Erythrocyte and Porcine Intestinal Glycosphingolipids Recognized by F4 Fimbriae of Enterotoxigenic Escherichia coli

Enterotoxigenic F4-fimbriated Escherichia coli is associated with diarrheal disease in neonatal and postweaning pigs. The F4 fimbriae mediate attachment of the bacteria to the pig intestinal epithelium, enabling an efficient delivery of diarrhea-inducing enterotoxins to the target epithelial cells. There are three variants of F4 fimbriae designated F4ab, F4ac and F4ad, respectively, having different antigenic and adhesive properties. In the present study, the binding of isolated F4ab, F4ac and F4ad fimbriae, and F4ab/ac/ad-fimbriated E. coli, to glycosphingolipids from erythrocytes and from porcine small intestinal epithelium was examined, in order to get a comprehensive view of the F4-binding glycosphingolipids involved in F4-mediated hemagglutination and adhesion to the epithelial cells of porcine intestine. Specific interactions between the F4ab, F4ac and F4ad fimbriae and both acid and non-acid glycosphingolipids were obtained, and after isolation of binding-active glycosphingolipids and characterization by mass spectrometry and proton NMR, distinct carbohydrate binding patterns were defined for each fimbrial subtype. Two novel glycosphingolipids were isolated from chicken erythrocytes, and characterized as GalNAcα3GalNAcß3Galß4Glcß1Cer and GalNAcα3GalNAcß3Galß4GlcNAcß3Galß4Glcß1Cer. These two compounds, and lactosylceramide (Galß4Glcß1Cer) with phytosphingosine and hydroxy fatty acid, were recognized by all three variants of F4 fimbriae. No binding of the F4ad fimbriae or F4ad-fimbriated E. coli to the porcine intestinal glycosphingolipids occurred. However, for F4ab and F4ac two distinct binding patterns were observed. The F4ac fimbriae and the F4ac-expressing E. coli selectively bound to galactosylceramide (Galß1Cer) with sphingosine and hydroxy 24:0 fatty acid, while the porcine intestinal glycosphingolipids recognized by F4ab fimbriae and the F4ab-fimbriated bacteria were characterized as galactosylceramide, sulfatide (SO3-3Galß1Cer), sulf-lactosylceramide (SO3-3Galß4Glcß1Cer), and globotriaosylceramide (Galα4Galß4Glcß1Cer) with phytosphingosine and hydroxy 24:0 fatty acid. Finally, the F4ad fimbriae and the F4ad-fimbriated E. coli, but not the F4ab or F4ac subtypes, bound to reference gangliotriaosylceramide (GalNAcß4Galß4Glcß1Cer), gangliotetraosylceramide (Galß3GalNAcß4Galß4Glcß1Cer), isoglobotriaosylceramide (Galα3Galß4Glcß1Cer), and neolactotetraosylceramide (Galß4GlcNAcß3Galß4Glcß1Cer)

Prognostic gene expression analysis in a retrospective, multinational cohort of 155 multiple myeloma patients treated outside clinical trials

10.1111/ijlh.13691INTERNATIONAL JOURNAL OF LABORATORY HEMATOLOGY441127-13

Prognostic gene expression analysis in a retrospective, multinational cohort of 155 multiple myeloma patients treated outside clinical trials

Objectives: Typically, prognostic capability of gene expression profiling (GEP) is studied in the context of clinical trials, for which 50%-80% of patients are not eligible, possibly limiting the generalizability of findings to routine practice. Here, we evaluate GEP analysis outside clinical trials, aiming to improve clinical risk assessment of multiple myeloma (MM) patients. Methods: A total of 155 bone marrow samples from MM patients were collected from which RNA was analyzed by microarray. Sixteen previously developed GEP-based markers were evaluated, combined with survival data, and studied using Cox proportional hazard regression. Results: Gene expression profiling-based markers SKY92 and the PR-cluster were shown to be independent prognostic factors for survival, with hazard ratios and 95% confidence interval of 3.6 [2.0-6.8] (P <.001) and 5.8 [2.7-12.7] (P <.01) for overall survival (OS). A multivariate model proved only SKY92 and the PR-cluster to be independent prognostic factors compared to cytogenetic high-risk patients, the International Staging System (ISS), and revised ISS. A substantial number of high-risk individuals could be further identified when SKY92 was added to the cytogenetic, ISS, or R-ISS. In the cytogenetic standard-risk group, ISS I/II, and R-ISS I/II, 13%, 23%, and 23% of patients with adverse survivals were identified. Conclusions: For the first time, this study confirmed the prognostic value of GEP markers outside clinical trials. Conventional prognostic models to define high-risk MM are improved by the incorporation of GEP markers

SecA Is Not Required for Signal Recognition Particle-mediated Targeting and Initial Membrane Insertion of a Nascent Inner Membrane Protein

In Escherichia coli, signal recognition particle (SRP)-dependent targeting of inner membrane proteins has been described. In vitro cross-linking studies have demonstrated that short nascent chains exposing a highly hydrophobic targeting signal interact with the SRP. This SRP, assisted by its receptor, FtsY, mediates the transfer to a common translocation site in the inner membrane that contains SecA, SecG, and SecY. Here we describe a further in vitro reconstitution of SRP-mediated membrane insertion in which purified ribosomenascent chain-SRP complexes are targeted to the purified SecYEG complex contained in proteoliposomes in a process that requires the SRP-receptor FtsY and GTP. We found that in this system SecA and ATP are dispensable for both the transfer of the nascent inner membrane protein FtsQ to SecY and its stable membrane insertion. Release of the SRP from nascent FtsQ also occurred in the absence of SecYEG complex indicating a functional interaction of FtsY with lipids. These data suggest that SRP/FtsY and SecB/SecA constitute distinct targeting routes.

The myeloma stem cell concept, revisited:from phenomenology to operational terms

The concept of the myeloma stem cell may have important therapeutic implications, yet its demonstration has been hampered by a lack of consistency in terms and definitions. Here, we summarize the current documentation and propose single-cell in vitro studies for future translational studies. By the classical approach, a CD19–/CD45low/–/CD38high/CD138+ malignant plasma cell, but not the CD19+/CD38low/–memory B cell compartment, is enriched for tumorigenic cells that initiate myeloma in xenografted immunodeficient mice, supporting that myeloma stem cells are present in the malignant PC compartment. Using a new approach, analysis of c-DNA libraries from CD19+/CD27+/CD38–single cells has identified clonotypic memory B cell, suggested to be the cell of origin. This is consistent with multiple myeloma being a multistep hierarchical process before or during clinical presentation. We anticipate that further characterization will require single cell geno-and phenotyping combined with clonogenic assays. To implement such technologies, we propose a revision of the concept of a myeloma stem cell by including operational in vitro assays to describe the cellular components of origin, initiation, maintenance, and evolution of multiple myeloma. These terms are in accordance with recent (2012) consensus statements on the definitions, assays, and nomenclature of cancer stem cells, which is technically precise without completely abolishing established terminology. We expect that this operational model will be useful for future reporting of parameters used to identify and characterize the multiple myeloma stem cells. We strongly recommend that these parameters include validated standard technologies, reproducible assays, and, most importantly, supervised prospective sampling of selected biomaterial which reflects clinical stages, disease spectrum, and therapeutic outcome. This framework is key to the characterization of the cellular architecture of multiple myeloma and its use in precision medicine

The myeloma stem cell concept, revisited: From phenomenology to operational terms

The concept of the myeloma stem cell may have important therapeutic implications, yet its demonstration has been hampered by a lack of consistency in terms and definitions. Here, we summarize the current documentation and propose single-cell in vitro studies for future translational studies. By the classical approach, a CD19–/CD45/CD38/CD138+ malignant plasma cell, but not the CD19/CD38–memory B cell compartment, is enriched for tumorigenic cells that initiate myeloma in xenografted immunodeficient mice, supporting that myeloma stem cells are present in the malignant PC compartment. Using a new approach, analysis of c-DNA libraries from CD19/CD27/CD38–single cells has identified clonotypic memory B cell, suggested to be the cell of origin. This is consistent with multiple myeloma being a multistep hierarchical process before or during clinical presentation. We anticipate that further characterization will require single cell geno-and phenotyping combined with clonogenic assays. To implement such technologies, we propose a revision of the concept of a myeloma stem cell by including operational in vitro assays to describe the cellular components of origin, initiation, maintenance, and evolution of multiple myeloma. These terms are in accordance with recent (2012) consensus statements on the definitions, assays, and nomenclature of cancer stem cells, which is technically precise without completely abolishing established terminology. We expect that this operational model will be useful for future reporting of parameters used to identify and characterize the multiple myeloma stem cells. We strongly recommend that these parameters include validated standard technologies, reproducible assays, and, most importantly, supervised prospective sampling of selected biomaterial which reflects clinical stages, disease spectrum, and therapeutic outcome. This framework is key to the characterization of the cellular architecture of multiple myeloma and its use in precision medicine.This work on MMSC and myeloma pathogenesis and classification was supported by research funding from the EU 6th FP to MSCNET (LSHC-CT-2006-037602), the Danish Research Agency (grants no. 99 00 771, 271-05-0286, 271-05-0537, and 22-00-0314; to CHEPRE, #2101-07-0007), the Danish Cancer Society (grants no. DP 07014, DR 07017, and DP 9810009), Novo Nordic Foundation (Senior Fellowship 2001-4), KE Jensen Foundation (2005-2013), the Multiple Myeloma Research Foundation (senior grant 2003-4, contract no. 14).Peer Reviewe