the world prospectively enrolled 197 patients (mean age 71±11 years, range 41-93; 129 men) from May 2012 to July 2013 who met the inclusion criterion of at least one CTO for which a retrograde crossing procedure was planned or became necessary. The population consisted of 64 (32.5%) claudicants (Rutherford categories 2/3) and 133 (67.5%) patients with critical limb ischemia (Rutherford category ≥4). A primary antegrade attempt to cross had been made prior to the tibiopedal attempt in 132 (67.0%) cases. Techniques used for access, retrograde lesion crossing, and treatment were at the operator’s discretion. Follow-up data were obtained 30 days after the procedure. Results:Technical tibiopedal access success was achieved in 184 (93.4%) of 197 patients and technical occlusion crossing success in 157 (85.3%) of the 184 successful tibial accesses. Failed access attempts were more common in women (9 of 13 failures). The rate of successful crossing was roughly equivalent between sexes [84.7% (50/59) women compared to 85.6% (107/125) men]. Technical success did not differ significantly based on a prior failed antegrade attempt: the access success rate was 92.4% (122/132) after a failed antegrade access vs 95.4% (62/65) in those with a primary tibiopedal attempt (p=0.55). Similarly, crossing success was achieved in 82.8% (101/122) after a failed antegrade access vs 90.3% (56/62) for patients with no prior antegrade attempt (p=0.19). Minor complications related to the access site occurred in 11 (5.6%) cases; no patient had access vessel thrombosis, compartment syndrome, or surgical revascularization. Conclusion:Tibiopedal access appears to be safe and can be used effectively for the crossing of infrainguinal lesions in patients with severe lower limb ischemia.

The high mortality risk of patients with type 2 diabetes mellitus may well be explained by the several comorbidities and/or complications. Also the intrinsic genetic component predisposing to diabetes might have a role in shaping the risk of diabetes-related mortality. Among type 2 diabetes mellitus SNPs, rs1801282 is of particular interest because (i) it is harbored by peroxisome proliferator-activated receptor-γ2 (PPARγ2), which is the target for thiazolidinediones which are used as antidiabetic drugs, decreasing all-cause mortality in type 2 diabetes mellitus, and (ii) it is associated with insulin resistance and related traits, risk factors for overall mortality in type 2 diabetes mellitus. We investigated the role of PPARγ2P12A, according to a dominant model (PA + AA vs. PP individuals) on incident all-cause mortality in three cohorts of type 2 diabetes mellitus, comprising a total of 1672 patients (462 deaths) and then performed a meta-analysis of ours and all available published data. In the three cohorts pooled and analyzed together, no association between PPARγ2P12A and all-cause mortality was observed (HR 1.02, 95 % CI 0.79–1.33). Similar results were observed after adjusting for age, sex, smoking habits, and BMI (HR 1.09, 95 % CI 0.83–1.43). In a meta-analysis of ours and all studies previously published (n= 3241 individuals; 666 events), no association was observed between PPARγ2P12A and all-cause mortality (HR 1.07, 95 % CI 0.85–1.33). Results from our individual samples as well as from our meta-analysis suggest that the PPARγ2P12A does not significantly affect all-cause mortality in patients with type 2 diabetes mellitus.

Purpose:To test the safety, efficacy, and diagnostic accuracy of automated carbon dioxide (CO2) angiography (ACDA) for the evaluation of diabetic patients with critical limb ischemia (CLI) and baseline renal insufficiency and compare ACDA with iodinated contrast medium (ICM) during endovascular treatment. Methods:From November 2014 to January 2015, 36 consecutive diabetic patients (mean age 74.8±5.8 years; 27 men) with stage ≥3 chronic kidney disease (CKD ≥3) and CLI underwent lower limb angiography with both CO2and ICM followed by balloon angioplasty in a prospective single-center study. The primary outcome measure was the safety and efficacy of ACDA as the exclusive agent to guide angioplasty in this cohort. The secondary outcomes were the safety and diagnostic accuracy of ACDA injection as compared with ICM digital subtraction angiography (DSA) for invasive evaluation of these patients. Results:ACDA safely and effectively guided angioplasty in all patients without complications. Transcutaneous oxygen pressure improved from 11.8±6.3 to 58.4±7.6 mm Hg (p<0.001). There were no complications related to ACDA during diagnostic imaging and no significant changes in the estimated glomerular filtration rate from baseline to 24 hours (44.7±13.3 vs 47.0±0.8 mL/min/1.73 m2; nonsignificant). The diagnostic accuracy of CO2was 89.8% (sensitivity 92.3%; specificity 75%; positive predictive value 95.5%; negative predictive value 63.1%). There was no statistically significant difference in the qualitative diagnostic accuracy between the media (p=0.197). Conclusion:ACDA is an accurate, safe, and effective technique that can be utilized to guide endovascular interventions in diabetics with CLI and baseline CKD ≥3. Larger multicenter randomized studies are needed to validate these results.

The current study is focused on physicochemical characterization and in vitrodrug delivery evaluation of self-organized nanoparticles based on drug-interpolyelectrolyte complexes (DIPEC). The interaction between two oppositely charged polymethacrylates, Eudragit®EPO and L100, and four non-steroidal anti-inflammatory drugs, Salicylic Acid, Benzoic Acid, Ketoprofen and Naproxen was studied.

Brachyury is a transcription factor of the T-box family typically expressed in notochord and chordoma. Some studies report brachyury as highly specific for chordoma, whereas others have concluded that brachyury is expressed in many types of common carcinomas by reverse transcription polymerase chain reaction and immunohistochemistry and could be involved in the epithelial-mesenchymal transition and metastatic process. In this study, we immunohistochemically evaluated 5229 different tumors for nuclear brachyury expression using a new rabbit monoclonal antibody and automated immunostaining (Leica Bond Max). Only nuclear labeling was scored, and antibody dilution of 1:2000 was used. In normal tissues, only rare cells in seminiferous tubules were labeled; all other organs were negative. All chordomas (75/76), except a sarcomatous one, were positive, whereas chondrosarcomas were negative. Among epithelial tumors, positivity was often detected in embryonal carcinoma (74%) and seminoma (45%). Pulmonary small cell carcinoma was often positive (41%), whereas pulmonary and pancreatic adenocarcinomas only rarely showed nuclear brachyury positivity (3% to 4%). Common carcinomas such as ductal carcinomas of the breast or adenocarcinomas of the prostate only exceptionally showed nuclear positivity (<1%). No colorectal, hepatocellular, renal cell, squamous cell, thyroid or urothelial carcinoma, or mesothelioma showed nuclear brachyury positivity. Among mesenchymal and neuroectodermal tumors, only isolated cases of melanoma, malignant peripheral nerve sheath tumor, rhabdomyosarcoma, synovial sarcoma, and follicular lymphoma showed nuclear expression. However, as shown previously with lung carcinoma, experiments with lower antibody dilutions (1:200 to 1:500) showed weak cytoplasmic and nuclear labeling in breast cancers. In addition to chordoma, we show here for the first time that nuclear brachyury expression is prevalent in embryonal carcinoma, seminoma, and small cell carcinoma of the lung but very rare in common carcinomas, sarcomas, and melanoma. With these reservations, we have demonstrated the presence of nuclear brachyury immunoreactivity to be a sensitive and fairly specific marker for chordoma.

Model checkers and sequential equivalence checkers have become essential tools for the semiconductor industry in recent years. The Hardware Model Checking Competition (HWMCC) was founded in 2006 with the purpose of intensifying research interest in these technologies, and establishing more of a science behind them. For example, the competition provided a standardized benchmark format, a challenging and diverse set of industrially-relevant public benchmarks, and, as a consequence, a significant motivation for additional research to advance the state-of-the-art in model checkers for these verification problems. This paper provides a historical perspective, and an analysis of the tools and benchmarks submitted to the competition. It also presents a detailed analysis of the results collected in the 2014 edition of the contest, showing relations among tools, and among tools and benchmarks. It finally proposes a list of considerations, lessons learned, and hints for both future organizers and competitors.

Purpose: To describe a novel technical strategy to approach below-the-ankle chronic total occlusions after failed antegrade recanalization in patients who are not candidates for retrograde puncture.Technique: After a failure of antegrade recanalization of the tibial and foot vessels, an antegrade pedal access can be performed in the pedal or plantar artery. After crossing the plantar arch and the occlusions in the opposing circulation pathway of the foot, a rendezvous with the antegrade catheter is performed, followed by final angioplasty and hemostasis.Conclusion: An antegrade pedal approach to the opposing circulation in the foot may be considered for below-the-ankle recanalization after standard antegrade recanalization failure in patients who are not candidates for a retrograde puncture.

Purpose To describe advanced retrograde access (transmetatarsal or transplantar arch) for endovascular treatment of critical limb ischemia (CLI) and foot salvage.Methods From September 2011 to March 2012, 28 CLI patients (24 men; mean age 71.9±10.6 years) being treated for foot salvage had failed antegrade recanalization, and percutaneous retrograde access at the pedal or plantar artery was unavailable. Advanced retrograde access techniques were required to recanalize the target vessel to restore blood flow to the compromised tissue. After local administration of verapamil to control spasm, the first dorsal metatarsal artery was preferentially accessed with a 21-G needle. When the first metatarsal artery was occluded and not fluoroscopically viewable, the plantar arch was punctured directly. After puncture, a 0.018- or 0.014-inch guidewire and microsheath were inserted for retrograde recanalization of the foot and tibial arteries with balloons sized to the target vessels.Results Retrograde transmetatarsal artery access was performed in 25 cases and direct transplantar arch access in 3. Technical success (ability to deliver the balloon across the lesion and inflate it at nominal pressure) was achieved in 24 (86%) cases, with <50% residual stenosis and no complications. The 4 technical failures were due to spasm or no true lumen re-entry after successful transmetatarsal (n=3) and transplantar arch access. During a mean 5-month follow-up (range 1–8), clinical improvement was obtained in the patients having technically successful tibial and foot artery recanalization; the transcutaneous pressure improved from 12.5±6.7 to 49.8±9.5 mmHg. There were no major and only 8 minor amputations. Amputation-free survival estimated by Kaplan-Meier analysis was 71% at 6 months. In patients with failed advanced access, the clinical condition did not improve.Conclusion The advanced retrograde access technique appears feasible and beneficial as a rescue strategy in challenging patients with a failed antegrade approach who are unsuitable for retrograde pedal/plantar access.

The epithelial-mesenchymal transition (EMT) is thought to be a critical step along the metastasis of carcinomas. In addition to gaining motility and invasiveness, tumor cells that undergo EMT also acquire increased resistance to many traditional cancer treatment modalities, including chemotherapy and radiation. As such, EMT has become an attractive, potentially targetable process for therapeutic interventions against tumor metastasis. The process of EMT is driven by a group of transcription factors designated as EMT transcription factors, such as Snail, Slug, Twist, and the recently identified T-box family member, Brachyury. In an attempt to determine which of these drivers of EMT is more amenable to targeted therapies and, in particular, T-cell–mediated immunotherapeutic approaches, we have examined their relative expression levels in a range of human and murine normal tissues, cancer cell lines, and human tumor biopsies. Our results demonstrated that Brachyury is a molecule with a highly restricted human tumor expression pattern. We also demonstrated that Brachyury is immunogenic and that Brachyury-specific CD8+T cells expanded in vitro are able to lyse Brachyury-positive tumor cells. We thus propose Brachyury as an attractive target for vaccination strategies designed to specifically target tumor cells undergoing EMT.

This article reports dynamic light scattering (DLS) data, and electrokinetic ζ-potentials of aqueous dispersions of two linear flexible polymethacrylic acid derivatives Eudragit® L100 (EuL) and S100 (EuS) loaded with two model drugs (D), lidocaine (Ld) and atenolol (At). Dispersions of EuL and EuS at 1,0 % neutralized with increasing percentages (50, 75 and 100 mole %) of each D exhibited a unimodal scattering distribution rendering diffusion coefficients (DC) in the interval of 4 to 9. 10-9cm2/s. All dispersions 50% neutralized exhibited quite similar DC. However, the effect of increasing neutralization followed a different pattern in each Eudragit® . All dispersions exhibited high negative ζ-potentials that were lower at 100% with respect to 50% loading. Both, DC and ζ-potentials, of redispersed lyophilized samples of (EuL-D) and (EuS-D) remained identical to those of in situ prepared while those of analog samples prepared by solvent evaporation exhibited some slight differences. The behavior of (EuL-D) and (EuS-D) systems examined here through DLS appears to be quite similar to that reported for polyelectrolytes neutralized with monovalent inorganic cations. Last, it has been shown that DLS provides valuable information about the reversibility from solid to dispersion states of these nanometric drug carrier systems.

This article reports dynamic light scattering (DLS) data, and electrokinetic ζ-potentials of aqueous dispersions of two linear flexible polymethacrylic acid derivatives Eudragit® L100 (EuL) and S100 (EuS) loaded with two model drugs (D), lidocaine (Ld) and atenolol (At). Dispersions of EuL and EuS at 1,0 % neutralized with increasing percentages (50, 75 and 100 mole %) of each D exhibited a unimodal scattering distribution rendering diffusion coefficients (DC) in the interval of 4 to 9. 10 cm/s. All dispersions 50% neutralized exhibited quite similar DC. However, the effect of increasing neutralization followed a different pattern in each Eudragit® . All dispersions exhibited high negative ζ-potentials that were lower at 100% with respect to 50% loading. Both, DC and ζ-potentials, of redispersed lyophilized samples of (EuL-D) and (EuS-D) remained identical to those of in situ prepared while those of analog samples prepared by solvent evaporation exhibited some slight differences. The behavior of (EuL-D) and (EuS-D) systems examined here through DLS appears to be quite similar to that reported for polyelectrolytes neutralized with monovalent inorganic cations. Last, it has been shown that DLS provides valuable information about the reversibility from solid to dispersion states of these nanometric drug carrier systems.

This study is a retrospective investigation of the role of non contrast-enhanced CT (NCCT) in the diagnosis of cerebral venous thrombosis (CVT) in clinical practice. We retrospectively identified 24 patients discharged with a diagnosis of CVT between January 2002 and December 2008 who had undergone NCCT as the first imaging modality. NCCT had been evaluated by a general radiologist and subsequently by a neuroradiologist in five cases. Final diagnosis was established by CT angiography (CTA), magnetic resonance (MR) and digital subtraction angiography (DSA). NCCT diagnosis was defined as “positive” when the neuroradiologist suggested the diagnosis of CVT on the report, as opposed to the “negative” diagnosis group. All NCCT examinations were reviewed by a neuroradiologist experienced in cerebrovascular pathology. We compared his evaluation and analysed the location and number of direct signs found on NCCT. The neuroradiologist strongly suggested an NCCT diagnosis of CVT in 63% (15/24) of patients: 80% (4/5) with deep venous thrombosis (DVT) and 57% (11/19) with sinus venous thrombosis (SVT). The general radiologist's NCCT evaluation was incorrect in four cases, subsequently diagnosed at NCCT by the neuroradiologist. After reviewing the NCCT examination the experienced neuroradiologist identified the CVT direct sign in two that belonged to the NCCT negative diagnosis group. Thus the direct sign was present in 71% (17/24) of the cases: all the patients with DVT and 63% (12/19) of the patients with SVT. NCCT still plays an important role for fast and accurate diagnosis of CVT in the emergency setting. NCCT displayed the CVT direct sign more frequently than previously thought and it was correctly interpreted in most cases. Neuroradiological consultation adds value to the general radiologist's evaluation.

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CD4+CD25+Foxp3+ regulatory T (Treg) cells have been implicated in the lack of effective antitumor immunity. Denileukin diftitox (DAB389IL-2), a fusion protein of interleukin 2 (IL-2) and diphtheria toxin, provides a means of targeting Treg cells. In this study, we examined (1) the effect of denileukin diftitox on the deletion of Treg cells in various lymphoid compartments and (2) the dose scheduling of denileukin diftitox in combination with a recombinant poxviral vaccine to enhance antigen-specific immune responses. Treg cells in spleen, peripheral blood, and bone marrow of normal C57BL/6 mice were variously reduced after a single intraperitoneal injection of denileukin diftitox; the reduction was evident within 24 hours and lasted approximately 10 days. Injection of denileukin diftitox 1 day before vaccination enhanced antigen-specific T-cell responses above levels induced by vaccination alone. These studies show for the first time in a murine model (1) the differential effects of denileukin diftitox on Treg cells in different cellular compartments, (2) the advantage of combining denileukin diftitox with a vaccine to enhance antigen-specific T-cell immune responses, (3) the lack of inhibition by denileukin diftitox of host immune responses directed against a live viral vector, and (4) the importance of dose scheduling of denileukin diftitox when used in combination with a vaccine.

Chronic lymphocytic leukemia (CLL) is a disease of CD5+ B lymphocytes (designated as CLL cells) that are inefficient antigen-presenting cells. Their poor ability to present antigens to the T cells, largely due to an inadequate costimulatory capacity, is manifested as a failure to stimulate proliferation of both allogeneic and autologous T cells. We have investigated the ability of in vitro manipulated CLL cells, via hyperexpression of a triad of costimulatory molecules (B7-1, intercellular adhesion molecule 1 [ICAM-1], and leukocyte-function–associated antigen 3 [LFA-3], designated TRICOM), to stimulate effective antitumor T-cell responses. A recombinant modified vaccinia virus strain Ankara (MVA), which is a highly attenuated, replication-impaired virus variant, was successfully used to infect and deliver the simultaneous expression of the 3 human costimulatory molecules in TRICOM on the surface of the CLL cells. Proliferation of allogeneic and autologous T cells was observed when MVA-TRICOM–infected CLL cells were used as stimulators in proliferation assays. Cytotoxic T lymphocytes, generated in vitro by stimulation of autologous T cells with MVA-TRICOM–infected CLL cells, showed cytotoxicity against unmodified/uninfected CLL cells. Therefore, our findings suggest that the use of CLL cells infected ex vivo with MVA-TRICOM or direct injection of MVA-TRICOM in patients with CLL has potential for the immunotherapy of CLL.

We recently demonstrated that the p53 oncosuppressor associates to centrosomes in mitosis and this association is disrupted by treatments with microtubule-depolymerizing agents. Here, we show that ATM, an upstream activator of p53 after DNA damage, is essential for p53 centrosomal localization and is required for the activation of the postmitotic checkpoint after spindle disruption. In mitosis, p53 failed to associate with centrosomes in two ATM-deficient, ataxiatelangiectasia–derived cell lines. Wild-type ATM gene transfer reestablished the centrosomal localization of p53 in these cells. Furthermore, wild-type p53 protein, but not the p53-S15A mutant, not phosphorylatable by ATM, localized at centrosomes when expressed in p53-null K562 cells. Finally, Ser15 phosphorylation of endogenous p53 was detected at centrosomes upon treatment with phosphatase inhibitors, suggesting that a p53 dephosphorylation step at centrosome contributes to sustain the cell cycle program in cells with normal mitotic spindles. When dissociated from centrosomes by treatments with spindle inhibitors, p53 remained phosphorylated at Ser15. AT cells, which are unable to phosphorylate p53, did not undergo postmitotic proliferation arrest after nocodazole block and release. These data demonstrate that ATM is required for p53 localization at centrosome and support the existence of a surveillance mechanism for inhibiting DNA reduplication downstream of the spindle assembly checkpoint

We recently demonstrated that the p53 oncosuppressor associates to centrosomes in mitosis and this association is disrupted by treatments with microtubule-depolymerizing agents. Here, we show that ATM, an upstream activator of p53 after DNA damage, is essential for p53 centrosomal localization and is required for the activation of the postmitotic checkpoint after spindle disruption. In mitosis, p53 failed to associate with centrosomes in two ATM-deficient, ataxiatelangiectasia-derived cell lines. Wild-type ATM gene transfer reestablished the centrosomal localization of p53 in these cells. Furthermore, wild-type p53 protein, but not the p53-S15A mutant, not phosphorylatable by ATM, localized at centrosomes when expressed in p53-null K562 cells. Finally, Ser15 phosphorylation of endogenous p53 was detected at centrosomes upon treatment with phosphatase inhibitors, suggesting that a p53 dephosphorylation step at centrosome contributes to sustain the cell cycle program in cells with normal mitotic spindles. When dissociated from centrosomes by treatments with spindle inhibitors, p53 remained phosphorylated at Ser15. AT cells, which are unable to phosphorylate p53, did not undergo postmitotic proliferation arrest after nocodazole block and release. These data demonstrate that ATM is required for p53 localization at centrosome and support the existence of a surveillance mechanism for inhibiting DNA reduplication downstream of the spindle assembly checkpoint

Dendritic cells (DCs) are the most potent of the antigen-presenting cells (APCs). Preparation of sufficient numbers of mature DCs, however, is both costly and time-consuming. We have examined here the possibility of using an alternative source of APCs that would be easier to obtain, would not require extensive culture, and thus would be more applicable to human immunotherapy protocols. We show here that freshly isolated human B cells can be efficiently infected by a replication-defective fowlpox recombinant vector, designated rF-TRICOM (TRIad of COstimulatory Molecules), to markedly increase surface expression of the human costimulatory molecule B7-1 and moderately increase expression of intercellular adhesion molecule-1 (ICAM-1) and leukocyte function-associated antigen-3 (LFA-3). Peptide-pulsed rF-TRICOM–infected B cells were highly efficient in activating antigen-specific human T cells and shown to be superior to the use of CD40L in enhancing APC potency. Moreover, when infection of freshly isolated B cells with rF-TRICOM was combined with CD40L, a still further marked enhancement of the antigen-presenting potency was observed. Ex vivo–generated antigen-specific T cells activated in this manner might be applied to experimental protocols or used for adoptive transfer in immunotherapy protocols.

Plant homeodomain-leucine zipper proteins, unlike most animal homeodomains, bind DNA efficiently only as dimers. In the present work, we report that the deletion of the homeodomain N-terminal arm (first nine residues) of the homeodomain-leucine zipper protein Hahb-4 dramatically affects its DNA-binding affinity, causing a 70-fold increase in dissociation constant. The addition of the N-terminal arm of DrosophilaAntennapedia to the truncated form restores the DNA-binding affinity of dimers to values similar to those of the native form. However, the Antennapedia N-terminal arm is not able to confer increased binding affinity to monomers of Hahb-4 lacking the leucine zipper motif, indicating that the inefficient binding of monomers must be due to structural differences in other parts of the molecule. The construction of proteins with modifications at residues 5 to 7 of the homeodomain suggests strongly that positively charged amino acids at these positions play essential roles in determining the DNA-binding affinity. However, the effect of mutations at positions 6 and 7 can be counteracted by introducing a stretch of positively charged residues at positions 1 to 3 of the homeodomain. Sequence comparisons indicate that all homeodomain-leucine zipper proteins might use contacts of the N-terminal arm with DNA for efficient binding. The occurrence of a homeodomain with a DNA-interacting N-terminal arm must then be an ancient acquisition in evolution, earlier than the separation of lines leading to metazoa, fungi and plants.

We have examined the murine genes encoding transcription factors E2F1, -3, -5 and -6 in gametes and early embryos. All genes are expressed as maternal transcripts and all are efficiently transcribed after the blastocyst stage. Between those two stages, each E2F mRNA is transcribed with a distinctive and unique pattern. E2F proteins are also differentially expressed and compartmentalized in pre-implantation embryos.

We identified in the EST database murine and human sequences similar, but not identical, to the members of the PC3/BTG/TOB family of cell cycle inhibitors. A conserved domain (aa 50–68) of the PC3 protein, the prototype member of the family, was used as a query. That domain has been shown by us to be necessary for the antiproliferative activity of PC3. A murine EST clone and a highly homologous human EST clone, containing the entire ORF, were chosen for sequencing. Comparison to databases and a phylogenetic tree analysis indicated that these EST clones are the mouse and human homologues of a gene that represents a novel member of the PC3/BTG/TOB family. This gene, named PC3B, is endowed with marked antiproliferative activity, being able to induce G1arrest, and is highly expressed in testis, in oocyte, and in preimplantation embryos. Analysis of its expression during murine development indicated a specific localization in the olfactory epithelium at midgestation, suggesting that PC3B might be involved in the differentiation of this neuronal structure. Human PC3B mapped to chromosome 11q23, as indicated by radiation hybrid analysis.

This doubleblind, randomized study was aimed at evaluating comparatively, in postmenopausal women, the activity of a standardized soy extract SOYSELECT and placebo when given alone or in combination with conjugated equine estrogens CEE on early climacteric symptoms. Lipid profile, pituitary hormones, osteocalcin and endothelin levels, and vaginal and endometrial parameters were also evaluated.

SCLC is a highly aggressive tumor with a 5-year survival rate of less than 6%. A heterogeneous disease, SCLC is classified into four subtypes that include tumors with neuroendocrine and non-neuroendocrine features. Immune checkpoint blockade has been recently added for the frontline treatment of SCLC; however, this therapy has only led to modest clinical improvements. The lack of clinical benefit in a cancer type known to have a high tumor mutational burden has been attributed to poor T-cell infiltration and low expression of MHC-class I in most SCLC tumors. In an attempt to devise a more effective immunotherapeutic regimen, this study investigated an alternate approach on the basis of the use of the clinical-stage interleukin-15 superagonist, N-803.

We have analysed the interaction of the sunflower homeodomain leucine-zipper (Hd-Zip) protein Hahb-4 with DNA. The complete Hd-Zip domain from Hahb-4 was able to select specific sequences from a random oligonucleotide mixture that contained a 9-bp core with four fixed and five degenerate positions. Analysis of the binding of some of the selected sequences suggests that Hahb-4 preferentially binds the dyad-symmetrical sequence CAAT(A/T)ATTG. Single-nucleotide replacements at positions 1, 5 or 9 of this sequence produced a decrease in binding of 2-4-fold. DNA binding as a function of protein concentration was non-hyperbolic. This behaviour could be explained by an equation in which dimer formation is a pre-requisite for DNA binding. A global dissociation constant (Kd) of 1.31×10-14 M2 could be calculated. The removal of the leucine zipper promoted a change in specificity and a decrease in binding affinity (Kd = 5.03×10-5 M). Mutation of Phe-20 of the homeodomain into Leu completely abolished DNA binding. The mutant protein, however, was able to inhibit DNA binding by the non-mutant form, presumably through the formation of heterodimers. The analysis of this inhibitory effect at different mutant concentrations allowed the estimation of the Kd for the dimer-monomer equilibrium [about (2-4)×10-6 M]; from this, a Kd of 3-6×10-9 M for the dimer-DNA complex could be estimated. The results obtained indicate that the formation of dimers is the main factor influencing the interaction of Hahb-4 with DNA. It is proposed that shifts in a dimer-monomer equilibrium could be used within the cell to modulate the interaction of this protein with target genes.

The CpG-rich promoter of the retinoblastoma tumor suppressor gene (Rb-1) is normally unmethylated. However, aberrant methylation of CpG dinucleotides within the Rb-1 promoter has been depicted in certain tumors, which determines transcriptional inactivity of the gene and absence of the pRb retinoblastoma protein. Here we have concentrated on an E2F-binding site in the Rb-1 promoter. We show that the E2F site is required for cell-cycle regulated Rb-1 transcription in non-transformed cells. The function of the E2F site is associated with its ability to interact with several activating factors of the E2F family. In contrast, in vitro methylation of two tandemly arranged CpGs in the E2F recognition site prevents binding by E2F factors, and determines instead the recruitment of the general repressor methylcytosine-binding protein 2 (MeCP2). These results suggest that the interaction of MeCP2 with the methylated version of the E2F site may represent a step towards Rb-1 promoter inactivity in tumor cells.

The CpG-rich promoter of the retinoblastoma tumor suppressor gene (Rb-1) is normally unmethylated. However, aberrant methylation of CpG dinucleotides within the Rb-1 promoter has been depicted in certain tumors, which determines transcriptional inactivity of the gene and absence of the pRb retinoblastoma protein. Here we have concentrated on an E2F-binding site in the Rb-1 promoter. We show that the E2F site is required for cell-cycle regulated Rb-1 transcription in non-transformed cells. The function of the E2F site is associated with its ability to interact with several activating factors of the E2F family. In contrast, in vitro methylation of two tandemly arranged CpGs in the E2F recognition site prevents binding by E2F factors, and determines instead the recruitment of the general repressor methylcytosine-binding protein 2 (MeCP2). These results suggest that the interaction of MeCP2 with the methylated version of the E2F site may represent a step towards Rb-1 promoter inactivity in tumor cells.

The gene encoding Ran-binding protein 1 (RanBP1) is transcribed in a cell cycle-dependent manner. The RanBP1 promoter contains two binding sites for E2F factors, named E2F-c, located proximal to the transcription start, and E2F-b, falling in a more distal promoter region. We have now induced site-directed mutagenesis in both sites. We have found that the distal E2F-b site, together with a neighboring Sp1 element, actively controls up-regulation of transcription in S phase. The proximal E2F-c site plays no apparent role in cycling cells yet is required for transcriptional repression upon growth arrest. Protein binding studies suggest that each E2F site mediates specific interactions with individual E2F family members. In addition, transient expression assays with mutagenized promoter constructs indicate that the functional role of each site is also dependent on its position relative to other regulatory elements in the promoter context. Thus, the two E2F sites play opposite genetic functions and control RanBP1 transcription through distinct molecular mechanisms.

The results of an epidemiological survey on Huntington's disease in the Lazio Region, Central Italy, and of linkage studies in a subset of families are reported. From a total of 99 ascertained families and 491 patients, a prevalence of 25.6 × 10-6 was obtained, with distributions of age at onset and age at death similar to those described in the literature. No relationship with the sex of the transmitting parent was observed. Analysis of 10 chromosome 4 restriction fragment length polymorphisms in 11 families showed consistent linkage between the genetic loci D4S10, D4S43 and D4S95, and the disease. A recombination rate of 0.08 for D4S10 markers was obtained in this sample. Allelic frequencies of DNA markers in the general population are also reported.

The haplotypes at four polymorphic loci of theY chromosome were determined in 245 Caucasian males from 12 subpopulations. The data show that haplotype radiation occurred among Caucasians. Haplotype radiation was accompanied by recurrent mutations at STR loci that caused partial randomization of haplotype structure. The present distribution of alleles at short tandem repeats (STRs) can be explained by a mutation pattern similar to those described for autosomal STRs. The degree of variation among groups of subpopulations was assayed by using the Analysis of Molecular Variance. The results confirm a faster divergence of the Y chromosome as compared to the rest of the genome.

Abstract: Naturally occurring deletion mutations within the human β-globin cluster lead to specific, phenotypically discrete syndromes (i.e., δβ-thalassemias and hereditary persistence of fetal hemoglobin, HPFH), characterized by increased production of fetal hemoglobin in adult life. We have previously characterized an enhancer element, which is juxtaposed to the fetal Gγ-gene, by means of a deletion first described in a Thai family. To obtain further insights into the mechanisms involved in this deletion, we have now characterized several of its novel features. Following amplification by the polymerase chain reaction and sequencing of the 1.5-kb bridging fragment, we have shown that the 5′ breakpoint of the deletion occurs 1260 bp 3′ of the fetal Gγ-globin gene, whereas the 3′ breakpoint lies 521 bp upstream of the EcoRI site of the enhancer element and 2845 bp upstream of the 3′ breakpoint of the Chinese (Aγδβ)°-thalassemia deletion. The total length of the deletion is 101 kb, which resembles that of HPFH-1 and HPFH-2 deletions and a set of two γδβ-thalassemia deletions. Our data further support the hypothesis that these sets of large deletions with almost identical lengths are generated via the loss of a complete chromatin loop. To elucidate further the mechanisms leading to the deletion, we have sequenced the novel 0.5-kb region residing immediately 3′ to the breakpoint and shown that it contains putative binding sites for several transcription factors, such as HNF-1, AP-1, and TFIID. Sequence comparison of the deletion breakpoints reveals no junctional homology, indicating an end-to-end joining of blunted ends; a pair of 7-nt complementary repeats adjacent to a set of a direct CCCT repeat flanks the breakpoints. This limited homology constitutes a frequent characteristic of a non-homologous recombination mechanism. All these features of the HPFH-6 deletion suggest that this mutation has resulted from a non-homologous recombination event.

A novel deletion in the human β-globin gene cluster associated with increased levels of fetal hemoglobin (HbF) in adult life was molecularly characterized in a member of a family of Eastern European descent. The phenotype of the deletion, documented in five members of the family, shows mild hypochromia and microcytosis (mean corpuscular Hb, 24 to 25.9 pg; mean corpuscular volume, 74 to 78.5 fL) but high production of HbF (13% to 24%) with heteroceilular distribution (36% to 86% F cells). Extensive restriction enzyme mapping of the β-globin cluster and sequencing of the region encompassing the breakpoints showed that the deletion starts 1,612 bp upstream of the cap site of the δ-globin gene and terminates within the first intron of the β-globin gene, deleting 9.1 kb of DNA. This length is definitely shorter than the average 12.0 kb of the previously characterized (δβ)° thalassemias. The 5‘ breakpoint of the new deletion is close to that of the Yugoslavian δβ-thalassemia deletion, whereas the 3‘ breakpoint is very close to those of the Turkish and the Greek βo-thalassemia deletions. The breakpoints of the deletion occur within a direct repeat containing a tetranucleotide exhibiting homology to a donor-splice site, and is symmetrically flanked by a set of 13- and 14-bp homologous complementary sequences, respectively. It is likely that the deletion may be the result of an “illegitimate” or “nonhomologous” recombination event to which these two short sequences may have contributed. It is of interest that the novel deletion (9.1 kb) is comparable to the Italian HPFH-5 deletion (12.9 kb), regarding both the size and the position of the breakpoints. However, the HPFH-5 deletion includes sequences flanking the breakpoints that are preserved in the new deletion. Considering the resulting two discrete phenotypes (ie, δβ-thalassemia vHPFH), it can be hypothesized that the deleted sequences in the Italian HPFH-5 mutation may harbor regulatory elements that exert a negative control on the γ-globin gene expression.

RanBP1 is a molecular partner of the Ran GTPase, which is implicated in the control of several processes, including DNA replication, mitotic entry and exit, cell cycle progression, nuclear structure, protein import and RNA export. While most genes encoding Ran-interacting partners are constitutively active, transcription of the RanBP1 mRNA is repressed in non proliferating cells, is activated at the G1/S transition in cycling cells and peaks during S phase. We report here that forced expression of the RanBP1 gene disrupts the orderly execution of the cell division cycle at several stages, causing inhibition of DNA replication, defective mitotic exit and failure of chromatin decondensation during the telophase-to-interphase transition in cells that achieve nuclear duplication and chromosome segregation. These results suggest that deregulated RanBP1 activity interferes with the Ran GTPase cycle and prevents the functioning of the Ran signalling system during the cell cycle.

A novel deletion in the human beta-globin gene cluster associated with increased levels of fetal hemoglobin (HbF) in adult life was molecularly characterized in a member of a family of Eastern European descent. The phenotype of the deletion, documented in five members of the family, shows mild hypochromia and microcytosis (mean corpuscular Hb, 24 to 25.9 pg; mean corpuscular volume, 74 to 78.5 fL) but high production of HbF (13% to 24%) with heterocellular distribution (36% to 86% F cells). Extensive restriction enzyme mapping of the beta-globin cluster and sequencing of the region encompassing the breakpoints showed that the deletion starts 1,612 bp upstream of the cap site of the delta-globin gene, and terminates within the first intron of the beta-globin gene, deleting 9.1 kb of DNA. This length is definitely shorter than the average 12.0 kb of the previously characterized (delta beta) zero-thalassemias. The 5' breakpoint of the new deletion is close to that of the Yugoslavian delta beta-thalassemia deletion, whereas the 3' breakpoint is very close to those of the Turkish and the Greek beta zero-thalassemia deletions. The breakpoints of the deletion occur within a direct repeat containing a tetranucleotide exhibiting homology to a donor-splice site, and is symmetrically flanked by a set of 13- and 14-bp homologous complementary sequences, respectively. It is likely that the deletion may be the result of an “illegitimate” or “nonhomologous” recombination event to which these two short sequences may have contributed. It is of interest that the novel deletion (9.1 kb) is comparable to the Italian HPFH-5 deletion (12.9 kb), regarding both the size and the position of the breakpoints. However, the HPFH-5 deletion includes sequences flanking the breakpoints that are preserved in the new deletion. Considering the resulting two discrete phenotypes (ie, delta beta-thalassemia v HPFH), it can be hypothesized that the deleted sequences in the Italian HPFH-5 mutation may harbor regulatory elements that exert a negative control on the gamma-globin gene expression.

Complementary DNA sequences encoding different portions of two sunflower homeodomain proteins were cloned in-frame in the expression vectors pRSET and pGEX-3X. When introduced into competentEscherichia colicells and induced, the resulting plasmids directed the expression of large amounts (5–10% of total cellular protein) of the encoded polypeptides. As a rule, fusions in pRSET rendered insoluble proteins, while fusions in pGEX were soluble and could be purified in a single step by selective absorption onto glutathione–agarose beads, followed by elution with free glutathione. The purified proteins showed both glutathioneS-transferase and DNA-binding activity, indicating that they retain their native conformation. The expression–purification protocol that was employed allowed the isolation of up to 0.7 mg of protein per gram of transformed cells. One of the fusion proteins, RH11 (which is a fusion of the homeodomain protein HAHR1 in pRSET), though insoluble, was able to bind DNA when spotted onto a nitrocellulose filter. This protein could also be simply purified in large amounts by electroelution from sodium dodecyl sulfate–polyacrylamide gels and used to elicit antibodies which recognized both the transgenic fusion and the native protein from sunflower nuclei. Our results clearly show that vector choice is a critical parameter for obtaining large amounts of a desired protein for particular purposes.

Pressure applied to the eyeball increases the intraocular pressure and impairs retinal circulation, resulting in field loss and finally, ocular blackout. To quantitate this phenomenon, the plethysmographic goggle ophthalmodynamometer was used to vary pressures in normal healthy male patients, and the time onset of blackout was recorded. The results could be divided into the following three groups: (1) Induced pressures lower than that of the retinal artery diastolic pressure caused a transient field defect, but no ocular blackout. (2) At pressures between diastolic and systolic, as the induced pressure increased, the time onset to blackout decreased in a logarithmic manner. (3) At induced pressures systolic and above, the time onset until blackout remained constant. These results did not change with varying illumination.

ABSTRACTMesenchymalization is a cellular and molecular program in which epithelial cells progressively lose their well-differentiated phenotype and adopt mesenchymal characteristics. Tumor mesenchymalization occurs during the progression of cancer to metastatic disease, and is also associated with resistance to multiple therapeutics, including killing by cytotoxic immune cells. Furthermore, tumor cells can evade immune destruction by upregulating the checkpoint molecule PD-L1, and emerging research has found higher PD-L1 expression in mesenchymalized tumors. Here, the association between TGF-β1-mediated mesenchymalization and PD-L1 was investigated in non-small cell lung cancer cells (NSCLC). TGF-β1 was found to upregulate PD-L1 gene transcription in a Smad2-dependent manner, and a positive association between PD-L1 and phosphorylated Smad2 was found in NSCLC tumors. The potential to target these 2 negative immune regulators with a single agent was investigated using M7824, a novel clinical-stage bifunctional agent that targets both PD-L1 and TGF-β. Treatment of NSCLC cells with M7824 in vitroand in vivoattenuated features of TGF-β1-mediated mesenchymalization, including mesenchymal marker expression, proliferation suppression, and chemoresistance. These findings demonstrate that upregulation of tumor cell PD-L1 is a novel mechanism of TGF-β1-induced immunosuppression in NSCLC, and that treatment with M7824 has the potential to simultaneously block both tumor mesenchymalization and PD-L1-dependent immunosuppression.