anti-P Antibodies Research Articles

Lack of Association between Anti-Ribosomal-P Antibody and Chemokines in Neuropsychiatric Syndromes of Systemic Lupus Erythematosus

Anti-ribosomal P protein antibody (anti-P) in cerebral spinal fluids (CSF) has been reported to associate with NPSLE and IgG anti-P in CSF was suggested to be a useful marker for the diagnosis of neuropsychiatric syndromes of systemic lupus erythematosus (NPSLE). There is growing evidence that chemokines, such as MCP-1/CCL2 and IP- 10/CXCL10 are involved in the pathogenesis of NPSLE. To study the relationship between these markers in CSF from pa- tients with NPSLE, we measured the levels of IgG anti-P, MCP-1/CCL2 and IP-10/CXCL10 in the same samples. We found no correlations between the titer of IgG anti-P antibody and IP-10/CXCL10 or MCP-1/CCL2, suggesting that IgG anti-P antibody and chemokine levels might discriminate the different subset of NPSLE symptoms.

Association of ribosomal anti-P antibodies with different parameters of lupus

Systemic Lupus Erythematosus is an autoimmune multisystem disorder with protean manifestations and variable behavior. The main objectives of this study are to verify the association of ribosomal anti-P antibodies with neuropsychiatric lupus manifestations and to find out the relationship of ribosomal anti-P antibodies with other autoimmune parameters of lupus. Ribosomal anti-P antibodies were evaluated in the serum of 41 systemic lupus erythematosus (SLE) patients as well as ANA, dsDNA, anti- Sm, anti-SSA, anti-SSB, anti-histone by an indirect ELISA technique. Apart from Immunological tests, we also studied hematological parameters and biochemical parameters. Of the 41 SLE patients, ribosomal anti-P antibodies were significantly elevated in the sera of 6 lupus patients suffering from severe depression, anxiety, headache and lupus psychosis. Immunological parameters like ANA, dsDNA were positive in 100% of the Anti-Rib-P positive patients; but 66.6% were positive for SSA, only 33.3% for Sm, and none of them were positive for anti-histone and SSB. Biochemical parameters like urea, creatinine were higher than the normal range in 66.6% of the anti-P positive lupus patients, suggestive of lupus nephritis.

Antiribosomal-P autoantibodies from psychiatric lupus target a novel neuronal surface protein causing calcium influx and apoptosis

The interesting observation was made 20 years ago that psychotic manifestations in patients with systemic lupus erythematosus are associated with the production of antiribosomal-P protein (anti-P) autoantibodies. Since then, the pathogenic role of anti-P antibodies has attracted considerable attention, giving rise to long-term controversies as evidence has either contradicted or confirmed their clinical association with lupus psychosis. Furthermore, a plausible mechanism supporting an anti-P–mediated neuronal dysfunction is still lacking. We show that anti-P antibodies recognize a new integral membrane protein of the neuronal cell surface. In the brain, this neuronal surface P antigen (NSPA) is preferentially distributed in areas involved in memory, cognition, and emotion. When added to brain cellular cultures, anti-P antibodies caused a rapid and sustained increase in calcium influx in neurons, resulting in apoptotic cell death. In contrast, astrocytes, which do not express NSPA, were not affected. Injection of anti-P antibodies into the brain of living rats also triggered neuronal death by apoptosis. These results demonstrate a neuropathogenic potential of anti-P antibodies and contribute a mechanistic basis for psychiatric lupus. They also provide a molecular target for future exploration of this and other psychiatric diseases.

Prevalence of depressive and anxiety disorders in systemic lupus erythematosus and their association with anti-ribosomal P antibodies

Objective To estimate the prevalence of psychiatric disorders in patients with systemic lupus erythematosus (SLE) and explore their association with anti-ribosomal P (anti-P) antibodies. Methods Seventy-one consecutive female SLE patients without neurological manifestations were evaluated for psychiatric disorders using the Structured Clinical Interview for DSM-IV (SCID). Anti-P antibodies were measured by enzyme-linked immunosorbent assay (ELISA)/immunoblot analysis. Results The mean age of subjects was 34.8 years (SD: 10.1 years), and the mean duration of SLE was 9.8 years (SD: 6.5 years). The 30-day prevalences of psychiatric disorders were: mood disorders 26.8%, anxiety disorders 46.5%, adjustment disorders 8.4%, alcohol abuse 1.4%, and somatoform disorder 1.4%. The lifetime prevalences of psychiatric disorders were: mood disorders 69%, anxiety disorders 52.1%, alcohol abuse 1.4%, and somatoform disorder 1.4%. Subjects with and without psychiatric manifestations did not differ regarding SLE clinical and laboratorial parameters including presence or absence of anti-P antibodies (23.1% vs. 20%, respectively, p = 1.0), disease activity, as measured by the Systemic Lupus Erythematosus Disease activity Index (4.08 ± 5.7 vs. 4.95 ± 6.3 respectively, p = 0.60) and cumulated damage, as measured by the Systemic Lupus International Collaborating Clinics/American College of Rheumatology Damage Index (0.7 ± 2.3 vs. 0.3 ± 0.7 respectively, p = 0.33). Conclusions Mood and anxiety disorders are the most frequently observed psychiatric disorders in female SLE patients without concomitant neurological manifestations. These mild/moderate forms of psychiatric disorders are not associated with anti-P antibodies in SLE patients. Our findings reinforce the importance of systematic psychiatric evaluation for these patients in order to provide adequate and comprehensive care.

Antibodies directed against ribosomal P proteins cross-react with phospholipids

Anti-ribosomal P protein (anti-P) antibodies are marker antibodies in systemic lupus erythematosus (SLE). Their association with psychiatric or neurological manifestations has been proposed, but remains controversial. Anti-phospholipid antibodies are the hallmark of a syndrome that may comprise a number of neurological manifestations. Thus, anti-P and anti-phospholipid antibodies have both been associated with central nervous system involvement and their co-existence in the same sera was reported. We verified the ability of purified anti-P antibodies to bind different phospholipids and phospholipid-binding proteins in solid-phase assays. Anti-P antibodies from five of eight patients bound cardiolipin (CL) when saturated with fetal calf serum (FCS); in three cases anti-CL antibodies were also detected in the flow-through. No anti-P eluate, nor any corresponding flow-through, bound beta(2)-glycoprotein I alone or prothrombin. Moreover, no anti-P eluate bound CL when the plates were blocked with bovine serum albumin in the absence of FCS. Anti-P antibodies with anti-CL activity bound both ssDNA and dsDNA and also nucleosomes in three patients. Our data indicate a great heterogeneity of anti-P antibodies that appear to be overlapped partially with the other autoantibody populations detected frequently in SLE.

Liver aspirate from a Shar Pei dog

A 5-year-old, neutered male, Shar Pei dog was presented with weight loss, anorexia, lethargy, stranguria, and distal limb edema. Clinicopathologic abnormalities included anemia, an inflammatory leukogram, azotemia, icterus, urinary tract infection, and hepatomegaly with a markedly hypoechoic liver. Cytologic findings in a fine-needle aspirate of the liver included large amounts of amorphous, pink, extracellular matrix between hepatocytes. The amorphous material was congophilic using Congo red stain on a hepatic cytology specimen and green birefringent areas were observed under polarized light, confirming the presence of amyloid. The dog was euthanized and a necropsy was done. Histopathologic evaluation using H&E and Congo red staining confirmed amyloid deposits within the liver, kidneys, intestinal vessels, pancreas, and mesenteric ganglia. Immunohistochemical staining of liver and kidney sections using anti-AA amyloid and anti-P component antibodies confirmed the presence of AA amyloid. In this case, we demonstrated that Congo red staining and polarized light microscopy are a useful diagnostic technique in cytologic specimens of suitable thickness for confirming the presence of amyloid.

Are anti-ribosomal P protein antibodies relevant in systemic lupus erythematosus?

Systemic lupus erythematosus (SLE) is a prototypal auto-immune disorder characterized with multiple organ involvement resulting in disability and increased mortality. Immune regulatory disturbances cumulate in activation of B cells and consequent auto-antibody production. Antigens for these auto-antibodies can be nuclear components and cytoplasmic elements. Anti-P antibodies react against acidic phosphorylated ribosomal proteins P0, P1, and P2 (with molecular mass of 38, 19, and 17 kDa, respectively) and are located on the S60 subunit of ribosomes. Ribosomal P proteins share a common 22-amino acid sequence that is present in the carboxyl-terminal. Anti-P antibodies can be detected in approx 15 to 20% of patients with lupus by several immunoassays, most frequently by enzyme-linked immunosorbent assay (ELISA) and/or Western blotting. However, no standardized assay is available. Auto-antibodies against eukaryotic P proteins appear highly specific for SLE; therefore, they can be used as diagnostic marker for the disease. Furthermore, association has been described with particular manifestations of lupus, especially with neuropsychiatric, renal, and hepatic involvements. Anti-P positivity and the titer of anti-P antibodies also fluctuate with clinical disease activity. Despite several lines of evidence, results are conflicting regarding the existence of such associations. Discrepancies can be explained by different study set-up or study population; it also can be attributed to the different sensitivity of tests used for the detection of anti-P antibody.

On the molecular mechanisms for the highly procoagulant pattern of C6 glioma cells.

That there is a correlation between cancer and procoagulant states is well-known. C6 glioma cell line was originally induced in random-bred Wistar-Furth rats and is morphologically similar to glioblastoma multiforme, the most common aggressive glioma resistant to therapeutic interventions. In this study we analyzed the molecular mechanisms responsible for the highly procoagulant properties of C6 glioma cells. The presence of tissue factor (TF) and phosphatidylserine (PS) in C6 cells was investigated by flow-cytometric and functional analyses. The assembly of extrinsic tenase, intrinsic tenase and prothrombinase complexes on these cells was studied using enzymatic assays employing plasma or purified proteins. TF was identified by flow-cytometric and functional [factor (F) Xa formation in the presence of cells and FVIIa] assays. Alternatively, conversion of FX into FXa was also observed in the presence of C6 cells, FIXa and FVIIIa. This effect was both cell- and FVIIIa-dependent, being consistent with formation of the intrinsic tenase complex. C6 cells were also able to activate prothrombin in the presence of FXa and FVa, thus supporting formation of the prothrombinase complex. This ability was similar to positive controls performed with PS-containing vesicles. Accordingly, exposure of PS on C6 cells was demonstrated by flow cytometry employing specific anti-PS antibodies. In addition, annexin V, which blocks PS binding sites, inhibited FX and prothrombin conversion by their respective C6-assembled activating complexes. C6 glioma cells support all procoagulant reactions leading to robust thrombin formation. This ability results from concomitant TF exposure and from the presence of the anionic lipid PS at the outer leaflet of cell membrane. Therefore, this animal cell line may be used to explore new aspects concerning the role of blood coagulation proteins in tumor biology, especially those affecting the central nervous system.

Autoantibodies to the ribosomal P proteins in systemic lupus erythematosus

This paper describes the clinical significance of antibodies to the ribosomal P proteins in systemic lupus erythematosus. It appears that liver disease due to the lupus process and not attributable to viral infection, alcohol or drugs is associated with anti-ribosomal P. In addition, there is a strong relationship to central nervous system disease and nephritis of antibodies to ribosomal P proteins. The prevalence of the anti-P antibodies is strongly related to disease activity wherein disease remission is associated with disappearance of anti-P antibodies. These phenomena taken together suggest an immunopathogenic role for anti-P antibodies. This idea is strongly supported by the observation that immunoglobulin G containing antiribosomal P activity binds and penetrates living cells with profoundly inhibitory effects on protein synthesis. Finally, a new era of research has been uncovered by the observation that in 54 of 55 instances normal sera passed over a ribosome-sepharose column unmasks anti-P antibodies, which can be eluted from the ribosome column with 3.0 M magnesium chloride. This suggests that anti-idiotypes regulate the expression of anti-P antibodies in normal persons and in lupus patients this regulation is ineffective, with the development of free anti-P antibodies in a proportion of patients with active disease.

Antibodies to ribosomal P proteins: A potential serologic marker for lupus membranous glomerulonephritis

To evaluate the relevance of antibodies to ribosomal P proteins (anti-P antibodies) in discriminating histopathologic patterns of lupus nephritis. The study group comprised 81 consecutive patients with systemic lupus erythematosus who underwent renal biopsy and for whom frozen serum was available at the time of biopsy. All biopsy specimens were reviewed in a blinded manner, according to the 2004 criteria of the International Society of Nephrology and the Renal Pathology Society. Anti-P antibodies were detected by enzyme-linked immunosorbent assay (ELISA)/immunoblot analysis, and anti-double-stranded DNA (anti-dsDNA) was detected by indirect immunofluorescence/ELISA. Anti-P antibodies were detected in 18 patients (22%). The demographic and clinical features of patients with and those without anti-P antibodies were similar. Remarkably, analyses of biopsy specimens revealed that the frequency of anti-P antibodies in patients with class V lupus nephritis was higher than the frequency among patients with other classes of renal disease (72% versus 28%; P = 0.005). Accordingly, anti-P antibody-positive patients had a higher mean (+/-SD) proteinuria level compared with anti-P antibody-negative patients (6.4 +/- 4.8 versus 4.7 +/- 3.9 gm/dl; P = 0.046). Renal function was preserved in 6 of 7 patients who had both isolated anti-P antibodies and class V lupus nephritis. In contrast, anti-dsDNA was associated with proliferative-class lupus nephritis (P = 0.050) and higher creatinine levels (P = 0.014). Furthermore, 7 of 9 patients with isolated anti-P antibodies had class V lupus nephritis, and, more importantly, 5 of these 7 patients (71%) displayed a pure membranous pattern. Conversely, a tendency toward the predominance of class V lupus nephritis (67%) with concomitant proliferative lesions was observed when anti-P antibody was associated with anti-dsDNA. Our data introduce anti-P antibody as a novel serologic marker for membranous lupus nephritis and support the notion that the presence of isolated anti-P antibodies may discriminate patients with pure class V lupus nephritis, whereas the simultaneous presence of anti-dsDNA antibodies suggests class V disease with concomitant proliferative lesions.

Accuracy of anti–ribosomal P protein antibody testing for the diagnosis of neuropsychiatric systemic lupus erythematosus: An international meta‐analysis

To quantitatively evaluate the diagnostic accuracy of antibodies to ribosomal P proteins (anti-P) for neuropsychiatric systemic lupus erythematosus (NPSLE) in general, for psychosis, mood disorder, or both, and for other diffuse manifestations. This international meta-analysis combined standardized data from 1,537 lupus patients contributed by 14 research teams. Weighted estimation of sensitivity and specificity with fixed-effects and random-effects models, as well as summary receiver operating characteristic (SROC) curve analysis, was used to summarize test performance. The robustness of the overall estimates was examined in sensitivity analyses that included additional studies published up to November 1, 2004 in the Medline, EMBase, and Cochrane databases. Combining the data from the 14 teams, the weighted sensitivity and specificity estimates for the diagnosis of NPSLE were 26% (95% confidence interval [95% CI] 15-42%) and 80% (95% CI 74-85%), respectively. For psychosis, mood disorder, or both, the sensitivity and specificity were 27% (95% CI 14-47%) and 80% (95% CI 74-85%), respectively. For other diffuse manifestations, the sensitivity was 24% (95% CI 12-42%), and the specificity was 80% (95% CI 73-85%). The proportion of patients with anti-P antibodies did not vary markedly across different presentations of NPSLE. Between-study heterogeneity was substantial, but the SROC curves were consistent with the weighted estimates. In further analyses that included another 24 published studies, only the sensitivity for psychosis and/or mood disorder was slightly improved, but it was still suboptimal (42% [95% CI 30-53%]); the specificity remained essentially the same (81% [95% CI 76-85%]). Anti-P antibody testing has limited diagnostic value for NPSLE, and it is not helpful in differentiating among various disease phenotypes.

An Anti-Phosphatidylserine (PS) Antibody, Tarvacin™ That Targets Tumor Blood Vessels Does Not Affect Platelet Function In Vitro.

Tarvacin™ is a chimeric anti-PS antibody that is currently in Phase I clinical trials in cancer patients. It acts by targeting PS that becomes exposed on vascular endothelium in tumors in response to oxidative stress in the tumor microenvironment. Tarvacin™ recognizes a complex of PS and the PS-binding protein, β2 glycoprotein I. Host leukocytes are induced to bind to the complex in tumor vessels and destroy tumor vessels by antibody-dependent cellular cytotoxity. However, antibodies directed against PS-associated proteins are also known to elicit anti-phospholipid syndromes (APS). Anti-PS antibodies possibly cause APS by displacing anticoagulant proteins from PS on activated cell or by enhancing the binding of prothrombin; another explanation might be a direct activation of endothelial cells and platelets. The aim of the study was to determine whether Tarvacin ™ induces or interferes with platelet activation caused by ADP, collagen type I or calcimycin in vitro. Blood was drawn from 3 healthy volunteers, aged 31–54, who have not taken any antiplatelet medication for 14 days prior to the study. Dual channel whole blood aggregometer (Chronolog, Havertown, PA, USA) was employed for platelet aggregation studies in whole blood (WB/impedance method) and platelet rich plasma (PRP/optical method). Platelet count in PRP was adjusted to 200 K/μL. Platelet agonists (PS exposure triggers) used in the experiments were as follows: collagen (0.5, 1, 2 μg/mL), ADP (1.25, 2.5, 5, 10 μM), Calcimycin (10, 20, 30 μM) and Calcium ions (1, 2 mmol/L). Tarvacin™ was provided by Peregrine Pharmaceuticals Inc, Tustin, CA. The Anti-CD 20 antibody, Rituxan ™ and physiologic saline were used as controls. Specimens (WB diluted with saline in 1:1 ratio or PRP) with the addition of Tarvacin™ (100 μg/mL) or Rituxan ™ (100 μg/mL) or saline were first incubated on a gentle mixer for 10 minutes; incubation was then continued at 37 ° in the aggregometer well for another 5 minutes. Agonist-induced platelet aggregation was subsequently examined. Platelet aggregation studies in both WB and PRP showed that Tarvacin™ neither induced platelet activation, nor inhibited platelet activation in response to ADP, collagen or calcimycin in vitro. In conclusion, Tarvacin™ does not affect platelet function in the present in vitro assays. Possibly, the epitope on the PS -β2 glycoprotein I complex does not orientate the antibody in a manner that interferes with platelet activation. Alternatively, activated endothelial cells or other factors may be critical to support platelet activation.

Anti-ribosomal P-protein and its role in psychiatric manifestations of systemic lupus erythematosus: myth or reality?

Systemic lupus erythematosus (SLE) is an autoimmune disease that may involve the central nervous system (CNS) resulting in neuropsychiatric manifestations. The associated psychiatric disorders include depression, psychosis, mood disorders, anxiety, cognitive dysfunction, and delirium/ encephalopathy. Several autoantibodies may play a role in the pathogenesis of psychiatric complications of SLE, particularly antibodies against ribosomal P-proteins (anti-P) and possibly antibodies against endothelial cells (AECA). The reported prevalence of anti-P is highly variable in SLE patients and is dependent on different ethnic backgrounds, sensitivity and specificity of the assays employed for autoantibody detection, and the time at which sera were analysed in relation to the clinical event. Controversial data exist on the association of anti-P with psychiatric manifestations of SLE. These autoantibodies have been suggested to be specific markers of the psychiatric manifestations of SLE, particularly of the psychosis and depression, and the antibody level varied with the clinical activity of the disease. Some studies have confirmed the hypothesis of an association of anti-P antibodies with psychiatric manifestations of neuropsychiatric SLE (NPSLE) while others have disputed this relationship. This review summarizes the recent studies about relationship between anti-P antibodies and psychiatric manifestation of SLE.

Serological correlations with nephritis in systemic lupus erythematosus

Autoantibodies have long been thought to participate in the pathogenesis of lupus nephritis. In this regard, antibodies to double stranded (ds)DNA and ribosomal P protein have been studied the most intensively. We now report a new specificity, antibodies to lipoprotein lipase (LPL) that is strongly associated with lupus nephritis and has a powerful synergistic effect with anti-ribosomal P antibodies in its association with nephritis. The recognition of anti-LPL antibodies and their synergy with anti-P antibodies are discussed in terms of the pathogenesis of lupus nephritis.

Clinical Relevance of Antiphospholipid Antibodies in Primary Biliary Cirrhosis

Primary biliary cirrhosis (PBC) is a cholestatic liver disease characterized by the presence of antimitochondrial autoantibodies (AMAs), but also with reactivities to other autoantigens. Recent studies showed that antibodies to phospholipids (APAs) represent an important group of autoantibodies identified in patients with PBC. In this study different types of APAs were identified in the sera of patients with PBC and autoimmune hepatitis (AIH) and control subjects. Sera from patients with PBC and AIH were tested for the presence of antibodies directed against cardiolipin (CL), phosphatidylserine (PS), and to beta(2)-glycoprotein I (beta(2)-GPI). Furthermore, an in-house test for antithromboplastin antibodies was performed. Antinuclear antibodies (ANAs) and antimitochondrial antibodies (AMAs) were tested with standard tests. IgM anti-PS antibodies were found in 75% of the 51 PBC patients, but only in 4% of the 48 AIH patients (P < 0.0001). IgM anti-CL antibodies were more frequently detected in AIH than in PBC (75% vs. 89%; P = 0.045). IgM anti-beta(2)-GPI antibodies were observed more frequently in patients with AIH (83%) than with PBC (59%; P = 0.007). The APAs of the IgG type did not differ significantly between the groups of patients. Considering the clinical/laboratory parameters, the levels of alkaline phosphatase (P = 0.017), glutamate pyruvate transaminase (P = 0.035), and glutamate oxalacetate transaminase (P = 0.034) were significantly higher in PBC patients who were positive for IgM anti-PS antibodies than in the anti-PS antibody-negative patients. In conclusion, APAs are present in PBC patients with a higher level of the disease or more intense liver damage than in patients without APAs. Thus IgM anti-PS antibodies represent a new marker of activity in PBC patients.

Antiphospholipid antibodies are induced by in vitro fertilization and correlate with paraoxonase activity and total antioxidant capacity of plasma in infertile women

The objectives of this study were to determine whether antiphosholipid antibodies are associated with in vitro fertilization (IVF), and assess the potential biological effects of these antibodies. Sera from seventy infertile women (18 before IVF, 13 submitted to one IVF cycle and 39 after three cycles) and 28 healthy controls were collected. Anticardiolipin (anti-CL) and antiphosphatidylserine (anti-PS) antibodies, paraoxonase (PON) and Total Anti-oxidant Capacity of plasma (TAC) were measured. Anti-CL and anti-PS titres were significantly increased in treated patients compared with patients before treatment or controls (P < 0.001). There were no differences regarding anti-CL and anti-PS titres between controls and untreated patients nor when different types of infertility were considered. PON activity and TAC were significantly reduced in treated patients when compared to untreated and controls (P < 0.001; P < 0.002). PON correlated inversely with anti-CL and anti-PS IgG (r = -0.734; P < 0.001) and directly with TAC (r = 0.720, P < 0.001). In conclusion PON activity is decreased in women submitted to IVF treatment and is associated with the presence of antiphospholipid antibodies. These factors might contribute to the increased oxidative status found in these patients.

Anti–ribosomal P protein antibody in human systemic lupus erythematosus up‐regulates the expression of proinflammatory cytokines by human peripheral blood monocytes

Autoantibodies to ribosomal P proteins (anti-P antibodies) are detected in 12-16% of patients with systemic lupus erythematosus (SLE) and have been found to be associated with some manifestations of the disease, including lupus psychosis and hepatitis. Recent studies have disclosed that anti-P antibodies react with activated T cells but not with B cells, suggesting possible direct effects of anti-P antibodies on immune regulation. The present study was designed to explore the presence of the epitope recognized by anti-P antibodies on human peripheral blood monocytes. Highly purified peripheral blood monocytes obtained from healthy donors were cultured with or without interferon-gamma (IFNgamma) in the presence of either anti-P antibodies purified by affinity chromatography from the sera of patients with SLE or control IgG. Flow cytometry analysis disclosed that fresh (day 0) monocytes did not express the ribosomal P epitope, whereas expression of the ribosomal P epitope was induced on annexin V-negative monocytes after activation through plastic adherence for 48 hours. More important, anti-P antibodies (compared with normal IgG or IgG from SLE patients devoid of anti-P antibodies) enhanced the production of tumor necrosis factor alpha (TNFalpha) and interleukin-6 (IL-6) by activated monocytes. Accordingly, anti-P antibodies also up-regulated the expression of TNFalpha and IL-6 messenger RNA in activated monocytes. Of note, F(ab')(2) fragments of anti-P antibodies, which do not result in Fcgamma receptor (FcgammaR) crosslinking, also effectively up-regulated the expression of TNFalpha and IL-6. These results indicate that human peripheral blood monocytes express the ribosomal P epitope upon activation, irrespective of induction of apoptosis. Moreover, the data suggest that anti-P antibodies might modify a variety of inflammatory responses through up-regulation of the expression of proinflammatory cytokines in monocytes, in a manner that does not involve FcgammaR crosslinking.

Mesenchymal Stem Cells (MSC) Display Coordinated Rolling and Adhesion Behaviour on Endothelial Cells under Shear Flow.

Although MSC have been applied in a number of transplantation protocols, the mechanisms which are responsible for their extravasation and tissue specific homing are less well understood. To elucidate the potential of MSC to undergo coordinated interaction with endothelial cells, we investigated MSC under controlled shear flow using a parallel plate flow chamber. MSC were isolated from bone marrow of 6 different donors, depleted of hematopoietic cells by plastic adherence to yield >99% CD45− populations and further expanded up to 106 fold in DMEM/FCS/bFGF over 35 days. Their osteogenic, chondrogenic and adipogenic differentiation potential was maintained during the expansion period. Flow cytometric analysis showed that MSC expressed integrins alpha 1, alpha 4, alpha 5 and beta 1, the chemokine receptor CCR6 and, in a small subpopulation also CXCR4, but that they did not express beta 2 integrin, L-selectin or PSGL-1. During continuous laminar flow on early passage HUVEC at wall shear stresses between 0.1 and 1 dyn/cm2, MSC extended cytoplasmic podia towards endothelium, resulting in capturing and subsequent rolling. This resulted in firm adherence of MSC which resisted shear stresses of 2 dyn/cm2 for >20min. This was, in a similar way, also observed with peripheral blood mononuclear cells or CD34+ primary hematopoietic progenitor cells, whereas erythrocytes and primary endothelial cells did not interact with the HUVEC monolayer. Shear stresses of 2 dyn/cm² and more resulted in a decrease of MSC rolling. Prestimulation of endothelial cells with TNF-alpha increased both MSC rolling (3.7-fold ± 0.6 fold) and firm adhesion (3.3 ± 0.3 fold; means ± SD, n=5; p<0.05). Preincubation of endothelial cells with function-blocking anti-P selectin antibodies reduced the number of rolling MSC by 37.7% ± 5.9% and of firmly adhering MSC by 54.6% ± 11.3% (means ± SD; n=4; p<0.05). Although preincubation with function-blocking antibodies against PSGL-1 did not detectably affect MSC rolling and adhesion, preincubation with O-sialoglycoprotease and with fucosidase but not with neuaminidase significantly reduced MSC rolling and adhesion, indicating that a selectin-type ligand is responsible for the binding. Preincubation of MSC with function-blocking anti-beta 1 integrin antibody also led to a statistically significant reduction of MSC firm adhesion by 21.3% ± 5.7%. Taken together, our results demonstrate that MSC can undergo coordinated rolling and adhesion on endothelium under shear flow involving (i) stimulation in a pro-inflammatory environment, (ii) engagement of P-selectin and a fucosylated ligand on MSC inducing capturing and rolling, and (iii) activation of beta 1 integrins mediating firm adhesion to endothelial cells

Production and characterization of a human monoclonal anti-idiotype to anti-ribosomal P antibodies

Autoantibodies to ribosomal P protein (anti-P) are a specific hallmark of systemic lupus erythematous (SLE). Several authors found significant associations of anti-P antibodies with neuropsychiatric, hepatic, and renal disease. We now report the isolation by phage display of human anti-idiotype (Id) monoclonal antibody fragments as single-chain Fv fragment (scFv) against anti-P antibodies. The V gene repertoires were derived from the RNA obtained from the B cells of a SLE patient. Affinity-purified anti-P antibodies were used for the selection of bacterial clones producing anti-P-specific scFv antibody fragments and little reactivity with normal IgG and other IgG antibodies. The anti-Id antibody recognizes a public idiotope broadly cross-reactive with polyclonal anti-P antibodies and inhibited binding of anti-P to ribosomal P antigen in immunoassays and on Jurkat cells. The anti-Id scFv antibody fragment may have therapeutic implications in SLE. They may also be used as probes in the study of the structure of the idiotype.

Characterization of ribosomal P autoantibodies in relation to cell destruction and autoimmune disease.

Autoantibodies against the ribosomal P proteins are related to cell death and tissue destruction and are frequently exhibited in patients with systemic lupus erythematosus (SLE). In an attempt to explore the effect of tissue destruction on the induction of anti-P autoantibodies, we searched for anti-P autoantibodies by enzyme-linked immunosorbent assay in 201 antinuclear antibody (ANA)-positive individuals, in 10 patients with treated kidney SLE and in 45 acute leukaemia patients undergoing intensive chemotherapy. The autoantibody reactivity was further characterized using one- and two-dimensional immunoblot analysis and immunofluorescence. Anti-P were detected in 5.5% (11/201) of ANA-positive individuals, but not in kidney-affected SLE patients or in patients with leukaemia. Seven of 11 anti-P-positive patients had SLE (3/11), primary Sjögrens's syndrome (1/11) and other autoimmune diseases (3/11). A relation between disease activity and anti-P was suggested by follow-up examinations in one SLE patient, supported by the absence of anti-P autoantibodies in the 10 treated kidney SLE patients. Anti-P autoantibodies were detected by immunoblot in one patient with SLE indicating anti-P2 predominance and in the patient with Sjögrens's syndrome indicating anti-P1 predominance. Diverging humoral responses in these ANA- and anti-P-positive patients were further illustrated by immunofluorescence, elucidating varying nuclear reactivity and anti-P pattern. The observation of anti-P in individuals with active autoimmune disease, but not in patients with chemotherapy-induced cell damage, suggests that anti-P antibodies are part of a specific disease process, and not elicited as a response to cell destruction per se.