Identification and Characterization of Microcin S, a New Antibacterial Peptide Produced by Probiotic Escherichia coli G3/10

Escherichia coli G3/10 is a component of the probiotic drug Symbioflor 2. In an in vitro assay with human intestinal epithelial cells, E. coli G3/10 is capable of suppressing adherence of enteropathogenic E. coli E2348/69. In this study, we demonstrate that a completely novel class II microcin, produced by probiotic E. coli G3/10, is responsible for this behavior. We named this antibacterial peptide microcin S (MccS). Microcin S is coded on a 50.6 kb megaplasmid of E. coli G3/10, which we have completely sequenced and annotated. The microcin S operon is about 4.7 kb in size and is comprised of four genes. Subcloning of the genes and gene fragments followed by gene expression experiments enabled us to functionally characterize all members of this operon, and to clearly identify the nucleotide sequences encoding the microcin itself (mcsS), its transport apparatus and the gene mcsI conferring self immunity against microcin S. Overexpression of cloned mcsI antagonizes MccS activity, thus protecting indicator strain E. coli E2348/69 in the in vitro adherence assay. Moreover, growth of E. coli transformed with a plasmid containing mcsS under control of an araC PBAD activator-promoter is inhibited upon mcsS induction. Our data provide further mechanistic insight into the probiotic behavior of E. coli G3/10

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

IRSp53 links the enterohemorrhagic E. coli effectors Tir and EspFU for actin pedestal formation

Actin pedestal formation by pathogenic E. coli requires signaling by the bacterial intimin receptor Tir, which induces host cell actin polymerization mediated by N-WASP and the Arp2/3 complex. Whereas canonical enteropathogenic E. coli (EPEC) recruit these actin regulators through tyrosine kinase signaling cascades, enterohemorrhagic E. coli (EHEC) O157:H7 employ the bacterial effector EspF(U) (TccP), a potent N-WASP activator. Here, we show that IRSp53 family members, key regulators of membrane and actin dynamics, directly interact with both Tir and EspF(U). IRSp53 colocalizes with EspF(U) and N-WASP in actin pedestals. In addition, targeting of IRSp53 is independent of EspF(U) and N-WASP but requires Tir residues 454-463, previously shown to be essential for EspF(U)-dependent actin assembly. Genetic and functional loss of IRSp53 abrogates actin assembly mediated by EHEC. Collectively, these data indentify IRSp53 family proteins as the missing host cell factors linking bacterial Tir and EspF(U) in EHEC pedestal formation

Metastatic status of sentinel lymph nodes in melanoma determined noninvasively with multispectral optoacoustic imaging.

Sentinel lymph node (SLN) excision is included in various cancer guidelines to identify microscopic metastatic disease. Although effective, SLN excision is an invasive procedure requiring radioactive tracing. Novel imaging approaches assessing SLN metastatic status could improve or replace conventional lymph node excision protocols. In our first-in-human study, we used noninvasive multispectral optoacoustic tomography (MSOT) to image SLNs ex vivo and in vivo in patients with melanoma, to determine metastatic status. MSOT significantly improved the tumor metastasis detection rate in excised SLN (506 SLNs from 214 melanoma patients) compared with the conventional EORTC (European Organisation for Research and Treatment of Cancer) Melanoma Group protocol (22.9% versus 14.2%). MSOT combined with the near-infrared fluorophore indocyanine green reliably visualized SLNs in vivo in 20 patients, up to 5-cm penetration and with 100% concordance with (99m)Tc-marked SLN lymphoscintigraphy. MSOT identified cancer-free SLNs in vivo and ex vivo without a single false negative (189 total lymph nodes), with 100% sensitivity and 48 to 62% specificity. Our findings indicate that a noninvasive, nonradioactive MSOT-based approach can identify and determine SLN status and confidently rule out the presence of metastasis. The study further demonstrates that optoacoustic imaging strategies can improve the identification of SLN metastasis as an alternative to current invasive SLN excision protocols

Risk-adapted postremission therapy in acute myeloid leukemia: results of the German multicenter AML HD93 treatment trial

The objective of the AML HD93 treatment trial was to evaluate the outcome in young adults with acute myeloid leukemia (AML) after postremission therapy was stratified according to cytogenetically defined risk. The rationales for the study design were based (i) on previous favorable results with high-dose cytarabine in AML with t(8;21), inv/t(16q22) and in AML with normal karyotype, and ii) on encouraging results obtained in several phase II trials using autologous stem cell transplantation (SCT). Between July 1993 and January 1998, 223 eligible patients, 16–60 years of age with newly diagnosed AML other than French–American–British type M3/M3v, were entered into the trial. Risk groups were defined as follows: low risk: t(8;21) or inv/t(16q22); intermediate risk: normal karyotype; high risk: all other chromosomal abnormalities. Following intensive double induction therapy with idarubicin, cytarabine and etoposide, all patients in complete remission (CR) received a first consolidation therapy with high-dose cytarabine and mitoxantrone (HAM). A second consolidation therapy was stratified according to the risk group: low risk: HAM; intermediate risk: related allogeneic SCT or sequential HAM; high risk: related allogeneic or autologous SCT. Double induction therapy resulted in a high CR rate of 74.5%, and 90% of the responding patients were eligible for consolidation therapy. Survival for all 223 trial entrants was 40%, and for the 166 patients who entered CR, disease-free (DFS) and overall survival were 40 and 51% after 5 years, respectively. Within the low-, intermediate- and high-risk groups, DFS and survival after 5 years were 62.5 and 87, 40 and 49 and 17 and 26% respectively, without advantage for allogeneic transplantation in the intermediate- and high-risk groups. Postremission therapy-related mortality was 0, 7 and 14%, respectively. This study demonstrates the feasibility of cytogenetically defined risk-adapted consolidation therapy. The overall trial results are at least equivalent to those of published trials supporting the risk-adapted treatment strategy