Apoptotic cells and clonally expanded cytotoxic T cells in bone marrow trephines of patients with myelodysplastic syndrome.

AIMS: There is increasing evidence that autoimmunity is involved in the pathogenesis of myelodysplastic syndromes (MDS). We examined the number of apoptotic cells, and analysed the T cells and the T cell receptor gene rearrangements in bone marrow trephines of patients with low-grade MDS [refractory anaemia (RA), refractory anaemia with ringed sideroblasts (RAS) and refractory cytopenia with multilineage dysplasia (RCMD)] to investigate the correlation between T cells and apoptosis. METHODS AND RESULTS: Bone marrow trephines from 30 patients with RA, seven patients with RCMD, four patients with RAS and 11 normal bone marrow donors were stained for CD3 and for apoptotic cells using immunohistochemistry and terminal deoxynucleotidyl transferase 2'-deoxyuridine, 5'-triphosphate (dUTP) nick end labelling (TUNEL) technique, respectively. The positive cells were quantified by computer-assisted image analysis. In addition, CD 8 and T cell-restricted intracellular antigen-1 (TIA-1)-positive cells were analysed by single staining and evaluated semiquantitatively by light microscopy. Junctional diversity of the T cell receptor (TCR) α-, ß- and γ-chains were analysed in 24 cases of RA and RCMD by reverse transcription-polymerase chain reaction (RT-PCR). In all cases of RA, RCMD and RAS an increase of apoptotic cells was accompanied by an increase of T cells, when compared to normal donors (P < 0.001). Expression of TIA-1 was found in 33 of 41 patients with low-grade MDS. In contrast, normal controls showed either no or only very weak expression. Furthermore, 14 of 24 cases with low-grade MDS showed clonal TCR gene rearrangement. CONCLUSION: These findings provide evidence that increased apoptosis in low-grade MDS correlates with increased numbers of cytotoxic T cells. A considerable proportion of the MDS cases showed clonal TCR rearrangement suggesting an antigen-driven selection of the T cells. We therefore speculate that cases of MDS can be accompanied by a presumably autoreactive T cell-mediated apoptosis induction in bone marrow cells.

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 beta1 integrin ligation or alphaCD3/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 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.

Measles virus-induced block of transendothelial migration of T lymphocytes and infection-mediated virus spread across endothelial cell barriers.

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.

Alteration of B-cell subsets enhances neuroinvasion in mouse scrapie infection.

Acquired forms of prion diseases or transmissible spongiform encephalopathies are believed to occur following peripheral exposure. Prions initially accumulate in the lymphoid system before spreading to the nervous system, but the underlying mechanisms for prion transfer between the two systems are still elusive. Here we show that ablation of the B-cell-specific transmembrane protein CD19, a coreceptor of the complement system, results in an acceleration of prion neuroinvasion. This appears to be due to an alteration of the follicular dendritic cell (FDC) network within the lymphoid tissue, thereby reducing the distance between FDCs and adjacent nerve fibers that mediate prion neuroinvasion.

Cardiac allograft vasculopathy after cardiac transplantation and hormone therapy: positive effects?

BACKGROUND: There is a great deal of controversy surrounding the issue of hormone replacement therapy after transplantation. The question whether or not this therapy has effects in cardiac allograft vasculopathy (CAV), the Achilles heel of cardiac transplantation or other unique aspects of allograft function is still unknown. METHODS: We investigated the long-term effect of 17beta-estradiol as well as phytoestrogen Coumestrol, a synthetically produced phytoestrogen, on the development of CAV and the degree of fibrosis in an ovariectomized female heterotopic chronic allograft model (LEW-F344). RESULTS: We found that, 150 days after transplantation, no significant effect of estrogen application on intimal thickening of coronary arteries was observed. 17beta-estradiol and phytoestrogen Coumestrol did significantly reduce the perivascular immune reaction. However, the immune effect had no consequence on the intensity of CAV. Although neither 17beta-estradiol nor phytoestrogen Coumestrol revealed a positive effect on CAV, the group of animals treated with 17beta-estradiol showed the highest decline in heart function and the most distinct fibrosis. CONCLUSIONS: 17beta-estradiol does not affect CAV positively, but worsens cardiac allograft function and leads to increased fibrosis. This is the first study showing a negative effect of 17-beta-estradiol after heart transplantation in the long term.

Formation of 4-hydroxy-2-nonenal protein adducts in the ischemic rat heart after transplantation.

BACKGROUND: Free radicals formed during ischemia and reperfusion can lead to lipid peroxidation (LPO) and the formation of 4-hydroxy-2-nonenal (4-HNE), one of the most toxic products of LPO. Using a heterotopic rat heart transplantation model we investigated endogenous 4-HNE formation as a response to cold storage of the transplant and warm blood reperfusion in the recipient. METHODS: Lewis rat hearts were subjected to 30, 240 or 480 minutes of 4 degrees C cold ischemia in Bretschneider cardioplegic solution without or with transplantation and 240-minute reperfusion in F344 recipients. The amount of 4-HNE modified proteins was quantified in rat heart cryosections with an antibody recognizing cysteine-, histidine- and lysine-4-HNE Michael adducts and image analysis of immunostained tissue. RESULTS: We detected 4-HNE-modified proteins in ischemic rat hearts after transplantation and reperfusion. In hearts submitted to ischemia only, 4-HNE-protein adducts comprised 0.7 +/- 0.3% (30 minutes), 0.7 +/- 0.4% (240 minutes) and 0.2 +/- 0.1% (480 minutes) (mean +/- SEM) of the tissue area. Transplantation and reperfusion in the recipient significantly increased the amount of protein adducts to 6.8 +/- 2.6% (p = 0.041), 5.2 +/- 1.4% (p = 0.009) and 5.7 +/- 0.9% (p = 0.002) in 30-, 240- and 480-minute ischemic hearts, respectively. CONCLUSIONS: Under the conditions applied in the present study, cold ischemia for >30 minutes did not significantly alter the amount of 4-HNE protein adducts. However, because after transplantation and reperfusion, 6% of heart tissue consisted of 4-HNE-modified proteins, it can be assumed that this damage negatively affects long-term survival of the transplant.

Heterotopic rat heart transplantation: severe loss of glutathione in 8-hour ischemic hearts.

BACKGROUND: Tissue damage caused by reactive oxygen species (ROS) formed during ischemia/reperfusion seems to be an important risk factor in the failure of transplanted hearts. Although endogenous anti-oxidants protect the myocardium against free radical attack under physiologic conditions, their capacity may become limited during severe oxidative stress. Thus, we investigated the effect of 8-hour cold ischemia on the myocardial anti-oxidative defense system in a heterotopic rat heart transplantation model. METHODS: Lewis rat hearts were subjected to 30 or 480 minutes of 4 degrees C cold ischemia in Bretschneider cardioplegic solution with or without transplantation and reperfusion (30 or 240 minutes) into F344 recipients. Activity levels of superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and glutathione S-transferase (GST), and concentrations of glutathione (GSH), glutathione disulfide (GSSG) and lipid hydroperoxides (LOOH) were analyzed in heart homogenates. For histology, cross-sections of the ventricles were stained with hematoxylin-eosin. RESULTS: Except for GST, enzyme activities and GSSG concentration increased and the glutathione redox ratio (GSH/GSH + 2GSSG) significantly decreased in 480-minute ischemic hearts compared with those with 30-minute ischemia. Reperfusion dramatically decreased both GSH and GSSG and increased LOOH formation but without severe histopathologic findings in the transplants. Applying a tree-structured classifier technique, GSH and LOOH were identified as significant features indicative of transplantation-induced oxidative stress. CONCLUSIONS: In the present study severe loss of glutathione and formation of LOOH indicated transplantation-induced oxidative stress in the rat heart; therefore, alterations of these parameters may hint at relevant deficits in the myocardial anti-oxidative defense and may also predict subsequent tissue damage.

Heterotopic rat heart transplantation (Lewis to F344): early ICAM-1 expression after 8 hours of cold ischemia.

BACKGROUND: Primary graft dysfunction is a still poorly understood complication after cardiac transplantation. Ischemia/reperfusion injury contributes to different disorders resulting in impaired graft function. METHODS: In a heterotopic rat heart transplantation model we extended graft ischemic time up to 8 hours. RESULTS: Using immunohistochemistry we detected an up to 4-fold increase in intracellular adhesion molecule-1 (ICAM-1) expression during 4 hours of reperfusion, independent of ischemic time (30-minute ischemia: 7.65 +/- 2.15 without reperfusion, 19.46 +/- 4.6 after 4-hour reperfusion; 240-minute ischemia: 5.6 +/- 1.99 and 22.3 +/- 3.77; 480-minute ischemia: 3.7 +/- 1.56 and 13.1 +/- 2.2). Eight-hour ischemic allografts had an increase in CD8-positive cells (1.37 +/- 0.5 and 2.3 +/- 0.77) and a significant increase in MHC II expression (11.48 +/- 2.1 and 18.27 +/- 1.34) during 4 hours of reperfusion. CONCLUSIONS: We hypothesize that these findings reflect an early inflammatory reaction in the allograft possibly triggered by oxidative stress. During therapeutic interventions, both of these pathways must be considered.

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 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 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.

Fine needle aspiration of the thyroid: can an image processing system improve differentiation?

OBJECTIVE: To differentiate thyroid nodules by means of fine needle aspiration with subsequent computer-aided diagnosis. STUDY DESIGN: Fine needle aspiration biopsies obtained from 137 patients for whom histopathologic diagnosis was available were investigated: 16 hyperplastic nodules (12%), 39 adenomas (28%), 25 follicular thyroid carcinomas (18%), 19 follicular variants of papillary thyroid carcinoma (14%) and 38 papillary thyroid carcinomas (28%). From each stained specimen 100 cell scenes were scanned and analyzed, constituting a total of 62,325 cell images. RESULTS: All the entities described could be well discriminated from each other by automated image analysis methods. Both the diagnosis of tumor type and the differentiation between benign and malignant could be achieved with a sensitivity of .98. CONCLUSION: With only 7-10 calculated cell features (texture line analysis) and classification with decision trees, a tool for high-quality cell image diagnosis is available. Subtypes of cells characterized by the calculated features could be found in all the specimens and could be assigned to the malignancies with high statistical significance. The method increases the relevance of image processing as an additional diagnostic tool for cytologic examination of thyroid nodules.

Synovial tissue of the hip at power Doppler US: correlation between vascularity and power Doppler US signal.

PURPOSE: To correlate power Doppler ultrasonographic (US) findings of the vascularity of synovial tissue of the hip joint with the results of histopathologic examination of the same tissue to assess the value of power Doppler US in the visualization of synovitis. MATERIALS AND METHODS: The hip joints of 24 patients with osteoarthritis (n = 15) or rheumatoid arthritis (n = 9) of the hip joint were examined with US before arthroplasty. The vascularity of the synovial membrane was classified qualitatively by using power Doppler US. During surgery, a section of the synovial tissue examined at power Doppler US preoperatively was resected. The vascularity of the tissue specimen was investigated and graded qualitatively by a pathologist who was not aware of the US findings. Visual qualitative grading was controlled by means of analysis of the US images and histopathologic specimens with a digital image evaluation system. Correlations between power Doppler US and histopathologic examination findings were calculated by using Spearman rank correlation and Pearson correlation tests. RESULTS: The correlation between the qualitative power Doppler US results and the qualitative vascularity grades was 0.92 (P <.01, Spearman rho). The correlation between quantitative and qualitative results was 0.93 (P <.01, Spearman rho) for US imaging and 0.97 (P <.01, Spearman rho) for histopathologic examination. CONCLUSION: Study results showed power Doppler US to be reliable for qualitative grading of the vascularity of synovial tissue of the hip.