Harry Harms

Correlation of power Doppler sonography with vascularity of the synovial tissue of the knee joint in patients with osteoarthritis and rheumatoid arthritis.

OBJECTIVE To examine the significance of power Doppler sonography (PDS) in the diagnosis of synovial hypertrophy of the knee joint by verifying and comparing the PDS findings with histopathologic findings of synovial membrane vascularity. METHODS The knee joints of 23 patients who were undergoing arthroplasty of the knee joint because of osteoarthritis or rheumatoid arthritis were examined with ultrasound before arthroplasty. The vascularity of the synovial membrane was classified semiquantitatively using PDS. A sample of synovial tissue was obtained during the arthroplasty, and the vascularity of the synovial tissue was evaluated by immunohistochemistry (factor VIII) and was graded qualitatively by a pathologist who was unaware of the PDS findings. The visual qualitative grading by the examiner was controlled by analyzing PDS images and histologic samples using a digital image evaluation system. RESULTS The correlation between the qualitative PDS results and the qualitative grading of the vascularity by the pathologist was 0.89 by Spearmans rho (P < 0.01). The Pearson correlation coefficient between the digital analysis of the PDS images and the digital analysis of the tissue sections was 0.81 (P < 0.01). Digital image analysis and qualitative grading by the examiner had a correlation of 0.89 by Spearmans p (P < 0.01) for the PDS images. The correlation between the qualitative estimation of vascularity by the pathologist and the digital image analysis was 0.88 by Spearmans rho (P < 0.01). CONCLUSION In the present study, PDS proved to be a reliable diagnostic method for qualitative grading of the vascularity of the synovial tissue. In clinical practice, PDS allows further differentiation of the hypertrophic synovium.

Improvement of monitoring of melanocytic skin lesions with the use of a computerized acquisition and surveillance unit with a skin surface microscopic television camera

BACKGROUND Photographic documentation of melanocytic skin lesions is important. Storage and retrieval of slides, however, take much time and space. OBJECTIVE Our purpose was to develop and clinically test a computerized acquisition and surveillance (CAS) unit with a television camera for monitoring including measurements of lesional areas. METHODS A CAS unit connected with a skin surface microscopic television camera was used for monitoring of melanocytic nevi (MN). The lesional area and the skin surface microscopic appearance (SMA) were analyzed after 10 to 21 months in 54 of 1355 MN. RESULTS In 19 MN (35.2%), changes were found. In eight cases, changes in size of more or less than 15% were detected; in five cases only the SMA changed. In six cases both characteristics changed. CONCLUSION In approximately 25% of MN, changes were only detectable in the SMA but not with area measurements. This favors the use of systems such as CAS because only they allow a time-saving comparison of actual and previous images.

Evaluation of different image acquisition techniques for a computer vision system in the diagnosis of malignant melanoma

BACKGROUND Digital image analysis was found to be a useful technique for improved accuracy of preoperative diagnosis of melanocytic lesions. In previous studies digitized color slides were used as input for digital image analysis. New technologies and smaller video cameras made it possible to develop a camera system that allows the digitization of skin lesions directly from the patient. OBJECTIVE We investigated whether conventional color slides or directly digitized images should be used for a reliable recognition of malignant melanoma. METHODS Computer features describing characteristics of the lesions were computed for 404 digitized color slides and for 309 directly acquired lesions. Statistical analysis and classifier construction was performed by the commercial statistical classification program CART. RESULTS With the data set derived either from the color slides or from the directly digitized lesions a sensitivity of about 90% for the recognition of malignant melanoma could be obtained. CONCLUSION Both image acquisition techniques allow a reliable detection of malignant melanoma and both are appropriate as input for an image analysis system regarding its efficiency as a diagnostic tool. However, none of the classifiers can be applied with reasonable significance to both techniques.

Combined local color and texture analysis of stained cells

Abstract Combined color and texture techniques provide new, useful features for blood cell analysis. After scene segmentation, texture line following programs define the borders of textons, the smallest texture grains. Size, color, and shape features are then calculated for each texton. The final feature data set includes: (1) The nucleus and cytoplasm features: size and shape, measurements of the texture lines from the green TV image, average CIE-PAL color coordinates and a color code. (2) The new texton features: total number of texton pixels with a specific color code, mean texton radius, size for each color code, and texton shape factors.

Measles Virus Contact with T Cells Impedes Cytoskeletal Remodeling Associated with Spreading, Polarization, and CD3 Clustering

CD3/CD28‐induced activation of the PI3/Akt kinase pathway and proliferation is impaired in T cells after contact with the measles virus (MV) glycoprotein (gp) complex. We now show that this signal also impairs actin cytoskeletal remodeling in T cells, which loose their ability to adhere and to promote microvilli formation. MV exposure results in an almost complete collapse of membrane protrusions associated with reduced phosphorylation levels of cofilin and ezrin/radixin/moesin (ERM) proteins. Consistent with their inability to activate Cdc42 and Rac1 in response to the ligation of CD3/CD28, T cells exposed to MV fail to acquire a morphology consistent with spreading and lamellopodia formation. In spite of these impairments of cytoskeleton‐driven morphological alterations, these cells are recruited into conjugates with dendritic cells as efficiently as control T cells. The signal elicited by MV, however, prevents T cells to polarize as documented by a failure to redistribute the microtubule organizing center toward the synapse. Moreover, CD3 cannot be efficiently clustered and redistributed to the central region of the immunological synapse. Thus, by inducing microvillar collapse and interfering with cytoskeletal remodeling, MV signaling disturbs the ability of T cells to adhere, spread, and cluster receptors essential for sustained T‐cell activation.

Induction of membrane ceramides: a novel strategy to interfere with T lymphocyte cytoskeletal reorganisation in viral immunosuppression.

Silencing of T cell activation and function is a highly efficient strategy of immunosuppression induced by pathogens. By promoting formation of membrane microdomains essential for clustering of receptors and signalling platforms in the plasma membrane, ceramides accumulating as a result of membrane sphingomyelin breakdown are not only essential for assembly of signalling complexes and pathogen entry, but also act as signalling modulators, e. g. by regulating relay of phosphatidyl-inositol-3-kinase (PI3K) signalling. Their role in T lymphocyte functions has not been addressed as yet. We now show that measles virus (MV), which interacts with the surface of T cells and thereby efficiently interferes with stimulated dynamic reorganisation of their actin cytoskeleton, causes ceramide accumulation in human T cells in a neutral (NSM) and acid (ASM) sphingomyelinase–dependent manner. Ceramides induced by MV, but also bacterial sphingomyelinase, efficiently interfered with formation of membrane protrusions and T cell spreading and front/rear polarisation in response to β1 integrin ligation or αCD3/CD28 activation, and this was rescued upon pharmacological or genetic ablation of ASM/NSM activity. Moreover, membrane ceramide accumulation downmodulated chemokine-induced T cell motility on fibronectin. Altogether, these findings highlight an as yet unrecognised concept of pathogens able to cause membrane ceramide accumulation to target essential processes in T cell activation and function by preventing stimulated actin cytoskeletal dynamics.

Measles virus induces expression of SIP110, a constitutively membrane clustered lipid phosphatase, which inhibits T cell proliferation

Interference of measles virus (MV) with phosphatidyl‐inositol‐3‐kinase (PI3K) activation in response to T cell receptor ligation was identified as important for the induction of T cell paralysis. We now show that MV exposure of unstimulated T cells induces expression of SIP110, an isoform of the lipid phosphatase SHIP145, which is translated from an intron‐derived sequences containing mRNA. We found that MV contact can regulate stimulated exon inclusion into pre‐mRNAs by targeting PI3K or MAPK‐dependent nuclear translocation and activation of splicing regulatory serine–arginine rich (SR) and Sam68 proteins. Induction of SIP110 in resting T cells relied on MV‐dependent interference with basal activity of the PI3K. SIP110 was cloned from MV‐exposed T cells, and, when transiently expressed in primary or Jurkat T cells, localized into membrane clusters independently of T cell activation. Confirming that SIP110 is a catalytically active lipid phosphatase, its transgenic expression abolished basal and impaired PMA/ionomycin‐stimulated phosphorylation of the Akt kinase which is important for T cell proliferation. Thus MV causes induction of SIP110 expression, which constitutively depletes the cellular phosphoinositol‐3,4,5‐phosphate pool suggesting that thereby the threshold for activation signals necessary for the induction of T cell proliferation is raised.

Measles Virus-Induced Block of Transendothelial Migration of T Lymphocytes and Infection-Mediated Virus Spread across Endothelial Cell Barriers

ABSTRACT In order to analyze whether measles virus (MV) is transported via transmigrating leukocytes across endothelial barriers or whether virus spreads via infection of endothelial cells and basolateral release, we investigated the migratory behavior of infected human primary T lymphocytes across polarized cell layers of human brain microvascular endothelial cells. We found that the capacity of lymphocytes to migrate through filter pores was only slightly affected by wild-type MV infection, whereas their capacity to migrate through endothelial barriers was drastically reduced. MV infection stimulated the expression and activation of the leukocyte integrins LFA-1 and VLA-4, mediating a strong adherence to the surface of endothelial cells. Furthermore, the formation of engulfing membrane protrusions by endothelial cells, so-called transmigratory cups, was induced, but transmigration was impaired. As a consequence of this close cell-cell contact, MV infection was transmitted from lymphocytes to the endothelium. MV envelope proteins were expressed on the apical and basolateral surfaces of infected polarized endothelial cells, and virus was released from both sides. Wild-type MV infection did not induce the formation of syncytia, suggesting virus spread from cell to cell via cell processes and contacts. Our data indicate that transendothelial migration of infected T cells is strongly inhibited, whereas virus can cross endothelial barriers by productive infection of the endothelium and subsequent bipolar virus release.

Immune synapses formed with measles virus‐infected dendritic cells are unstable and fail to sustain T cell activation

Interaction with dendritic cells (DCs) is considered as central to immunosuppression induced by viruses, including measles virus (MV). Commonly, viral infection of DCs abrogates their ability to promote T cell expansion, yet underlying mechanisms at a cellular level are undefined. We found that MV‐infected DCs only subtly differed from LPS‐matured with regard to integrin activation, acquisition of a migratory phenotype and motility. Similarly, the organization of MV‐DC/T cell interfaces was consistent with that of functional immune synapses with regard to CD3 clustering and MHC class II surface recruitment. The majority of MV‐DC/T cell conjugates was, however, unstable and only promoted abortive T cell activation. Thus, MV‐infected DCs retain activities required for initiating, but not sustaining T cell conjugation and activation. This is partially rescued if surface expression of the MV glycoproteins on DCs is abolished by infection with a recombinant MV encoding VSV G protein instead, indicating that these contribute directly to synapse destabilization and thereby act as effectors of T cell inhibition.

Measles virus M protein-driven particle production does not involve the endosomal sorting complex required for transport (ESCRT) system

Assembly and budding of enveloped RNA viruses rely on viral matrix (M) proteins and host proteins involved in sorting and vesiculation of cellular cargoes, such as the endosomal sorting complex required for transport (ESCRT). The measles virus (MV) M protein promotes virus-like particle (VLP) production, and we now show that it shares association with detergent-resistant or tetraspanin-enriched membrane microdomains with ebolavirus VP40 protein, yet accumulates less efficiently at the plasma membrane. Unlike VP40, which recruits ESCRT components via its N-terminal late (L) domain and exploits them for particle production, the M protein does this independently of this pathway, as (i) ablation of motifs bearing similarity to canonical L domains did not affect VLP production, (ii) it did not redistribute Tsg101, AIP-1 or Vps4A to the plasma membrane, and (iii) neither VLP nor infectious virus production was sensitive to inhibition by dominant-negative Vps4A. Importantly, transfer of the VP40 L domain into the MV M protein did not cause recruitment of ESCRT proteins or confer sensitivity of VLP release to Vps4A, indicating that MV particle production occurs independently of and cannot be routed into an ESCRT-dependent pathway.