[Ultrasound artifacts and their diagnostic significance in internal medicine and gastroenterology - Part 1: B-mode artifacts]. / Artefakte in der Sonografie und ihre Bedeutung für die internistische und gastroenterologische Diagnostik - Teil 1: B-Mode-Artefakte.

Artifacts in ultrasonographic diagnostics are a result of the physical properties of the ultrasound waves and are caused by interaction of the ultrasound waves with biological structures and tissues and with foreign bodies. On the one hand, they may be distracting and may lead to misdiagnosis. On the other hand, they may be diagnostically helpful. Ultrasound imaging suffers from artifacts, because in reality, parameters assumed to be constant values, such as sound speed, sound rectilinear propagation, attenuation, etc., are often different from the actual parameters. Moreover, inadequate device settings may cause artifacts. Profound knowledge of the causes, avoidance, and interpretation of artifacts is a necessary precondition for correct clinical appraisal of ultrasound images. Part 1 of this review comments on the physics of artifacts and describes the most important B-mode artifacts. Pitfalls, as well as diagnostic chances resulting from B-mode artifacts, are discussed.

[Ultrasound artifacts and their diagnostic significance in internal medicine and gastroenterology - part 2: color and spectral Doppler artifacts]. / Artefakte in der Sonografie und ihre Bedeutung für die internistische und gastroenterologische Diagnostik - Teil 2: Artefakte im Farb- und Spektraldoppler.

Artifacts in ultrasonographic diagnostics are a result of the physical properties of the ultrasound waves and are caused by interaction of the ultrasound waves with biological structures and tissues of the body and with foreign materials. On the one hand, they may be diagnostically helpful. On the other hand, they may be distracting and may lead to misdiagnosis. Profound knowledge of the causes, avoidance, and interpretation of artifacts is a necessary precondition for correct clinical appraisal of ultrasound images. Part 1 of this review commented on the physics of artifacts and described the most important B-mode artifacts. Part 2 focuses on the clinically relevant artifacts in Doppler and color-coded duplex sonography. Problems and pitfalls of interpretation arising from artifacts, as well as the diagnostic use of Doppler and colour-coded duplex sonography, are discussed.

Specificity of the proteasome and the TAP transporter.

The generation of antigenic peptides and their transport across the membrane of the endoplasmic reticulum for assembly with MHC class I molecules are essential steps in antigen presentation to cytotoxic T lymphocytes. Recent studies have characterized the substrate specificities of the proteasome and the transporter associated with antigen processing. It is interesting to compare the specificity of this transporter to the wide spectrum of peptides generated by the proteasome, to the binding motifs of MHC class I molecules and in particular to the principles of T cell recognition.

The active domain of the herpes simplex virus protein ICP47: a potent inhibitor of the transporter associated with antigen processing.

The herpes simplex virus type 1 (HSV-1) protein ICP47 binds specifically to the transporter associated with antigen processing (TAP), thereby blocking peptide-binding and translocation by TAP and subsequent loading of peptides onto MHC class I molecules in the endoplasmic reticulum. In consequence, HSV-infected cells are masked for immune recognition by cytotoxic T-lymphocytes. To investigate the molecular details of this, so far, unique transporter-inhibitor interaction, the active domain and critical amino acid residues were identified by using short overlapping fragments and systematic deletions of the viral inhibitor. A fragment of 32 amino acid residues, ICP47(3-34), was found to be the minimal region harboring an activity to inhibit peptide-binding to TAP comparable to the action of the full-length protein and therefore representing the active domain. Further N or C-terminal truncations cause an abrupt loss in activity. Within the identified active domain, various mutants and chimeras of ICP47 derived from HSV-1 and HSV-2 helped to identify amino acid residues critical for TAP inhibition. On the basis of these results, therapeutic drugs could be designed that are applicable in treatment of allograft rejection or in novel vaccination strategies against HSV, restoring the ability of the immune system to recognize HSV-infected cells.

Peptide binding and photo-crosslinking to detergent solubilized and to reconstituted transporter associated with antigen processing (TAP).

The transporter associated with antigen processing (TAP) is essential for peptide loading onto major histocompatibility (MHC) class I molecules by translocating peptides into the endoplasmic reticulum. We have explored the conditions for detergent solubilization of functionally active, heterologously expressed human TAP from microsomal membranes. The efficiency to solubilize TAP was tested for a variety of detergents as well as for different solubilization conditions. The activity of the solubilized TAP complex was analyzed over time, using a non-radioactive crosslinking assay with a photo-activateable peptide, in the presence or absence of external lipid. The detergent CHAPS was found optimally to retain activity and thus allowed us to reconstitute detergent-solubilized, active TAP into proteoliposomes.

Functional expression and purification of the ABC transporter complex associated with antigen processing (TAP) in insect cells.

Using the baculovirus expression system the gene products of human tap1 and tap2 were over-expressed as wild-type as well as oligohistidine fusion proteins in Spodoptera frugiperda (Sf9) insect cells. Both gene products were co-expressed within the same cells and were found enriched in microsomal membranes. Immunoprecipitation and immobilized metal affinity chromatography revealed complex formation between TAP1 and TAP2. The expressed TAP complex was shown to be functional by peptide translocation into microsomes of Sf9 cells. Peptide transport strictly requires TAP1 and TAP2 as well as ATP. For the first time the functional expression of the human TAP complex in insect cells has been demonstrated, indicating that additional cofactors of a highly developed immune system are not essential for peptide transport across microsomal membranes.

Structure of the viral TAP-inhibitor ICP47 induced by membrane association.

Herpes simplex virus type I protein ICP47 (IE12) turns off antigen presentation by specifically binding to and blocking the major histocompatibility complex- (MHC-) encoded transporter associated with antigen processing (TAP). Due to the lack of translocated peptides inside the endoplasmic reticulum, MHC class I molecules fail to assemble and therefore MHC-peptide complexes do not reach the cell surface for immune recognition by cytotoxic T-lymphocytes. Here we investigated the structure of ICP47 representing the first natural inhibitor of an ATP-binding-cassette (ABC) transporter identified so far. First, we demonstrate that the N-terminal half of ICP47 is as active in inhibition of human TAP as the full-length protein and therefore serves as an ideal model to investigate structural and functional aspects of the inhibitor. Second, from circular dichroism analysis, the viral inhibitor of TAP appears to be mainly unstructured in aqueous solution. However, in the presence of membrane mimetics or lipid membranes an alpha-helical structure is induced. Third, circular dichroism and fluorescence spectroscopy reveal that membrane adsorption and conformational change of ICP47 are directly dependent on the surface charge density of the lipid membrane. Therefore we conclude that docking to membranes induces a conformational change in ICP47 that may be prerequisite to blocking TAP function.

Intracellular location, complex formation, and function of the transporter associated with antigen processing in yeast.

Peptide transport across the membrane of the endoplasmic reticulum (ER) gains increasing importance in view of its potential function in selective protein degradation and antigen processing. An example for peptide transport in the ER is the transporter associated with antigen processing (TAP), which supplies peptides for the formation of major-histocompatibility-complex class-I complexes. Here, we have expressed human TAP1 and TAP2 in the yeast Saccharomyces cerevisiae. Expression of both genes resulted in the formation of a stable TAP heterodimer that was localized mainly in the ER. Although a minor fraction of TAP is found in the plasma membrane, TAP is unable to restore a-factor secretion in a mutant cell line that lacks the yeast mating-factor transporter Ste6. Nevertheless, in vitro studies with microsomal vesicles demonstrated that the TAP complex is fully functional in the ER membrane in terms of selective peptide binding, ATP-dependent transport, and specific inhibition by the viral protein of herpes simplex virus ICP47. This offers opportunities for topological, structural and mechanistic studies as well as genetic screenings for TAP functionality.

Downregulation of TAP1 in B lymphocytes by cellular and Epstein-Barr virus-encoded interleukin-10.

Virally infected cells degrade intracellular viral proteins proteolytically and present the resulting peptides in association with major histocompatibility complex (MHC) class I molecules to CD8+ cytotoxic T lymphocytes (CTLs). These cells are normally prone to CTL-mediated elimination. However, several viruses have evolved strategies to avoid detection by the immune system that interfere with the pathway of antigen presentation. Epstein-Barr virus (EBV) expresses a predominantly late protein, the BCRF1 gene product vIL-10, that is similar in sequence to the human interleukin-10 (hIL-10). We show here that vIL-10 affects the expression of one of the two transporter proteins (TAPs) associated with antigen presentation. Similarly, hIL-10 showed the same activity. Expression of the LMP2 and TAP1 genes but not expression of TAP2 or LMP7 is efficiently downregulated, indicating a specific IL-10 effect on the two divergently transcribed TAP1 and LMP2 genes. Downregulation of TAP1 by IL-10 hampers the transport of peptide antigens into the endoplasmatic reticulum, as shown in the TAP-specific peptide transporter assay, their loading onto empty MHC I molecules, and the subsequent translocation to the cell surface. As a consequence, IL-10 causes a general reduction of surface MHC I molecules on B lymphocytes that might also affect the recognition of EBV-infected cells by cytotoxic T cells.

Recognition principle of the TAP transporter disclosed by combinatorial peptide libraries.

Transport of peptides across the membrane of the endoplasmic reticulum for assembly with MHC class I molecules is an essential step in antigen presentation to cytotoxic T cells. This task is performed by the major histocompatibility complex-encoded transporter associated with antigen processing (TAP). Using a combinatorial approach we have analyzed the substrate specificity of human TAP at high resolution and in the absence of any given sequence context, revealing the contribution of each peptide residue in stabilizing binding to TAP. Human TAP was found to be highly selective with peptide affinities covering at least three orders of magnitude. Interestingly, the selectivity is not equally distributed over the substrate. Only the N-terminal three positions and the C-terminal residue are critical, whereas effects from other peptide positions are negligible. A major influence from the peptide backbone was uncovered by peptide scans and libraries containing D amino acids. Again, independent of peptide length, critical positions were clustered near the peptide termini. These approaches demonstrate that human TAP is selective, with residues determining the affinity located in distinct regions, and point to the role of the peptide backbone in binding to TAP. This binding mode of TAP has implications in an optimized repertoire selection and in a coevolution with the major histocompatibility complex/T cell receptor complex.

Requirements for peptide binding to the human transporter associated with antigen processing revealed by peptide scans and complex peptide libraries.

Antigenic peptides are translocated into the lumen of the endoplasmic reticulum by the action of the transporter associated with antigen processing (TAP), where they are subsequently needed for the correct assembly of major histocompatibility complex molecules. The transport function was reconstituted in insect cells by expression of both TAP genes. On the basis of this over-expression system, substrate selection was analyzed in detail by a direct biomolecular peptide binding assay. Competition assays with peptide variants, including substitutions of residues with alanine or structurally related amino acids, underline the broad peptide specificity of the human TAP complex. Steric requirements of the substrate-binding pocket were mapped using elongated peptides and scans with bulky, hydrophobic amino acids. Complex nonapeptide libraries were used to determine the contribution of each residue to stabilize peptide-TAP complexes. For the first time, this approach lets us directly evaluate the importance of peptide selection for the overall process of antigen presentation on the level of the peptide transporter.

Molecular mechanism and species specificity of TAP inhibition by herpes simplex virus ICP47.

The immediate early protein ICP47 of herpes simplex virus (HSV) inhibits the transporter for antigen processing (TAP)-mediated translocation of antigen-derived peptides across the endoplasmic reticulum (ER) membrane. This interference prevents assembly of peptides with class I MHC molecules in the ER and ultimately recognition of HSV-infected cells by cytotoxic T-lymphocytes, potentially leading to immune evasion of the virus. Here, we demonstrate that recombinant, purified ICP47 containing a hexahistidine tag inhibits peptide import into microsomes of insect cells expressing human TAP, whereas inhibition of peptide transport by murine TAP was much less effective. This finding indicates an intrinsic species-specificity of ICP47 and suggests that no additional proteins interacting specifically with either ICP47 or TAP are required for inhibition of peptide transport. Since neither purified nor induced ICP47 inhibited photocrosslinking of 8-azido-ATP to TAP1 and TAP2 it seems that ICP47 does not prevent ATP from binding to TAP. By contrast, peptide binding was completely blocked by ICP47 as shown both by photoaffinity crosslinking of peptides to TAP and peptide binding to microsomes from TAP-transfected insect cells. Competition experiments indicated that ICP47 binds to human TAP with a higher affinity (50 nM) than peptides whereas the affinity to murine TAP was 100-fold lower. Our data suggest that ICP47 prevents peptides from being translocated by blocking their binding to the substrate-binding site of TAP.