Selective infection of antigen-specific B lymphocytes by Salmonella mediates bacterial survival and systemic spreading of infection.

Background: The bacterial pathogen Salmonella causes worldwide disease. A major route of intestinal entry involves M cells, providing access to B cell-rich Peyer's Patches. Primary human B cells phagocytose Salmonella typhimurium upon recognition by the specific surface Ig receptor (BCR).

B cell receptor-mediated internalization of salmonella: a novel pathway for autonomous B cell activation and antibody production.

The present paradigm is that primary B cells are nonphagocytosing cells. In this study, we demonstrate that human primary B cells are able to internalize bacteria when the bacteria are recognized by the BCR. BCR-mediated internalization of Salmonella typhimurium results in B cell differentiation and secretion of anti-Salmonella Ab by the Salmonella-specific B cells.

Cryo-immunogold electron microscopy.

This unit describes subcellular localization of proteins/antigens using high-resolution cryo-immunogold electron microscopy, which allows study of topological biochemistry at the ultrastructural level. This is the most sensitive procedure for immunodetection of antigens on ultrathin sections prepared from chemically fixed cells or tissues, because aldehyde fixation is the only denaturation step. The omission of harsh organic solvents (such as those used for plastic embedding) ensures better preservation of protein antigenicity.

A genetic screen implicates miRNA-372 and miRNA-373 as oncogenes in testicular germ cell tumors.

Endogenous small RNAs (miRNAs) regulate gene expression by mechanisms conserved across metazoans. While the number of verified human miRNAs is still expanding, only few have been functionally annotated. To perform genetic screens for novel functions of miRNAs, we developed a library of vectors expressing the majority of cloned human miRNAs and created corresponding DNA barcode arrays.

PKA-induced resistance to tamoxifen is associated with an altered orientation of ERalpha towards co-activator SRC-1.

Resistance to tamoxifen is observed in half of the recurrences in breast cancer, where the anti-estrogen tamoxifen acquires agonistic properties for transactivating estrogen receptor alpha (ERalpha). In a previous study, we showed that protein kinase A (PKA)-mediated phosphorylation of serine 305 (S305) of ERalpha results in resistance to tamoxifen. Now, we demonstrate that phosphorylation of S305 in ERalpha by PKA leads to an altered orientation between ERalpha and its coactivator SRC-1, which renders the transcription complex active in the presence of tamoxifen.

Classification of anti-estrogens according to intramolecular FRET effects on phospho-mutants of estrogen receptor alpha.

Anti-estrogen resistance is a major clinical problem in the treatment of breast cancer. In this study, fluorescence resonance energy transfer (FRET) analysis, a rapid and direct way to monitor conformational changes of estrogen receptor alpha (ERalpha) upon anti-estrogen binding, was used to characterize resistance to anti-estrogens. Nine different anti-estrogens all induced a rapid FRET response within minutes after the compounds have liganded to ERalpha in live cells, corresponding to an inactive conformation of the ERalpha.

Visualizing the action of steroid hormone receptors in living cells.

Transcription controlled by Steroid Hormone Receptors (SHRs) plays a key role in many important physiological processes like organ development, metabolite homeostasis, and response to external stimuli. Understandably, the members of this family have drawn a lot of attention from the scientific community since their discovery, four decades ago. Still, after many years of research we are only beginning to unravel the complex nature of these receptors.

Radiation modulates the peptide repertoire, enhances MHC class I expression, and induces successful antitumor immunotherapy.

Radiotherapy is one of the most successful cancer therapies. Here the effect of irradiation on antigen presentation by MHC class I molecules was studied. Cell surface expression of MHC class I molecules was increased for many days in a radiation dose-dependent manner as a consequence of three responses.

A genetic screen implicates miRNA-372 and miRNA-373 as oncogenes in testicular germ cell tumors.

Endogenous small RNAs (miRNAs) regulate gene expression by mechanisms conserved across metazoans. While the number of verified human miRNAs is still expanding, only few have been functionally annotated. To perform genetic screens for novel functions of miRNAs, we developed a library of vectors expressing the majority of cloned human miRNAs and created corresponding DNA barcode arrays.

MHC class I alleles and their exploration of the antigen-processing machinery.

At the cell surface, major histocompatibility complex (MHC) class I molecules present fragments of intracellular antigens to the immune system. This is the end result of a cascade of events initiated by multiple steps of proteolysis. Only a small part of the fragments escapes degradation by interacting with the peptide transporter associated with antigen presentation and is translocated into the endoplasmic reticulum lumen for binding to MHC class I molecules.

Presenting antigen presentation in living cells using biophysical techniques.

The combination of genetically encoded fluorescent probes and advanced microscopic techniques has dramatically propelled the understanding of cell biology. Highly complex reactions can now be studied in detail in a relatively cost-effective and easy manner and, perhaps most importantly, in the context of a single living cell. In the past decade, numerous reports have uncovered the localization of key molecules in virtually all cellular processes.

Chaperoning antigen presentation by MHC class II molecules and their role in oncogenesis.

Tumor vaccine development aimed at stimulating the cellular immune response focuses mainly on MHC class I molecules. This is not surprising since most tumors do not express MHC class II or CD1 molecules. Nevertheless, the most successful targets for cancer immunotherapy, leukemia and melanoma, often do express MHC class II molecules, which leaves no obvious reason to ignore MHC class II molecules as a mediator in anticancer immune therapy.

Spatial separation of HLA-DM/HLA-DR interactions within MIIC and phagosome-induced immune escape.

Major Histocompatibility Complex (MHC) class II molecules, including Human Leukocyte Antigen (HLA)-DR, present peptide fragments from proteins degraded in the endocytic pathway. HLA-DR is targeted to late-endocytic structures named MHC class II-containing Compartments (MIIC), where it interacts with HLA-DM. This chaperone stabilizes HLA-DR during peptide exchange and is critical for successful peptide loading.

Tamoxifen resistance by a conformational arrest of the estrogen receptor alpha after PKA activation in breast cancer.

Using a novel approach that detects changes in the conformation of ERalpha, we studied the efficacy of anti-estrogens to inactivate ERalpha under different experimental conditions. We show that phosphorylation of serine-305 in the hinge region of ERalpha by protein kinase A (PKA) induced resistance to tamoxifen. Tamoxifen bound but then failed to induce the inactive conformation, invoking ERalpha-dependent transactivation instead.

Peptide diffusion, protection, and degradation in nuclear and cytoplasmic compartments before antigen presentation by MHC class I.

Antigenic peptides generated by the proteasome have to survive a peptidase-containing environment for presentation by MHC class I molecules. We have visualized the fate and dynamics of intracellular peptides in living cells. We show that peptides are distributed over two different but interconnected compartments, the cytoplasm and the nucleus, and diffuse rapidly through and between these compartments.