A cross-sectional study on smartphone uses among pregnant women attending childbirth classes in the Metropolitan Area of Palermo, Italy: The Stop-Phone study.

Background: Prevalence of mobile device addiction has increased over the years; both women and men have assimilated the mobile phone as a central component of their personal existence: integrating it into their lifestyle or becoming so dependent on it that life without it has become unimaginable. Smartphones generate radio-frequency electromagnetic fields. While short-term exposure in adults was considered quite safe, effects of long-term exposure or exposure during pregnancy on fetuses or during breastfeeding on newborns are not well studied yet. The objective of the present study was to investigate the prevalence and usage characteristics of smartphones among a sample of pregnant women, and promote the correct and conscious use of the smartphone. Methods: A cross-sectional study was conducted, with a questionnaire administered during childbirth classes and - after the questionnaire administration - an educational intervention focused on promoting the correct and conscious use of smartphones was carried out by psychologists and psychotherapists. Results: The findings of our study suggest that a significant number of the participants suffered addiction to mobile phone usage, but were not aware of it. More than two third of the sample (67.2%) have not changed their smartphone use habits since the beginning of their pregnancy and even more significant data shows that almost all future moms (98.3%) never speak with their doctor about smartphone use during pregnancy. Conclusions: Data collected suggest a lack of attention to the proposed topic, especially in relation to pregnancy. It seems necessary to sensitize future mothers on this topic. The promotion of a more conscious and controlled use of electronic devices can help reduce the radiation to which the unborn child may be exposed, but has a fundamental role even after birth, to ensure an adequate psychomotor and relational development of the child and do not affect, due to uncontrolled use of smartphones, the mother-child relationship.

Actions of thrombin in the interstitium.

Thrombin is a pleiotropic enzyme best known for its contribution to fibrin formation and platelet aggregation during vascular hemostasis. There is increasing evidence to suggest a role for thrombin in the development of interstitial fibrosis, but interstitial thrombin has not been demonstrated by the direct determination of activity. Rather its presence is inferred by products of thrombin action such as fibrin and activated fibroblasts. This review will focus on possible mechanisms of thrombin formation in the interstitial space, the possible actions of thrombin, processes regulating thrombin activity in the interstitial space, and evidence supporting a role for thrombin in fibrosis.

Loss of cell surface TFII-I promotes apoptosis in prostate cancer cells stimulated with activated α2 -macroglobulin.

Receptor-recognized forms of α2 -macroglobulin (α2 M) bind to cell surface-associated GRP78 and initiate pro-proliferative and anti-apoptotic signaling. Ligation of GRP78 with α2 M also upregulates TFII-I, which binds to the GRP78 promoter and enhances GRP78 synthesis. In addition to its transcriptional functions, cytosolic TFII-I regulates agonist-induced Ca(2+) entry. In this study we show that down regulation of TFII-I gene expression by RNAi profoundly impairs its cell surface expression and anti-apoptotic signaling as measured by significant reduction of GRP78, Bcl-2, and cyclin D1 in 1-Ln and DU-145 human prostate cancer cells stimulated with α2 M. In contrast, this treatment significantly increases levels of the pro-apoptotic proteins p53, p27, Bax, and Bak and causes DNA fragmentation. Furthermore, down regulation of TFII-I expression activates agonist-induced Ca(2+) entry. In plasma membrane lysates p-PLCγ1, TRPC3, GRP78, MTJ1, and caveolin co-immunoprecipitate with TFII-I suggesting multimeric complexes of these proteins. Consistent with this hypothesis, down regulating TFII-I, MTJ1, or GRP78 expression by RNAi greatly attenuates cell surface expression of TFII-I. In conclusion, we demonstrate that not only does cell surface GRP78 regulate apoptosis, but it also regulates Ca(2+) homeostasis by controlling cell surface localization of TFII-I.

LMP-420: a novel purine nucleoside analog with potent cytotoxic effects for CLL cells and minimal toxicity for normal hematopoietic cells.

B-cell chronic lymphocytic leukemia (CLL) is characterized by slow accumulation of malignant cells, which are supported in the microenvironment by cell-cell interactions and soluble cytokines such as tumor necrosis factor (TNF). We evaluated the effect of the small molecule TNF inhibitor LMP-420 on primary CLL cells. The mean concentration of LMP-420 required to induce 50% cytotoxicity (ED50) at 72 h was 245 n. LMP-420-induced time- and dose-dependent apoptosis, as shown by annexin V staining, caspase activation and DNA fragmentation. These changes were associated with decreased expression of anti-apoptotic proteins Mcl-1, Bcl-xL and Bcl-2. CLL cells from patients with poor prognostic indicators showed LMP-420 sensitivity equal to that for cells from patients with favorable characteristics. In addition, LMP-420 potentiated the cytotoxic effect of fludarabine and inhibited in vitro proliferation of stimulated CLL cells. Gene expression profiling indicated that the mechanism of action of LMP-420 may involve suppression of nuclear factor-kappaB and immune response pathways in CLL cells. LMP-420 had minimal effects on normal peripheral blood mononuclear cell, B- and T-cell function, and hematopoietic colony formation. Our data suggest that LMP-420 may be a useful treatment for CLL with negligible hematologic toxicities.

Inhibition of NF-kappaB1 and NF-kappaB2 activation in prostate cancer cells treated with antibody against the carboxyl terminal domain of GRP78: effect of p53 upregulation.

Ligation of cancer cell surface GRP78 by activated alpha2-macroglobulin (alpha2M*) triggers pro-proliferative and anti-apoptotic signaling pathways. Cancer patients who develop autoantibodies to the alpha2M* binding site in GRP78 have a poor prognosis since these antibodies are receptor agonists. The NF-kappaB family of transcription factors induces expression of genes affecting cell growth and differentiation. NF-kappaB1 plays a major regulatory role in controlling innate immunity and inflammation, whereas NF-kappaB2 plays a greater role in cancer cell proliferation. Here we report that treatment of prostate cancer cells with antibody directed against the carboxyl terminal domain of GRP78 inhibits alpha2M*-induced activation of NF-kappaB2 by approximately 50% while exerting a lesser effect of approximately 20% on NF-kappaB1 activation. Treatment of these cells nearly abolished alpha2M*-induced activation of IKKalpha involved in the activation of NF-kappaB2. This antibody also suppressed alpha2M*-induced phosphorylation of IKKalpha, IKKalpha/beta, IkappaBalpha, and IkappaBbeta as well as levels of NIK. Antibody treatment of cancer cells elevated pro-apoptotic p21WAF and p27kip while reducing cyclin D1 levels. These studies demonstrate that antibody directed against the carboxyl terminal domain of GRP78 inhibits the pro-proliferative NF-kappaB signaling cascade in cancer cells.

Modulation of the unfolded protein response in prostate cancer cells by antibody-directed against the carboxyl-terminal domain of GRP78.

Receptor-recognized forms of alpha(2)-macroglobulin (alpha(2)M*) bind to cancer cell surface GRP78, which functions as a signaling receptor promoting proliferation and survival. Patients with prostate, ovary, and skin cancer may develop auto-antibodies to the alpha(2)M* binding site which are receptor agonists whose presence indicates a poor prognosis. By contrast, antibodies directed against the COOH-terminal domain of GPR78 (anti-CTD antibody), are antagonists which down regulate pro-proliferative signaling and upregulate p53. Unfolded protein response (UPR) signaling plays an important role in cell survival and proliferation as well as apoptosis. We, therefore, studied the effect of anti-CTD antibody on UPR signaling in 1-LN and DU-145 prostate cancer cells. Treatment of these cells, which express GRP78 on their cell surface, with this antibody significantly downregulated IRE1-alpha, PERK, and ATF6alpha-dependent UPR signaling. By contrast, the pro-apoptotic protein GADD153 was elevated. Anti-CTD antibody treatment also elevated apoptotic components, cleaved PARP-1, and Erdj5. In general, a two to threefold effect was observed for the parameters which were studied. These studies suggest that anti-CTD antibody induces growth inhibitory and pro-apoptotic effects by modulating UPR signaling in human prostate cancer cells.

PFT-alpha inhibits antibody-induced activation of p53 and pro-apoptotic signaling in 1-LN prostate cancer cells.

Antibodies against the COOH-terminal domain of cell surface GRP78 induce apoptosis in cancer cell lines via activation of p53 signaling. We now have studied the effects of PFT-alpha, an inhibitor of p53-mediated apoptotic pathways, on anti-GRP78 antibody-induced activation of p53 and pro-apoptotic signaling in 1-LN prostate cancer cells. Pretreatment of 1-LN cancer cells with this agent significantly inhibited antibody or doxorubicin-induced upregulation of p53. Concomitantly, PFT-alpha treatment prevented down regulation of ERK1/2 activation by either antibody or doxorubicin. Likewise, PFT-alpha prevented increases in the pro-apoptotic proteins BAD, BAK, BAX, PUMA, and NOXA as well as activation of caspases-3, -7, and -9. We conclude that antibody-induced apoptosis in prostate cancer cells is mediated predominantly by p53 using the mitochondrial pathway of apoptosis.

Transcription factor TFII-I causes transcriptional upregulation of GRP78 synthesis in prostate cancer cells.

Receptor-recognized forms of alpha(2)-macroglobulin (alpha(2)M*) bind to cell surface-associated GRP78 and induce proliferative and survival signaling in prostate cancer cells. As part of the cellular response to alpha(2)M*, GRP78 expression is itself upregulated. In response to other stimuli, the transcription factor TFII-I upregulates GRP78 by binding to its gene promoter. We have, therefore, studied the role of TFII-I in transcriptional upregulation of GRP78 in 1-LN human prostate cancer cells stimulated with alpha(2)M*. This treatment caused a two- to threefold increase in TFII-I and GRP78 synthesis from [(35)S]-labeled precursor amino acids. Synthesis of both TFII-I and GRP78 were significantly reduced by silencing TFII-I gene expression or pretreatment of cells with genistein or actinomycin D. Confocal microscopy was employed to demonstrate relocation of TFII-I to the nucleus. In alpha(2)M*-stimulated cells, moreover, TFII-I bound to the GRP78 promoter as determined by CHIP assay. We also demonstrate binding of TFII-I to the c-fos promoter, consistent with its role in upregulating c-fos gene expression. In non-lymphoid cells, phosphorylated c-Src is an activator of TFII-I. Ligation of GRP78 on 1-LN cells with alpha(2)M* was followed by tyrosine phosphorylation of c-Src as well as TFII-I. We conclude that alpha(2)M*-induced increase in GRP78 synthesis is caused by transcriptional upregulation of TFII-I which binds to the GRP78 promoter and thus potentiates its cell survival and antipoptotic functions in 1-LN prostate cancer cells.

Amyloid precursor protein and amyloid beta-peptide bind to ATP synthase and regulate its activity at the surface of neural cells.

Amyloid precursor protein (APP) and amyloid beta-peptide (Abeta) have been implicated in a variety of physiological and pathological processes underlying nervous system functions. APP shares many features with adhesion molecules in that it is involved in neurite outgrowth, neuronal survival and synaptic plasticity. It is, thus, of interest to identify binding partners of APP that influence its functions. Using biochemical cross-linking techniques we have identified ATP synthase subunit alpha as a binding partner of the extracellular domain of APP and Abeta. APP and ATP synthase colocalize at the cell surface of cultured hippocampal neurons and astrocytes. ATP synthase subunit alpha reaches the cell surface via the secretory pathway and is N-glycosylated during this process. Transfection of APP-deficient neuroblastoma cells with APP results in increased surface localization of ATP synthase subunit alpha. The extracellular domain of APP and Abeta partially inhibit the extracellular generation of ATP by the ATP synthase complex. Interestingly, the binding sequence of APP and Abeta is similar in structure to the ATP synthase-binding sequence of the inhibitor of F1 (IF(1)), a naturally occurring inhibitor of the ATP synthase complex in mitochondria. In hippocampal slices, Abeta and IF(1) similarly impair both short- and long-term potentiation via a mechanism that could be suppressed by blockade of GABAergic transmission. These observations indicate that APP and Abeta regulate extracellular ATP levels in the brain, thus suggesting a novel mechanism in Abeta-mediated Alzheimer's disease pathology.

Behçet's disease patients present high levels of deglycosylated anti-lipoteichoic acid IgG and high IL-8 production after lipoteichoic acid stimulation.

OBJECTIVES: Lipoteichoic acid (LTA), induces some of the clinical symptoms of Behçet's disease (BD) in a rat animal model. These results led to the hypothesis that LTA may also trigger BD in humans. We investigated the humoral and cellular immune response against LTA and lipopolysaccharide (LPS) in patients with BD, and compared these responses with those of patients with active chronic oral ulcers (OU) and normal controls. METHODS: Samples were obtained from 12 active BD, 12 inactive BD, 12 active OU and 12 normal controls. Anti-LTA, anti-LPS antibodies levels and the capacity of immune complexes anti-LTA IgG-LTA to activate complement were studied. Exposed mannose residues in anti-LTA IgG were analyzed in the four groups. The interleukin-8 (IL-8) production by peripheral blood mononuclear cells cultures after LTA and LPS stimulation was also studied in all groups. RESULTS: The capacity to bind mannan binding protein (MBP) of anti-LTA IgGs was significantly higher in BD and active OU patients relative to normal controls (p < 0.001). However, only active BD patients generated significantly higher levels of C5a than controls (p < 0.0001). The IgGs purified from the sera of BD patients showed a high specificity for LTA from Streptococcus sanguis or Streptococcus faecalis. LTA also stimulates the secretion of IL-8 in peripheral blood mononuclear cells isolated from active BD patients. Anti-LPS IgA and IgG titers were significantly higher only in active OU patients relative to normal controls (p < 0.0018). CONCLUSION: These results suggest a mechanism involving LTA from streptococci in the pathogenesis of BD.

Potentiation of signal transduction mitogenesis and cellular proliferation upon binding of receptor-recognized forms of alpha2-macroglobulin to 1-LN prostate cancer cells.

The alpha2-macroglobulin signalling receptor is upregulated in highly metastatic 1-LN prostate cancer cells. Stimulation of 1-LN cells with activated alpha2-macroglobulin (alpha2M*) caused a two- to threefold increase in [3H]thymidine uptake and cell number. These events require the Ras-dependent MAPK and PI 3-kinase/Akt signalling cascades. Incubation of 1-LN cells with alpha2M* induced Grb2, shc, sos and Raf-1 expression, as well as phosphorylation of MEK 1/2, ERK 1/2, p38 MAPK and JNK. This treatment also increased PI 3-kinase activation, PDK1 expression, Akt phosphorylation and p70s6k phosphorylation. Levels of the early gene products c-fos protein and thymidylate synthase were comparably increased. Exposure of 1-LN cells to alpha2M* significantly raised the levels of phosphorylated CREB by about 15-20 min and phosphorylated p53 by about 60-90 min of incubation. We conclude that the growth regulatory effects of ligating the alpha2M* signalling receptor on 1-LN cells are exerted via the onset and crosstalk between the Ras-dependent MAPK and PI 3-kinase/Akt signalling cascades.

Receptor-associated protein binding blocks ubiquitinylation of the low density lipoprotein receptor-related protein.

The low density lipoprotein receptor-related protein (LRP) consists of two subunits, M(r) approximately 515,000 and 85,000. LRP is a receptor for activated alpha2-macrogobulin (alpha2M*), Pseudomonas exotoxin A, and many other proteins. We now report that ubiquitinylation of the LRP heavy chain occurred when either Pseudomonas exotoxin A or alpha2M* bound to LRP on macrophages. Ubiquitinylation was dose-dependent and maximal about 30 min after ligation of the receptor. Addition of the proteosome inhibitor MG-132 sustained the level of ubiquitin-LRP for longer time intervals in macrophages treated with either alpha2M* or Pseudomonas exotoxin A. By contrast, when receptor associated protein (RAP) bound to LRP, ubiquitinylation did not occur. While RAP is not found in the extracellular environment it binds to LRP and is believed to function as an intracellular chaperone. The presence of RAP within the cell may, therefore, contribute to the recycling of intact LRP which has ligated and internalized its ligands.

Apolipoprotein E and mimetic peptide initiate a calcium-dependent signaling response in macrophages.

Apolipoprotein E (ApoE) is a 34-kDa cholesterol transport protein that also possesses immunomodulatory properties. In this study, we demonstrate that ApoE initiates a signaling cascade in murine peritoneal macrophages that leads to increased production of inositol triphosphate with mobilization of intracellular Ca(2+) stores. This cascade is inhibited by pretreatment with receptor-associated protein and Ni(2+), and it is mediated by a pertussis toxin-sensitive G protein. These properties are characteristic of signal transduction induced via ligand binding to the cellular receptor, lipoprotein receptor-related protein. A peptide derived from the receptor-binding region of ApoE also initiates signal transduction in a manner similar to that of the intact protein, suggesting that this isolated region is sufficient for signal transduction. The ApoE-mimetic peptide competed for binding with the intact protein, confirming that they both interact with the same site. ApoE-dependent signal transduction might play a role in mediating the functional properties of this lipoprotein.

Covalent complexes of antigen and alpha(2)-macroglobulin: evidence for dramatically-increased immunogenicity.

A safe, effective, more potent adjuvant than currently available would be beneficial in developing new therapeutics and diagnostic reagents. We report here a technique for the rapid, efficient incorporation of non-proteolytic antigens into alpha(2)-macroglobulin (alpha(2)M; tradename, SynerVax), allowing us to covalently couple much larger subunit antigens to alpha(2)M than previously possible. Our goal was to determine if incorporation of HB, the monomeric form of Hepatitis B virus (HBV) surface antigen (HBsAg), into alpha(2)M would result in increased immune reactivity. Earlier attempts to immunize animals using HB did not generate significant levels of antibodies. Using HB complexes prepared with alpha(2)M we now report dramatically-increased immunogenicity of HB in BALB/c mice. Combining these soluble complexes with a depot-generating agent (alum), titers>1:1,000,000 are obtained with a single injection. This novel adjuvant technology should provide a valuable tool for the development of either prophylactic and therapeutic vaccines, or monoclonal antibodies against hitherto poorly-immunogenic subunit antigens.

Induction of cyclooxygenase-2 synthesis by ligation of the macrophage alpha(2)-macroglobulin signalling receptor.

We have studied the induction of cyclooxygenase-2 (COX-2) in macrophages consequent to ligating the alpha(2)-macroglobulin (alpha(2)M) signalling receptor (alpha(2)MSR) with receptor-recognized forms of alpha(2)M (alpha(2)M*). Macrophage stimulation with alpha(2)M* increased total cellular and nuclear COX-2 two- to threefold. The maximal increase in COX-2 occurred at a ligand concentration of 50-100 pM and after 2 h. Modulation of intracellular Ca(2+) levels or incubation of [35S] methionine-labelled macrophages with actinomycin D, prior to treatment with alpha(2)M*, markedly reduced the induction of total cellular and nuclear COX-2. Protein kinase C (PKC) or phospholipase A(2) (PLA(2)) inhibition in alpha(2)M*-stimulated macrophages or inhibition of the p21(ras)-dependent mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI 3-kinase) signalling pathways also significantly reduced alpha(2)M*-induced total cellular and nuclear COX-2 expression. Thus, COX-2 induction is dependent on cPLA(2) activity, Ca(2+) mobilization, and PKC activity and requires participation of both the p21(ras)-dependent MAPK and PI 3-kinase signalling pathways. COX-2 activation may mediate alpha(2)M*-induced mitogenesis, which we have previously observed in this and other cell types.

Kinetics of nonproteolytic incorporation of a protein ligand into thermally activated alpha 2-macroglobulin: evidence for a novel nascent state.

We have previously shown that antigens complexed to the receptor-recognized form of alpha(2)-macroglobulin (alpha(2)M*) demonstrate enhanced immune responsiveness mediated by the low density lipoprotein receptor-related protein LRP/CD91. Recently, we developed a proteinase-independent method to covalently bind antigens to alpha(2)M*. Given the potential applications of this chemistry, we analyzed the kinetics, thermodynamics, and pH dependence of this reaction. The incorporation of lysozyme into alpha(2)M* was a mixed bimolecular second-order reaction with a specific rate constant of 91.0 +/- 6.9 m(-1) s(-1), 50.0 degrees C, pH 7.4. The activation energy, activation entropy, and Gibbs' free energy at 50.0 degrees C were 156 kJ mol(-1), 266 J mol(-1) K(-1), and 70 kJ mol(-1), respectively. The rate of incorporation increased as a function of pH from pH 5.0 to 7.0 and was unchanged thereafter. Furthermore, the reaction between alpha(2)M* and lysozyme was irreversible. The data are consistent with a two-step mechanism. In the first step, alpha(2)M* reforms its thiol ester bond, entering a reactive state that mimics the proteolytically induced "nascent state." In the rate-limiting second step, the reformed bond quickly undergoes nucleophilic attack by lysozyme. The kinetic equations derived in this study are the basis for optimizing the formation of stable alpha(2)M*.antigen complexes.

Inducible expression of the alpha2-macroglobulin signaling receptor in response to antigenic stimulation: a study of second messenger generation.

Thioglycollate (TG)-elicited murine, peritoneal macrophages express two receptors for activated forms of the proteinase inhibitor alpha2-macroglobulin (alpha2M*)--namely, the low density lipoprotein receptor-related protein (LRP) and the alpha2M signaling receptor (alpha2MSR). We now report that resident peritoneal macrophages express only 400+/-50 alpha2MSR receptors/cell compared to 5000+/-500 receptor/TG-elicited macrophage. By contrast, LRP expression is only 2-2.5-fold greater on elicited cells. The low level of alpha2MSR expression by resident cells is insufficient to trigger signal transduction in contrast to TG-elicited cells which when exposed to alpha2M* demonstrate a rapid rise in inositol 1,4,5-trisphosphate and a concomitant increase in cytosolic free Ca2+. We then studied a variety of preparations injected subcutaneously for their ability to upregulate alpha2MSR. Macroaggregated bovine serum albumin (macroBSA) injection upregulated alpha2MSR and triggered signaling responses by splenic macrophages. Nonaggregated BSA injection alone or in the presence of alum, by contrast, did not alter alpha2MSR expression. Recombivax (hepatitis B antigen adsorbed to alum) injection also upregulated alpha2MSR on splenic macrophages while the alum carrier had no effect. We conclude that macrophage alpha2M* receptors are inducible and their expression may be regulated, in part, by potential antigens.

Unusual brain growth patterns in early life in patients with autistic disorder: an MRI study.

OBJECTIVE: To quantify developmental abnormalities in cerebral and cerebellar volume in autism. METHODS: The authors studied 60 autistic and 52 normal boys (age, 2 to 16 years) using MRI. Thirty autistic boys were diagnosed and scanned when 5 years or older. The other 30 were scanned when 2 through 4 years of age and then diagnosed with autism at least 2.5 years later, at an age when the diagnosis of autism is more reliable. RESULTS: Neonatal head circumferences from clinical records were available for 14 of 15 autistic 2- to 5-year-olds and, on average, were normal (35.1 +/- 1.3 cm versus clinical norms: 34.6 +/- 1.6 cm), indicative of normal overall brain volume at birth; one measure was above the 95th percentile. By ages 2 to 4 years, 90% of autistic boys had a brain volume larger than normal average, and 37% met criteria for developmental macrencephaly. Autistic 2- to 3-year-olds had more cerebral (18%) and cerebellar (39%) white matter, and more cerebral cortical gray matter (12%) than normal, whereas older autistic children and adolescents did not have such enlarged gray and white matter volumes. In the cerebellum, autistic boys had less gray matter, smaller ratio of gray to white matter, and smaller vermis lobules VI-VII than normal controls. CONCLUSIONS: Abnormal regulation of brain growth in autism results in early overgrowth followed by abnormally slowed growth. Hyperplasia was present in cerebral gray matter and cerebral and cerebellar white matter in early life in patients with autism.

Endothelial cell surface F1-F0 ATP synthase is active in ATP synthesis and is inhibited by angiostatin.

Angiostatin blocks tumor angiogenesis in vivo, almost certainly through its demonstrated ability to block endothelial cell migration and proliferation. Although the mechanism of angiostatin action remains unknown, identification of F(1)-F(O) ATP synthase as the major angiostatin-binding site on the endothelial cell surface suggests that ATP metabolism may play a role in the angiostatin response. Previous studies noting the presence of F(1) ATP synthase subunits on endothelial cells and certain cancer cells did not determine whether this enzyme was functional in ATP synthesis. We now demonstrate that all components of the F(1) ATP synthase catalytic core are present on the endothelial cell surface, where they colocalize into discrete punctate structures. The surface-associated enzyme is active in ATP synthesis as shown by dual-label TLC and bioluminescence assays. Both ATP synthase and ATPase activities of the enzyme are inhibited by angiostatin as well as by antibodies directed against the alpha- and beta-subunits of ATP synthase in cell-based and biochemical assays. Our data suggest that angiostatin inhibits vascularization by suppression of endothelial-surface ATP metabolism, which, in turn, may regulate vascular physiology by established mechanisms. We now have shown that antibodies directed against subunits of ATP synthase exhibit endothelial cell-inhibitory activities comparable to that of angiostatin, indicating that these antibodies function as angiostatin mimetics.

alpha(2)-Macroglobulin from rheumatoid arthritis synovial fluid: functional analysis defines a role for oxidation in inflammation.

A hallmark of inflammation is the release of oxidants, proteinases, and cytokines, all important mediators of the inflammatory cascade. alpha(2)-Macroglobulin (alpha(2)M) is a high-affinity, broad-specificity proteinase inhibitor that also binds and regulates the biological activities of a number of cytokines. We demonstrated recently that hypochlorite-oxidized alpha(2)M has decreased ability to inhibit proteinases and regulate cytokines in vitro. The role of oxidation in regulating alpha(2)M functions in vivo is largely unknown. To determine the extent and biological consequence of in vivo alpha(2)M oxidation, we measured the degree of oxidative alpha(2)M modification from rheumatoid arthritis (RA) synovial fluid and compared this with osteoarthritis (OA) as noninflammatory controls. We found that RA synovial fluid alpha(2)M is significantly more oxidized than that from OA. RA synovial fluid also contains a twofold higher median alpha(2)M level than OA, while having only half the alpha(2)M-proteinase inhibitory activity. Detailed biochemical analysis demonstrates proteolytically degraded alpha(2)M in RA greater than in OA synovial fluid. Additionally, the hypochlorite-mediated oxidation product, chlorotyrosine, is present in RA more than in OA or plasma alpha(2)M samples. Taken together, these findings confirm a role for oxidative regulation of inflammation by altering the functions of extracellular mediators such as alpha(2)M.