Effects of Lactococcus lactis on Composition of Intestinal Microbiota: Role of Nisin

This study examined the ability of (i) pure nisin, (ii) nisin-producing Lactococcus lactis strain CHCC5826, and (iii) the non-nisin-producing L. lactis strain CHCH2862 to affect the composition of the intestinal microbiota of human flora-associated rats. The presence of both the nisin-producing and the non-nisin-producing L. lactis strains significantly increased the number of Bifidobacterium cells in fecal samples during the first 8 days but decreased the number of enterococci/streptococci in duodenum, ileum, cecum, and colon samples as detected by selective cultivation. No significant changes in the rat fecal microbiota were observed after dosage with nisin. Pearson cluster analysis of denaturing gradient gel electrophoresis profiles of the 16S rRNA genes present in the fecal microbial population revealed that the microbiota of animals dosed with either of the two L. lactis strains were different from that of control animals dosed with saline. However, profiles of the microbiota from animals dosed with nisin did not differ from the controls. The concentrations of nisin estimated by competitive enzyme-linked immunosorbent assay (ELISA) were approximately 10-fold higher in the small intestine and 200-fold higher in feces than the corresponding concentrations estimated by a biological assay. This indicates that nisin was degraded or inactivated in the gastrointestinal tract, since fragments of this bacteriocin are detected by ELISA while an intact molecule is needed to retain biological activity.     

Photochemical internalization (PCI) of HER2-targeted toxins: Synergy is dependent on the treatment sequence

Background

Photochemical internalization (PCI) is a modality for cytosolic release of drugs trapped in endocytic vesicles. The method is based upon photosensitizers localized in the membranes of endocytic vesicles which create membrane rupture upon light exposure by generating reactive oxygen species (ROS), predominantly singlet oxygen (¹O₂).

Methods

The human epidermal growth factor receptor 2 (HER2)-targeted immunotoxin (IT), trastuzumab–saporin, was evaluated in combination with PCI using TPCS_2a (Amphinex®), a new photosensitizer approved for clinical use.

Results

PCI synergistically enhanced the cytotoxicity of trastuzumab–saporin on trastuzumab-resistant HER2⁺ Zr-75-1 cells. The PCI effect was only observed when the IT was administered prior to the photochemical treatment (“light after” strategy), while administration of a non-targeted drug may equally well be performed after light exposure. Mechanistic studies showed reduced ligand-induced HER2 phosphorylation and receptor-mediated endocytosis after TPCS_2a-PDT. Photochemical disruption of the cytoplasmic domain of HER2 was found to be induced by ¹O₂ generated both by photosensitizer located in the endocytic vesicles and in the outer leaflet of the plasma membrane.

Conclusions

Administration of the HER2-targeted toxin prior to light exposure is a prerequisite for successful PCI-mediated delivery of HER2-targeted toxins.

General significance

PCI of HER2-targeted toxins is demonstrated as a highly effective treatment modality which may overcome trastuzumab resistance. The mechanistic studies of the lack of PCI effect of the “light first” procedure is of outermost importance when designing a clinical PCI treatment protocol for delivery of HER2-targeted therapies.     

Mapping of potent and specific binding motifs, GLOGEN and GVOGEA, for integrin α1β1 using collagen toolkits II and III

Integrins are well characterized cell surface receptors for extracellular matrix proteins. Mapping integrin-binding sites within the fibrillar collagens identified GFOGER as a high affinity site recognized by α2β1, but with lower affinity for α1β1. Here, to identify specific ligands for α1β1, we examined binding of the recombinant human α1 I domain, the rat pheochromocytoma cell line (PC12), and the rat glioma Rugli cell line to our collagen Toolkit II and III peptides using solid-phase and real-time label-free adhesion assays. We observed Mg(2+)-dependent binding of the α1 I domain to the peptides in the following rank order: III-7 (GLOGEN), II-28 (GFOGER), II-7 and II-8 (GLOGER), II-18 (GAOGER), III-4 (GROGER). PC12 cells showed a similar profile. Using antibody blockade, we confirmed that binding of PC12 cells to peptide III-7 was mediated by integrin α1β1. We also identified a new α1β1-binding activity within peptide II-27. The sequence GVOGEA bound weakly to PC12 cells and strongly to activated Rugli cells or to an activated α1 I domain, but not to the α2 I domain or to C2C12 cells expressing α2β1 or α11β1. Thus, GVOGEA is specific for α1β1. Although recognized by both α2β1 and α11β1, GLOGEN is a better ligand for α1β1 compared with GFOGER. Finally, using biosensor assays, we show that although GLOGEN is able to compete for the α1 I domain from collagen IV (IC(50) ～3 μm), GFOGER is much less potent (IC(50) ～90 μm), as shown previously. These data confirm the selectivity of GFOGER for α2β1 and establish GLOGEN as a high affinity site for α1β1.     

5-alpha-reductase type I (SRD5A1) is up-regulated in non-small cell lung cancer but does not impact proliferation, cell cycle distribution or apoptosis

Background  

Non-small cell lung cancer (NSCLC) is one of the most frequent malignancies and has a high mortality rate due to late detection and lack of efficient treatments. Identifying novel drug targets for this indication may open the way for new treatment strategies. Comparison of gene expression profiles of NSCLC and normal adjacent tissue (NAT) allowed to determine that 5-alpha-reductase type I (SRD5A1) was up-regulated in NSCLC compared to NAT. This raised the question whether SRD5A1 was involved in sustained proliferation and survival of NSCLC.     

Treatment of Visceral Leishmaniasis: Model-Based Analyses on the Spread of Antimony-Resistant L. donovani in Bihar,India

Background

Pentavalent antimonials have been the mainstay of antileishmanial therapy for decades, but increasing failure rates under antimonial treatment have challenged further use of these drugs in the Indian subcontinent. Experimental evidence has suggested that parasites which are resistant against antimonials have superior survival skills than sensitive ones even in the absence of antimonial treatment.

Methods and Findings

We use simulation studies based on a mathematical L. donovani transmission model to identify parameters which can explain why treatment failure rates under antimonial treatment increased up to 65% in Bihar between 1980 and 1997. Model analyses suggest that resistance to treatment alone cannot explain the observed treatment failure rates. We explore two hypotheses referring to an increased fitness of antimony-resistant parasites: the additional fitness is (i) disease-related, by causing more clinical cases (higher pathogenicity) or more severe disease (higher virulence), or (ii) is transmission-related, by increasing the transmissibility from sand flies to humans or vice versa.

Conclusions

Both hypotheses can potentially explain the Bihar observations. However, increased transmissibility as an explanation appears more plausible because it can occur in the background of asymptomatically transmitted infection whereas disease-related factors would most probably be observable. Irrespective of the cause of fitness, parasites with a higher fitness will finally replace sensitive parasites, even if antimonials are replaced by another drug.     

Virulence of a Klebsiella pneumoniae strain carrying the New Delhi metallo-beta-lactamase-1 (NDM-1)

The aim of the study was to compare and evaluate virulence in five strains of Klebsiella pneumoniae, including an isolate carrying New Delhi metallo-beta-lactamase-1 (NDM-1). In vivo virulence was assessed using a murine sepsis model and using the nematode Caenorhabditis elegans killing model, and in vitro virulence by assessing various virulence factors. The NDM-1 carrying K. pneumoniae isolate was the most virulent in the murine sepsis model but there was no clear cut correlation to in vitro virulence factors or killing in C. elegans. It is concluded that K. pneumoniae carrying NDM-1 have an intrinsic virulence potential, which in coexistence with its multiresistance could promote and partly explain its epidemiological success.     

Mammalian SEPT9 isoforms direct microtubule-dependent arrangements of septin core heteromers

Septin-family proteins assemble into rod-shaped heteromeric complexes that form higher-order arrangements at the cell cortex, where they serve apparently conserved functions as diffusion barriers and molecular scaffolds. There are 13 confirmed septin paralogues in mammals, which may be ubiquitous or tissue specific. Septin hetero-oligomerization appears homology subgroup directed, which in turn determines the subunit arrangement of six- to eight-subunit core heteromers. Here we address functional properties of human SEPT9, which, due to variable mRNA splicing, exists as multiple isoforms that differ between tissues. Myeloid K562 cells express three SEPT9 isoforms, all of which have an equal propensity to hetero-oligomerize with SEPT7-containing hexamers to generate octameric heteromers. However, due to limiting amounts of SEPT9, K562 cells contain both hexameric and octameric heteromers. To generate cell lines with controllable hexamer-to-octamer ratios and that express single SEPT9 isoforms, we developed a gene product replacement strategy. By this means we identified SEPT9 isoform–specific properties that either facilitate septin heteromer polymerization along microtubules or modulate the size range of submembranous septin disks—a prevalent septin structure in nonadhered cells. Our findings show that the SEPT9 expression level directs the hexamer-to-octamer ratio, and that the isoform composition and expression level together determine higher-order arrangements of septins.