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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.Preclinical models of SCLC spanning all molecular subtypes were used to evaluate the susceptibility of SCLC to natural killer (NK)-mediated lysis in vitro, including NK cells activated by N-803. Antitumor activity of N-803 was evaluated in vivo with a xenograft model of SCLC.In vitro and in vivo data revealed differences in susceptibility of SCLC subtypes to lysis by NK cells and that NK cells activated by N-803 effectively lyse SCLC tumor cells across all variant subtypes, regardless of their expression of MHC-class I.These findings highlight the potential of a novel immune-based intervention using a cytokine-based therapeutic option for the treatment of SCLC. We hypothesize that N-803 may provide benefit to most patients with SCLC, including those with immunologically cold tumors lacking MHC expression.

To evaluate the feasibility and early outcomes of below the ankle orbital atherectomy in chronic limb-threatening ischemia in patients with calcified foot arteries.12 patients (mean age 69.4 ± 14.7; range 57 to 85 years) who were affected by diabetes underwent orbital atherectomy below the knee and ankle arterial segments. Technical success was defined as orbital atherectomy passage and debulked the calcified lesion, delivery of adjunctive therapy, and30% residual stenosis at final angiogram. The procedural outcome included complications, amputation-free survival, and freedom from clinically-driven target lesion revascularization at 30-days and 6-months of follow-up.Orbital atherectomy was performed in 3 cases in Anterior tibial (AT) and dorsalis pedis (Ped) arteries + Posterior tibial (PT) and Lateral Plantar (Lat Plan), 5 cases in PT and Lat Plan arteries, 1 case of PT and Medial Plantar, 1 case of Peroneal and Plan Lat, and 2 cases of AT and Ped. After atherectomy, we used a drug-coated balloon (DCB) angioplasty. Technical success was achieved in 11 (91.6%) cases. No deaths were registered during the follow-up. The limb salvage rate was 100%, and no major amputations were registered. Amputation-free survival was 50%. Freedom from CD-TLR was 100% at 30 days and 91.7% at 6-months. One patient underwent a TLR at three months. No major cardiovascular events, limb events, or significant procedure-related complications were registered.CSI orbital atherectomy and DCB angioplasty appear a feasible and promissor treatment options in diabetic CLTI patients.

Diabetic foot ulcer treatment is a challenge for the healthcare world. Widespread infection and the presence of critical ischemia (especially with end-stage renal disease) can lead to major amputation rather than amenable to conservative treatment. Surgical strategies of the diabetic foot have been changing over the past 10 years and are now focused on reconstructive treatment and limb salvage. These goals were achieved, thanks to an evolution of distal revascularization techniques and a distinct approach, which integrates various methods focused on limb salvage. Podoplastic techniques of the diabetic foot are focused on infection clearance, the surgical treatment of corrective deformities, soft tissue coverage and limb ischemia correction along with the management of diabetes and the comorbidities that compromise tissue repair processes. The reconstructive techniques used in diabetic foot treatment owe their effectiveness in part to the results of technological improvements such as the circular external fixator as a tool for stabilization and surgical site protection. In the last decade, many studies have shown that circular external fixation should be considered as the most useful method to protect the reconstructive surgical site in limb salvage of the diabetic foot. The objective of this review is to highlight the role of surgical offloading using circular external fixation as an adjunct to the podoplastic diabetic foot reconstruction procedures.

Bone metastases are devastating complications of cancer. They are particularly common in prostate cancer (PCa), represent incurable disease, and are refractory to immunotherapy. We seek to define distinct features of the bone marrow (BM) microenvironment by analyzing single cells from bone metastatic prostate tumors, involved BM, uninvolved BM, and BM from cancer-free, orthopedic patients, and healthy individuals. Metastatic PCa is associated with multifaceted immune distortion, specifically exhaustion of distinct T cell subsets, appearance of macrophages with states specific to PCa bone metastases. The chemokine CCL20 is notably overexpressed by myeloid cells, as is its cognate CCR6 receptor on T cells. Disruption of the CCL20-CCR6 axis in mice with syngeneic PCa bone metastases restores T cell reactivity and significantly prolongs animal survival. Comparative high-resolution analysis of PCa bone metastases shows a targeted approach for relieving local immunosuppression for therapeutic effect.

Objective Antegrade crossing techniques via transfemoral access are often challenging and may be associated with technical and clinical failure when treating patients with critical limb ischaemia (CLI). The objective of this study was to summarise all available literature regarding retrograde endovascular treatment of patients with CLI and to investigate the technical success and complication rate of retrograde access. Methods A systematic literature search was performed in PubMed, Scopus, and Cochrane Central until May 2020. A meta-analysis of 31 observational studies (29 retrospective and two prospective; 26 and five studies with low and moderate risk of bias, respectively) was conducted with random effects modelling. The incidence of adverse events peri-procedurally and during follow up were calculated. Results The 31 studies enrolled 1 910 patients who were treated endovascularly for femoropopliteal and/or infrapopliteal lesions causing CLI. Most of the patients had diabetes while more than half of the overall population had coronary artery disease and dyslipidaemia. All lesions were located in the infra-inguinal segment and most were chronic total occlusions (96%; 95% CI 85%–100%). Seven studies reported moderate or severe calcification in approximately half of the cases (45%; 95% CI 30%–60%). The overall technical success of the retrograde approach was 96% (18 studies; 95% CI 92%–100%). Perforation, flow limiting dissection, distal embolisation, and local haematoma at the retrograde access site were infrequent and observed in 2.1%, 0.6%, 0.1%, and 1.3% of the patients, respectively. The six month primary patency rate was 78% (five studies; 95% CI 46%–99%), the six month limb salvage rate was 77% (four studies; 95% CI 70%–84%). Conclusion The results indicated that the retrograde or bidirectional antegrade/retrograde approach is safe and effective and facilitates angioplasty when antegrade treatment fails. However, prospective studies with standardised wound care and surveillance protocols are needed to investigate retrograde techniques in patients with CLI who failed antegrade revascularisation, to improve long term limb salvage and survival.

Human papillomavirus (HPV) is associated with the etiology of cervical carcinoma, head and neck squamous cell carcinoma, and several other cancer types. Vaccines directed against HPV virus-like particles and coat proteins have been extremely successful in the prevention of cervical cancer through the activation of host HPV-specific antibody responses; however, HPV-associated cancers remain a major public health problem. The development of a therapeutic vaccine will require the generation of T-cell responses directed against early HPV proteins (E6/E7) expressed in HPV-infected tumor cells. Clinical studies using various vaccine platforms have demonstrated that both HPV-specific human T cells can be generated and patient benefit can be achieved. However, no HPV therapeutic vaccine has been approved by the Food and Drug Administration to date. One method of enhancing the potential efficacy of a therapeutic vaccine is the generation of agonist epitopes. We report the first description of enhancer cytotoxic T lymphocyte agonist epitopes for HPV E6 and E7. While the in silico algorithm revealed six epitopes with potentially improved binding to human leukocyte antigen–A2 allele (HLA-A2)–Class I, 5/6 demonstrated enhanced binding to HLA-Class I in cell-based assays and only 3/6 had a greater ability to activate HPV-specific T cells which could lyse tumor cells expressing native HPV, compared to their native epitope counterparts. These agonist epitopes have potential for use in a range of HPV therapeutic vaccine platforms and for use in HPV-specific adoptive T- or natural killer–cell platforms.

Tumor progression relies on the ability of cancer cells to effectively invade surrounding tissues and propagate. Among the many mechanisms that contribute to tumor progression is the epithelial-to-mesenchymal transition (EMT), a phenotypic plasticity phenomenon that increases the cancer cells' motility and invasiveness and influences their surrounding microenvironment by promoting the secretion of a variety of soluble factors. One such factor is IL-8, a chemokine with multiple pro-tumorigenic roles within the tumor microenvironment (TME), including stimulating proliferation or transformation of tumor cells into a migratory or mesenchymal phenotype. Further, IL-8 can increase tumor angiogenesis or recruit larger numbers of immunosuppressive cells to the tumor. Prognostically, observations in many tumor types show that patients with higher levels of IL-8 at baseline experience worse clinical outcomes. Additionally, studies have shown that the chemokine directly contributes to the development of resistance to both chemotherapy and molecularly targeted agents. More recently, clinical studies evaluating levels of IL-8 in patients receiving immune checkpoint inhibition (ICI) therapy deduced that myeloid tumor infiltration driven by IL-8 contributes to resistance to ICI agents and that peripheral IL-8 can predict outcomes to ICI therapy. Further, pre-clinical data demonstrate that targeting IL-8 or its receptors enables improved tumor killing by immune cells, and treatment strategies combining blockade of the IL-8/IL-8R axis with ICI ultimately improve anti-tumor efficacy. Based on these results and the prognostic capacity of IL-8, there are a number of ongoing clinical trials evaluating the addition of IL-8 targeting strategies to immune-based therapies.

Background Therapeutic cancer vaccines targeting tumour associated antigens (TAAs) offer a potential method to activate cytotoxic T-cells. A vaccine using a novel Ad5 vector (E1-, E2b-) targeting 3 TAAs, PSA, MUC-1 and Brachyury, has been constructed. Both the C-terminus of MUC-1 and Brachyury have been shown to play an integral role in epithelial-to-mesenchymal transition, metastasis, and chemotherapy resistance. Both antigens are overexpressed in mCRPC. The transgenes for PSA, MUC-1 and Brachyury contain modifications for the expression of CD8+ T-cell enhancer agonist epitopes. This vaccine has not been previously tested in humans. Methods Pts with mCRPC were treated with the combination of 3 vaccines targeting PSA, MUC-1 and Brachyury at 5 x 1011 viral particles (VP) each, SQ every 3 weeks for maximum of 3 doses (dose de-escalation cohort) and followed by boost every 8 weeks for 1 year (dose expansion cohort only). The primary objective was to determine the safety and tolerability and to establish the recommended phase 2 dose. Immune assays were conducted in the first 5 enrolled patients. Results 12 pts were enrolled (6 in each cohort) between 07/2018 and 04/2019 and received at least 1 dose. Median PSA was 37.8 (range, 5.81 – 1006 ng/mL). Vaccine was safe and tolerable, no DLTs or grade 3 or higher treatment-related adverse events (TRAEs) were observed. All other TRAEs were Grade 1 or 2; the most common was injection-site reaction in all pts. Two chemotherapy naive pts had confirmed PSA declines (89% and 50%, respectively) observed after only 1 dose. Third had unconfirmed 12% PSA decline at week 3. 5/5 patients mounted responses to at least 1 TAA while 3/5 mounted immune responses to all 3 TAAs. Multifunctional T-cell responses to PSA, MUC-1 and Brachyury were also detected post-vaccination. Conclusions Ad5 PSA/MUC-1/Brachyury vaccine is safe and well tolerated. The recommended Phase 2 dose is 5 x 1011 VP. Confirmed PSA decline was observed in 2 pts. Multifunctional TAA specific T-cell responses to all 3 antigens were seen in a patient with 89% PSA decline. Further research is warranted to evaluate immunogenicity and eventual clinical benefit. Future trials will involve the use of this vaccine in combination with other immuno-oncology agents. Clinical trial identification NCT03481816. Editorial acknowledgement Debra Weingarten. Legal entity responsible for the study Center for Cancer Research, National Cancer Institute, National Institutes of Health. Funding Intramural Research Program of the Center for Cancer Research, National Cancer Institute (NCI), National Institutes of Health (NIH), and by Cooperative Research and Development Agreements (CRADAs) between the NCI and NantCell/Etubics, and the NCI and NantBioscience. Disclosure E.S. Gabitzsch: Full / Part-time employment: Etubics Corporation. F.R. Jones: Full / Part-time employment: Etubics Corporation. J.P. Balint: Full / Part-time employment: Etubics Corporation. P. Soon-Shiong: Full / Part-time employment, Founder and an executive: NantCell; Full / Part-time employment, Founder and an executive: NantBioscience. S. Rabizadeh: Full / Part-time employment: NantCell. All other authors have declared no conflicts of interest.

B-Laser™ is a novel atherectomy device that uses a solid-state third harmonic pulsed Nd:YAG laser with an output of 355 nm. Early data showed that the B-Laser™ is safe in treating a broad range of infrainguinal arterial lesions. We present the results of the EX-PAD-03 U.S. pivotal trial of the

The number of diabetic patients is actually increasing all around the world, consequently, critical limb ischemia and ischemic diabetic foot disorders related to the presence of diabetic occlusive arterial disease will represent in the next few years a challenging issue for vascular specialists. Revascularization represents one step in the treatment for the multidisciplinary team, reestablishing an adequate blood flow to the wound area, essential for healing and avoiding major amputations. The targets of revascularization can be established to obtain a "complete" revascularization, treating all tibial and foot vessels or following the angiosome and wound-related artery model, obtaining direct blood flow for the wound. In this article, we summarize our experience in endovascular treatment of diabetic critical limb ischemia, focusing on the angiosome and wound-related artery model of revascularization and the technical challenges in treating below-the-knee and below-the-ankle vessels.

Purpose To evaluate the efficacy and safety of "Direct Stent Puncture" technique for intraluminal stent recanalization in the femoro-popliteal segments. Methods and Materials A cohort of diabetics who had symptomatic in-stent occlusion of the superficial femoral or popliteal arteries underwent endovascular recanalization. After antegrade failure, direct stent puncture technique was performed. The primary end-point was to efficacy assessment, intended as technical success and clinical improvement. The secondary end-point was safety assessment, intended as free of complication rate. Results Fifty-four patients (37 men; 73.6±8.5years) underwent direct stent puncture technique, after several unsuccessful antegrade attempts to cross the occluded stent. Technical success for intraluminal stent recanalization was achieved in 53/54 (98.2%) of cases and failed in 1/54 (1.8%). Clinical improvement was obtained in 51/54 (94.4%) of cases, with regression of the clinical symptoms and improvement of the TcPO 2 , from 3±18mmHg to 43±11mmHg after 15days ( p Free of complications rate was 92.5%. In 2/54 (3.7%) of cases distal embolization occurred, in 1/54 (1.9%) case a sudden vessel thrombosis was diagnosed after 12hours and in 1/54 (1.9%) case hematoma at the stent puncture site was observed. Conclusions Direct Stent Puncture technique is an efficacy and safety option for intraluminal stent recanalization in the femoro-popliteal segment in-stent occlusion.

Drug delivery by layered double hydroxides (LDHs) is extensively studied due to the capacity of these solids to preserve the active agent and to modify its release rate and bioavailability. In this work, an ibuprofen–LDH complex was synthesized with high drug content (54% w/w), incorporated both in the interlayer and on the surface. The mechanisms involved in the ibuprofen release process were studied as a function of the anion type (chloride, acetate, and phosphate) and concentration, and the pH of the release media. The experimental conditions selected were based on the composition of pharmaceutically relevant fluids such as gastric, intestinal and lysosomal simulated media. Three release mechanisms accounted for the rate and extent of the drug release: ion exchange (highly dependent on the anions), desorption and weathering (dependent on the pH). These mechanisms, together with the different solubility of the anionic and non-ionic ibuprofen species, also explained the release behavior for particulate solid and hydrogel formulations. Anion exchange determined ibuprofen release in intestinal medium (phosphate anions pH 6.8) whereas surface reactions mediated by solid weathering or drug solubility ruled the release in acid media (gastric fluid and lysosomal medium).

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.

In centrosome-containing cells, spindle assembly relies on microtubules (MTs) nucleated from both centrosomes and chromosomes [1, 2]. Recent work has suggested that additional spindle MTs can be nucleated by ?-tubulin ring complexes (?-TuRCs) that associate laterally with preexisting spindle MTs, leading to spindle amplification. It has been proposed that in Drosophila S2 cells, ?-TuRCs are anchored to the spindle MTs by augmin, a multiprotein complex that contains at least eight subunits [3-5]. Here we show that the Dgt6 component of augmin is primarily required for kinetochore fiber (k-fiber) formation. An analysis of MT regrowth after cold exposure showed that formation of kinetochore-driven k-fibers is severely impaired in Dgt6-depleted cells. In control cells, these fibers are enriched in Dgt6, ?-tubulin, and Msps/XMAP215. Consistent with these results, Dgt6 coprecipitates with Msps, D-TACC, ?-tubulin, Ndc80, and Nuf2. However, RNA interference (RNAi)-mediated inhibition of ?-tubulin, Msps/XMAP215, or Ndc80/Hec1 reduced but did not abolish k-fiber regrowth. These results indicate that Dgt6 plays a pivotal role in kinetochore-driven k-fiber formation, mediating nucleation and/or initial stabilization of chromosome-induced MTs. We propose that Dgt6 binds and stabilizes nascent chromatin-induced MTs, facilitating their interaction with the Ndc80-Nuf2 complex. Dgt6 may also promote elongation of kinetochore-driven k-fibers through its interaction with ?-tubulin and Msps. © 2009 Elsevier Ltd. All rights reserved.

The ozonolysis of carbohydrate derived norbornene systems in participating solvents demonstrated that different remote controlling factors could induce the regioselective fragmentation of the primary ozonide. The exo norbornene series afforded a complete regioselective process.

The 2-oxoazetidinylacetate sodium salt was synthesized as a model of a minimal beta-lactam drug. This compound and the monobactam aztreonam were assayed as substrates of the Metallo-beta-lactamase BcII. None of them was hydrolyzed by the enzyme. While the azetidinone was not able to bind BcII, aztreonam was shown to bind in a nonproductive mode. These results provide an explanation for the unability of Metallo-beta-lactamases to inactive monobactams and give some clues for inhibitor design.

Computer-based systems (CBS) can provide information to help individuals analyze their health care needs and make decisions about management of health problems. This article discusses the development of a CBS that delivers an individualized educational intervention for continence health promotion. System development included cycles of prototype design, testing, analysis, and redesign. Knowledge acquisition, representation methods, and design decisions are discussed. Participants completed 4 rounds of usability testing and a pilot test, which resulted in enhancements to both the CBS and the expert system feature that produced individualized feedback. This iterative design process involved users throughout system development. User involvement resulted in a tutorial to explain navigation and other features of the CBS, graphics to enhance the written message, and clarification of continence-related content. The procedures resulted in an informative, usable product; they can be used to develop systems that provide information about symptom self-management for other health conditions.

Human tumor-associated antigens (TAAs) are weak immunogens. One strategy for increasing the immunogenicity of TAAs is to generate altered peptide ligands. In the studies reported here, microarray technology has been used to compare gene expression profiles of a human T cell line that was derived from the peripheral blood of a cancer patient vaccinated with a carcinoembryonic antigen (CEA)-based vaccine. We compared the gene expression profiles of this CEA-specific CD8 T cell line when (a) stimulated with the native peptide used to derive this line vs. no peptide, and (b) stimulated with its TCR enhancer agonist epitope vs. no peptide. The results demonstrate that the effect on the T cell line, when stimulated with the agonist peptide, is not an enhanced quantitative expression of the same genes or gene sets induced by the native peptide, but is rather a nearly reciprocal upregulation of different gene sets. The gene for the chemokine lymphotactin, which was overexpressed only in T cells stimulated with the agonist peptide, stood out above all others. This finding was extended using other T cell lines, and another set of agonist and native peptides from another TAA. ELISPOT and ELISA assays for lymphotactin confirmed and extended these findings. These studies suggest a potential role for lymphotactin in the T-cell activation processes and subsequent anti-tumor events.

The ELISPOT assay is used to detect T-cell responses in patients enrolled in vaccine clinical trials; it is a relatively sensitive assay that can be performed without in vitro stimulation (IVS) of PBMC. However, this assay may be limited in some cases because of a weak signal 1 (as is the case for tumor-associated antigens [TAA]), or by a limited number of PBMC available from patients. The objective of this study is to enhance the sensitivity of the ELISPOT by increasing the level of signal 2, in this case, B7-1 expression on antigen-presenting cells (APC), allowing for a more sensitive detection of antigen-specific T-cell precursors. C1RA2 cells were used as APC and were uninfected, or infected with either recombinant avipox (fowlpox)-B7-1 (rF-B7-1) or control fowlpox wild-type (FP-WT) vector. The expressions of B7-1, MHC Class II, as well as HLA-A2 on the infected cells were analyzed by flow cytometry. A nearly threefold increase in B7-1 mean fluorescence intensity (MFI) occurred in the rF-B7-1-infected C1RA2 cells with no changes in MHC Class II or HLA-A2 expression. Various PBMC/APC ratios were used to further analyze the use of rF-B7-1-infected C1RA2 cells as APC in the ELISPOT assay. Fewer APC were required to activate PBMC when C1RA2 infected with rF-B7-1 were used as APC. Furthermore, using a PBMC/APC ratio of 1:1, similar detection was achieved using fewer PBMC. In addition, we demonstrated that the reactivity can be blocked by adding anti-B7-1 antibody. We performed the assay using APC on five normal healthy donors. All five donors showed substantial increases in PF to the Flu matrix peptide (Flu peptide) using the rF-B7-1-infected C1RA2 cells. Finally, we evaluated five cancer patients who received a carcinoembryonic antigen (CEA) vaccine-based therapy. Increases in CEA peptide precursors were noted in all five patients using the B7-1-infected APC. In conclusion, we have demonstrated the ability to enhance the sensitivity of the ELISPOT assay through infection of C1RA2 with rF-B7-1.

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 Drosophila Antennapedia 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.