Michigan Manufacturing Technology Center

Ecological interactions between bacteria and nematodes in many environments provide a basis for the prediction that diverse bacteria produce anti-nematode compounds. The discovery of microbial secondary metabolites with broad-spectrum nematostatic or nematicidal properties can be hastened by drug screening approaches that include several nematode species and phenotypes. We cultured a collection of 22 soil-derived bacterial isolates that carry in their genomes putative pathways for production of unknown secondary metabolites. Isolates were cultured in various media to enhance natural product diversity and yield, and we evaluated culture filtrates for activity against two evolutionarily distinct nematode species: Clade V free-living nematode Caenorhabditis elegans and Clade III mammalian parasitic nematodes in the genus Brugia. Partitioned extracts from Pseudomonas sp. strain TE4607 stunted C. elegans development and caused motility defects in both blood-circulating larval and adult stages of Brugia. The primary active compound was identified as labradorin 1, an indole with known antibacterial and anticancer properties that had not been previously described as affecting nematodes. Notably, filtrates of Pseudomonas sp. TE4607 cultures attracted free-living nematodes in sensory assays, adding to evidence that certain Pseudomonas species modulate the behavior of free-living nematodes. These findings underscore the need to further explore the link between nematode sensory responses and whole-organism effects of microbial metabolites, with potential applications in anthelmintic discovery.

Introduction/Background: Current knowledge of a Stage 1 shunted single ventricle (SSV) physiology is predominantly based on clinical data. Development of a reliable and reproducible SSV animal model can facilitate pre-clinical research on strategies to improve outcomes. Research Questions/ Hypothesis: We hypothesize that a functional SSV animal model can be created by percutaneously creating a large atrial communication, placing a systemic to pulmonary artery shunt and excluding the right ventricle (RV) from the circulation. Goals/Aims: We aimed to create a neonatal porcine SSV model without using cardiopulmonary bypass. Methods/Approach: Neonatal pigs (n=8; weight 6.1±2.6 kgs) were used. A median sternotomy was performed. A 6-mm ring enforced PTFE graft was anastomosed end-side to the main pulmonary artery (MPA), end-end to the innominate artery and occluded with a vascular clamp. Under epicardial echocardiography guidance, the atrial septum was percutaneously perforated using electrocautery. (Figure 1A). Electrocautery was applied using a coaxial system of 4 Fr JR 3 catheter and a 0.014-inch coronary wire with its denuded tip at the atrial septum. Balloon atrial septostomy was performed using B. Braun Z-5 13.5 mm and/or the Edwards Miller septostomy balloon. To prevent RV distension, a per-ventricular Tricuspid valve (TV) avulsion was done using a Rumel tourniquet guide as a snare placed through a 7 Fr per-ventricular sheath (Figure 1B) . The systemic to pulmonary artery shunt (SPS) was unclamped, and antegrade pulmonary blood (APF) was interrupted by the occlusion of the MPA. Inotropic support was initiated. Donor pig blood was transfused for volume replacement. Hemodynamic and laboratory data were collected. The circulation was maintained for a minimum duration of 1 hour. Results/Data: The SSV was successfully created in 6 out of 8 animals. The SSV circulation had a mean arterial pressure of 37.5±12 mmHg and a common atrial pressure of 12±6 mmHg. The mean aortic flow was 0.76±0.34 L/min, and the systemic to pulmonary artery shunt flow was 0.36±0.23 L/min. The mean Qp/Qs ratio was 1.27±1.14. The systemic saturation was 82±16%, and the mixed venous saturation was 58±17%. Conclusions: A porcine animal model of an SSV circulation can be created without using cardiopulmonary bypass by performing a percutaneous atrial septostomy, per ventricular TV avulsion, placing a SPS and interrupting APF. This model effectively replicates the stage 1 SSV physiology.

To evaluate safety- and seizure-related outcomes with repeated intermittent use of a novel formulation of midazolam administered as a single-dose nasal spray (MDZ-NS) in the outpatient treatment of patients experiencing seizure clusters (SCs). In this open-label extension trial (ClinicalTrials.gov NCT01529034), patients aged ≥12years and on a stable regimen of antiepileptic drugs who completed the original phase III, randomized controlled trial were enrolled. Caregivers administered MDZ-NS 5mg when patients experienced SCs; a second dose could be given if seizures did not terminate within 10minutes or recurred within 10minutes-6hours. Patients were monitored for treatment-emergent adverse events (TEAEs) throughout, and the main seizure-related outcome was treatment success, defined as seizure termination within 10minutes and no recurrence 10minutes-6hours after drug administration. Of 175 patients enrolled, 161 (92.0%) received ≥1 MDZ-NS dose, for a total of 1998 SC episodes. Median time spent by patients in the trial was 16.8months (range = 1-55.7months). TEAEs were experienced by 40.4% of patients within 2days of drug administration and 57.1% overall. TEAEs reported by most patients (within 2days and overall) were nasal discomfort (12.4%) and somnolence (9.3%). One patient each discontinued due to treatment-related nasal discomfort and somnolence. There were no patients with treatment-related respiratory depression, and none with TEAEs indicative of drug abuse or dependence. Treatment success criteria were met in 55% (1108/1998) of SC episodes after administration of a single 5-mg dose and in 80.2% (617/769) with the second dose. Treatment success was consistent over treated episode number. Repeated, intermittent, acute treatment of patients experiencing SCs with MDZ-NS in the outpatient setting was well tolerated over an extended period, with maintenance of efficacy suggesting lack of development of tolerance.

DAA, direct-acting antiviral agents; HCV, hepatitis C virus; ISDR, interferon-alpha sensitivity determining region. Hepatitis C virus (HCV) is a major cause of liver disease and mortality affecting 170 million people worldwide.1 Chronic infection with HCV can lead to liver fibrosis, cirrhosis, and hepatocellular carcinoma2 and is the most common reason for liver transplantation in the United States.3 HCV is an enveloped single-strand RNA virus that belongs to the flaviviridae family. It has a 9.6-kb RNA genome encoding a single polyprotein of 3,010 amino acids. This polyprotein is composed of structural and nonstructural proteins. HCV entry into hepatocytes is mediated by the structural proteins, including glycoproteins E1 and E2, which interact with cellular receptors. Once HCV enters the host cell, replication of its genome is orchestrated by the nonstructural proteins, including proteases (NS2-3, NS3-4A), helicases (NS3), polymerases (NS5B), and NS5A, a protein with no known enzymatic activity.4-6 NS5A is essential to the replication machinery of the virus and critical in the assembly of infectious viral particles. However, its specific role remains unclear.6 A number of properties of this viral protein have been described. NS5A interacts with a variety of host proteins, including some which modulate host cellular signaling pathways.7, 8 Studies have demonstrated that NS5A interacts with p53 and p21, affecting cell cycle control.7 More recently, NS5A was found to interact with proteins related to focal adhesions, gap-junction, and host signaling pathways.7, 8 NS5A also interacts with other nonstructural viral proteins. During HCV viral replication, NS5A interacts with the RNA- dependent RNA-polymerase NS5B, which is essential to maintain HCV replication in cell culture. Through its domain 1 NS5A has RNA-binding capacity, a property that can be critical for RNA replication.9 In addition to being involved in HCV replication, NS5A is involved in the assembly of the virus by forming stable complexes with NS2, the main protein involved in HCV assembly.10 NS5A attracted attention due to its interferon-alpha sensitivity determining region (ISDR), which can confer resistance to interferon treatment. Moreover, NS5A has an effect on interferon activity by up-regulating interleukin (IL)-8, which has been reported to attenuate the antiviral properties of interferon.11 These observations suggest that NS5A plays a critical role in many aspects of the life cycle of HCV and is therefore an attractive target for antiviral therapy (Fig. 1). The potential effects of targeting NS5A. Increase in interferon sensitivity by direct effect of blocking NS5A ISDR or by down-regulation of IL-8. Alteration of NS5A interaction with NS5B and NS2 affecting HCV replication and viral assembly. Downregulation of host signaling pathways such as those involving p21, p53, and Cdk2. Until recently, the standard treatment for HCV infection has been pegylated interferon-alpha and ribavirin. These drugs have significant and often serious side effects. Moreover, in patients with genotype 1 (the most prevalent HCV genotype), sustained viral clearance is achieved in less than 50% of cases.12 In recent years, several viral inhibitors targeting nonstructural proteins of HCV have been developed (direct-acting antiviral agents [DAA]). Most of the research has been focused on inhibitors of the NS3-NS4A and NS5B proteins, mainly because of a better understanding of the role of these proteins. A variety of NS3-NS4A and NS5B inhibitors are currently in various stages of development in preclinical and clinical studies.13 Telaprevir and boceprevir, which are both inhibitors of the NS3-NS4A serine protease, have recently been approved for use in combination with pegylated interferon and ribavirin in patients with genotype 1 HCV infection.14, 15 Treatment with pegylated interferon, ribavirin, and either of these protease inhibitors results in improved rates of viral eradication (both in previously untreated and in those who have failed prior treatment). However, the recommended treatment regimens are complex and are associated with significant side effects from interferon and ribavirin, as well as adverse events from the protease inhibitors. Furthermore, these protease inhibitors need to be administered every 8 hours with food and there is also a very real problem of interaction with other drugs. In addition, the rapid replication of HCV makes the appearance of viral resistance a potential problem, requiring the need for combination therapies with drugs directed against a variety of viral targets BMS-790052 is an agent with activity against the NS5A protein. This agent was identified in 2010 using a screening strategy that was designed to identify new agents that inhibit HCV replication with no effect on NS3-4A or N5B (which already had known inhibitors). The compound was found to inhibit all HCV genotypes at low concentrations. Through coprecipitation it was demonstrated that BMS-790052 interacted with NS5A.16 BMS-790052 inhibits a number of aspects of viral replication17, 18 and has been shown in the replicon system to be additive or synergistic with other DAAs.16 Early clinical testing of BMS-790052 showed that a single dose of the NS5A inhibitor was able to markedly decrease HCV viral levels in patients with chronic HCV infection.16 Nettles et al.19 showed a marked decline in HCV RNA after administration of BMS-790052 in a double-blind, placebo-controlled, multiple ascending dose study in 30 patients infected with HCV genotype 1. The study demonstrated a dose-dependent effect on HCV RNA. There were no significant side effects observed in this study. Pharmacokinetic studies showed that this drug is effective in a once-a-day dose. Most patients had viral rebound by day 7. This viral rebound was associated with the emergence of mutations associated with viral resistance, suggesting that treatment with this drug alone will be unlikely to result in a sustained suppression of HCV RNA. Preliminary studies with BMS-790052 combined with another antiviral, with and without pegylated interferon and ribavirin, have been shown to successfully lower HCV levels for a longer period of time.20 BMS-790052 is a new, promising agent in the treatment of chronic HCV infection. It is the first drug with activity against NS5A. Further studies with longer courses of therapy of BMS-790052 in combination with other DAAs and pegylated interferon and ribavirin are required to establish the long-term efficacy and safety of BMS-790052. BMS 790052 is one of over 50 new DAAs that are in different stages of development from preclinical to phase 3 clinical trials. These agents act on one of three targets on the HCV. They inhibit NS3-NS4A protease, NS5B polymerase (nucleoside or nonnucleoside), or the NS5A protein. Many of these agents result in viral suppression in over 80% of cases. These classes of agents exhibit different properties, with respect to development of resistance mutations, genotypic coverage, potency, and toxicity (Table 1). Most of these molecules appear to be well tolerated and exhibit pharmacokinetics that allow for daily or twice-daily administration. The pattern of resistance mutations induced by these agents varies among the different classes of agents. This suggests that a combination of agents may be effective in treating hepatitis C and minimizing the development of resistance mutations. Studies are under way with single DAAs and combinations of these agents with interferon and ribavirin. Preliminary results appear promising. It has now been shown that treatment of hepatitis C with a combination of DAAs (including BMS 790052) alone, without interferon, may result in sustained viral clearance.20 There are a number of ongoing studies utilizing combinations of DAAs without interferon that are showing promise. It still remains to be seen whether ribavirin will be needed as part of these regimens to produce a sustained viral response. Oral, interferon-free treatment of hepatitis C appears a possibility. It will require a combination of agents aimed at different targets on the HCV. We are approaching the dawn of a new era in the treatment of HCV with the possibility of oral, effective, and well-tolerated combination therapy. These regimens have the possibility of eradicating HCV in the majority of patients. Much work is still needed to define the most clinically effective and least toxic regimens.

Role of Statistical Methods Implementing Statistical Methods Organization of the Book How to Use this Book

Cancer BiotherapyVol. 10, No. 4 Whole Body Hyperthermia as a Potential Therapeutic OptionL.M. Shecterle and J.A. St. CyrL.M. ShecterleSearch for more papers by this author and J.A. St. CyrSearch for more papers by this authorPublished Online:17 Jul 2009https://doi.org/10.1089/cbr.1995.10.253AboutSectionsPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail FiguresReferencesRelatedDetailsCited byCell-Based Targeting of Anticancer Nanomaterials to Tumors30 January 2014T-cell receptor repertoires of tumor-infiltrating lymphocytes after hyperthermia using functionalized magnetite nanoparticlesNanomedicine, Vol. 8, No. 6Whole-body hyperthermia: a review of theory, design and application30 June 2016 | Perfusion, Vol. 17, No. 4Enhanced cytotoxicity and suppression of glucose transport rate by combined treatment of recombinant human tumour necrosis factor-α and hyperthermia on L929 cellsLife Sciences, Vol. 65, No. 8Regulation of Leukocyte-Endothelial Cell Interactions in Tumor ImmunityExtracorporeal whole body hyperthermia treatment of HIV patients, a feasibility study9 July 2009 | International Journal of Hyperthermia, Vol. 13, No. 6 Volume 10Issue 4Jan 1995 To cite this article:L.M. Shecterle and J.A. St. Cyr.Whole Body Hyperthermia as a Potential Therapeutic Option.Cancer Biotherapy.Jan 1995.253-256.http://doi.org/10.1089/cbr.1995.10.253Published in Volume: 10 Issue 4: July 17, 2009PDF download