Irrelevant Antibody Research Articles

Differential Activation of ERK and Rac Mediates the Proliferative and Anti-proliferative Effects of Hyaluronan and CD44

Hyaluronan, a widely distributed component of the extracellular matrix, exists in a high molecular weight (native) form and lower molecular weight form (HMW- and LMW-HA, respectively). These different forms of hyaluronan bind to CD44 but elicit distinct effects on cellular function. A striking example is the opposing effects of HMW- and LMW-HA on the proliferation of vascular smooth muscle cells; the binding of HMW-HA to CD44 inhibits cell cycle progression, whereas the binding of LMW-HA to CD44 stimulates cell cycle progression. We now report that cyclin D1 is the primary target of LMW-HA in human vascular smooth muscle cells, as it is for HMW-HA, and that the opposing cell cycle effects of these CD44 ligands result from differential regulation of signaling pathways to cyclin D1. HMW-HA binding to CD44 selectively inhibits the GTP loading of Rac and Rac-dependent signaling to the cyclin D1 gene, whereas LMW-HA binding to CD44 selectively stimulates ERK activation and ERK-dependent cyclin D1 gene expression. These data describe a novel mechanism of growth control in which a ligand-receptor system generates opposing effects on mitogenesis by differentially regulating signaling pathways to a common cell cycle target. They also emphasize how a seemingly subtle change in matrix composition can have a profound effect on cell proliferation.

Passive immunization reduces murine cytomegalovirus-induced brain pathology in newborn mice.

Human cytomegalovirus (HCMV) is the most frequent cause of congenital viral infections in humans and frequently leads to long-term central nervous system (CNS) abnormalities that include learning disabilities, microcephaly, and hearing loss. The pathogenesis of the CNS infection has not been fully elucidated and may arise as a result of direct damage of CMV-infected neurons or indirectly secondary to inflammatory response to infection. We used a recently established model of mouse CMV (MCMV) infection in newborn mice to analyze the contribution of humoral immunity to virus clearance from the brain. In brains of MCMV-infected newborn mice treated with immune serum, the titer of infectious virus was reduced below detection limit, whereas in the brains of mice receiving control (nonimmune) serum significant amounts of virus were recovered. Moreover, histopathological and immunohistological analyses revealed significantly less CNS inflammation in mice treated with immune serum. Treatment with MCMV-specific monoclonal antibodies also resulted in the reduction of virus titer in the brain. Recipients of control serum or irrelevant antibodies had more viral foci, marked mononuclear cell infiltrates, and prominent glial nodules in their brains than mice treated with immune serum or MCMV-specific antibodies. In conclusion, our data indicate that virus-specific antibodies have a protective role in the development of CNS pathology in MCMV-infected newborn mice, suggesting that antiviral antibodies may be an important component of protective immunological responses during CMV infection of the developing CNS.

Dlx5, a Positive Regulator of Osteoblastogenesis, is Essential for Osteoblast-Osteoclast Coupling

The homeodomain protein Dlx5 is an activator of Runx2 (a key regulator of osteogenesis) and is thought to be an important regulator of bone formation. At present, however, the perinatal lethality of Dlx5-null mice has hampered the elucidation of its function in osteogenesis. Here we provide the first analysis of the effects of Dlx5 inactivation on bone development. Femurs of Dlx5-null mouse embryos at the end of gestation exhibit a reduction in both total and trabecular bone volume associated with increased trabecular separation and reduced trabecular number. These parameters are often associated with pathological conditions characterized by reduced osteoblast activity and increased bone resorption. Dlx5(-/-) osteoblasts in culture display reduced proliferation and differentiation rate and reduction of Runx2, Osx, Osteocalcin and Bone Sialoprotein expression. In addition to impaired osteoblast function, Dlx5(-/-) femurs exhibit significant increases in osteoclast number. As Dlx5 is not expressed by osteoclasts, we suggest that its osteoblastic expression might control osteoblast/osteoclast coupling. Cultured Dlx5(-/-) osteoblasts displayed a higher RANKL/OPG ratio. Furthermore, Dlx5(-/-) osteoblasts induced a higher number of TRAP-positive multinucleated cells in normal spleen cultures with a globally increased resorption activity. These findings suggest that Dlx5 is a central regulator of bone turnover as it activates bone formation directly and bone resorption indirectly.

Expression and Suppressive Effects of Interleukin-19 on Vascular Smooth Muscle Cell Pathophysiology and Development of Intimal Hyperplasia

Anti-inflammatory cytokines may play a protective role in the progression of vascular disease. The purpose of this study was to characterize interleukin (IL)-19 expression and function in the development of intimal hyperplasia, and discern a potential mechanism of its direct effects on vascular smooth muscle cells (VSMCs). IL-19 is an immunomodulatory cytokine, the expression of which is reported to be restricted to inflammatory cells. In the present study, we found that IL-19 is not expressed in quiescent VSMCs or normal arteries but is induced in human arteries by injury and in cultured human VSMCs by inflammatory cytokines. Recombinant IL-19 significantly reduced VSMC proliferation (37.1 +/- 4.8 x 10(3) versus 72.2 +/- 6.1 x 10(3) cells/cm(2)) in a dose-dependent manner. IL-19 adenoviral gene transfer significantly reduced proliferation and neointimal formation in balloon angioplasty-injured rat carotid arteries (0.172 +/- 29.9, versus 0.333 +/- 71.9, and 0.309 +/- 56.6 microm(2)). IL-19 induced activation of STAT3 as well as the expression of the suppressor of cytokine signaling 5 (SOCS5) in VSMCs. IL-19 treatment significantly reduced the activation of p44/42 and p38 MAPKs in stimulated VSMCs. Additionally, SOCS5 was found to interact with both p44/42 and p38 MAPKs in IL-19-treated human VSMCs. This is the first description of the expression of both IL-19 and SOCS5 in VSMCs and of the functional interaction between SOCS5 and MAPKs. We propose that through induction of SOCS5 and inhibition of signal transduction, IL-19 expression in VSMCs may represent a novel, protective, autocrine response of VSMCs to inflammatory stimuli.

The ATP-gated P2X1 Receptor Plays a Pivotal Role in Activation of Aspirin-treated Platelets by Thrombin and Epinephrine

Human platelets express protease-activated receptor 1 (PAR1) and PAR4 but limited data indicate for differences in signal transduction. We studied the involvement of PAR1 and PAR4 in the cross-talk between thrombin and epinephrine. The results show that epinephrine acted via alpha(2A)-adrenergic receptors to provoke aggregation, secretion, and Ca(2+) mobilization in aspirin-treated platelets pre-stimulated with subthreshold concentrations of thrombin. Incubating platelets with antibodies against PAR4 or the PAR4-specific inhibitor pepducin P4pal-i1 abolished the aggregation. Furthermore, platelets pre-exposed to the PAR4-activating peptide AYPGKF, but not to the PAR1-activating peptide SFLLRN, were aggregated by epinephrine, whereas both AYPGKF and SFLLRN synergized with epinephrine in the absence of aspirin. The roles of released ATP and ADP were elucidated by using antagonists of the purinergic receptors P2X(1), P2Y(1), and P2Y(12) (i.e. NF449, MRS2159, MRS2179, and cangrelor). Intriguingly, ATP, but not ADP, was required for the epinephrine/thrombin-induced aggregation. In Western blot analysis, a low concentration of AYPGKF, but not SFLLRN, stimulated phosphorylation of Akt on serine 473. Moreover, the phosphatidyl inositide 3-kinase inhibitor LY294002 antagonized the effect of epinephrine combined with thrombin or AYPGKF. Thus, in aspirin-treated platelets, PAR4, but not PAR1, interacts synergistically with alpha(2A)-adrenergic receptors, and the PI3-kinase/Akt pathway is involved in this cross-talk. Furthermore, in PAR4-pretreated platelets, epinephrine caused dense granule secretion, and subsequent signaling from the ATP-gated P2X(1)-receptor and the alpha(2A)-adrenergic receptor induced aggregation. These results suggest a new mechanism that has ATP as a key element and circumvents the action of aspirin on epinephrine-facilitated PAR4-mediated platelet activation.

Control of T Lymphocyte Signaling by Ly108, a Signaling Lymphocytic Activation Molecule Family Receptor Implicated in Autoimmunity

The signaling lymphocytic activation molecule family of receptors has been implicated in the pathophysiology of autoimmunity in humans and mice. One member of the family, Ly108, was strongly linked to lupus susceptibility in mice. High expression of a Ly108 isoform, Ly108-1, was observed in lymphocytes of lupus-prone mice. Herein, we examined the molecular basis for the influence of Ly108 on lupus susceptibility by studying Ly108 signal transduction in T cells. We observed that Ly108 was able to mediate a tyrosine phosphorylation signal implicating Ly108, Vav-1, and c-Cbl in a manner strictly dependent on engagement of the extracellular domain of Ly108 and co-expression of the Src homology 2 (SH2) domain-containing adaptor signaling lymphocytic activation molecule (SLAM)-associated protein (SAP). Evaluation of T cells from mice carrying mutations in the SAP-FynT pathway indicated that Ly108-triggered protein tyrosine phosphorylation was due to the capacity of SAP to recruit FynT. Importantly, Ly108-1 was more apt at triggering tyrosine phosphorylation signals in T cells when compared with the predominant Ly108 isoform found in non-lupus-prone mice, Ly108-2. This difference was due in part to the presence in Ly108-1 of a unique intra-cytoplasmic tyrosine-based motif that promoted Ly108 signal transduction. Together these data provided a molecular explanation for the involvement of Ly108 in lupus susceptibility in mice.

Using Human CD20-Transfected Murine Lymphomatous B Cells to Evaluate the Efficacy of Intravitreal and Intracerebral Rituximab Injections in Mice

The treatment of primary central nervous system lymphoma (PCNSL) and its subset, primary intraocular lymphoma (PIOL), remains of limited efficiency, and salvage therapies are often used without prior testing in adequate animal models. Most PNCSL/PIOL are aggressive B-cell malignancies. Two animal models that closely mimic the human situation were established to evaluate the efficiency of intravitreal and intracerebral anti-CD20 monoclonal antibody (rituximab) injections. Human CD20-transfected murine B-lymphoma cells (38C13 CD20(+)) were inoculated in the vitreous through the pars plana or in the caudate nucleus with the use of a stereotaxic frame in immunocompetent syngeneic mice. Animals were monitored clinically and by funduscopic and histologic examination. Rituximab was injected intravitreally or intracerebrally. Occurrences of exophthalmia, neurologic disturbance, and weight loss were monitored over 2 months. Inoculation of 38C13 CD20(+) cells in the eye or the brain resulted in tumor occurrence after a median of 15 days or 22 days, respectively, with histologic characteristics closely resembling those of PIOL and PCNSL. Local rituximab injections eradicated tumor colonization in more than half the graft recipients and inhibited tumor progression significantly in the others compared with progression in mice that underwent grafting with the control 38C13 cell line (no human CD20 expression) and in mice that underwent grafting with 38C13 CD20(+) cells that received local injections of an irrelevant antibody (trastuzumab). Inoculation of native or human CD20-transfected murine 38C13 cells in the vitreous or the brain of immunocompetent mice provides useful novel models for evaluating the biology and treatment of PIOL and PCNSL. Intravitreal and intracerebral rituximab injections reduced tumor occurrence and growth in each model.

The PDZ Protein Mupp1 Promotes Gi Coupling and Signaling of the Mt1 Melatonin Receptor

Intracellular signaling events are often organized around PDZ (PSD-95/Drosophila Disc large/ZO-1 homology) domain-containing scaffolding proteins. The ubiquitously expressed multi-PDZ protein MUPP1, which is composed of 13 PDZ domains, has been shown to interact with multiple viral and cellular proteins and to play important roles in receptor targeting and trafficking. In this study, we show that MUPP1 binds to the G protein-coupled MT(1) melatonin receptor and directly regulates its G(i)-dependent signal transduction. Structural determinants involved in this interaction are the PDZ10 domain of MUPP1 and the valine of the canonical class III PDZ domain binding motif DSV of the MT(1) carboxyl terminus. This high affinity interaction (K(d) approximately 4 nm), which is independent of MT(1) activation, occurs in the ovine pars tuberalis of the pituitary expressing both proteins endogenously. Although the disruption of the MT(1)/MUPP1 interaction has no effect on the subcellular localization, trafficking, or degradation of MT(1), it destabilizes the interaction between MT(1) and G(i) and abolishes G(i)-mediated signaling of MT(1). Our findings highlight a previously unappreciated role of PDZ proteins in promoting G protein coupling to receptors.

Inhibition of the Proteolytic Activity of Pregnancy-associated Plasma Protein-A by Targeting Substrate Exosite Binding

The metalloproteinase pregnancy-associated plasma protein-A (PAPP-A) cleaves both insulin-like growth factor (IGF)-binding protein 4 (IGFBP-4) and -5 at a single site in their central domain causing the release of bioactive IGF. Inhibition of IGF signaling is relevant in human disease, and several drugs in development target the IGF receptor. However, inhibition of PAPP-A activity may be a valuable alternative. We have generated monoclonal phage-derived single chain fragment variable (scFv) antibodies which selectively inhibit the cleavage of IGFBP-4 by PAPP-A, relevant under conditions where cleavage of IGFBP-4 represents the final step in the delivery of IGF to the IGF receptor. None of the antibodies inhibited the homologous proteinase PAPP-A2, which allowed mapping of antibody binding by means of chimeras between PAPP-A and PAPP-A2 to the C-terminal Lin12-Notch repeat module, separated from the proteolytic domain by almost 1000 amino acids. Hence, the antibodies define a substrate binding exosite that can be targeted for the selective inhibition of PAPP-A proteolytic activity against IGFBP-4. In addition, we show that the Lin12-Notch repeat module reversibly binds a calcium ion and that bound calcium is required for antibody binding, providing a strategy for the further development of selective inhibitory compounds. To our knowledge these data represent the first example of differential inhibition of cleavage of natural proteinase substrates by exosite targeting. Generally, exosite inhibitors are less likely to affect the activity of related proteolytic enzymes with similar active site environments. In the case of PAPP-A, selective inhibition of IGFBP-4 cleavage by interference with exosite binding is a further advantage, as the activity against other known or unknown PAPP-A substrates, whose cleavage may not depend on binding to the same exosite, is not targeted.

Autoimmuninduktion durch pegyliertes Interferon-alpha-2b in Korrelation zur Überlebenszeit bei Mittel- und Hochrisikomelanompatienten

BACKGROUND: The appearance of autoimmunity in tumor patients seems to be associated with a better prognosis. The aim of this retrospective study was to detect autoimmunity under adjuvant treatment with pegylated interferon-alpha-2b and to determine a correlation between autoimmune induction and survival. PATIENTS AND METHODS: To detect autoimmunity clinical and serological parameters were examined in 25 patients with R0-resected histological proven stage Ib-IV melanoma before and every three months during an 18-month adjuvant treatment with 2 μg/kg pegylated interferon-alpha-2b given once a week. RESULTS: Under treatment 16 of 25 patients (64 %) developed autoantibodies. There was no significant difference regarding survival data in patients with or without induction of autoantibodies (31,6 versus 31,9 months). However, it is remarkable that all 6 patients developing a clinically significant autoimmune thyroiditis and/or specific antinuclear antibodies were still alive 36 months after treatment beginning, with two of these patients being in complete remission from stage IV melanoma, compared to a mean survival time of 26,73 months for the remaining patient group with physiological irrelevant positive antibodies (n = 10/16). CONCLUSIONS: In our cohort, the number of patients who developed autoimmunity under pegylated interferon-alpha-2b is significantly higher as published data of non-pegylated interferon-alpha. Nevertheless, the significance of specific autoimmune induction as a prognostic indicator for a disease controlling antitumor immune response still needs to be confirmed.

Characterization of 111In and 177Lu‐labeled antibodies binding to CD44v6 using a novel automated radioimmunoassay

Targeted cancer therapies rely on bifunctional molecules, typically a protein that specifically recognizes tumor cells and a toxic component which is linked to the protein. Therefore, development of such therapies includes detailed characterizations of protein-cell interactions in order to find a good targeting agent. Knowledge of factors such as antibody-antigen specificity, as well as cellular uptake, retention and affinity of the antibody are necessary in order to be successful. In this paper, we have used a novel instrument, LigandTracer Yellow, to characterize the interactions of (111)In and (177)Lu-labeled monoclonal antibodies (MAbs) with CD44v6. Uptake studies with varying specific radioactivity of the chimeric MAb U36 and with an irrelevant antibody for the CD44v6 receptor verified the reliability of the method, as well as the specificity of the antibody-receptor binding. Uptake, retention, and affinity were very similar for the (111)In and (177)Lu-labeled conjugate, and were in line with earlier studies using manual methods. The fact that no adverse effects from labeling were seen, together with the high retention, could make these conjugates promising candidates for imaging and therapy of certain cancer types in the future. The novel LigandTracer technology reduced the workload and reagent spending while providing data with superior time resolution. The obtained results were in agreement with previously reported findings. In addition the real-time detection and higher time resolution made more detailed studies of the interactions possible.

Hzf regulates adipogenesis through translational control of C/EBPα

Adipocyte differentiation requires a well-defined programme of gene expression in which the transcription factor C/EBPalpha (CCAAT/enhancer-binding protein) has a central function. Here, we show that Hzf (haematopoietic zinc-finger), a previously identified p53 transcriptional target, regulates C/EBPalpha expression. Hzf is induced during differentiation of preadipocyte cell lines, and its suppression by short hairpin RNA disrupts adipogenesis. In Hzf's absence, expression of C/EBPalpha is severely impaired because of reduced translation of its mRNA. Hzf physically interacts with the 3' untranslated region of C/EBPalpha mRNA to enhance its translation. Taken together, these findings underscore a critical role of Hzf in the adipogenesis regulatory cascade.

The Dystonia-associated Protein TorsinA Modulates Synaptic Vesicle Recycling

The loss of a glutamic acid residue in the AAA-ATPase (ATPases associated with diverse cellular activities) torsinA is responsible for most cases of early onset autosomal dominant primary dystonia. In this study, we found that snapin, which binds SNAP-25 (synaptosome-associated protein of 25,000 Da) and enhances the association of the SNARE complex with synaptotagmin, is an interacting partner for both wild type and mutant torsinA. Snapin co-localized with endogenous torsinA on dense core granules in PC12 cells and was recruited to perinuclear inclusions containing mutant DeltaE-torsinA in neuroblastoma SH-SY5Y cells. In view of these observations, synaptic vesicle recycling was analyzed using the lipophilic dye FM1-43 and an antibody directed against an intravesicular epitope of synaptotagmin I. We found that overexpression of wild type torsinA negatively affects synaptic vesicle endocytosis. Conversely, overexpression of DeltaE-torsinA in neuroblastoma cells increases FM1-43 uptake. Knockdown of snapin and/or torsinA using small interfering RNAs had a similar inhibitory effect on the exo-endocytic process. In addition, down-regulation of torsinA causes the persistence of synaptotagmin I on the plasma membrane, which closely resembles the effect observed by the overexpression of the DeltaE-torsinA mutant. Altogether, these findings suggest that torsinA plays a role together with snapin in regulated exocytosis and that DeltaE-torsinA exerts its pathological effects through a loss of function mechanism. This may affect neuronal uptake of neurotransmitters, such as dopamine, playing a role in the development of dystonic movements.

3D MR neovascular mapping of rabbit Vx2 tumor response to Avastin (bevacizumab)

The objective of the present study was to characterize angiogenesis and growth response to bevacizumab in the Vx2 rabbit tumor model using MR molecular imaging and 3D neovascular mapping.MethodsRabbits (n=20) implanted with the Vx2 adenocarcinoma were administered bevacizumab (5mg/kg) or an irrelevant IgG1 antibody IV on days 8, 10, 12, and 14. MRI (3T) and 3D MR neovascular mapping were performed with ανβ3‐targeted nanoparticles on days 8 and 16.ResultsSerial treatment with bevacizumab decreased Vx2 tumor size relative to control (2.1 vs. 1.2 cm3, p<0.05), while angiogenesis was diminished by 16% from baseline (p>0.05). 3D neovascular maps illustrated the “angiogenic switch” between days 8 and 16, and revealed peripheral distributed neovasculature with densely coalesced regions interspersed with reticular network of angiogenic regions (Fig). Incomplete suppression of neovascularity with bevacizumab may reflect an under dosing of the drug, given its lower VEGF binding affinity in rabbits and suggests that further benefit of bevacizumab could be obtained with continued treatment at higher dosages.Conclusion3D MR molecular imaging permits unique assessments of tumor neovasculature, which could have an important role in patient stratification and longitudinal management of patients receiving antiangiogenic treatment strategies. Support: NIH/NCI U54 CA 119342

The Interferon Consensus Sequence-binding Protein (ICSBP/IRF8) Represses PTPN13 Gene Transcription in Differentiating Myeloid Cells

The interferon consensus sequence-binding protein (ICSBP/IRF8) is an interferon regulatory factor that is expressed in myeloid and B-cells. ICSBP-deficient mice develop a myeloproliferative disorder characterized by cytokine hypersensitivity and apoptosis resistance. To identify ICSBP target genes involved in these effects, we screened a CpG island microarray with chromatin that co-immunoprecipitated with ICSBP from myeloid cells. Using this technique, we identified PTPN13 as an ICSBP target gene. PTPN13 encodes Fas-associated phosphatase 1 (Fap-1), a ubiquitously expressed protein-tyrosine phosphatase. This was of interest because interaction of Fap-1 with Fas results in Fas dephosphorylation and inhibition of Fas-induced apoptosis. In this study, we found that ICSBP influenced Fas-induced apoptosis in a Fap-1-dependent manner. We also found that ICSBP interacted with a cis element in the proximal PTPN13 promoter and repressed transcription. This interaction increased during myeloid differentiation and was regulated by phosphorylation of conserved tyrosine residues in the interferon regulatory factor domain of ICSBP. ICSBP deficiency was present in human myeloid malignancies, including chronic myeloid leukemia. Therefore, these studies identified a mechanism for increased survival of mature myeloid cells in the ICSBP-deficient murine model and in human myeloid malignancies with decreased ICSBP expression.

Inhibition of Multidrug Resistance by AdamantylGb3, a Globotriaosylceramide Analog

Multidrug resistance (MDR) via the ABC drug transporter (ABCB1), P-glycoprotein (P-gp/MDR1) overexpression, is a major obstacle in cancer chemotherapy. Many inhibitors reverse MDR but, like cyclosporin A (CsA), have significant toxicities. MDR1 is also a translocase that flips glucosylceramide inside the Golgi to enhance neutral glycosphingolipid (GSL) synthesis. We observed partial MDR1/globotriaosylceramide (Gb3) cell surface co-localization, and GSL removal depleted cell surface MDR1. MDR1 may therefore interact with GSLs. AdamantylGb3, a water-soluble Gb3 mimic, but not other GSL analogs, reversed MDR1-MDCK cell drug resistance. Cell surface MDR1 was up-regulated 1 h after treatment with CsA or adaGb3, but at 72 h, cell surface expression was lost. Intracellular MDR1 accumulated throughout, suggesting long term defects in plasma membrane MDR1 trafficking. AdaGb3 or CsA rapidly reduced rhodamine 123 cellular efflux. MDR1 also mediates gastrointestinal epithelial drug efflux, restricting oral bioavailability. Vinblastine apical-to-basal transport in polarized human intestinal C2BBe1 cells was significantly increased when adaGb3 was added to both sides, or to the apical side only, comparable with verapamil, a standard MDR1 inhibitor. Disulfide cross-linking of mutant MDR1s showed no binding of adaGb3 to the MDR1 verapamil/cyclosporin-binding site between surface proximal helices of transmembrane segments (TM) 6 and TM7, but rather to an adjacent site nearer the center of TM6 and the TM7 extracellular face, i.e. close to the bilayer leaflet interface. Verotoxin-mediated Gb3 endocytosis also up-regulated total MDR1 and inhibited drug efflux. Thus, a functional interplay between membrane Gb3 and MDR1 provides a more physiologically based approach to MDR1 regulation to increase the bioavailability of chemotherapeutic drugs.

A Plasmodium falciparum antigen increases HIV-1 replication in a human placenta-derived cell line

Material and methods The placenta-derived choriocarcinoma cell line BeWo and monocyte-derived macrophages (MDM) were infected with varying doses of luciferase reporter HIV-1 pseudotyped with the vesicular stomatitis virus protein G. A recombinant DBL3γ domain, derived from a placental Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) adhesin domain (DBL3γ-732), that binds to chondroitin sulfate A (CSA) and a non-CSA-binding PfEMP1 adhesin domain, DBL1α-varO domain were used to stimulate infected cells. In some experiments, DBL3γ732 was preincubated with the Fab fragment of a specific or an irrelevant monoclonal antibodies (mAb) that inhibits, or not, binding to CSA. TNF-α was measured in the culture supernatants and luciferase activity was quantified at different time points in cell lysates to evaluate viral replication. Results Addition of DBL3γ-732 to BeWo cells, led to a dosedependent increase of HIV-1 replication of up to 400 times the control level in the absence of DBL3γ-732. This enhancement was specific since it was inhibited by Fab fragment of an antiDBL3γ-732 monoclonal antibody but not by the Fab of an irrelevant mAb. In contrast, the addition of DBL1α-varO domain does not increase viral replication. In MDM which presents surface CSA, both DBL domains strongly inhibit viral replication. The effect of DBL3γ-732 on HIV-1 replication is most likely mediated by TNF-α as this cytokine is significantly increased by DBL3γ-732 binding to BeWo cells and MDM.

Vimentin autoantibodies induce platelet activation and formation of platelet-leukocyte conjugates via platelet-activating factor

Anti-vimentin antibodies (AVA) are associated with autoimmunity and solid organ transplantation, conditions associated with vascular disease, but their contribution to disease pathogenesis is unknown. Here, we have examined interactions between AVA (mAb and serum from patients) and various leukocyte populations using whole blood and flow cytometry. Normal blood treated with patient sera containing high AVA-IgM titers or with a vimentin-specific monoclonal IgM led to activation of platelets and other leukocytes, as demonstrated by induced expression of P-selectin, fibrinogen, tissue factor, and formation of platelet:leukocyte (P:L) conjugates and a reduction in platelet counts. This activity was antigen (vimentin)-specific and was not mediated by irrelevant IgM antibodies. Flow cytometry demonstrated that AVA do not bind directly to resting platelets in whole blood, but they bind to approximately 10% of leukocytes. Supernatant, derived from AVA-treated leukocytes, induced platelet activation, as measured by the generation of platelet microparticles, when added to platelet-rich plasma. When AVA were added to whole blood in the presence of CV-6209, a platelet-activating factor (PAF) receptor inhibitor, platelet depletion was inhibited. This suggests that PAF is one of the mediators released from AVA-activated leukocytes that leads to P:L conjugation formation and platelet activation. In summary, AVA bind to leukocytes, resulting in release of a PAF and prothrombotic factor that exert a paracrine-activating effect on platelets. Overall, this proposed mechanism may explain the pathogenesis of thrombotic events in autoimmune diseases associated with AVA.

Retinoschisin (RS1), the Protein Encoded by the X-linked Retinoschisis Gene, Is Anchored to the Surface of Retinal Photoreceptor and Bipolar Cells through Its Interactions with a Na/K ATPase-SARM1 Complex

Retinoschisin or RS1 is a discoidin domain-containing protein encoded by the gene responsible for X-linked retinoschisis (XLRS), an early onset macular degeneration characterized by a splitting of the retina. Retinoschisin, expressed and secreted from photoreceptors and bipolar cells as a homo-octameric complex, associates with the surface of these cells where it serves to maintain the cellular organization of the retina and the photoreceptor-bipolar synaptic structure. To gain insight into the role of retinoschisin in retinal cell adhesion and the pathogenesis of XLRS, we have investigated membrane components in retinal extracts that interact with retinoschisin. Unlike the discoidin domain-containing blood coagulation proteins Factor V and Factor VIII, retinoschisin did not bind to phospholipids or retinal lipids reconstituted into unilamellar vesicles or immobilized on microtiter plates. Instead, co-immunoprecipitation studies together with mass spectrometric-based proteomics and Western blotting showed that retinoschisin is associated with a complex consisting of Na/K ATPase (alpha3, beta2 isoforms) and the sterile alpha and TIR motif-containing protein SARM1. Double labeling studies for immunofluorescence microscopy confirmed the co-localization of retinoschisin with Na/K ATPase and SARM1 in photoreceptors and bipolar cells of retina tissue. We conclude that retinoschisin binds to Na/K ATPase on photoreceptor and bipolar cells. This interaction may be part of a novel SARM1-mediated cell signaling pathway required for the maintenance of retinal cell organization and photoreceptor-bipolar synaptic structure.

Imaging of the Distribution of 90Y-Ibritumomab Tiuxetan in Bone Marrow and Comparison with Pathology

Radioimmunotherapy with anti-CD20 antibodies (Abs) labeled with beta-emitters is now used in the treatment of non-Hodgkin's lymphoma (NHL). Because (90)Y is a pure beta-emitter, no direct image of its distribution can be obtained in humans. In this paper, we present in this study imaging data of (90)Y-Ab distribution in human-mantle-cell lymphoma within a mouse model. Describing the actual distribution of the radionuclide at the level of particles range may have important impact on patient dosimetry and therapy treatment planning. NOD/SCID mice were grafted with a human NHL cell line that involves the bone marrow. The mice were treated with (90)Y-ibritumomab tiuxetan (Zevalin); Schering AG, Germany) and sacrificed 2 hours after Zevalin administration. Tissue sections were then prepared and viewed under conventional microscopy. The distribution of the radioactivity in mouse femur was determined by using digital autoradiography and subsequently correlated with immunohistochemical results. Various extent of bone marrow infiltration was investigated and found to be reproducible. Zevalin uptake was heterogeneous within the bone marrow. However, unspecific mouse monoclonal uptake by accessory myeloid cells gave nonspecific background radioactivity. Treating mice with an irrelevant mouse IgG1 monoclonal antibody (mAb) before Zevalin injection controlled this unspecific uptake, and images were strongly correlated with bone marrow infiltration on histologic analysis. Our model was reproducible, and allows for the study of various bone marrow involvement with good sensitivity. We demonstrated that imaging of the beta-emitter was possible with good image quality and that (90)Y-Zevalin is distributed heterogeneously within bone marrow. These data suggest that detailed pharmacokinetics may be developed with this model.