A Phase I Trial of DFMO Targeting Polyamine Addiction in Patients with Relapsed/Refractory Neuroblastoma

BackgroundNeuroblastoma (NB) is the most common cancer in infancy and most frequent cause of death from extracranial solid tumors in children. Ornithine decarboxylase (ODC) expression is an independent indicator of poor prognosis in NB patients. This study investigated safety, response, pharmacokinetics, genetic and metabolic factors associated with ODC in a clinical trial of the ODC inhibitor difluoromethylornithine (DFMO) ± etoposide for patients with relapsed or refractory NB.ConclusionsDFMO doses of 500-1500mg/m2/day are safe and well tolerated in children with relapsed NB. Children with the minor T allele at rs2302616 of the ODC gene with relapsed or refractory NB had higher levels of urinary polyamine markers and responded better to therapy containing DFMO, compared to those with the major G allele at this locus. These findings suggest that this patient subset may display dependence on polyamines and be uniquely susceptible to therapies targeting this pathway.

Trial Registration

Clinicaltrials.gov NCT#01059071     

Milk chloride level as an indicator of bovine mastitis

The aim of this study was to determine an influence of udder infection on milk chloride level and on milk productivity of cows of black and white race. Bacteriological analysis was performed by bacterial isolation from milk collected in sterile conditions from single lobes of mammary gland. The study was aimed to detect the following bacteria: Streptococcus agalactiae, Streptococcus uberis, Streptococcus agalactiae, Staphylococcus aureus, Staphylococcus epidermidis, Actinomyces pyogenes, Enterobacteriaceae rods, Corynebacterium bovis, and Micrococcus sp. Milk chloride level was determined by burette method in 1250 milk samples collected from entire udder. Milk productivity was determined on the day of bacterial isolation. Statistical analysis of the results of the study on udder infection, milk chloride level, and milk productivity of mammary gland did reveal lack of a simple correlations between those parameters and in the indirect manner indicated an influence of inappropriate maintenance conditions of tested cows on the health condition of their udder. It seems possible that alkalosis and acidosis in cows, taken into consideration in discussion section, could constitute a factor influencing the frequency of mastitis incidence.     

Etanercept attenuates TNBS-induced experimental colitis: role of TNF-α expression

Crohn′s disease (CD) is associated with gut barrier dysfunction. Tumour necrosis factor-α (TNF-α) plays an important role into the pathogenesis of several inflammatory diseases because its expression is increased in inflamed mucosa of CD patients. Anti-TNF therapy improves significantly mucosal inflammation. Thus, this study aimed to evaluate the effect of Etanercept (ETC), a tumour necrosis factor alpha (TNF-α) antagonist on the 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced experimental colitis. A total of 18 Wistar rats were randomized into four groups, as follows: (1) Sham: sham induced-colitis; (2) TNBS: non-treated induced-colitis; (3) ETC control; (4) ETC-treated induced-colitis. Rats from group 4 presented significant improvement either of macroscopic or of histopathological damage in the distal colon. The gene expression of TNF-α mRNA, decreased significantly in this group compared to the TNBS non-treated group. The treatment with etanercept attenuated the colonic damages and reduced the inflammation caused by TNBS. Taken together, our results suggest that ETC attenuates intestinal colitis induced by TNBS in Wistar rats by TNF-α downregulation.     

Heterogeneity in White Blood Cells Has Potential to Confound DNA Methylation Measurements

Epigenetic studies are commonly conducted on DNA from tissue samples. However, tissues are ensembles of cells that may each have their own epigenetic profile, and therefore inter-individual cellular heterogeneity may compromise these studies. Here, we explore the potential for such confounding on DNA methylation measurement outcomes when using DNA from whole blood. DNA methylation was measured using pyrosequencing-based methodology in whole blood (n = 50–179) and in two white blood cell fractions (n = 20), isolated using density gradient centrifugation, in four CGIs (CpG Islands) located in genes HHEX (10 CpG sites assayed), KCNJ11 (8 CpGs), KCNQ1 (4 CpGs) and PM20D1 (7 CpGs). Cellular heterogeneity (variation in proportional white blood cell counts of neutrophils, lymphocytes, monocytes, eosinophils and basophils, counted by an automated cell counter) explained up to 40% (p<0.0001) of the inter-individual variation in whole blood DNA methylation levels in the HHEX CGI, but not a significant proportion of the variation in the other three CGIs tested. DNA methylation levels in the two cell fractions, polymorphonuclear and mononuclear cells, differed significantly in the HHEX CGI; specifically the average absolute difference ranged between 3.4–15.7 percentage points per CpG site. In the other three CGIs tested, methylation levels in the two fractions did not differ significantly, and/or the difference was more moderate. In the examined CGIs, methylation levels were highly correlated between cell fractions. In summary, our analysis detects region-specific differential DNA methylation between white blood cell subtypes, which can confound the outcome of whole blood DNA methylation measurements. Finally, by demonstrating the high correlation between methylation levels in cell fractions, our results suggest a possibility to use a proportional number of a single white blood cell type to correct for this confounding effect in analyses.     

Signaling signatures and functional properties of anti-human CD28 superagonistic antibodies

Superagonistic CD28 antibodies (CD28SAs) activate T lymphocytes without concomitant perturbation of the TCR/CD3-complex. In rodents these reagents induce the preferential expansion of regulatory T cells and can be used for the treatment of autoimmune diseases. Unexpectedly, the humanized CD28 superagonist TGN1412 caused severe and life threatening adverse effects during a recently conducted phase I clinical trail. The underlying molecular mechanisms are as yet unclear. We show that TGN1412 as well as the commercially available CD28 superagonist ANC28.1 induce a delayed but extremely sustained calcium response in human naïve and memory CD4⁺ T cells but not in cynomolgus T lymphocytes. The sustained Ca⁺⁺-signal was associated with the activation of multiple intracellular signaling pathways and together these events culminated in the rapid de novo synthesis of high amounts of pro-inflammatory cytokines, most notably IFN-γ and TNF-α. Importantly, sustained transmembranous calcium flux, activation of Src-kinases as well as activation of PI3K were found to be absolutely required for CD28SA-mediated production of IFN-γ and IL-2. Collectively, our data suggest a molecular basis for the severe side effects caused by TGN1412 and impinge upon the relevance of non-human primates as preclinical models for reagents that are supposed to modify the function of human T cells.     

Evaluation of live attenuated influenza a virus h6 vaccines in mice and ferrets

Avian influenza A virus A/teal/HK/W312/97 (H6N1) possesses seven gene segments that are highly homologous to those of highly pathogenic human influenza H5N1 viruses, suggesting that a W312-like H6N1 virus might have been involved in the generation of the A/HK/97 H5N1 viruses. The continuous circulation and reassortment of influenza H6 subtype viruses in birds highlight the need to develop an H6 vaccine to prevent potential influenza pandemics caused by the H6 viruses. Based on the serum antibody cross-reactivity data obtained from 14 different H6 viruses from Eurasian and North American lineages, A/duck/HK/182/77, A/teal/HK/W312/97, and A/mallard/Alberta/89/85 were selected to produce live attenuated H6 candidate vaccines. Each of the H6 vaccine strains is a 6:2 reassortant ca virus containing HA and NA gene segments from an H6 virus and the six internal gene segments from cold-adapted A/Ann Arbor/6/60 (AA ca), the master donor virus that is used to make live attenuated influenza virus FluMist (intranasal) vaccine. All three H6 vaccine candidates exhibited phenotypic properties of temperature sensitivity (ts), ca, and attenuation (att) conferred by the internal gene segments from AA ca. Intranasal administration of a single dose of the three H6 ca vaccine viruses induced neutralizing antibodies in mice and ferrets and fully protected mice and ferrets from homologous wild-type (wt) virus challenge. Among the three H6 vaccine candidates, the A/teal/HK/W312/97 ca virus provided the broadest cross-protection against challenge with three antigenically distinct H6 wt viruses. These data support the rationale for further evaluating the A/teal/HK/W312/97 ca vaccine in humans.     

Identification of compendial nonionic detergents for the replacement of Triton X-100 in bioprocessing

We have systematically investigated six compendial nonionic detergents as potential replacements for Triton ×-100 in bioprocessing applications. Use of compendial raw materials in cGMP bioprocessing is advantageous for a variety of reasons including material specifications developed to meet stringent pharmaceutical product quality requirements, regulatory familiarity and comfort, and availability from vendors experienced supplying the biopharmaceutical industry. We first examine material properties of the detergents themselves including melting point and viscosity. Process performance and product contact in real-world bioprocess applications are then investigated. Lastly, we test the detergents in virus inactivation (VI) experiments with recombinant proteins and adeno-associated virus. Two of the detergents tested, PEG 9 Lauryl Ether and PEG 6 Caprylic/Capric Glycerides, showed favorable properties that make them attractive for use as potential Triton X-100 replacements. Process performance testing indicated negligible impact of the detergents on product yield, purity, and activity compared to a control with no detergent. Importantly, both PEG 9 Lauryl Ether and PEG 6 Caprylic/Capric Glycerides demonstrated very fast VI kinetics with complete inactivation of XMuLV observed in less than 1 min at a target 1% detergent concentration. Potential advantages and disadvantages of both candidate detergents for use in cGMP bioprocessing are summarized and discussed.     

Effect of COX-2 inhibitor after TNBS-induced colitis in wistar rats

Inflammatory bowel disease (IBD) is a common chronic gastrointestinal disorder characterized by alternating periods of remission and active intestinal inflammation. Some studies suggest that antiinflammatory drugs are a promising alternative for treatment of the disease. Thus, this study aimed to evaluate the effect of lumiracoxib, a selective-cyclooxygenase-2 (COX-2) inhibitor, on 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced experimental colitis. Wistar rats (n = 25) were randomized into four groups, as follows: Group (1) Sham group: sham induced-colitis rats; Group (2) TNBS group: nontreated induced-colitis rats; Group (3) Lumiracoxib control group; and Group (4) Lumiracoxib-treated induced-colitis rats. Our results showed that rats from groups 2 and 4 presented similar histopathological damage and macroscopic injury in the distal colon as depicted by significant statistically differences (P < 0.01; P < 0.05) compared to the other two groups. Weak expression of COX-2 mRNA was detected in normal colon cells, while higher levels of COX-2 mRNA were detected in group 2 and group 4. Therapy with lumiracoxib reduced COX-2 expression by 20–30%, but it was still higher and statistically significant compared to data obtained from the lumiracoxib control group. Treatment with the selective COX-2 inhibitor lumiracoxib did not reduce inflammation-associated colonic injury in TNBS-induced experimental colitis. Thus, the use of COX-2 inhibitors for treating IBD should be considered with caution and warrants further experimental investigation to elucidate their applicability.