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Human α 2 -macroglobulin (hα 2 M) is a large homotetrameric protein involved in the broad inhibition of endopeptidases. Following cleavage within a bait region, hα 2 M undergoes stepwise transitions from its native, expanded, highly flexible, active conformation to an induced, compact, triggered conformation. As a consequence, the peptidase is entrapped by an irreversible Venus flytrap mechanism. Given the importance of hα 2 M, biochemical studies galore over more than seven decades have attempted to ascertain its role, typically using authentic hα 2 M purified from frozen and non-frozen fresh blood plasma, and even outdated plasma. However, hα 2 M is sensitive once isolated and purified, and becomes heterogeneous during storage and/or freezing, raising concerns about the functional competence of frozen plasma-derived hα 2 M. We therefore used a combination of native and sodium dodecylsulfate polyacrylamide gel electrophoresis, affinity and ion-exchange chromatography, multi-angle laser light scattering after size-exclusion chromatography, free cysteine quantification, and peptidase inhibition assays with endopeptidases of two catalytic classes and three protein substrates, to characterize the biochemical and biophysical properties of hα 2 M purified ad hoc either from fresh plasma or frozen fresh plasma after thawing. We found no differences in the molecular or functional properties of the preparations, indicating that protective components in plasma maintain native hα 2 M in a functionally competent state despite freezing., (© 2023. The Author(s).)

Studies in the field of microelement speciation in the body of farm animals, in particular dairy cattle, are almost completely absent. The average concentration of Mn in the blood serum of all the studied animals (n = 80) was 2.5 μg/L, which corresponds to normal values. Of the total number of animals, 21% were the cows with the low normal values (serum Mn concentration ≤ 2 µg/L, i.e. less than Q 25 of the total sample) and 25% were the animals with the high normal values (serum Mn concentration ≥ 2.72 µg/L, i.e. more than Q 75 of the total sample). The data obtained in the course of the study indicate that the change in the Mn level, even in the range of normal values, is accompanied by the redistribution of this element over various protein fractions. The six found Mn blood serum forms are presumably represented by α2-macroglobulin (tetramer, dimer, and monomer), transferrin/albumine, manganese citrates, and "free" metal ions. The analyzed fractions of Mn found in the blood serum of cows had the following hierarchy of concentrations: in the group with low-normal values of Mn ("free" Mn >> tetrameric form of α2-macroglobulin >> transferrin/albumine >> dimeric form of α2-macroglobulin >> monomeric form of α2-macroglobulin >> citrate), in the group with high normal manganese values ("free" Mn >> monomeric form of α2-macroglobulin >> transferring/albumine >> citrate >> tetrameric form of α2-macroglobulin >> dimeric form of α2-macroglobulin). In the group with high normal Mn values relative to the group with low normal values, there was a percentage decrease in the tetrameric fraction of a2-macroglobulin from 17.2 to 4.4%, dimeric fraction of a2-macroglobulin from 6.9 to 2.2%, "free" Mn from 54.3 to 44.4% and an increase in monomeric fraction of a2-macroglobulin from 6.7 to 23.1%, transferrin/albumine from 10.1 to 17.7%, citrate from 4.8 to 8.2%. Our data demonstrate the features of Mn redistribution of dairy cows, which can be used for an extended assessment of the microelement status of animals., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Alpha-2-macroglobulin ( α 2 -MG) is a multifunctional protein involved in neurodegeneration, inflammation and neovascularization, which are key processes in the pathogenesis of age-related macular degeneration (AMD) and proliferative diabetic retinopathy (PDR). AMD and PDR are two of the main causes of vision loss and blindness, are difficult to treat, and are generally diagnosed at the stage of irreversible changes., Purpose: This study estimates the activity of α 2 -MG in the blood serum and tears of patients with AMD and PDR in order to reveal the relation of its levels with the intensity of the pathological process in the retina., Material and Methods: The study included 17 patients (34 eyes) with AMD, 15 patients (30 eyes) with PDR, and 15 healthy adults (30 eyes) of the similar age. The activity of α 2 -MG in serum and tears was measured enzymatically using the specific substrate N-benzoyl-DL-arginine-p-nitroanilide (BAPNA)., Results: The activity of α 2 -MG in tears of patients with AMD was on the average 3.5 times higher than in healthy controls, and in patients with PDR - 1.5 times higher. Patients with AMD at the submacular fibrosis stage showed decreased α 2 -MG activity in tears. The activity of α 2 -MG in serum of patients with AMD and PDR was on the average 25% higher than in healthy persons. No correlation was revealed between serum and tear levels of α 2 -MG activity., Conclusion: This study revealed for the first time that in AMD and PDR the activity of α 2 -MG in tears is increased, and that in AMD the increase is higher than in PDR. An increase of α 2 -MG activity in serum confirms the presence of systemic inflammation. Absence of correlation between the serum and tear activity of α 2 -MG confirms its local origin. The high level of α 2 -MG activity in tears reflects the presence of an active destructive process in the retina, justifying its further investigation as a predictor of AMD and PDR course, as well as an indicator of therapy effectiveness.

The proteolytic cleavage of C3 to generate C3b is the central and most important step in the activation of complement, a major component of innate immunity. The comparison of the crystal structures of C3 and C3b illustrates large conformational changes during the transition from C3 to C3b. Exposure of a reactive thio-ester group allows C3b to bind covalently to surfaces such as pathogens or apoptotic cellular debris. The displacement of the thio-ester-containing domain ( TED) exposes hidden surfaces that mediate the interaction with complement factor B to assemble the C3-convertase of the alternative pathway ( AP). In addition, the displacement of the TED and its interaction with the macroglobulin 1 ( MG1) domain generates an extended surface in C3b where the complement regulators factor H ( FH), decay accelerating factor ( DAF), membrane cofactor protein ( MCP) and complement receptor 1 ( CR1) can bind, mediating accelerated decay of the AP C3-convertase and proteolytic inactivation of C3b. In the last few years, evidence has accumulated revealing that the structure of C3b in solution is significantly more flexible than anticipated. We review our current knowledge on C3b structural flexibility to propose a general model where the TED can display a collection of conformations around the MG ring, as well as a few specialized positions where the TED is held in one of several fixed locations. Importantly, this conformational heterogeneity in C3b impacts complement regulation by affecting the interaction with regulators. [ABSTRACT FROM AUTHOR]

Nerve growth factor (NGF) is generated from a precursor, proNGF, that is proteolytically processed. NGF preferentially binds a trophic tyrosine kinase receptor, TrkA, while proNGF binds a neurotrophin receptor (NTR), p75NTR, that can have neurotoxic activity. Previously, we along with others showed that the soluble protein α2-macroglobulin (α2M) is neurotoxic. Toxicity is due in part to α2M binding to NGF and inhibiting trophic activity, presumably by preventing NGF binding to TrkA. However, the mechanisms remained unclear. Here, we show ex vivo and in vivo three mechanisms for α2M neurotoxicity. First, unexpectedly the α2M-NGF complexes do bind TrkA receptors but do not induce TrkA dimerization or activation, resulting in deficient trophic support. Second, α2M makes stable complexes with proNGF, conveying resistance to proteolysis that results in more proNGF and less NGF. Third, α2M-proNGF complexes bind p75NTR and are more potent agonists than free proNGF, inducing tumor necrosis factor alpha (TNF-α) production. Hence, α2M regulates proNGF/p75NTR positively and mature NGF/TrkA negatively, causing neuronal death ex vivo. These three mechanisms are operative in vivo, and α2M causes neurodegeneration in a p75NTR- and proNGF-dependent manner. α2M could be exploited as a therapeutic target, or as a modifier of neurotrophin signals. [ABSTRACT FROM AUTHOR]

The effects of Ec-α 2 MR ( Epinephelus coiodes -α 2 -macroglobulin receptor) on growth performance, enzymatic activity, respiratory burst, MDA level, total antioxidant capacity, DPPH radical scavenging percentage and immune-related gene expressions of the juvenile orange-spotted grouper were evaluated. The commercial diet supplemented with α 2 MR additive was used to feed the orange-spotted grouper for six weeks. Although a slight increase was observed in the specific growth rate, survival rate and weight gain, no significance was observed among different group. After the feeding trial, the groupers were exposed to cold stress. Respiratory burst activity and MDA level decreased significantly in α 2 MR additive group by comparing with the control and additive control group, while a sharp increase of ACP activity, ALP activity, total antioxidant capacity and DPPH radial scavenging percentage was observed in α 2 MR additive group. qRT-PCR analyses confirmed that the up-regulated mRNA expressions of C3, TNF1, TNF2, IL-6, CTL, LysC, SOD1 and SOD2 were observed in α 2 MR additive group at 20 °C. These results showed that α 2 MR additive may moderate the immune response in grouper following cold shock challenge. [ABSTRACT FROM AUTHOR]

The survival of commensal bacteria requires them to evade host peptidases. Gram-negative bacteria from the human gut microbiome encode a relative of the human endopeptidase inhibitor, α2-macroglobulin (α2M). Escherichia coli α2M (ECAM) is a ~180-kDa multidomain membrane-anchored pan-peptidase inhibitor, which is cleaved by host endopeptidases in an accessible bait region. Structural studies by electron microscopy and crystallography reveal that this cleavage causes major structural rearrangement of more than half the 13-domain structure from a native to a compact induced form. It also exposes a reactive thioester bond, which covalently traps the peptidase. Subsequently, peptidase-laden ECAM is shed from the membrane and may dimerize. Trapped peptidases are still active except against very large substrates, so inhibition potentially prevents damage of large cell envelope components, but not host digestion. Mechanistically, these results document a novel monomeric "snap trap." [ABSTRACT FROM AUTHOR]

Bacterial α-2-macroglobulins have been suggested to function in defence as broad-spectrum inhibitors of host proteases that breach the outer membrane. Here, the X-ray structure of protease-cleaved Escherichia coliα-2-macroglobulin is described, which reveals a putative mechanism of activation and conformational change essential for protease inhibition. In this competitive mechanism, protease cleavage of the bait-region domain results in the untethering of an intrinsically disordered region of this domain which disrupts native interdomain interactions that maintain E. coliα-2-macroglobulin in the inactivated form. The resulting global conformational change results in entrapment of the protease and activation of the thioester bond that covalently links to the attacking protease. Owing to the similarity in structure and domain architecture of Escherichia coliα-2-macroglobulin and human α-2-macroglobulin, this protease-activation mechanism is likely to operate across the diverse members of this group. [ABSTRACT FROM AUTHOR]

Treatment of rats with mercazolyl (25 mg/kg for 20 days), which decreases blood levels of iodine-containing thyroid hormones (ITH), decreased trypsin-like activity (TLA) and α1-antitrypsin (α1-AT) and α2-macroglobulin (α2-MG) activities in the liver and blood; in the stage of anxiety associated with the stress reaction (1 h after swimming in a vessel for 1 h), experimental rats showed more marked stimulation of proteolysis than seen in euthyroid animals, due to decreases in α1-AT and α2-MG activities, while in the resistance stage (at 48 h), the normalization of TLA and α1-AT and α2-MG levels occurring in stressed euthyroid rats was blocked; in the exhaustion stage (stressing for 1 h for 10 days), there was greater activation of proteolysis in experimental rats due to profound suppression of α1-AT and α2-MG activities. Administration of L-thyroxine (1.5-3.0 μg/kg for 28 days, which did not alter the blood ITH concentration, had no effect on the proteolysis system; in the anxiety and exhaustion stages, the increase in TLA was limited, while in the resistance stage this was prevented, with elimination of the depression of α1-AT and α2-MG activities. These results demonstrated a novel aspect of the involvement of ITH in the body's antistress system, i.e., their influences on the proteinase/inhibitors system. [ABSTRACT FROM AUTHOR]

Background: Gluten-free foods have increased in popularity over the past decade and are now being consumed by individuals without celiac disease. However, the physiologic effects of gluten intake in individuals without celiac disease remain unknown. High-abundance plasma proteins involved in inflammation, endothelial function, and other physiologic pathways may represent potential biomarkers of biological effects of gluten intake. Objective: The objective was to examine the association between gluten intake and plasma proteomic biomarkers in a population of adults without clinically diagnosed celiac disease. Methods: Subjects (n = 1095) were participants of the Toronto Nutrigenomics and Health Study, a cross-sectional examination of young adults aged 20-29 y. Dietary gluten intake was estimated by using a 1-mo, 196-item semiquantitative food-frequency questionnaire. The concentrations of 54 plasma proteins were measured simultaneously by liquid chromatography/ multiple-reaction monitoring mass spectrometry. The association between gluten intake and each proteomic biomarker was examined by using general linear models. Analyses were then conducted in individuals who do not have the human leukocyte antigen (HLA)-DQ2 or DQ8 risk variants required for the development of celiac disease to determine whether any associations observed could have been due to undiagnosed cases of celiac disease. Results: Increased gluten intake was associated with increased concentrations of plasma α2-macroglobulin (P = 0.01), a marker of inflammation and cytokine release. The association remained after adjusting for age, sex, BMI, ethnicity, physical activity, energy intake, fiber intake, and hormonal contraceptive use among women. This relation was not modified by HLA risk variants. Conclusion: Gluten consumption is associated with increased plasma a2-macroglobulin in young adults, which appears to be independent of celiac disease, suggesting possible effects of gluten on inflammation. [ABSTRACT FROM AUTHOR]

The diffusion of materials from systemic circulation to the central nervous system (CNS) is restricted by the blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (BCSFB). Choroid plexus epithelial cells (CPECs) of the brain ventricles constitute the BCSFB and regulate the infiltration of plasma proteins as well as immune cells into the interstitium of the CNS. The barrier function is altered in pathologic conditions. However, the regulatory mechanism of BCSFB is not fully understood. Here, we investigated the function of transient receptor potential vanilloid 4 (TRPV4), a polymodally gated divalent cation channel that is highly expressed in CPECs. TRPV4 was localized broadly on the apical membrane in swine CPECs, in contrast with an intense ciliary localization found on other cell types. Treatment with the TRPV4-specific agonist, GSK1016790A (GSK; EC50 34 nM), induced a robust calcium influx and an immediate serine/threonine protein phosphorylation. The agonist treatment induced a marked decrease in the amount of filamentous actin and disintegrated the cell junctions in 10-20 minutes. In contrast, inhibition of the basal TRPV4 activity with the TRPV4-specific antagonist, HC067047 (HC; IC50 74 nM), reduced the basolateral-to-apical transport of α-2-macroglobulin (A2M). Overall, this study demonstrated a novel physiologic function of TRPV4 in the regulation of BCSFB permeability. [ABSTRACT FROM AUTHOR]

The α 2 -macroglobulin receptor (α 2 MR) is a major domain of complement component 3b, which may play an important role in regulating the downstream complement system in teleosts. In order to characterize the domain thoroughly larger than currently available quantities are required. Thus, in this study the Epinephelus coioides α2MR (Ec-α2MR) was expressed and secreted by the methylotrophic yeast Pichia pastoris with variations in pH and induction time to identify optimal production conditions. At pH 5.5 with 48 h induction 13 mg of Ec-α2MR (ca. 90% purity) was obtained from 500 ml of culture. The Ec-α2MR protein product was validated by MALDI-TOF MS sequence analysis, and both Western blotting and ELISAs demonstrated that it possessed the expected activity, binding to C3b or C3b homolog antibodies, and thus can be used for future studies of the interactions and functions of complement proteins in teleosts. [ABSTRACT FROM AUTHOR]

Ligation of cell surface GRP78 by activated α2-macroglobulin (α2M*) promotes cell proliferation and suppresses apoptosis. α2M*-treated human prostate cancer cells exhibit a 2-3-fold increase in glucose uptake and lactate secretion, an effect similar to insulin treatment. In both α2M* and insulin-treated cells, the mRNA levels of SREBP1-c, SREBP2, fatty-acid synthase, acetyl-CoA carboxylase, ATP citrate lyase, and Glut-1 were significantly increased together with their protein levels, except for SREBP2. Pretreatment of cells with α2M* antagonist antibody directed against the carboxyl-terminal domain of GRP78 blocks these α2M*-mediated effects, and silencing GRP78 expression by RNAi inhibits up-regulation of ATP citrate lyase and fatty-acid synthase. α2M* induces a 2-3-fold increase in lipogenesis as determined by 6-[14C]glucose or 1-[14C]acetate incorporation into free cholesterol, cholesterol esters, triglycerides, free fatty acids, and phosphatidylcholine, which is blocked by inhibitors of fatty-acid synthase, PI 3-kinase, mTORC, or an antibody against the carboxyl-terminal domain of GRP78. We also assessed the incorporation of [14CH3]choline into phosphatidylcholine and observed similar effects. Lipogenesis is significantly affected by pretreatment of prostate cancer cells with fatostatin A, which blocks sterol regulatory element-binding protein proteolytic cleavage and activation. This study demonstrates that α2M* functions as a growth factor, leading to proliferation of prostate cancer cells by promoting insulin-like responses. An antibody against the carboxyl-terminal domain of GRP78 may have important applications in prostate cancer therapy. [ABSTRACT FROM AUTHOR]

The plasma protein C3 is a central element in the activation and effector functions of the complement system. A hereditary dysfunction of C3 that prevents complement activation via the alternative pathway (AP) was described previously in a Swedish family, but its genetic cause and molecular consequences have remained elusive. In this study, we provide these missing links by pinpointing the dysfunction to a point mutation in the ß-chain of C3 (c.1180T > C; p.Met373Thr). In the patient's plasma, AP activity was completely abolished and could only be reconstituted with the addition of normal C3. The M373T mutation was localized to the macroglobulin domain 4 of C3, which contains a binding site for the complement inhibitor compstatin and is considered critical for the interaction of C3 with the AP C3 convertase. Structural analyses suggested that the mutation disturbs the integrity of macroglobulin domain 4 and induces conformational changes that propagate into adjacent regions. Indeed, C3 M373T showed an altered binding pattern for compstatin and surface-bound C3b, and the presence of Thr373 in either the C3 substrate or convertase-affiliated C3b impaired C3 activation and opsonization. In contrast to known gain-of-function mutations in C3, patients affected by this loss-of-function mutation did not develop familial disease, but rather showed diverse and mostly episodic symptoms. Our study therefore reveals the molecular mechanism of a relevant loss-of-function mutation in C3 and provides insight into the function of the C3 convertase, the differential involvement of C3 activity in clinical conditions, and some potential implications of therapeutic complement inhibition. [ABSTRACT FROM AUTHOR]

Background. Subjects with type 2 diabetes mellitus (DM2) require an adequate glycemic control to avoid diabetic complications. Currently, saliva biomarkers are used as a diagnostic tool and can be indicative of the degree of progression and control of various diseases. Several studies indicate that α-2-macroglobulin levels are elevated in diabetic patients. Methods. 120 subjects with DM2 were enrolled and classified into two groups according to their glycemic control (percentage of glycated hemoglobin-A1c (HbA1c), <7% adequate glycemic control group; >7% inadequate glycemic control group). The relationship between α-2-macroglobulin levels from saliva samples and HbA1c was subsequently evaluated. Results. We found a positive correlation between α-2-macroglobulin and HbA1c (r=0.778 and P<0.0001). Area under the receivers operating characteristic (ROC) curve of α-2-macroglobulin indicated a positive discrimination threshold of α-2-macroglobulin (AUC = 0.903, CI 95%: 0.847–0.959, P<0.0001) to diagnose glycemic control. Conclusions. Our data strongly suggest that the level of saliva α-2-macroglobulin is an indicator for the degree of glycemic control in diabetic patients and represents a promising alternative method to evaluate this parameter. [ABSTRACT FROM AUTHOR]

This retrospective analysis was conducted in 64 patients diagnosed with type I cryoglobulinaemia ( CG) followed at two French centres. Median follow-up was 6·75 years. CG was IgG in 60% and IgM in 40% of all cases and was asymptomatic in 16 patients (25%). Cold-triggered ischaemic skin manifestations were observed in 33 patients (51%). Neurological manifestations were observed in 15 patients and renal manifestations in 13. Most of the patients with necrotic purpura (14/16, P = 0·009) and renal manifestations (11/13, P = 0·057) had IgG CG. IgG CG was associated with monoclonal gammopathy of undetermined significance ( MGUS), myeloma, chronic lymphocytic leukaemia and lymphoplasmocytic lymphoma in 18, 13, 5 and 2 patients, respectively. IgM CG was associated with MGUS and Waldenström macroglobulinaemia in 8 and 18 cases, respectively. One third of patients did not receive any specific treatment. Various treatments, including rituximab, were administered to 25/31 patients with IgG CG and 6/25 patients with IgM CG due to CG-related symptoms. Rituximab was ineffective in all cases associated with a predominantly plasmacytic proliferation. To conclude, type I CG has specific clinico-biological characteristics compared to type II CG. Furthermore, there are differences in terms of related manifestations between type I IgG and type I IgM CG. [ABSTRACT FROM AUTHOR]

CXCR4 WHIM frameshift and nonsense mutations follow MYD88L265P as the most common somatic variants in Waldenström Macroglobulinaemia ( WM), and impact clinical presentation and ibrutinib response. While the nonsense ( CXCR4S338X) mutation has been investigated, little is known about CXCR4 frameshift ( CXCR4FS) mutations. We engineered WM cells to express CXCR4FS mutations present in patients, and compared their CXCL12 ( SDF-1a) induced signalling and ibrutinib sensitivity to CXCR4wild-type (WT) and CXCR4S338X cells. Following CXCL12 stimulation, CXCR4FS and CXCR4S338X WM cells showed impaired CXCR4 receptor internalization, and enhanced AKT1 (also termed AKT) and MAPK1 (also termed ERK) activation versus CXCRWT cells ( P < 0·05), though MAPK1 activation was more prolonged in CXCR4S338X cells ( P < 0·05). CXCR4FS and CXCR4S338X cells, but not CXCR4WT cells, were rescued from ibrutinib-triggered apoptosis by CXCL12 that was reversed by AKT1, MAPK1 or CXCR4 antagonists. Treatment with an inhibitor that blocks MYD88L265P signalling triggered similar levels of apoptosis that was not abrogated by CXCL12 treatment in CXCR4WT and CXCR4WHIM cells. These studies show a functional role for CXCR4FS mutations in WM, and provide a framework for the investigation of CXCR4 antagonists with ibrutinib in CXCR4WHIM-mutated WM patients. Direct inhibition of MYD88L265P signalling overcomes CXCL12 triggered survival effects in CXCR4WHIM-mutated cells supporting a primary role for this survival pathway in WM. [ABSTRACT FROM AUTHOR]

The solution structure of complement C3b is crucial for the understanding of complement activation and regulation. C3b is generated by the removal of C3a from C3. Hydrolysis of the C3 thioester produces C3u, an analog of C3b. C3b cleavage results in C3c and C3d (thioester-containing domain; TED). To resolve functional questions in relation to C3b and C3u, analytical ultracentrifugation and x-ray and neutron scattering studies were used with C3, C3b, C3u, C3c, and C3d, using the wild-type allotype with Arg102. In 50 MM NaCl buffer, atomistic scattering modeling showed that both C3b and C3u adopted a compact structure, similar to the C3b crystal structure in which its TED and macroglobulin 1 (MG1) domains were connected through the Arg102-Glu1032 salt bridge. In physiological 137 MM NaCl, scattering modeling showed that C3b and C3u were both extended in structure, with the TED and MG1 domains now separated by up to 6 nm. The importance of the Arg102-Glu1032 salt bridge was determined using surface plasmon resonance to monitor the binding of wild-type C3d(E1032) and mutant C3d(A1032) to immobilized C3c. The mutant did not bind, whereas the wild-type form did. The high conformational variability of TED in C3b in physiological buffer showed that C3b is more reactive than previously thought. Because the Arg102-Glu1032 salt bridge is essential for the C3b-Factor H complex during the regulatory control of C3b, the known clinical associations of the major C3S (Arg102) and disease-linked C3F (Gly102) allotypes of C3b were experimentally explained for the first time. [ABSTRACT FROM AUTHOR]

The excessive activities of the serine proteinases neutrophil elastase and proteinase 3 are associated with tissue damage in chronic obstructive pulmonary disease. Reduced concentrations and/or inhibitory efficiency of the main circulating serine proteinase inhibitor α-1-antitrypsin result from point mutations in its gene. In addition, α-2- macroglobulin competes with α-1-antitrypsin for proteinases, and the α-2-macroglobulin-sequestered enzyme can retain its catalytic activity. We have studied how serine proteinases partition between these inhibitors and the effects of α-1-antitrypsin mutations on this partitioning. Subsequently, we have developed a three-dimensional reaction- diffusion model to describe events occurring in the lung interstitium when serine proteinases diffuse from the neutrophil azurophil granule following degranulation and subsequently bind to either α-1-antitrypsin or α-2-macroglobulin. We found that the proteinases remained uninhibited on the order of 0.1 s after release and diffused on the order of 10 µm into the tissue before becoming sequestered. We have shown that proteinases sequestered to α-2-macroglobulin retain their proteolytic activity and that neutrophil elastase complexes with α-2-macroglobulin are able to degrade elastin. Although neutrophil elastase is implicated in the pathophysiology of emphysema, our results highlight a potentially important role for proteinase 3 because of its greater concentration in azurophil granules, its reduced association rate constant with all α-1-antitrypsin variants studied here, its greater diffusion distance, time spent uninhibited following degranulation, and its greater propensity to partition to α-2-macroglobulin where it retains proteolytic activity. [ABSTRACT FROM AUTHOR]

This study aims to assess in residual periodontal pockets the clinical, microbiological, and local biological effects of antimicrobial photodynamic therapy (PDT), delivered after ultrasonic instrumentation either once or twice in a 1-week interval. A single center, three-arm randomized longitudinal study was carried out for 6 months. Twenty-eight systemically healthy patients on periodontal maintenance with residual pockets (pocket depth (PD) ≥5 mm, clinical attachment loss ≥2 mm, and bleeding upon probing (BOP+)) were included. Residual pockets on three teeth, separated from each other by at least two other teeth, served as study sites. After ultrasonic debridement, they were randomly assigned to either PDT delivered twice within 1 week (group A), PDT delivered only once (group B), or sham treatment without activating the laser (group C). Methylene blue was applied with a blunt irrigator tip into the pockets. Sites were irradiated with laser light at a wavelength of 670 nm using a light-diffusing tip introduced into the pocket. Initial PD was 5.9 ± 0.9, 6.3 ± 1.3, and 6.3 ± 1.5 mm in groups A, B, and C, respectively, differences being nonsignificant. PD was significantly reduced in all groups. At month 3, PD was significantly lower in groups A (2.9 ± 1.1 mm; p = 0.04) and B (2.8 ± 1.1 mm; p = 0.03) compared to group C (3.5 ± 1.2 mm). At month 6, none of the sites in group A had persisting pockets PD >4 mm and BOP+, whereas two sites in group B and four sites in group C stayed in this category. Detection frequencies of the studied microorganisms at >1,000 and >100.000 cells/ml did not change significantly from baseline to months 3 or 6 in any group. A significant overall decrease was observed from baseline to month 6 for C-reactive protein, serum amyloid A, fibrinogen, procalcitonin, and α-2 macroglobulin. When looking at the groups separately, C-reactive protein was significantly lower only if the laser had been activated twice ( p < 0.05). Other differences between groups were not significant. A single or double episodes of PDT had some additional benefit over ultrasonic instrumentation alone. [ABSTRACT FROM AUTHOR]

The ability of ticks to act as vectors for a wide range of serious human and animal infectious diseases is apparently linked to the insufficiency of the tick immune system to effectively eliminate pathogens they transmit. At the tick-pathogen interface, an important role is presumably played by components of an ancient complement system that includes a repertoire of thioester-containing proteins (TEPs), which in Ixodes sp. comprises three α 2 -macroglobulins (A 2 M), three C3 complement component-related molecules (C3), two macroglobulin complement-related (Mcr) and one insect-type TEPs (Tep). In order to assess the function of TEPs in tick immunity, a quantitative real-time PCR expression analysis of tick TEPs was performed at various developmental stages of Ixodes ricinus , and in tissues dissected from adult females. Expression of TEP genes was mostly tissue specific; IrA 2 M1, IrC3-1, IrC3-3 were found to be expressed in cells of tick fat body adjacent to the tracheal trunks, IrA 2 M2 in hemocytes, IrTep in ovaries, IrMcr1 in salivary glands and only IrA 2 M3, IrC3-2 and IrMcr2 mRNAs were present in multiple organs. Expression of tick TEPs was further examined in response to injection of model microbes representing Gram-negative, Gram-positive bacteria and yeast. The greatest expression induction was observed for IrA 2 M1 and IrC3-1 after challenge with the yeast Candida albicans . Phagocytosis of the yeast was strongly dependent on an active thioester bond and the subsequent silencing of individual tick TEPs by RNA interference demonstrated the involvement of IrC3-1 and IrMcr2. This result suggests the existence of a distinct complement-like pathway, different from that leading to phagocytosis of Gram-negative bacteria. Understanding of the tick immune response against model microbes should provide new concepts for investigating interactions between ticks and relevant tick-borne pathogens. [ABSTRACT FROM AUTHOR]

Posttranslationally modified proteins serve as autoimmunogenic targets in a wide spectrum of autoimmune diseases. Here, we identified a posttranslationally modified paraprotein target (paratargs) in monoclonal gammopathies of undetermined significance (MCUS), multiple myelomas (MM), and Waldenstrom's macroglobulinemias (WM) using protein macroarrays that were sumoylated and screened for reactivity with paraproteins from MGUS, MM, and WM patients. We found that paraproteins from a proportion of European, African-American, and Japanese patients specifically reacted with the sumoylated heat-shock protein 90 β isoform-α (HSP90-SUM01, where SUMO indicates small ubiquitin-like modifier), while no reactivity with HSP90-SUM01 was detected in over 800 controls. HSP90-SUMO1 was present in blood ceils from all patients with HSP90-SUMO1-binding paraproteins. We determined that the HSP90-SUM01 carrier state is autosomal-dominantly inherited and caused by the inability of SUMO peptidase sentrin/SUMO-specific protease 2 (SENP2) to desumoylate HSP90-SUM01. HSP90-SUM01 was detected in a small percentage of healthy individuals from all backgrounds; however, only MCUS, MM, and WM patients who were HSP90-SUM01 carriers produced HSP90-SUM01-specific paraproteins, suggesting that sumoylated HSP90 promotes pathogenesis of these diseases through chronic antigenic stimulation. This study demonstrates that harboring HSP90-5UM01 identifies healthy individuals at risk for plasma cell dyscrasias and that dominant inheritance of posttranslationally modified autoantigenic paratargs is one of the strongest molecular defined risk factors for MGUS, MM, and WM. [ABSTRACT FROM AUTHOR]

A case study is presented on virus inactivation by gamma irradiation. It states that in the manufacturing process of 𝛼2-Macroglobulin which is a glycoprotein there are risks of blood-borne infectious agent transmission. It also states the virus inactivation that includes enveloped viruses and non-enveloped viruses including pseudorabies virus, vesicular stomatitis virus, porcine parvovirus and murine encephalomyocarditis virus. The application of gamma irradiation is dicussed.

During rat pregnancy, some of the foetoplacental units undergo complete spontaneous resorption while the adjacent units remain unaffected. In an attempt to clarify the mechanisms implicated in this spontaneous resorption, implantation units from days 14 and 16 of pregnancy were examined. The number of implantation sites and resorption units was recorded, and uterine paraffin sections were stained with haematoxylin and eosin for the evaluation of tissue morphology. The incidence of resorption was about 9.2 % on day 14 and 8.2 % on day 16. Perforin and active caspase-3 immunostaining were performed for localization and characterization of uterine natural killer (uNK) and apoptotic cells, respectively. The α2-macroglobulin (α2-MG) expression was examined by in situ hybridization, immunohistochemistry and its levels quantified by enzyme-linked immunosorbent assay. A reduction in α2-MG decidual levels in resorpted units was observed when compared to normal implantation units in both days. This potent protease inhibitor is the major product secreted by the mesometrial decidual tissue and may constitute an indicator of maternal tissues remodelling abnormalities. Besides the decreased α2-MG levels, an increase in uNK cell number was found in resorption units. The decreased α2-MG levels may be related to the aberrant control of trophoblast invasion that may activate uNK cells. The elucidation of the mechanisms underlying natural pregnancy loss in rat may contribute for the clarification of the 'vanishing twin' phenomenon that occurs in human pregnancy. [ABSTRACT FROM AUTHOR]

α2- Macroglobulin (α2 M) has many functions in vertebrate physiology. To understand the basis of such functions, high-resolution structural models of its conformations and complexes with interacting partners are required. In an attempt to grow crystals that diffract to high or medium resolution, we isolated native human α2 M (hα2 M) and its counterpart from chicken egg white (ovostatin) from natural sources. We developed specific purification protocols, and modified the purified proteins either by deglycosylation or by conversion to their induced forms. Native proteins yielded macroscopically disordered crystals or crystals only diffracting to very low resolution (>20 Å), respectively. Optimization of native hα2 M crystals by varying chemical conditions was unsuccessful, while dehydration of native ovostatin crystals improved diffraction only slightly (10 Å). Moreover, treatment with several glycosidases hindered crystallization. Both proteins formed spherulites that were unsuitable for X-ray analysis, owing to a reduction of protein stability or an increase in sample heterogeneity. In contrast, transforming the native proteins to their induced forms by reaction either with methylamine or with peptidases (thermolysin and chymotrypsin) rendered well-shaped crystals routinely diffracting below 7 Å in a reproducible manner. [ABSTRACT FROM AUTHOR]

Bing-Neel syndrome (BNS), a rare neurological syndrome associated with Waldenström macroglobulinaemia (WM), is a direct involvement of the central nervous system by lymphoplasmacytoid cells characterized with an adverse prognostic. The MYD88 L265P mutation has been identified in the vast majority of patients with WM. The diagnosis of BNS is often challenging because of the variety of clinical presentations associated with difficult histological techniques. We hypothesized that identification of MYD88 L265P mutation in the cerebrospinal fluid (CSF) would contribute to the diagnosis of BNS in addition to imaging, flow cytometry and cytology. We identified MYD88 L265P mutation in the CSF and the bone marrow of all cases of BNS using quantitative polymerase chain reaction q PCR and Sanger sequencing. Copy neutral loss of heterozygosity including MYD88 was observed in one case. No mutation of CXCR4, CD79A and CD79B was observed in parallel. We further showed that monitoring the quantitative expression of MYD88 L265P mutation might be a useful molecular tool to monitor response to chemotherapy using q PCR. In conclusion, identification of MYD88 L265P mutation might be a new molecular-based biomarker tool to add to the diagnostic and monitoring armamentarium for BNS. [ABSTRACT FROM AUTHOR]

Aging is considered to be an adaptive mechanism to altered needs of an organism and/or to altered stimuli. Plasma concentrations of insulin-like growth factor binding protein-2 (IGFBP-2) increase with age and it is generally assumed that IGFBP-2 is a negative predictor of healthy aging. The aim of this study was to examine the distribution of IGFBP-2 molecular forms in different age groups and, specifically, the relationship between IGFBP-2 and alpha-2-macroglobulin (α2M). The relative amount of monomer IGFBP-2 was the highest in young persons, making up approximately 2/3 of the total circulating IGFBP-2. This gradually decreased with age down to 1/3 of total IGFBP-2 in elderly individuals. Fragmented IGFBP-2 increased with age and contributed almost 60% to the total immunoreactive IGFBP-2 in the age group 61–80 years. IGFBP-2/α2M complexes represented 10–12% of the total IGFBP-2 in the two younger groups but half this level in the oldest group. The significance of these changes and whether they affect more IGF-dependent or independent interactions are unknown. Due to drastic proteolysis of IGFBP-2, it may be postulated that either over-release of IGFBP-2-bound IGFs causes unwanted events or IGFBP-2 fragments are able to over-stimulate cellular processes. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the levels of α2-macroglobulin (α2M) in serum of patients with various types of glaucoma, given that it is a good indicator of inflammation and inflammatory processes in the body, which may be associated with the pathogenesis and progression of glaucoma. Materials and methods: the study included 180 eyes of patients of both sexes, aged from 40 to 70 years who based on the diagnostic tests were found to be suffering from glaucoma. Patients are, based on the type of glaucoma, divided into three groups: 60 eyes with glaucoma simplex (SG), 60 eyes with normotensive glaucoma (UE) and 60 eyes with glaucoma angular (GA). Fourth group, as a control group, consisted of 60 eyes with senile cataract without glaucoma. Each participant underwent a detailed ophthalmic examination which included: measurement of visual acuity, applanation tonometry, gonioskopy, ophthalmoscopy, field of vision and coherent tomography of the retina (OCT). The α2M concentration in patients serum, was determined by laser nephelometry (BN II analyzer apparatus). Results: the α2M concentration in serum of patients with different types of glaucoma was not significantly different compared to the control group, or between patients with different types of glaucoma. In patients with GA a significant positive correlation between the concentration of α2M in serum and OCT1OD was noted (rho=0.363, p<0.05), α2M in serum and OCT3OD (rho=0.470, p<0.01) and α2M in serum and OCT1OS (rho=0.438, p<0.05). Conclusion: in patients with GA a significant positive correlation between the concentration of α2M values and right eye PNO was noted (rh0=0.461, p<0.05) (Figure 2), and a statistically significant positive correlation between the concentration of α2M and LVD values (rh0=0.387, p<0.05). Statistically significant positive correlation between the concentration of α2M in serum and OCT1, OCT3 and OCT1, statistically significant positive correlation between the concentration of α2M values and PNO, and a significant positive correlation between the concentration of α2M and values of LV in patients with GA could suggest the adverse effects of the acute phase proteins in a group of patients with GA. [ABSTRACT FROM AUTHOR]