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Complement sensitizes pathogens for phagocytosis and lysis. We use electron microscopy to examine the structural transitions in the activation of the pivotal protein in the complement pathway, C3. In the cleavage product C3b, the position of the thioester domain moves ≈100 Å, which becomes covalently coupled to antigenic surfaces. In the iC3b fragment, cleavage in an intervening domain creates a long flexible linker between the thioester domain and the macroglobulin domain ring of C3. Studies on two products of nucleophile addition to C3 reveal a structural intermediate in activation, and a final product, in which the anaphylatoxin domain has undergone a remarkable movement through the macroglobulin ring. [ABSTRACT FROM AUTHOR]

The four mammalian bone morphogenetic protein 1 (BMP1)- like proteinases act to proteolytically convert procollagens to the major fibrous components of the extracellular matrix. They also activate lysyl oxidase, an enzyme necessary to the covalent cross-linking that gives collagen fibrils much of their tensile strength. Thus, these four proteinases are attractive targets for interventions designed to limit the excess formation of fibrous collagenous matrix that characterizes fibrosis. Although it has previously been reported that the serum protein α2-macroglobulin is unable to inhibit the astacin-like proteinases meprin a and meprin β, we herein demonstrate α2-macroglobulin to be a potent inhibitor of the similar BMP1-like proteinases. BMP1 is shown to cleave the α2-macroglobulin "bait" region, at a single specific site, which resembles the sites at which BMP1-like proteinases cleave the C-propeptides of procollagens I-Ill. α2-Macroglobulin is an irreversible inhibitor that is shown to bind bone morphogenetic protein 1 in a covalent complex. It is also demonstrated that genetically modified α2-macroglobulin, in which the native bait region is replaced by sequences flanking the probiglycan BMP1 cleavage site, is enhanced ~24-fold in its ability to inhibit BMP1, and is capable of inhibiting the biosynthetic processing of procollagen I by cells. These findings suggest possible therapeutic interventions involving ectopic expression of modified versions of α2-macroglobulin in the treatment of fibrotic conditions. [ABSTRACT FROM AUTHOR]

Given the importance of the low density lipoprotein receptor-related protein (LRP) as an essential endocytosis and signaling receptor for many protein ligands, and of α2-macroglobulin (α2M)-proteinase complexes as one such set of ligands, an understanding of the specificity of their interaction with LRP is an important goal. A starting point is the known role of the 138-residue receptor binding domain (RBD) in binding to LRP. Previous studies have localized high affinity α2M binding to the eight complement repeat (CR)-containing cluster 2 of LRP, In the present study we have identified the minimum CR domains that constitute the full binding site for RBD and, hence, for α2M on LRP. We report on the ability of the triple construct of CR3- 4-5 to bind RBD with an affinity (K2 = 130 nM) the same as for isolated RBD to intact LRP. This K2 is 30-fold smaller than for RBD to CR 5-6-7, demonstrating the specificity of the interaction with CR3-4-5. Binding requires previously identified critical lysine residues but is almost pH-independent within the range of pH values encountered between extracellular and internal compartments, consistent with an earlier proposed model of intracellular ligand displacement by intramolecular YWTD domains. The present findings suggest a model to explain the ability of LRP to bind a wide range of structurally unrelated ligands in which a nonspecific ligand interaction with the acidic region present in most CR domains is augmented by interactions with other CR surface residues that are unique to a particular CR cluster. [ABSTRACT FROM AUTHOR]

Resistance to infection and clearance of cell debris in mammals depend on the activation of the complement system, which is an important component of innate and adaptive immunity. Central to the complement system is the activated form of C3, called C3b, which attaches covalently to target surfaces to amplify complement response, label cells for phagocytosis and stimulate the adaptive immune response. C3b consists of 1,560 amino-acid residues and has 12 domains. It binds various proteins and receptors to effect its functions. However, it is not known how C3 changes its conformation into C3b and thereby exposes its many binding sites. Here we present the crystal structure at 4-Å resolution of the activated complement protein C3b and describe the conformational rearrangements of the 12 domains that take place upon proteolytic activation. In the activated form the thioester is fully exposed for covalent attachment to target surfaces and is more than 85 Å away from the buried site in native C3 (ref. 5). Marked domain rearrangements in the α-chain present an altered molecular surface, exposing hidden and cryptic sites that are consistent with known putative binding sites of factor B and several complement regulators. The structural data indicate that the large conformational changes in the proteolytic activation and regulation of C3 take place mainly in the first conversion step, from C3 to C3b. These insights are important for the development of strategies to treat immune disorders that involve complement-mediated inflammation. [ABSTRACT FROM AUTHOR]

The complement system is a key part of the innate immune system, and is required for clearance of pathogens from the bloodstream. After exposure to pathogens, the third component of the complement system, C3, is cleaved to C3b which, after recruitment of factor B, initiates formation of the alternative pathway convertases. CRIg, a complement receptor expressed on macrophages, binds to C3b and iC3b mediating phagocytosis of the particles, but it is unknown how CRIg selectively recognizes proteolytic C3-fragments and whether binding of CRIg to C3b inhibits convertase activation. Here we present the crystal structure of C3b in complex with CRIg and, using CRIg mutants, provide evidence that CRIg acts as an inhibitor of the alternative pathway of complement. The structure shows that activation of C3 induces major structural rearrangements, including a dramatic movement (>80 Å) of the thioester-bond-containing domain through which C3b attaches to pathogen surfaces. We show that CRIg is not only a phagocytic receptor, but also a potent inhibitor of the alternative pathway convertases. The structure provides insights into the complex macromolecular structural rearrangements that occur during complement activation and inhibition. Moreover, our structure–function studies relating the structural basis of complement activation and the means by which CRIg inhibits the convertases provide important clues to the development of therapeutics that target complement. [ABSTRACT FROM AUTHOR]

Many benthic marine invertebrates, like barnacles, have a planktonic larval stage whose primary purpose is dispersal. How these species colonize suitable substrata is fundamental to understanding their evolution, population biology, and wider community dynamics. Unlike larval dispersal, settlement occurs on a relatively small spatial scale and involves larval behavior in response to physical and chemical characteristics of the substratum. Biogenic chemical cues have been implicated in this process. Their identification, however, has proven challenging, no more so than for the chemical basis of barnacle gregariousness, which was first described >50 years ago. We now report that a biological cue to gregarious settlement, the settlement-inducing protein complex (SIPC), of the major fouling barnacle Balanus amphitrite is a previously undescribed glycoprotein. The SIPC shares a 30% sequence homology with the thioester-containing family of proteins that includes the α2-macroglobulins. The cDNA (5.2 kb) of the SIPC encodes a protein precursor comprising 1,547 aa with a 17-residue signal peptide region. A number of structural characteristics and the absence of a thioester bond in the SIPC suggest that this molecule is a previously undescribed protein that may have evolved by duplication from an ancestral α2-macroglobulin gene. Although the SIPC is regarded as an adult cue that is recognized by the cyprid at settlement, it is also expressed in the juvenile and in larvae, where it may function in larva-larva settlement interactions. [ABSTRACT FROM AUTHOR]

The differential diagnosis between multiple myeloma (MM) and Waldenström's macroglobulinemia (WM) is generally well defined. Consistent with a diagnosis of MM is the presence of a non-IgM monoclonal gammopathy associated to multiple osteolytic lesions and plasma cell infiltration of the bone marrow. Characteristic of WM is the presence of an IgM monoclonal gammopathy associated to lymphoadenopathy, hepatosplenomegaly, anemia and hyperviscosity syndrome in the presence of a monoclonal lymphoplasmacytoid proliferation in the bone marrow. Nonetheless, few cases of IgM myeloma have been reported that display clinico-pathologic features intermediate between MM and WM. Here, this study describes four of 317 (1.2%) patients with an IgM monoclonal gammopathy in whom the morphologic and clinical features were consistent with a diagnosis of IgM myeloma. [ABSTRACT FROM AUTHOR]

α2-Macroglobulin (α2M) inhibits diverse extracellular proteases, binds growth factors such as platelet-derived growth factor-BB (PDGF-BB) and transforming growth factor-β1 (TGF-β1), and carries β-amyloid peptide. α2M may also trigger cell signaling by binding to the low density lipoprotein receptor-related protein (LRP-1) and/or other cell surface receptors. Based on studies with recombinant α2M fragments expressed in bacteria and synthetic peptides, we previously localized a growth factor-binding site near the center of the α2M subunit. However, because intact α2M forms a hollow cylinder structure, an alternative model for growth factor binding involves nonspecific entrapment within the α2M core. To distinguish between these two models, we engineered mutations in the putative growth factor binding sequence of full-length α2M. These mutations did not perturb the tetrameric structure of α2M, reaction with proteases, the thiol ester bonds, or binding to LRP-1. A single mutation (E730R) completely blocked binding of platelet-derived growth factor-BB to intact α2M. E730R did not alter TGF-β1 binding; however, this mutation in combination with mutations at Glu714 and Asp719 eliminated the increase in TGF-β1 binding associated with α2M conformational change. These studies demonstrate that growth factor binding to intact α2M is specific, involving a defined region of the α2M subunit. The exact sequences required for binding different growth factors may be non-identical, mimicking the model of the bait region in which different proteases target adjacent and sometimes overlapping sequences, [ABSTRACT FROM AUTHOR]

Interaction between a serine proteinase from bovine duodenum and human serum α2-macroglobulin (α2-MG) was studied. α2-MG is established to be one of the most effective duodenase inhibitors. The enzyme is completely inhibited in less than 30 sec at equimolar ratio of the inhibitor and enzyme (concentration 2·10−8 M). Under identical conditions, the rate of duodenase association with α2-MG is at least 2.5-fold higher than the rate of chymotrypsin association with this inhibitor. The interaction with duodenase results in proteolysis of the inhibitor subunit in the “bait region”. Similarly to other proteases, duodenase in the complex with α2-MG retains the intact catalytic apparatus and ability to hydrolyze some small substrates. But the duodenase-inhibitor complex is fully inactive to proteins (bovine serum albumin). The stoichiometry of the enzyme interaction with the inhibitor is 2: 1 (mol/mol). Based on the association rate constant and the termination time of the duodenase and α2-MG in vivo association, α2-MG is suggested to be a physiological regulator of the enzyme. [ABSTRACT FROM AUTHOR]

Abstract: There has been a lack of consensus among results of assays for insulin autoantibody (IAA) carried out by different laboratories, despite a reduction in the non-specific effect using a cold insulin competitor in radioimmunoassays (RIAs) for IAA in type I diabetes. We speculated that the discrepancies are partly a result of the non-specific binding (NSB) of [125I]insulin to unidentified molecules in serum on polyethylene glycol separation, and tried to improve IAA RIAs. The molecular weight of a candidate for the factor causing NSB was estimated to be about 700kDa by gel filtration analysis, resembling that of alpha 2-macroglobulin (a2M). Further, the addition of purified a2M to the assay resulted in an increase in NSB. Screening revealed that heterocyclic compounds, such as isothiazolinone derivatives (ProClin300), were greatly effective at reducing NSB in control subjects from 2.904±0.909% to 1.347±0.254% (n =283, mean±SD, p <0.0001). Using our newly developed IAA RIA with ProClin300, the sensitivity for newly diagnosed type I diabetes patients (n =55) was 32.7% and 30.9% with or without insulin competition, respectively, whereas that of the former assay without ProClin300 was only 20.0%. [Copyright &y& Elsevier]

In the course of a large scale analysis of late-expressed genes in the human epidermis, we identified a new member of the α2-macroglobulin (α2M) protease inhibitor family, A2ML1 (for α2-macroglobulin-like 1). Like A2M and PZP, A2ML1 is located on chromosome 12p13.31. A2ML1 encodes a protein of 1454 amino acids, which fits the characteristics of α2Ms: 1) strong conservation in amino acid sequence including most of cysteine positions with α2M; 2) a putative central bait domain; 3) a typical thiol ester sequence. Northern blot and reverse transcriptase-PCR studies revealed a single 5-kb A2ML1 mRNA, mainly in the epidermis granular keratinocytes. A2ML1 is also transcribed in placenta, thymus, and testis. By Western blot analysis, α2ML1 is detected as a monomeric, ∼180-kDa protein in human epidermis. In vitro keratinocyte differentiation is associated with increased expression levels. By immunohistochemistry, α2ML1 was detected within keratinosomes in the granular layer of the epidermis, and as a secreted product in the extracellular space between the uppermost granular layer and the cornified layer. Recombinant α2ML1 displayed inhibitory activity toward chymotrypsin, papain, thermolysin, subtilisin A, and to a lesser extent, elastase but not trypsin. Incubation with chymotrypsin and the chymotrypsin-like kallikrein 7 protease indicated that α2ML1 binds covalently to these proteases, a feature shared with other members of the family. Therefore, α2ML1 is the first α2M family member detected in the epidermis. It may play an important role during desquamation by inhibiting extracellular proteases. [ABSTRACT FROM AUTHOR]

Abstract: Plasma extravasation is a common endothelium response to tissue injury provoked by pathogens. Herein I will review studies showing that host proteinase inhibitors (e.g., α2-macroglobulin (α2M) or kininogens) interact with protozoan cysteine proteinases (CPs) in extravascular infection sites, linking inflammation to innate immunity by different mechanisms. Using human monocytes as antigen presenting cells, we first demonstrated that α2M entrapment of cruzipain, a Trypanosoma cruzi CP, reduced the activation threshold of cruzipain-specific CD4 T cells due to facilitated uptake of α2M–cruzipain complexes by the multiscavenger receptor (CD91). More recently, studies of the mechanisms underlying inflammation elicited by T. cruzi revealed that kininogens, once bound to glycosaminoglycans, are not able to efficiently inactivate cruzipain via their inhibitory cystatin-like domains. Instead, we found that cruzipain readily processes surface-bound kininogens, liberating bioactive kinins. Acting as paracrine hormones, kinins vigorously activate host cells through bradykinin (BK) receptors, thus stimulating endocytic uptake of the pathogen. Rather than unilaterally enhancing parasite infectivity, the liberated kinins activate innate immunity by potently stimulating dendritic cell maturation via the BK B2 receptor. The discovery of chagasin, a novel family of endogenous inhibitors expressed by trypanosomatids, is likely another regulatory player involved in the dynamics of the inflammatory response. [Copyright &y& Elsevier]

Proteins of the macroglobulin family are an ancient and evolutionarily conservative link of the immune system, which is actively involved in both inhibition of tumor growth cells and proliferation of tumor cells. Two basically different binding sites and a great conformational plasticity of all representatives of the macroglobulin family, as well as the presence of two to four representatives of the family in the blood of most species allow them to transport diverse substances and exert various regulatory influences on both the tumor and the entire organism. For example, the capacity of macroglobulins for binding hydrolases makes it possible to inhibit enzyme mediated tumor invasion. At the same time, an excess of macroglobulin/hydrolase complexes can activate apoptosis. The tumor is able of using macroglobulins, especially pregnancy-associated proteins, for its own protection. Specifically, pregnancy-associated α2-glycoprotein, which is actively produced by human tumor cells, blocks the his to compatibility complex antigens. On the contrary, the capacity of binding zinc stimulates the thymulin-dependent activation of natural killer cells. Nevertheless, the actively growing tumor expresses many receptors to macroglobulins, which are the main carriers of some cytokines and growth factors essential for proliferation. [ABSTRACT FROM AUTHOR]

Abstract: α2-Macroglobulin is a major serum protein with diverse functions, including inhibition of protease activity and binding of growth factors, cytokines, and disease factors. We have cloned and characterized Panza, a new Xenopus laevis α2-macroglobulin. Panza has 56-60% amino acid similarity with previously identified Xenopus, mouse, rat and human α2-macroglobulins, indicating that Panza is a new member of the α2-macroglobulin family. Panza mRNA is first detected at the beginning of neurulation in the dorsal endoderm lining the primitive gut (archenteron roof). At the completion of neurulation and continuing through the late tadpole stage, Panza is restricted to a dorsal domain of the gut endoderm adjacent to the notochord and extending along the entire anterior–posterior axis. With outgrowth of the tailbud, Panza expression persists in the chordaneural hinge at the posterior end of the differentiating notochord and extends into the floor plate of the posterior neural tube. As gut coiling commences, Panza expression is initiated in the liver, and the dorsal domain of Panza expression becomes limited to the midgut and hindgut. With further gut coiling, strong Panza expression persists in the liver, but is lost from other regions of the gut. The expression of Panza in endodermal cells adjacent to the notochord points to a potential role for Panza in signal modulation and/or morphogenesis of the primitive gut. [Copyright &y& Elsevier]

We have characterized native and activated forms of rabbit α1M and compared them to rabbit and human α2M. Similar to human α2M, rabbit α1M is a tetramer associated via disulfide bonds and non-covalent interactions that exhibits autolysis into two fragments when heated. Like human α2M, rabbit α1M is cleaved by trypsin at one site; however, rabbit α1M shares characteristics with rabbit α2M that are different from the properties of human α2M. Amine or trypsin treatment of rabbit α-macroglobulins does not result in a significant conformational change or cleavage of four thiolester bonds. Full thiolester cleavage is only observed for rabbit α1M after exposure to both trypsin and a small amine. Additionally, amine-treated rabbit α-macroglobulins retain trypsin inhibitory potential and do not fully shield bound proteinases. Methylamine and trypsin treatment of rabbit α1M results in two dissimilar conformations that display differing exposure of the receptor-recognition site. While ammonia- and methylamine-modified rabbit α1M bind to macrophages with similar affinity to that of human α2M, trypsin-treated rabbit α1M exhibits dramatically lower affinity. This suggests that rabbit α1M may not play the same proteinase-inhibiting physiological role as human α2M. [ABSTRACT FROM PUBLISHER]

In this work we evaluate the proteolytic state of plasma α2-macroglobulin in acute pancreatitis. In addition, the plasma activity of matrix metalloproteinase-2 (MMP-2), MMP-9 and serine proteinases were analyzed. A total of 33 patients with acute pancreatitis were studied, of whom 16 were diagnosed as having mild and 17 as having severe acute pancreatitis. In the latter group, three patients progressed to multi-organ failure and died as a consequence of these complications. The proteolytic fragmentation of α2-macroglobulin was evaluated by Western blotting, whereas the plasma activity of MMP-2, MMP-9 and serine proteinases was evaluated by gelatin zymography. Enhanced fragmentation of α2-macroglobulin was detected in severe acute pancreatitis patients with multiple organ failure and lethal complications. In this same patient group, increased plasma activity of the active forms of MMP-2 and MMP-9, as well as serine proteinases, was apparent. In addition, we demonstrate that chymotrypsin-like proteinases could be the principal cause of α2-macroglobulin degradation in this group of patients. Our results indicate that secondary proteolysis of α2-macroglobulin promotes impaired control of extracellular proteolytic activity, leading to local and distant tissue injuries during severe acute pancreatitis. Finally, the structural evaluation of plasma α2-macroglobulin could be used as a prognostic marker of the severity of acute pancreatitis. [ABSTRACT FROM AUTHOR]

Background: Low-density lipoprotein receptor-related protein 1 (LRP1) and alpha-2-macroglobulin (A2M) are two plausible candidate genes for Alzheimer disease (AD) based on their important biological function and positional information. To date, numerous studies have investigated their possible association with AD but the results are controversial. Methods: To investigate the potential genetic contribution of the two genes in the Han Chinese population, we performed a case-control association study using 10 polymorphisms (4 in LRP1 and 6 in A2M) that span approximately the whole corresponding gene. Results: Comparison of allele, genotype, and haplotype frequencies for polymorphisms in A2M revealed no significant differences between patients and control subjects. For the LRP1 gene, however, we found an overrepresentation of the CTCG haplotype in the control group (p = .002). The difference was still of statistical significance in the apolipoprotein E (APOE) epsilon 4 negative subjects (pCTCG = .003). Multiple logistic regression analysis did not show any evidence of synergism between A2M, LRP1, and APOE. Conclusions: Our results indicate that the CTCG haplotype of LRP1 may reduce the risk of late-onset AD, but A2M is not associated with this disease in the Han Chinese population. [Copyright &y& Elsevier]

A case-control study was nested within two maternity cohorts with a total of 7 million years of follow-up for assessment of the role of bacterial infections in childhood leukemia. Offspring of 550,000 mothers in Finland and Iceland were combined to form a joint cohort that was followed for cancer up to age 15 years during 1975–1997 through national cancer registries. For each index mother-case pair, three or four matched control mother-control pairs were identified from population registers. First-trimester serum samples were retrieved from mothers of 341 acute lymphoblastic leukemia cases and 61 other leukemia cases and from 1,212 control mothers. Sera were tested for antibodies to the genus Chlamydia, Helicobacter pylori, and Mycoplasma pneumoniae. Odds ratios and 95% confidence intervals, adjusted for sibship size, were calculated as estimates of relative risk. M. pneumoniae immunoglobulin M appeared to be associated with increased risk (odds ratio (OR) = 1.6), but the association lost statistical significance when the specificity of the immunoglobulin M was considered (OR = 1.5, 95% confidence interval: 0.9, 2.4). In Iceland, H. pylori immunoglobulin G was associated with increased risk of childhood leukemia in offspring (OR = 2.8, 95% confidence interval: 1.1, 6.9). Since H. pylori immunoglobulin G indicates chronic carriage of the microorganism, early colonization of the offspring probably differs between Iceland and Finland, two affluent countries. [ABSTRACT FROM AUTHOR]

OBJECTIVES: IgM memory B cells that are responsible for the protection against infections by encapsulated bacteria, require the spleen for their generation and/or survival. Since the association between inflammatory bowel disease and functional hyposplenism is well described, our aim was to verify whether IgM memory B cells mirror the reduced splenic function in Crohn's disease and ulcerative colitis patients. METHODS: Peripheral blood samples were obtained from 32 Crohn's disease and 29 ulcerative colitis patients, 33 healthy controls, and 27 splenectomized patients. Perendoscopic intestinal biopsies were also collected from 15 of 32 Crohn's disease patients, 14 of 29 ulcerative colitis patients and 13 of 33 control subjects. Counting of erythrocytes with membrane abnormalities (pitted red cells) was used as an indicator of splenic function and flow cytometry was performed to analyze both peripheral and mucosal B cells. RESULTS: Twelve of 32 Crohn's disease patients and 13 of 29 ulcerative colitis patients had pitted red cell values >4% and were considered to be hyposplenic. In inflammatory bowel disease patients circulating IgM memory B cells were significantly lower than in control subjects. We observed a significant inverse correlation between the frequency of circulating IgM memory B cell and the pitted red cell values in inflammatory bowel disease patients with hyposplenism. To exclude the possibility that the reduction of circulating IgM memory B cells reflected their recruitment in the inflamed bowel mucosa, lamina propria B-cell populations were also characterized. We found that the frequency of IgM memory B cells was similar in the blood and in the lamina propria of the same patient. CONCLUSIONS: Our findings show that peripheral IgM memory B cells are reduced in inflammatory bowel disease patients and this defect seems to be related to the impairment of splenic function. (Am J Gastroenterol 2005;100:1–8) [ABSTRACT FROM AUTHOR]

Bacterial porins enhance the thrombin activity upon chromogen substrate chromozym. Should porin-dependent enhancement of thrombin activity take place also upon fibrinogen in vivo, this might greatly increase the fibrin production which, in turn, might lead to blood vessel obstruction. In this study, we demonstrate fibrin hyperproduction in a simplified coagulative system, consisting of fibrinogen and thrombin-pure molecules, in the presence of bacterial porins. In particular, bacterial porins, in the presence of thrombin, significantly increased the fibrin production compared with thrombin alone. Also, fibrin hyperproduction took place even in the presence of the thrombin inhibitors antithrombin III (AT III) or α2 macroglobulin (α2M). However, the thrombin–fibrinogen reaction in the presence of AT III or α2M did not generate fibrin, unless porins were present. In conclusion, porins not only enhance thrombin activity but also inhibit the antithrombin activity exerted by AT III or α2M. We hypothesize that, because of porins activity, fibrin is largely generated due to thrombin hyperactivation. Moreover, further fibrin is produced by thrombin, which is not blocked by two serpins for the presence of porins. These results might be relevant as to the occurrence of disseminated intravascular coagulation in sepsis by gram-negative bacteria, which are known to produce porins. [ABSTRACT FROM AUTHOR]

Two characteristics of highly malignant cells are their increased motility and secretion of proteinases allowing these cells to penetrate surrounding basement membranes and metastasize. Activation of 21-kDa activated kinases (PAKs) is an important mechanism for increasing cell motility. Recently, we reported that binding of receptor-recognized forms of the proteinase inhibitor α2-macroglobulin (α2M∗) to GRP78 on the cell surface of 1-LN human prostate cancer cells induces mitogenic signalling and cellular proliferation. In the current study, we have examined the ability of α2M∗ to activate PAK-1 and PAK-2. Exposure of 1-LN cells to α2M∗ caused a 2- to 3-fold increase in phosphorylated PAK-2 and a similar increase in its kinase activity toward myelin basic protein. By contrast, the phosphorylation of PAK-1 was only negligibly affected. Silencing the expression of the GRP78 gene, using either of two different mRNA sequences, greatly attenuated the appearance of phosphorylated PAK-2 in α2M∗-stimulated cells. Treatment of 1-LN cells with α2M∗ caused translocation of PAK-2 in association with NCK to the cell surface as evidenced by the coimmunoprecipitation of PAK-2 and NCK in the GRP78 immunoprecipitate from plasma membranes. α2M∗-indueed activation of PAK-2 was inhibited by prior incubation of the cells with specific inhibitors of tyrosine kinases and phosphatidylinositol 3-kinase. PAK-2 activation was accompanied by significant increases in the levels of phosphorylated LIMK and phosphorylated cofilin. Silencing the expression of the PAK-2 gene greatly attenuated the phosphorylation of LIMK. In conclusion, we show for the first time the activation of PAK-2 in 1-LN prostate cancer cells by a proteinase inhibiter, α2-macroglobullin, These studies suggest a mechanism by which α2M∗ enhances the metastatic potential of these cells. [ABSTRACT FROM AUTHOR]

Secreted IgM is a potent adjuvant that concentrates antigen into secondary lymphoid organs and initiates antibody responses, germinal centre formation and is crucial in resolving infections. The current studies investigated the influence of specific IgM on both the quantity and quality of antibody produced in response to T-dependent and T-independent antigens. The addition of IgM to either antigen had no significant effect on the titre or duration of antibody responses. However, the presence of specific IgM led to accelerated affinity maturation when mice were challenged with low doses of IgM-containing immune complexes (IgM-IC) of T-dependent antigens compared with antigen alone. Interestingly, the administration of higher concentrations of IgM-IC increased follicular deposition of antigen but did not result in accelerated affinity maturation or in higher antibody affinities. The administration of IgM complexed with T-independent antigens had no effect on antibody titre, duration or affinity maturation, despite increased antigen deposition in lymphoid follicles. Together, these results demonstrate that IgM accelerates affinity maturation in immune responses to T-dependent antigens and implies an antigen optimum exists for the generation of high affinity antibodies. The data also suggest IgM plays an important role in the induction of CD4 T cells, facilitating germinal centre formation and function. [ABSTRACT FROM AUTHOR]

Waldenström Macroglobulinemia is a low-grade immunosecretory disorder associated with lymphoid tumours, which is rarely reported in veterinary medicine. In this study, we describe two clinical cases of this rare syndrome in dogs, each characterized by a different onset and clinical course. In one case, a hyperacute onset and aggressive behaviour of the neoplasm was observed. Absolute serum viscosity (SV) was retrospectively evaluated in order to explain clinical findings. Rotational viscosimetry showed good precision in measuring SV. Both dogs had SV values higher than a control groups of healthy dogs although only one subject developed hyperviscosity symptoms and complications. At high paraprotein concentrations, a slight reduction of the M-component was associated with a marked decrease in SV. Thus, this work suggests that SV assessment is a relevant tool for managing monoclonal gammopathies, whose usefulness should be further confirmed in larger cohorts of dogs. [ABSTRACT FROM AUTHOR]

Macromolecules that bindβ-amyloid peptide (Aβ) and neutralize Aβ cytotoxicity offer a promising new approach for treating Alzheimer's disease. When the plasma protein,α2-macroglobulin (α2M), is treated with methylamine (α2M-MA), it undergoes conformational change and acquires Aβ-binding activity. In this study, we demonstrate that a chemically stabilized preparation of humanα2M conformational intermediates (α2M-cis-Pt/MA) binds Aβ with greatly increased affinity, compared withα2M-MA.α2M-cis-Pt/MA was generated by reactingα2M with the protein cross-linking reagent,cis-Pt, followed by methylamine. Increased Aβ-binding toα2M-cis-Pt/MA was demonstrated by co-migration of radio-iodinated proteins in non-denaturing PAGE, chemical cross-linking, and co-immunoprecipitation. The apparentKD for Aβ-binding toα2M-cis-Pt/MA was decreased 10-fold, compared withα2M-MA, to 29 nm. Nativeα2M demonstrated negligible Aβ-binding, as anticipated.α2M-cis-Pt/MA markedly counteracted Aβ-induced C6 cell apoptosis. Essentially complete inhibition of apoptosis was observed even when the Aβ was present at fourfold molar excess toα2M-cis-Pt/MA. Under equivalent conditions,α2M-MA inhibited apoptosis by 25 ± 6%. When Aβ andα2M-cis-Pt/MA were added to human plasmain vitro, significant binding was detected. No binding was observed when an equivalent concentration of nativeα2M orα2M-MA was added to plasma. We propose thatα2M-cis-Pt/MA is a novel alternative to Aβ-specific antibodies, for studying the efficacy of Aβ-binding agentsin vitroandin vivo. [ABSTRACT FROM AUTHOR]

Cathepsin E is an intracellular aspartic proteinase of the pepsin family predominantly expressed in cells of the immune system and believed to contribute to homeostasis by participating in host defense mechanisms. Studies on its enzymatic properties, however, have been limited by a lack of sensitive and selective substrates. For a better understanding of the importance of this enzyme in vivo , we designed and synthesized a highly sensitive peptide substrate for cathepsin E based on the sequence of the specific cleavage site of a2-macroglobulin. The substrate constructed, MOCAc-Gly-Ser-Pro-Ala-Phe-Leu-Ala-Lys(Dnp)-D-Arg-NH 2 [where MOCAc is (7-methoxycoumarin-4-yl)acetyl and Dnp is dinitrophenyl], derived from the cleavage site sequence of human a2-macroglobulin, was the most sensitive and selective for cathepsin E, with k cat / K m values of 8-11 µM -1 S -1 , whereas it was resistant to hydrolysis by the analogous aspartic proteinases cathepsin D and pepsin, as well as the lysosomal cysteine proteinases cathepsins B, L, and H. The assay allows the detection of a few fmol of cathepsin E, even in the presence of plasma and cell lysate, and gives accurate results over a wide enzyme concentration range. This substrate might represent a useful tool for monitoring and accurately quantifying cathepsin E, even in crude enzyme preparations. [ABSTRACT FROM AUTHOR]

CD91, the endocytic receptor for a2-macroglobulin (a2M), mediates the internalization of certain heat shock proteins (hsps) and the cross-presentation of peptides they chaperone by antigen-presenting cells. The phylogenetic conservation of the immunologically active CD91 ligands, a2M and hsps, is consistent with the idea of an ancestral system of immune surveillance. We have further explored this hypothesis by taking advantage of the frogXenopus, and asked how conserved is CD91 and whether the expression of CD91 is differentially modulated during immune responses of class I-positive adult and naturally class I-negative larvae. We have identified aXenopus CD91gene homologue that displays high sequence identity (>65%) with other CD91 homologues and contains an additional distinctive cytoplasmic NPXY motif. Phylogenetic analysis indicates that CD91 homologues branch as a monophyletic group distinct from other LDLRs; this suggests an origin of CD91 contemporary with that of metazoans. A 14-kb transcript is detected by Northern blotting in most adult and larval tissues, including lymphoid tissues. RT-PCR study reveals that CD91 is expressed in most cell types, including adult macrophages, B and T cells as well as in splenocytes and thymocytes from naturally MHC class I negative larvae. CD91 is markedly up-regulated in vivo by adult peritoneal leukocytes following bacterial and viral stimulation; it is constitutively expressed on class I-negative larval peritoneal leukocytes at high levels and cannot be further upregulated by such stimulation. These data are in agreement with a conserved role of CD91 in immunity. [ABSTRACT FROM AUTHOR]

The significant human bacterial pathogen Streptococcus pyogenes expresses GRAB, a surface protein that binds α2-macroglobulin (α2M), a major proteinase inhibitor of human plasma. α2M inhibits proteolysis by trapping the proteinase, which, however, still remains proteolytically active against smaller peptides that can penetrate the α2M-proteinase complex. Here we report that SpeB, a cysteine proteinase secreted by S. pyogenes, is trapped by α2M bound to protein GRAB. As a consequence, SpeB is retained at the bacterial surface and protects S. pyogenes against killing by the antibacterial peptide LL-37. [ABSTRACT FROM AUTHOR]

Unlabelled: Mutations in activation-induced cytidine deaminase can cause an autosomal recessive form of hyper-IgM syndrome. We have examined a Tunisian family composed of six members: two healthy parents, their two healthy daughters and two affected sons. We found a homozygous transversion G to T in the two sons while heterozygosity for the mutation was found in all other family members. This alteration is localised in intron 2 at the +1 position resulting in defective splicing. Use of various intronic cryptic splice-sites led to expression of various aberrant mRNA species.Conclusion: This is a novel mutation found in the gene encoding for activation-induced cytidine deaminase in a Tunisian family with hyper-IgM type 2 syndrome. This alteration leads to the use of two cryptic splicing sites causing the formation of two different mRNA species. [ABSTRACT FROM AUTHOR]

Recent data have suggested that rituximab is an active agent for the treatment of Waldenstrom's macroglobulinemia (WM). However, the patients that are more likely to benefit have not been clearly defined. In order to address this question we evaluated 52 patients who were treated with single-agent rituximab in the context of prospective studies. Several clinical and laboratory variables were assessed for their correlation with response and time to progression. Twenty-three (44%) patients achieved a partial response after treatment with rituximab. Previously untreated and pretreated patients had the same probability for response. Higher response rates were noted in patients with serum monoclonal protein < 40 g/l, with serum albumin &ges; 35 g/l and with kappa light chain. The median time to progression for all patients was 13.8 months. A multivariate analysis indicated that elevated serum monoclonal protein levels and low serum albumin were the dominant variables associated with shorter progression. Presence of two, one or none of these adverse prognostic factors was associated with time to progression of 3.6 months, 11 months and more than 40 months, respectively. We conclude that rituximab is an effective treatment modality for patients with WM. Patients with both low levels of monoclonal protein and normal albumin are the best candidates for treatment with standard dose rituximab. [ABSTRACT FROM AUTHOR]

Waldenström macroglobulinemia (WM) is a low-grade lymphoplasmacytic lymphoma that strongly expresses CD20 on the cell surface. This study was undertaken to determine the response rate of patients with untreated or previously treated WM to the monoclonal antibody rituximab, which is directed against CD20+ expressed by B-cell lymphomas. Thirty-four untreated and thirty-five previously treated patients received rituximab 375 mg/m 2 weekly for 4 consecutive weeks by intravenous infusion on days 1, 8, 15, and 22. Sixty-nine patients were evaluable for response; 19 (27.5%) achieved an objective response and 17 (24.6%) achieved a minor response. The overall response rate was 52.2% (90% CI [41.6%, 62.6%]). Of previously untreated patients 35.3% vs. 20% of previously treated patients achieved an objective response. Median response duration was not significantly different between previously untreated (27 months) and previously treated patients (not reached, P  = 0.8). Rituximab produced objective or minor responses in 52.2% of patients and is an active agent in the treatment of WM. Grade 4 toxicity was seen in 11%. [ABSTRACT FROM AUTHOR]

Caprine alpha-2-macroglobulin (α2M) is a broad-spectrum, homotetrameric proteinase inhibitor that can maximally bind a single molecule of proteinase. Inhibition of proteinases by caprine α2M results from a series of conformational changes that are initiated by the proteinase and results in physical sequestration of the proteinase within the closed cage-like structure of conformationally altered α2M. In a previous study, uric acid-generated superoxide anion was identified as one of the physiologically relevant inactivators of α2M S.A. Khan, F.H. Khan [Free. Radic. Res. 34 (2001) 113]. We now demonstrate that hypochlorous acid (HOCl) and, to lesser extent, hydrogen peroxide (H2O2) destroy the antiproteolytic potential of caprine α2M. At physiologically attainable concentration, we found that HOCl significantly compromised functional integrity of the inhibitor. High concentrations of H2O2 also partially diminished proteinase inhibitory capacity of α2M by a mechanism not involving formation of hydroxyl radicals. For hydrogen peroxide, catalase completely protected α2M activity while the ability to protect the inhibitor from HOCl-induced inactivation was limited by availability of albumin. Structure function analysis demonstrated that oxidized caprine inhibitor, unlike its human counterpart, retained its tetrameric configuration as well as its characteristic ability to undergo major conformational change upon trypsinization. It is proposed that inhibition of α2M activity may be due to oxidation of essential residues of the inhibitor and/or structural rearrangement of the subunits. [Copyright &y& Elsevier]

LDL receptor-related protein/alpha2-macroglobulin receptor (LRP1/A2MR) a multiligand receptor is considered as not only being a possible risk factor of neurodegenerative diseases like Alzheimer’s disease but also as determining the progression of other complex diseases like atherosclerosis and cancer. Although a large number of in vitro studies have highlighted its functional importance, as yet not enough is known about the clinical importance of the genetic background of LRP1 in human diseases.The aim of this ex vivo/in vivo study of 448 subjects was to present data on genetic LRP1 variants of healthy European Caucasians from Central Germany. Genotype-dependent LRP1 expression was analyzed in a representative subgroup (gene expression: n = 127, protein expression: n = 44). These data were evaluated in comparison to other published clinical LRP1 studies. For 15 functionally interesting genetic variants the genotype and allele distributions of the German Caucasians were presented in relation to their in vivo LRP1 gene and protein expression. A direct influence of the LRP1 promoter polymorphism c.1-25C>G on the human in vivo LRP1 expression level was demonstrated. In an analysis of 48 further studies genomic and functional results were evaluated. The analysis especially on Alzheimers’s disease partly highlighted contradictory results, but suggested that ethnic as well as genomic characteristics determine LRP1 expression and must be considered in clinical investigations on human LRP1. [Copyright &y& Elsevier]

Several ligands of the endocytic low density lipoprotein receptor-related protein (LRP), such as apoE-containing lipoproteins and activated α2-macroglobulin (α2M∗) promote neurite outgrowth, suggesting that LRP may have signaling functions. In this study, we found that the treatment of neurons with α2M∗ significantly increased the individual length (by 71%) and numbers (by 139%) of neurites of primary mouse cortical neurons. These effects were blocked by the LRP antagonist, the receptor-associated protein. We found similar neurite outgrowth with purified apoE3 and a tandem apoE peptide containing only the receptor-binding domain. To investigate the intracellular pathway of the LRP signaling involved in neurite outgrowth, we tested the effects of α2M∗ on the phosphorylation of the mitogen-activated protein (MAP) extracellular signal-regulated kinases 1 and 2 (ERK1/2). We found that 1) phospho-MAP kinase levels were altered within 30 min after treatment with α2M∗, 2) the MAP kinase inhibitor, PD98059, specifically blocked the α2M∗-induced neurite outgrowth, 3) manipulating intracellular calcium by BayK or BAPTA altered the neurite outgrowth and associated changes in the phospho-MAP kinase levels, which were blunted by α2M∗, 4) α2M∗ promoted the phosphorylation of the transcription factor CREB through MAP kinase, and 5) LRP-specific antibodies increased levels of phosphorylated MAP kinase and phosphorylated CREB. The effects of α2M∗, apoE3, and apoE peptides increased LRP levels in the cortical neurons, whereas LRP receptor-associated protein reduced dendritic LRP expression. These results demonstrate that p44/42 MAP kinase plays an important role in LRP-mediated neurite outgrowth with activation involving the effects on calcium homeostasis and downstream effects involving the activation of gene transcription through CREB. [ABSTRACT FROM AUTHOR]

Triflamp, a 28 kDa snake venom metalloproteinase purified from the venom of Trimeresurus flavoviridis, possesses the proteolytic activities toward P-selectin glycoprotein ligand-1 (PSGL-1), glycoprotein Ib (GPIb) and fibrinogen. In human whole blood preparation, however, triflamp (6 μg/ml) failed to cleave neutrophil PSGL-1 and platelet GPIb. Human α2-macroglobulin (α2M) was mainly responsible for the neutralization of the proteolytic effects of triflamp on PSGL-1, GPIb and fibrinogen. Human α2M neutralized triflamp at a stoichiometry about 1.1:1 (molar basis) determined by azocaseinolysis. SDS-PAGE analysis revealed that triflamp cleaved the bait-region of α2M. Western blot demonstrated that triflamp interacted with the C-terminal half-subunits of truncated α2M resulting in the formation of high-molecular-weight species of α2M-triflamp complexes. In the presence of competing nucleophile, 0.2 M methylamine, the proteolytic activity of triflamp was conserved. In vivo we found that mice neutrophils were resistant to the cleavage of PSGL-1 by triflamp. However, mouse PSGL-1 and GPIb were susceptible to be cleaved by triflamp in washed mouse neutrophil and platelet preparation, respectively. Similarly, mouse serum was also responsible for the inactivation of the proteolytic activity of triflamp. This study provides direct evidences for the reasonable explanation regarding the reduced proteolytic activity of triflamp toward its substrates in whole blood preparation and in vivo model. [Copyright &y& Elsevier]

The cDNA encoding the kuruma shrimp, Marsupenaeus japonicus α2-macroglobulin (α2M) was obtained by screening a haemocyte cDNA library and 5′ RACE PCR amplification. The full length cDNA of 4748 bp contains an open reading frame of 4518 nucleotides that translates into a 1505-amino acid putative peptide, with a 5′untranslated region (UTR) of 59 bp and a 3′UTR of 171 bp. The open reading frame encodes an N-terminal signal sequence of 17 residues and a mature protein of 1488 residues. The entire amino acid sequence is similar to the α2M sequences of arthropods (30–31% identity), mammals (26–27% identity) and fish (25–28% identity). The M. japonicus α2M sequence contains putative functional domains including a bait region, an internal thiol ester site, and a receptor-binding domain, which are present in mammalian α2Ms. In a healthy shrimp, the mRNA of α2M was mainly expressed in haemocytes. In addition, the expression level of α2M mRNA was dramatically increased by through time upon oral administration of peptidoglycan (PG), which is an immune stimulant. The highest expression of α2M mRNA was observed 7 days after feeding with PG. These results suggest that the shrimp α2M is an important molecule in immune system. [Copyright &y& Elsevier]

Osteoarthritis is characterized by the loss of aggrecan and collagen from the cartilage extracellular matrix. The proteinases responsible for the breakdown of cartilage aggrecan include ADAMTS-4 (aggrecanase 1) and ADAMTS-5 (aggrecanase 2). Post-translational inhibition of ADAMTS-4/-5 activity may be important for maintaining normal homeostasis of aggrecan metabolism, and thus, any disruption to this inhibition could lead to accelerated aggrecan breakdown. To date TIMP-3 (tissue inhibitor of matrix metalloproteinases-3) is the only endogenous inhibitor of ADAMTS-4/-5 that has been identified. In the present studies we identify α2-macroglobulin (α2M) as an additional endogenous inhibitor of ADAMTS-4 and ADAMTS-5. α2M inhibited the activity of both ADAMTS-4 and ADAMTS-5 in a concentration-dependent manner, demonstrating 1:1 stoichiometry with second-order rate constants on the order of 106 and 105 M-1 s-1, respectively. Inhibition of the aggrecanases was mediated by proteolysis of the bait region within α2M, resulting in physical entrapment of these proteinases. Both ADAMTS-4 and ADAMTS-5 cleaved α2M at Met690/ Gly691, representing a novel proteinase cleavage site within α2M and a novel site of cleavage for ADAMTS-4 and ADAMTS-5. Finally, the use of the anti-neoepitope antibodies to detect aggrecanase-generated α2M-fragments in synovial fluid was investigated and found to be uninformative. [ABSTRACT FROM AUTHOR]

Waldenström’s macroglobulinemia is a rare B cell malignancy. All prognostic studies have identified the adverse prognostic effect of advanced age and low hemoglobin level for survival, whereas the prognostic value of a high level of monoclonal component remains controversial. Response to treatment is probably a favorable prognostic factor for overall survival. Conventional treatment is based on alkylating agents, and especially chlorambucil. Purine analogues, used initially for salvage treatment, are increasingly employed as front-line therapy. Purine analogues have not been compared with alkylating agents as first-line therapy in randomized trials, and it is unclear whether purine analogues extend survival. An anti-CD20 monoclonal antibody has given a response rate in about 30% of patients. Autologous and allogeneic stem cell transplantation may be considered for patients with refractory or relapsing disease. [ABSTRACT FROM AUTHOR]

The α2-macroglobulin signalling receptor is upregulated in highly metastatic 1-LN prostate cancer cells. Stimulation of 1-LN cells with activated α2-macroglobulin (α2M*) 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 α2M* 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 α2M* 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 α2M* 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. [Copyright &y& Elsevier]

Polymorphisms in alpha-2-macroglobulin (A2M) and angiotensin-converting enzyme (ACE) genes have been considered as risk factors for Alzheimer''s disease (AD) independently of the risk conferred by the apolipoprotein E &epsiv;4 allele (APOE&epsiv;4) in diverse populations. In the present study, we have analysed the distribution of genotypes and alleles of the insertion/deletion (I/D) polymorphisms of ACE and A2M in 83 AD patients and 69 normal controls in Colombia. Our results showed that there is no association between the I/D polymorphisms of ACE and A2M with AD (P=0.788 and P=0.538, respectively). Using logistic regression and multiple correlation analysis (MCA), we confirmed that the main risk factor associated and consistently grouped with AD patients in this population is APOE&epsiv;4, but this association was not observed with alleles and genotypes of ACE and A2M. [Copyright &y& Elsevier]

We report here the genomic organization and phylogenic relationships of CD109, a member of the the α2-macroglobulin/complement (AMCOM) gene family. CD109 is a GPI-linked glycoprotein expressed on endothelial cells, platelets, activated T-cells, and a wide variety of tumors. We cloned full-length CD109 cDNA from the mammalian U373 cell line by RT-PCR and performed analysis of its corresponding genomic sequence. The CD109 cDNA spans 128 kb of chromosome 6q with its 33 exons constituting approximately 3.3% of the total CD109 genomic sequence. Sequence analysis revealed that CD109 contains specific motifs in its N-terminus, that are highly conserved in all AMCOM members. CD109 also shares motifs with certain other AMCOM members including: (1) a thioester ‘GCGEQ” motif, (2) a furin site of four positively charged amino acids, and (3) a double tyrosine near the C-terminus. Based on a phylogenic analysis of human CD109 with other human homologs as well as orthologs from other mammalian species, C. elegans (ZK337.1) and E. coli homologs, we propose CD109 represents a novel and independent branch of the α2-macroglobulin/complement gene family (AMCOM) and may be its oldest member. [Copyright &y& Elsevier]

Cellular senescence is a well-established model system for studying the molecular basis of aging. To identify a reliable biomarker for cellular age and further study the gene expression of aging, we profiled the gene expression difference between aged and young cultured human embryonic lung fibroblasts by high-density complementary deoxyribonucleic acid (cDNA) arrays. Among the differentially expressed genes, alpha-2-macroglobulin (α2M) was selected for further study. Its gene expression level as a function of population doubling level (PDL) in cultured fibroblasts was determined by RT-PCR and northern hybridization. mRNA level of α2M showed a positive linear-correlation with cumulative PDL. Additional assays revealed that the levels of α2M increased in irreversible growth arrest induced by sublethal H2O2, but not in quiescent state of cultured fibroblasts induced by serum-deprivation, and remained stable in Hela cells. These results suggest that mRNA level of α2M can be used as a biomarker of aging in cultured fibroblasts. mRNA level of α2M showed significant difference between newborn and old human leucocytes, which suggest that the mRNA level of α2M may be used as a biomarker of aging in vivo. [Copyright &y& Elsevier]

Background: The genes encoding myeloperoxidase (MPO) and α[sub 2]-macroglobulin (A2M) are involved in molecular pathways leading to β-amyloid deposition. Two polymorphic sites in these genes (MPO-G/A and A2M-Ile/Val) have been associated with Alzheimer disease (AD), but conflicting findings have been reported in populations with different ethnic backgrounds. Objectives: To study the association of MPO-G/A and A2M-Ile/Val polymorphisms with sporadic AD and to investigate the interactions among the MPO, A2M, and apolipoprotein E (APOE) gene polymorphisms in determining the risk of the development of AD. Design: Case-control study. Setting: Referral center for AD in Calabria, southern Italy. Participants: One hundred forty-eight patients with sporadic AD and 158 healthy control subjects. Results: The MPO-G and A2M-Val alleles were found more frequently in cases than in controls, as were the MPO-G/G and A2M-Val/Val genotypes. The odds ratio (OR) for the MPO-G/G genotype was 1.78 (95% confidence interval [CI], 1.13-2.80); for the A2M-Val/Val genotype, 3.81 (95% CI, 1.66-8.75). The presence of MPO-G/G and A2M-Val/Val genotypes synergistically increased the risk of AD (OR, 25.5; 95% CI, 4.65-139.75). Stratification of cases by sex, age at onset of AD, and APOE-ε4 status did not show significant differences in the distribution of MPO or A2M polymorphisms. Conclusions: The MPO and A2M polymorphisms are associated with sporadic AD in southern Italy. Moreover, a genomic interaction between these polymorphisms increases the risk of the development of AD. [ABSTRACT FROM AUTHOR]

The liver plays an important role in a severe thermal injury by modulating immune function, inflammatory processes and the acute phase response, which are an orchestrated attempt to restore homeostasis. Using high-density oligonucleotide arrays, we examined the gene expression profile in the livers of rats between 2 and 240 h after a 40% total body surface area (TBSA) burn. Alterations in gene expression unique to a thermal injury were identified. Approximately 39 genes out of 8700 genes on each array across all the time points showed a significant change in expression patterns. Real time reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analyses verified significant changes in early growth response-1 (Egr-1) messenger RNA (mRNA) and protein levels corresponding to the array data. Significant increases in serum levels of α-2-macroglobulin that correspond to changes in its mRNA levels were observed at 6 and 24 h after burn, p<0.05. The genomic pattern for liver in the hypermetabolic phase after the burn injury involves transcription factors, stress and inflammatory responses, cytoskeletal and extracellular matrix modifications, and regulation of cell proliferation and differentiation. During the initial phase of thermal injury gene expression profiles in the liver may provide some insight into how cellular protection mechanisms and systemic hypermetabolism are initiated and controlled. The genome wide changes observed may provide a rational therapeutic strategy to improve burn care. [Copyright &y& Elsevier]

The aims of this study were to describe the changes in the nasal lavage fluid (NLF) protein pattern after exposure to the irritating epoxy chemical dimethylbenzylamine (DMBA) and to identify the affected proteins using a proteomic approach. The protein patterns of NLF from six healthy subjects and eight epoxy workers with airway irritation were analysed using two-dimensional gel electrophoresis (2-DE) before and after exposure to 100 μg m-3 DMBA for 2 h in an exposure chamber. NLF proteins were identified by (i) comparison with a 2-DE NLF reference database; (ii) N-terminal amino acid sequencing; and (iii) mass spectrometry. In NLF from healthy subjects, the levels of immunoglobulin A increased and the levels of Clara cell protein 16 (CC16) decreased after chamber exposure, while in NLF from epoxy workers, α2-macroglobulin and caeruloplasmin increased. Two previously unidentified proteins decreased in NLF from epoxy workers after exposure; these were identified as statherin and calgranulin B. In addition, the subjects who developed high counts of eosinophils in their nasal mucosa after chamber exposure had significantly lower levels of immunoglobulin-binding factor (IgBF) before exposure than subjects with low eosinophil infiltration. These results show that short-term exposure to DMBA causes distinct changes in NLF proteins. Moreover, three proteins that have previously not been associated with upper airway irritation were identified: statherin, calgranulin B and IgBF. Further studies are needed to investigate whether these proteins may be used as biomarkers of airway irritation and to give new insight into the ways in which occupational exposure to irritants causes inflammation of the airways. [ABSTRACT FROM AUTHOR]

To investigate the effect of modification of biological environmental conditions, one of the factors influencing the healing of anterior cruciate ligament rupture, we performed experimental anterior cruciate ligament ruptures on New Zealand rabbits. After experimental rupture, intra-articular alpha-2 macroglobulin was injected into the knees of the rabbits in the experiment group to prevent structural changes resulting from the enzymatic reactions in the ruptured anterior cruciate ligament. At the end of 10th day of the experiment, we observed that the anterior cruciate ligaments in the experiment group had retained their prerupture brightness and volume when compared with the control group in which intraarticular alpha-2 macroglobulin had not been injected. We also noted that the anterior cruciate ligaments in the experiment group had not retracted or swollen, the incision sites were regular and clean, and they did not show any signs of degeneration. In the histological examination, the anterior cruciate ligaments in the control groups showed disruption of the collagen network and a significant diminution in number of fibroblasts and fibrocytes ( p < .001). At the end of this study, we concluded that the necessary conditions for the healing and repair of ruptured anterior cruciate ligament could exist if the enzymatic and biological environments were under control. [ABSTRACT FROM AUTHOR]

Circulating level of α2-macroglobulin–β2-microglobulin complex in hemodialysis patients. Background. The presence of α2-macroglobulin (α2M) in amyloid tissue from patients with dialysis-related amyloidosis (DRA) was demonstrated by Argilés et al in 1989. Thereafter, the formation of the complex of β2-microglobulin (β2m) with α2m was confirmed directly by in vitro study. In Alzheimer's disease, complex formation of amyloid β-peptide and α2M is considered to play an important role in the pathogenesis by modifying the degradation processes of amyloid protein. Thus, we hypothesized that the α2M-β2m complex is an important factor in the pathogenesis of DRA as well. Here, we measured the circulating levels of α2M-β2m complex in the maintenance hemodialysis patients and discussed about its clinical significance in DRA. Methods. One hundred and thirty-seven hemodialysis patients and 11 prehemodialysis chronic renal failure (CRF) patients were included in this study. The affinity of purified α2M for β2m was confirmed by a highly sensitive 27 MHz quartz crystal microbalance (QCM). The presence of circulating α2M-β2m complex was analyzed by immunoblotting analysis. Furthermore, the serum levels of α2M-β2m complex were measured by sandwich enzyme immunoassay. Results. QCM analysis revealed the high affinity of α2M for β2m. The presence of circulating α2M-β2m complex was detected in two out of a total 11 prehemodialysis CRF patients and in 95 out of the total of 137 hemodialysis patients. None of the healthy subjects, however, were observed to present with any α2M-β2m complex. Serum levels of the α2M-β2m complex were correlated to the duration of hemodialysis . Serum levels of the α2M-β2m complex were significantly higher in patients with high DRA score than in patients with negative DRA score . Moreover, serum levels of the α2M-β2m complex showed significantly lower in the hemodiafiltration patients compared to the hemodialysis patients and showed a strong correlation with DRA score in hemodialysis patients excluding 11 hemodiafiltration patients . Conclusion. This study is the first to demonstrate the presence of circulating α2M-β2m complex in hemodialysis patients. Furthermore, we observed the correlation between serum levels of α2M-β2m complex and clinical characteristics of DRA. Thus we concluded that a formation of an α2M-β2m complex may be implicated in DRA. [ABSTRACT FROM AUTHOR]

We describe a detailed time course of the assembly and disassembly of a STAT3-dependent, glucocorticoid-supplemented enhanceosome for the α2-macroglobulin (α2-M) gene and compare this with a detailed time course of transcription of the gene by run-on analysis. The glucocorticoid receptor (GR) can associate with the enhanceosome without STAT3. Furthermore, the enhanceosome contains c-Jun/c-Fos and OCT-1 constitutively. All of these factors (GR, c-Jun, OCT-1) have transcription activation domains, but STAT3 is required for the observed transcriptional increase. The time course of enhanceosome occupation by GR and tyrosine-phosphorylated STAT3 shows that these transcription factors precede by ∼5-10 min the arrival of RNA polymerase II (Pol II). The enhanceosome remains assembled for ∼90 min in the continued presence of both inducers. When IL-6 and Dex are removed (after 30 min of treatment), the disappearance within an additional 30 rain of the established enhanceosome indicates that renewal of STAT3 and GR binding must occur in the continued presence of IL-6+Dex. Compared with the total nuclear tyrosine-phosphorylated STAT3 capable of binding DNA, the chromatin-associated STAT3 resists dephosphorylation and appears to recycle to maintain the enhanceosome. Run-on transcription shows a lag after full enhanceosome occupation that can be largely but not completely explained by the ∼30 min transit time of Poi II across the α2-M locus. [ABSTRACT FROM AUTHOR]

Bear serum alpha2 macroglobulin (α2M) was purified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and partially characterized by tryptic digestion of α2M and analysis of the peptides by peptide mass fingerprinting. The molecular weight of bear serum α2M was 181 kDa, same as for human serum α2M, on SDS-PAGE. However, the MALDI mass spectrum of the tryptic digested bear serum α2M showed that it is different from human α2M or other data bank proteins. Liquid chromatography (LC)/mass spectrometry (MS)/MS of the proteolytic products of bear serum α2M showed eight peptides that had similarities to human α2M suggesting that the protein of interest was indeed α2M of bear. The polyclonal antibody against bear serum α2M recognized only one protein from the western blot of bear serum proteins. It also recognized human α2M. The levels of serum α2M were significantly increased during hibernating state as compared to active state of bears indicating its protective role from the consequences of the metabolic depression during hibernation. [Copyright &y& Elsevier]

Background: Tobacco smoking has considerable negative effects on the outcome of periodontal treatment. The reason for the inferior therapeutical effect might be related to an altered neutrophil activity in terms of elastase and/or matrix metallo-proteinase-8 (MMP-8), as well as in the protease inhibitor alpha-1-antitrypsin (α-1-AT) and alpha-2-macroglobulin (α-2-MG) activities. The aim of the present study, therefore, was to elucidate the effect of tobacco smoking on gingival crevicular fluid (GCF) levels or these substances following surgical treatment. Methods: The study population included 15 smoking and 15 non-smoking patients with moderate to severe periodontitis receiving surgical treatment. Clinical examinations and collection of GCF were done prior to surgery and 1 and 5 weeks following treatment. The elastase activity was measured with a chromogenic low-molecular substrate and the levels of α-1-AT, α-2-MG, and MMP-8 with enzyme-linked immunosorbent assay. Results: The results showed unaltered levels of α-1-AT, α-2-MG, and MMP-8 in smokers following surgery. In non-smokers, the levels of α-1-AT and α-2-MG increased, whereas MMP-8 levels decreased. The levels of elastase remained unaltered in both smokers and non-smokers. Conclusions: These results indicate that in the presence of smoking, the levels of α-1-AT, α-2-MG, and MMP-8 remained unaltered during the recovery period following surgical treatment. This is interpreted as a possible interference of smoking with the treatment response and may, in part, explain the clinical evidence of an interior treatment outcome in smokers. [ABSTRACT FROM AUTHOR]

The aim of this study was to assess anthropometric variables and the lipid pattern in relation to alpha-2-macroglobulin in normal- and over-nourished Thai individuals, to further support the hypothesis that alpha-2-macroglobulin plays a beneficial role in the determination of nutritional status. The study sample comprised of 48 male and 166 female overweight and obese Thai volunteers and 26 male and 81 female normal subjects. The overweight individuals had statistically significant lower alpha-2-macroglobulin (A2M) serum levels. The total serum cholesterol, low density lipoprotein-cholesterol (LDL-C) and triglycerides were significantly higher and high density lipoprotein-cholesterol (HDL-C) lower in the over-nourished group as compared with the normal subjects. The LDL/HDL ratio was slightly but significantly higher in the over-nourished group, but still well below the value of 5 for both groups. In using a stepwise multiple linear regression, the model, which best explained the variation of A2M for all individuals including age, HDL-C, BMI, and gender. The relationship of A2M to the variables under study differed between males and females. For males, a model which includes cholesterol and BMI explained best the variation of the proteinase inhibitor. For the females, the best model includes age, HDL-C and BMI. The role of protease inhibitors has hardly been explored in human epidemiological studies despite its relationship to important public health issues including nutrition, smoking, cancer and cardiovascular diseases. The results of this study further support the hypothesis, that A2M might play a role in the interrelationship of the nutritional status with the occurrence and the prevention of diseases. [Copyright &y& Elsevier]