Efficacy of preconditioning with intracoronary diltiazem in preventing laser-induced spasm

Laser energy produces a multitude of effects, resulting both in therapeutic tissue ablation and complications such as laser-induced spasm (LIS). LIS can occur during lasing itself or during subsequent adjunctive angioplasty. Intracoronary diltiazem (ICD) can partially reverse LIS after it occurs. To determine whether pre-treatment with ICD might prevent LIS during laser interventions, 3 groups of 50 lesions each were studied. Group 1 served as controls receiving no ICD during the procedure. Group 2 received 2.5 mg ICD before lasing. Group 3 received ICD before lasing and then a second infusion of 2.5 mg ICD after lasing but before adjunctive therapy. There were no differences in clinical characteristics of the 3 groups. Over 75% of lesions in each group were complex (B2 or C) lesions, and average lesion length was 15 mm in all 3 groups. Procedural success was > or = 94% in all groups. There was no significant difference among groups in pre-procedure artery stenosis, post-procedure stenosis, laser power used or number of laser pulses delivered. Pretreatment with ICD produced vasodilation of the minimum lumen diameter from 0.86 +/- 0.1 to 1.0 +/- 0.1 mm (p < 0.01) and was well tolerated. Control patients exhibited a 12% incidence of LIS. Group 2 had an 80% reduction of LIS during lasing (p < 0.01) but had increased LIS during adjunctive therapy with the same 12% incidence of LIS overall. Group 3 had only a 2% incidence of LIS (p < 0.01). We concluded that pretreatment with ICD significantly reduces LIS. Multiple infusions of ICD are necessary to sustain this protective effect.     

Thermal Effects In Vivo from Holmium: YAG Lasing in the Intracoronary Setting

While the thermal effect of laser energy does ablate atheromatous plaque, thermal injury to adjacent tissue produces high rates of arterial thrombosis and spasm. Holmium:YAG lasers use a pulsed laser source to maximize photoblative effects while minimizing thermal effects. These lasers have been utilized clinically to ablate thousands of complex coronary lesions with low rates of spasm and thrombosis, suggesting that little or no thermal injury occurs with these devices. However, we have been able to detect thermal injury in patients angioscopically in coronary arteries after holmium:YAG lasing. Here we report the use of directional coronary atherectomy (DCA) to òbiopsyó arteries in patients following holmium:YAG laser treatment, allowing direct histologic examination of lased tissue. Thirty such lased DCA samples were matched for patient age, gender, target vessel, and lesion characteristics with thirty control DCA samples obtained from patients undergoing DCA without prior lasing. Blinded pathologic examination correctly identified 27/30 control samples but only 18/30 lased samples. Subsequent unblinded analysis, sometimes with recutting and restaining of tissue blocks, resulted in the detection of thermal effects in 27/30 lased samples. The thermal effects seen included edge disruption, charring, coagulation necrosis, and most commonly, vacuolization. We conclude that holmium:YAG lasing does produce detectable thermal effects in tissue in most patients. These effects can be quite subtle or can be extensive, but do not predict poor patient outcome.     

Biofilm Formation Enhances Fomite Survival of Streptococcus pneumoniae and Streptococcus pyogenes

Both Streptococcus pyogenes and Streptococcus pneumoniae are widely thought to rapidly die outside the human host, losing infectivity following desiccation in the environment. However, to date, all literature investigating the infectivity of desiccated streptococci has used broth-grown, planktonic populations. In this study, we examined the impact of biofilm formation on environmental survival of clinical and laboratory isolates of S. pyogenes and S. pneumoniae as both organisms are thought to colonize the human host as biofilms. Results clearly demonstrate that while planktonic cells that are desiccated rapidly lose viability both on hands and abiotic surfaces, such as plastic, biofilm bacteria remain viable over extended periods of time outside the host and remain infectious in a murine colonization model. To explore the level and extent of streptococcal fomite contamination that children might be exposed to naturally, direct bacteriologic cultures of items in a day care center were conducted, which demonstrated high levels of viable streptococci of both species. These findings raise the possibility that streptococci may survive in the environment and be transferred from person to person via fomites contaminated with oropharyngeal secretions containing biofilm streptococci.     

Novel Strategy To Protect against Influenza Virus-Induced Pneumococcal Disease without Interfering with Commensal Colonization

Streptococcus pneumoniae commonly inhabits the nasopharynx as a member of the commensal biofilm. Infection with respiratory viruses, such as influenza A virus, induces commensal S. pneumoniae to disseminate beyond the nasopharynx and to elicit severe infections of the middle ears, lungs, and blood that are associated with high rates of morbidity and mortality. Current preventive strategies, including the polysaccharide conjugate vaccines, aim to eliminate asymptomatic carriage with vaccine-type pneumococci. However, this has resulted in serotype replacement with, so far, less fit pneumococcal strains, which has changed the nasopharyngeal flora, opening the niche for entry of other virulent pathogens (e.g., Streptococcus pyogenes, Staphylococcus aureus, and potentially Haemophilus influenzae). The long-term effects of these changes are unknown. Here, we present an attractive, alternative preventive approach where we subvert virus-induced pneumococcal disease without interfering with commensal colonization, thus specifically targeting disease-causing organisms. In that regard, pneumococcal surface protein A (PspA), a major surface protein of pneumococci, is a promising vaccine target. Intradermal (i.d.) immunization of mice with recombinant PspA in combination with LT-IIb(T13I), a novel i.d. adjuvant of the type II heat-labile enterotoxin family, elicited strong systemic PspA-specific IgG responses without inducing mucosal anti-PspA IgA responses. This response protected mice from otitis media, pneumonia, and septicemia and averted the cytokine storm associated with septic infection but had no effect on asymptomatic colonization. Our results firmly demonstrated that this immunization strategy against virally induced pneumococcal disease can be conferred without disturbing the desirable preexisting commensal colonization of the nasopharynx.     

Hybrid biosynthetic gene therapy vector development and dual engineering capacity

Genetic vaccines offer a treatment opportunity based upon successful gene delivery to specific immune cell modulators. Driving the process is the vector chosen for gene cargo packaging and subsequent delivery to antigen-presenting cells (APCs) capable of triggering an immune cascade. As such, the delivery process must successfully navigate a series of requirements and obstacles associated with the chosen vector and target cell. In this work, we present the development and assessment of a hybrid gene delivery vector containing biological and biomaterial components. Each component was chosen to design and engineer gene delivery separately in a complimentary and fundamentally distinct fashion. A bacterial (Escherichia coli) inner core and a biomaterial [poly(beta-amino ester)]-coated outer surface allowed the simultaneous application of molecular biology and polymer chemistry to address barriers associated with APC gene delivery, which include cellular uptake and internalization, phagosomal escape, and intracellular cargo concentration. The approach combined and synergized normally disparate vector properties and tools, resulting in increased in vitro gene delivery beyond individual vector components or commercially available transfection agents. Furthermore, the hybrid device demonstrated a strong, efficient, and safe in vivo humoral immune response compared with traditional forms of antigen delivery. In summary, the flexibility, diversity, and potential of the hybrid design were developed and featured in this work as a platform for multivariate engineering at the vector and cellular scales for new applications in gene delivery immunotherapy.Vaccines are often regarded as the most cost-effective advancement of modern medicine (1). Gene therapy has emerged as an alternative approach to the classical vaccine paradigm by using the delivery of specific therapeutic cargo for the purpose of altering gene expression (2, 3). In this context, the delivery of nucleic acids is accomplished by a variety of biological or synthetic vectors. Through specific engineering tools, each vector is designed to influence antigen-presenting cell (APC) gene expression levels to modulate an immune response toward the generation of antigen reactivity and memory. Vector-mediated gene delivery efficacy is strongly correlated with overcoming APC barriers, such as cellular uptake, phagosomal/lysosomal escape, nucleic acid unpackaging, nuclear translocation (excluding RNA-based therapeutics), and sustained gene expression (4). To be clinically relevant, vectors must also exert minimal to no cytotoxicity.The ability to deliver nucleic acids and proteins to mammalian cells has been demonstrated independently by both biomaterial-based nanoparticles and bacteria. Of the biomaterial vectors, cationic polymers (CPs) generally feature simple and scalable synthetic schemes capable of being tailored to specific applications (5). Specifically, poly(beta-amino esters) (PBAEs) are a class of CPs recognized for their facile synthesis and elevated levels of gene delivery (compared with commercially available reagents) (6–10). CPs also typically demonstrate or can be designed to exhibit low cytotoxicity. Bacterial vectors provide an orthogonal set of engineering tools to influence gene delivery. In particular, Escherichia coli, a rod-shaped, Gram-negative bacterium that is ∼2 μm in length and 0.5 μm in diameter, has been identified as an ideal gene carrier due to simple and scalable culture protocols, a rapid growth rate, and a wealth of established molecular biology manipulation techniques (11). Nonpathogenic strains of E. coli have exhibited minimal toxicity and immunogenicity (12–15), further supported by in vivo and in vitro delivery of plasmid (12–14, 16, 17) and larger DNA constructs (18, 19), shRNA (20), and immunogenic peptides (17, 21, 22).An alternative option to address the requirements of gene delivery is to combine the individual advantages of vectors, such as E. coli and CPs, and to leverage dual vector-specific toolsets to overcome APC gene delivery barriers. For example, E. coli, which inherently maintains plasmid DNA (pDNA) and natively promotes phagocytic uptake by APCs, can be engineered to escape the lysosome by expression of a pore-forming listeriolysin O (LLO) protein (16, 17, 21–24). Conversely, CPs facilitate uptake by generalized endocytosis mechanisms and instigate lysosomal escape by the “proton sponge effect” (4, 5). Combined, the innate and malleable features of these normally disparate vectors offer the potential to complement and enhance individual capabilities while simultaneously minimizing limitations.Thus, we report the generation of a hybrid biosynthetic vector that combines the capabilities of both bacterial and CP components for targeted gene delivery to APCs (SI Appendix, Fig. S1). Ninety-one structurally diverse PBAEs were synthesized and screened after surface attachment to LLO-producing E. coli for in vitro gene delivery to murine macrophages. After multiple rounds of screening, an optimal PBAE and bacterial strain were identified that, when combined together, possessed gene delivery potency greater than either vector in isolation. To demonstrate the engineering potential of the hybrid device, each individual vector was modified using vector-associated tools. Specifically, the lethal lysis gene E (LyE) from bacteriophage ΦX174 was incorporated into bacterial strains, which resulted in significant improvements to APC gene delivery and cytotoxicity. Similarly, mannose was attached to the terminal end of the optimal PBAE before hybrid device formation, resulting in increased vector uptake and gene delivery. Finally, the hybrid vector was successfully tested in the context of in vivo humoral immune response. The results and combined features of this new vector offer a promising platform for future applications in genetic vaccination as a byproduct of the duality in vector composition and engineering capability.     

Phase 1 study of the novel vascular disrupting agent plinabulin (NPI-2358) and docetaxel

Background Plinabulin (NPI-2358) is a vascular disrupting agent (VDA) that destabilizes tumor vascular endothelial cell architecture resulting in selective collapse of established tumor vasculature producing anti-tumor activity alone or in combination with cytotoxic agents. The objective of this study was to assess the recommended Phase 2 dose (RP2D) of plinabulin combined with docetaxel. Patients and Methods Patients received 75 mg/m² docetaxel on day 1 and plinabulin on days 1 and 8 intravenously in 21 day cycles. Plinabulin was escalated from the biologically effective dose (BED) of 13.5 mg/m² to the standard single agent dose of 30 mg/m² using a “3+3” design. Results Thirteen patients were enrolled. Adverse events were consistent with those of both agents alone. Fatigue, pain, nausea, diarrhea and vomiting were the most common events. One dose limiting toxicity of nausea, vomiting, dehydration and neutropenia occurred. The RP2D was 30 mg/m² of plinabulin with 75 mg/m² docetaxel. Pharmacokinetics did not indicate drug-drug interactions. Of the 8 patients with NSCLC evaluable for response, 2 achieved a partial response and 4 demonstrated lesser decreases in tumor measurements. Conclusions The combination of full doses of plinabulin and docetaxel is tolerable. With encouraging antitumor activity, this supported further development of this combination.     

Clinical effects and utility of intracoronary diltiazem

Coronary artery spasm is a known complication of coronary interventions, for which intracoronary nitroglycerin (ICN) is the treatment of choice. Some forms of intense spasm are resistant to ICN. Calcium channel antagonists are also known to be effective for coronary artery spasm, including nitroglycerin-resistant spasm. Here we describe a protocol for the clinical use of intracoronary diltiazem (ICD). By this protocol, ICD can be safely given without disturbing the clinical status of patients. ICD (2.5 mg) given slowly over 1 minute produced no vasodilitation of normal vessel segments but did produce significant dilatation of stenotic segments above and beyond the effects of nitrates. Mean minimum lumen diameter increased 18%, from 0.89 ± 0.06 mm to 1.06 ± 0.07 mm (mean ± SEM, P < 0.001). ICD produced clinically Insignificant changes in systolic blood pressure, diastolic blood pressure, heart rate, and PR, QRS, and QT intervals. This protocol has been employed to safely use ICD to relieve both nitroglycerin-resistant epicardial artery spasm and nitroglycerin-resistant distal microvascular spasm (the no-reflow phenomenon). © 1995 Wiley-Liss, Inc.