Quantification of human plasma metalloproteins in multiple sclerosis, ischemic stroke and healthy controls reveals an association of haptoglobin-hemoglobin complexes with age.

Advanced analytical methods play an important role in quantifying serum disease biomarkers. The problem of separating thousands of proteins can be reduced by analyzing for a 'sub-proteome', such as the 'metalloproteome', defined as all proteins that contain bound metals. We employed size exclusion chromatography (SEC) coupled to an inductively coupled plasma atomic emission spectrometer (ICP-AES) to analyze plasma from multiple sclerosis (MS) participants (n = 21), acute ischemic stroke (AIS) participants (n = 17) and healthy controls (n = 21) for Fe, Cu and Zn-metalloproteins. Using ANOVA analysis to compare the mean peak areas among the groups revealed no statistically significant differences for ceruloplasmin (p = 0.31), α2macroglobulin (p = 0.51) and transferrin (p = 0.31). However, a statistically significant difference was observed for the haptoglobin-hemoglobin (Hp-Hb) complex (p = 0.04), being driven by the difference between the control group and AIS (p = 0.012), but not with the MS group (p = 0.13), based on Dunnes test. A linear regression model for Hp-Hb complex with the groups now adjusted for age found no statistically significant differences between the groups (p = 0.95), but was suggestive for age (p = 0.057). To measure the strength of association between the Hp-Hb complex and age without possible modifications due to disease, we calculated the Spearman rank correlation in the healthy controls. The latter revealed a positive association (r = 0.39, 95% Confidence Interval = (-0.05, 0.83), which suggests that either the removal of Hp-Hb complexes from the blood circulation slows with age or that the release of Hb from red blood cells increases with age. We also observed that the Fe-peak corresponding to the Hp-Hb complex eluted ~100 s later in ~14% of all study samples, which was not correlated with age or disease diagnosis, but is consistent with the presence of the smaller Hp (1-1) isoform in 15% of the population.

Fecal host biomarkers predicting severity of Clostridioides difficile infection.

Clostridioides difficile is a major cause of health care-associated diarrhea. Severity ranges from mild to life-threatening, but this variability remains poorly understood. Microbiologic diagnosis of C. difficile infection (CDI) is straightforward but offers little insight into the patient's prognosis or into pathophysiologic determinants of clinical trajectory. The aim of this study was to discover host-derived, CDI-specific fecal biomarkers involved in disease severity. Subjects without and with CDI diarrhea were recruited. CDI severity was based on Infectious Diseases Society of America/Society for Healthcare Epidemiology of America criteria. We developed a liquid chromatography tandem mass spectrometry approach to identify host-derived protein biomarkers from stool and applied it to diagnostic samples for cohort-wise comparison (CDI-negative vs. nonsevere CDI vs. severe CDI). Selected biomarkers were orthogonally confirmed and subsequently verified in a CDI mouse model. We identified a protein signature from stool, consisting of alpha-2-macroglobulin (A2MG), matrix metalloproteinase-7 (MMP-7), and alpha-1-antitrypsin (A1AT), that not only discriminates CDI-positive samples from non-CDI ones but also is potentially associated with disease severity. In the mouse model, this signature with the murine homologs of the corresponding proteins was also identified. A2MG, MMP-7, and A1AT serve as biomarkers in patients with CDI and define novel components of the host response that may determine disease severity.

Simple blood fibrosis tests reduce unnecessary referrals for specialized evaluations of liver fibrosis in NAFLD and ALD patients.

BACKGROUND: Liver fibrosis evaluation is mandatory in non-alcoholic fatty liver disease (NAFLD) and alcoholic liver disease (ALD) to decide the patient management. Patients with these diseases are usually under the care of non-liver specialists who refer them to specialized centers where the most accurate fibrosis tests are available. We aimed to evaluate whether simple blood fibrosis tests available to all physicians help to reduce the rate of unnecessary referral of NAFLD and ALD patients without advanced fibrosis. METHODS: NAFLD and/or ALD patients newly referred to our center for a non-invasive evaluation of liver fibrosis were retrospectively included. The FibroMeterVCTE (FMVCTE, combination of blood markers and Fibroscan results) was defined as the reference test for specialized evaluation of liver fibrosis. A FMVCTE result <0.384 indicated the absence of advanced fibrosis and thus an "unnecessary referral". RESULTS: 558 patients were included (NAFLD: 283, ALD: 156, mixed NAFLD+ALD: 119). FMVCTE was <0.384 (unnecessary referral) in 58.8% of patients. FIB4 was <1.30 in 45.2% and eLIFT <8 in 47.7% of the patients. 84.9% of patients with FIB4 <1.30 and 85.3% of patients with eLIFT <8 had also FMVCTE <0.384. Therefore, using FIB4 or eLIFT as first-line evaluation of liver fibrosis decreased by three-fold the rate of unnecessary referral. The negative predictive value of FIB4 and eLIFT was >80% whatever the underlying cause of chronic liver disease. CONCLUSION: The use of eLIFT by non-liver specialists for NAFLD and ALD patients can improve the relevance of referrals for specialized evaluation of liver fibrosis.

Performance evaluation of 14 specific proteins measurement checked by an External Quality Assessment Scheme.

AIMS OF THE STUDY: To evaluate the state-of-the-art of 14 specific proteins measurement; to evaluate the laboratories' performance and the degree of harmonization in reporting results of participants in the External Quality Assessment Program of the Centre of Biomedical Research (CRB). METHODS: Overall and system-related inter-laboratory analytical variability (mean CVs%) and between-system differences (mean bias%) were evaluated from data of six EQA cycles 2013-2018. Moreover, we evaluated the analytical performance of participants as well as the units used to express proteins results. RESULTS: Overall inter-laboratory variability ranged from 3.8% for haptoglobin (HPT) to 12.5% for α1-antitrypsin (AAT) and decreased for IgA, α2-macroglobulin (A2M) and transferrin (TRF). Mean CVs% were generally higher for Siemens BN and Beckman Immage immunonephelometric systems, but <7.0% for all proteins. Mean bias > 7.0% was observed for BN (IgA, C4, AAT, transthyretin TTR), Siemens Vista (IgA, C4) and Immage (C4), whereas mean bias < -7.0% was found for Immage (AAT), Beckman AU (IgM) and Roche Cobas (C4, TTR, C-reactive protein). The laboratories' performance within the limits ranged from 85.1% of albumin (ALB) to 97.2% of HPT. The census of units employed in 2018, demonstrated that ~ 70% of laboratories still express the results in mg/dL. CONCLUSIONS: Despite a reduction in inter-laboratory variability for some proteins, different analytical systems showed both proportional and constant bias between methods. Units used by participants have not been substantially changed and dL is still largely used. The CRB EQA Program, with its performance data sets, is a valuable resource for laboratories and IVD manufacturers and support the goals of harmonization.

Public health research needs for molecular epidemiology and to emphasize homeostasis - could the omnipotent endopeptidase inhibitor α-2-macroglobulin be a meaningful biomarker?

Public health authorities in low- and middle-income countries face dramatic challenges in handling rapidly increasing non-communicable diseases (NCDs), due to the epidemiological- and particularly nutritional transition. Among major reasons for the development of NCDs are smoking and alcohol, but overnutrition and obesity are also major threats to population health. Obesity is related to diabetes and cancer, but also has a genetic background. It is difficult to recommend a healthy nutrition. This is because of conflicting nutritional conceptions, and given the complexity of human metabolism understanding this topic can be difficult for the laymen.  Public health measures advocating physical activity and refraining from high intake of energy, sugar and soft drinks need to be enhanced by supporting the 'intrinsic motivation' to preserve a good health. The mission of public health should be to increase awareness about the complexity of human metabolism, and the involvement of genetic and epigenetics in health and diseases. To maintain homeostasis, means to keep an optimal relationship between catabolism and synthesis, seems to be of particular interest. Preconditions for this is, that public health institutions within the administration- and academic sector follow up developments in life science and molecular biology and conduct population-based research making use of molecular epidemiology, especially those related to key metabolic steps and maintenance of 'homeostasis', in balancing catabolism and anabolism. A prospective biomarker for this situation might be α-2-macroglobulin.

Molecular form and concentration of serum α2-macroglobulin in diabetes.

α2-Macroglobulin is a highly abundant serum protein involved in the development of atherosclerosis and cardiac hypertrophy. However, its circulating molecular form and exact concentrations in human health/diseases are not known. Blue native-polyacrylamide gel electrophoresis of human serum was used to confirm the native conformation of α2-macroglobulin. We created an enzyme-linked immunosorbent assay suitable for quantifying its circulating molecular form and undertook a cross-sectional study to measure its serum levels in 248 patients with diabetes mellitus and 59 healthy volunteers. The predominant circulating molecular form of α2-macroglobulin was the tetramer, whereas its dimer was detectable in patients with high serum levels of α2-macroglobulin. The serum α2-macroglobulin concentration was not associated with glycated hemoglobin or any other glycemic variable as evaluated from 48-h continuous glucose monitoring, but showed close correlation with left ventricular posterior wall thickness, carotid artery intima-media thickness, urinary albumin:creatinine ratio (ACR) and brachial-ankle pulse wave velocity (baPWV). Multivariate analysis revealed only the ACR and baPWV to be independent variables influencing serum levels of α2-macroglobulin. Thus, an increased ACR and baPWV are associated with higher serum concentrations of α2-macroglobulin, and the latter may contribute to the mechanism by which albuminuria increases the risk of developing cardiovascular diseases.

Age-Dependent Variation in Glycosylation Features of Alpha-2-Macroglobulin.

Alpha-2-macroglobulin (A2M) is a glycosylated broad spectrum inhibitor of numerous proteases, including those involved in blood coagulation. Glycosylation characteristics can affect protein structure and function. This study compares glycosylation characteristics of A2M in newborn umbilical cord (NUCP) and adult pooled plasmas. Peptide N-Glycosidase F treatment was used to evaluate the total N-glycan content of the molecules. Neuraminidase treatment, and affinity for Ricinus Communis Agglutinin I were used to examine terminal sialic acid and galactose content, respectively. Two-dimensional (2D) electrophoresis was used to determine charge-related isoform profiles and fluorophore-assisted carbohydrate electrophoresis (FACE) was used to characterize N-glycan profiles. Results revealed no difference in total N-glycan mass, however, a statistically significant difference was shown in the change in charge associated with sialic acid loss in the NUCP A2M population. 2D electrophoresis indicated a lower pI range for NUCP A2M isoforms. In addition, NUCP A2M displayed a trend toward higher terminal galactose quantities than adult A2M. FACE revealed an increased abundance of more branched, higher molecular weight glycans in NUCP A2M. These differences in glycan branching and charged residues may impact A2M receptor-based clearance and thus could be responsible for the increased A2M concentration seen in NUCP, and newborns.

[Immunoregulatory proteins and cytokines in the blood of patients with Graves' disease].

BACKGROUND: Immunoregulatory proteins (alpha-2-macroglobulin, lactoferrin) actively participate in inflammatory and autoimmune processes, affect synthesis and transport of hormones and cytokines, and control cell proliferation and apoptosis. However, the role of these proteins in the pathogenesis of Graves' disease (GD) is poorly understood. Objective - the study objective was to determine blood levels of alpha-2-macroglobulin (α2-MG), lactoferrin (LF), and cytokines (TNF-α, IL-6, IL-8, and IFN-γ) in GD. MATERIAL AND METHODS: We determined blood levels of TSH, free T4, TSH receptor antibodies, TNF-α, IL-6, IL-8, IFN-γ, and LF by ELISA as well as α2-MG by quantitative rocket immunoelectrophoresis in 50 patients with decompensated and compensated (4-6 months and 1.5-2 years after treatment onset) GD and 25 healthy females (control group). RESULTS: GD clinically manifested by body weight l in 84% of patients, sinus tachycardia in almost all patients, paroxysmal atrial fibrillation in 18% of cases, endocrine ophthalmopathy in 12% of patients, and neurological changes. In decompensated GD, there was a statistically significant increase in levels of IFN-γ, IL-6, and α2-MG and an especially significant increase in levels of IL-8 and LF. At 4-6 months after treatment onset, clinical manifestations were stopped in all patients, levels of IL-6 and α2-MG decreased, but the concentrations of TSH receptor antibodies (TSHR-Abs), IL-8, IFN-γ, and LF remained elevated. At 1.5-2 years, levels of the studied proteins and cytokines did not differ from those in the control group. CONCLUSION: An increase in blood levels of IL-8, LF, IL-6, and α2-MG in incident or recurrent GD and a decrease in the levels during treatment confirm involvement of immunoregulatory proteins in pathogenesis of the disease.

Comparative Serum Proteomic Analysis of the Effects of Sodium Selenate on a Mouse Model of Alzheimer's Disease.

Selenium (Se), as a nutritionally essential trace element, has been shown to decrease with age and is closely related to Alzheimer's disease (AD). To probe the effects of Se on AD pathology, two-dimensional fluorescence difference gel electrophoresis was applied to the serum samples collected from the wild-type (WT) mice and the triple transgenic (PS1M146V/AßPPSwe/TauP301L) AD mice (3xTg-AD), treated with or without sodium selenate in drinking water for 4 months beginning at 2 months of age. Proteomics results revealed 17 differentially expressed proteins between WT and 3xTg-AD mice. It was found that the administration of selenate reversed the alterations of the differentially expressed serum proteins by up-regulating 13 proteins and down-regulating 2 proteins which were reported to be involved in the key pathogenesis of AD, including regulation of Aß production, lipid metabolism regulation, and anti-inflammation. These results suggested that a dietary supplement with selenate is effective for prevention and treatment of AD, and the mechanism was maybe related to its role in Aß regulation, lipid metabolism, and anti-inflammation. Moreover, we also presented that α-2 macroglobulin, transthyretin, haptoglobin, alpha-2-HS-glycoprotein, and alpha-1-antitrypsin in the serum can be used to evaluate the effect of selenate on AD pathology.

Serum leucine-rich α2-glycoprotein is elevated in patients with systemic lupus erythematosus and correlates with disease activity.

BACKGROUND: We evaluated whether serum leucine-rich α2-glycoprotein (LRG) is associated with disease activity in patients with systemic lupus erythematosus (SLE). METHODS: We measured serum LRG in 194 SLE patients. SLE disease activity index-2000 (SLEDAI-2 K) was used to assess SLE activity, and patients with SLEDAI-2 K ≥5 were defined as having active SLE. Correlation between serum LRG, SLEDAI-2 K, and laboratory variables was estimated by Pearson's correlation analysis. The optimal serum LRG cut-off value for predicting active SLE was calculated using receiver operator characteristic (ROC) curve, and multivariable logistic regression was used to determine the odds ratio (OR) of laboratory variables. RESULTS: In total, 74 (38.1%) and 120 (61.9%) patients were classified as active and inactive SLE, respectively. Serum LRG was higher in patients with active SLE than in inactive SLE and healthy controls (26.6 vs. 14.4 vs. 1.2 ng/ml, p < .001). Serum LRG significantly correlated with SLEDAI-2 K (r = 0.340, p < .001) and laboratory variables. ROC analysis revealed that optimal serum LRG cut-off value for active SLE was >45.7 ng/ml. In multivariable logistic regression analysis, serum LRG >45.7 ng/ml (OR 4.089, 95% confidence interval 1.351, 12.376, p = .013) was an independent predictor of active SLE. CONCLUSIONS: Serum LRG might be a biomarker for estimating SLE disease activity.

Binding interaction of sheep alpha-2-macroglobulin and tannic acid: A spectroscopic and thermodynamic study.

Tannic acid is a polyphenol found in plant species commonly consumed by ruminants. It works as an important molecule in plant defense system to fight against environmental stressors. Tannic acid has number of effects on animals and humans. An attempt has been made to study the interaction of tannic acid with alpha-2-macroglobulin (α2M). α2M is a large tetrameric glycoprotein which function as a key serum anti-proteinase under physiological conditions. In the present study we explored the tannic acid-α2M interaction by number of spectroscopic techniques such as UV, fluorescence, CD and FTIR along with isothermal titration calorimetry. CD and FT-IR spectroscopy were mainly used to study the secondary structural change induced in the antiproteinase. Analysis of activity shows the antiproteolytic potential of protein was compromised. Data of UV spectroscopy shows formation of α2M-tannic acid complex. The thermodynamic signatures of this interaction reveals hydrogen bonding played a major role in the binding of α2M-tannic acid. Analysis of CD and FTIR results suggest a minor conformational change in α2M on tannic acid binding. Overall, tannic acid induces subtle conformation change in α2M structure resulting the loss of its proteinase inhibitory activity.

Alpha-2-macroglobulin as a Promising Biological Marker of Endothelial Function.

AIMS: Alpha-2-macroglobulin (α2MG) is thought to be associated with inflammatory reactions and procoagulant properties that might cause ischemic stroke. Endothelial dysfunction plays an important role in atherosclerosis development and in the occurrence of cardiovascular events. In this study, we investigated whether serum α2MG levels, endothelial function, and endothelial progenitor cell (EPC) number were associated in patients with chronic stroke or cardiovascular risk factors. METHODS: Patients with a history of stroke or any established cardiovascular risk factors were enrolled in this study (n=102; 69 men, 70.1±9.2 years). Endothelial function was assessed by flow-mediated dilation (FMD). EPC numbers (CD34＋/CD133＋) were measured using flow cytometry (n=91). Serum α2MG levels were measured by nephelometry. RESULTS: Patients in the highest tertile of serum α2MG levels were older (P=0.019) and more frequently exhibited dyslipidemia (P=0.021). Univariate-regression analysis revealed that increased α2MG levels were negatively associated with FMD values (r=-0.25; P=0.010), whereas increased EPC numbers were positively associated (r=0.21; P=0.044). Multivariate-regression analysis adjusted for male gender, hypertension, and severe white-matter lesions showed that serum α2MG levels were independently associated with FMD values (standardized partial regression coefficient [ß] -0.185; P=0.033), although not significantly associated with EPC numbers. CONCLUSION: Serum α2MG levels might reflect endothelial dysfunction evaluated by FMD in patients with chronic stroke or cardiovascular risk factors.

Impaired fibrinolysis and lower levels of plasma α2-macroglobulin are associated with an increased risk of severe asthma exacerbations.

Recently we have reported that asthma is associated with enhanced plasma thrombin formation, impaired fibrinolysis and platelet activation. In the present study we investigated whether described prothrombotic blood alterations might predispose to thromboembolic events or asthma exacerbations. In 164 adult asthmatics we assessed clinical events during 3-year follow-up and analyzed their associations with measured at baseline prothrombotic blood parameters. Data were obtained from 157 (95.7%) of the asthma patients. We documented 198 severe asthma exacerbations (64/year), which occurred in 53 subjects (34%). These patients were older (p = 0.004), had worse asthma control (p = 0.02) and lower spirometry values (p = 0.01), at baseline. Interestingly, this subgroup had longer clot lysis time (CLT), as well as lower α2-macroglobulin (p = 0.038 and p = 0.04, respectively, after adjustment for potential confounders). Increased CLT and lower α2-macroglobulin were demonstrated as independent predictors of asthma exacerbation in multiple regression model. Moreover, we documented two episodes of deep vein thrombosis (1.3%), and eight acute coronary syndromes (5.1%). Patients who experienced thromboembolic events (n = 10, 6.4%, 2.1%/year) had lower α2-macroglobulin (p = 0.04), without differences in efficiency of fibrinolysis and thrombin generation. Impaired fibrinolysis and lower levels of α2-macroglobulin might predispose to a higher rate of asthma exacerbations, suggesting new links between disturbed hemostasis and asthma.

Peripheral blood gene expression of acute phase proteins in people with first episode psychosis.

BACKGROUND: There is a growing interest in the association between schizophrenia and the activation of inflammatory system with signs of acute phase (AP) response. Majority of such studies had focused on C-reactive protein (CRP). The aims of the present study were (i) to examine the gene expression profiles of other acute phase proteins (APP), namely haptoglobin (HP), alpha-1 antitrypsin (A1T), and alpha-2 macroglobulin (A2M) in patients with first episode psychosis (FEP) over a period of three months and (ii) to explore the association between APP levels and severity of symptoms. METHODS: In this study, HP, A1T and A2M gene expression levels from whole blood were measured at recruitment, 1- and 3-month follow-up visits using quantitative PCR (qPCR) in 43 patients with FEP and in 57 healthy controls. Diagnoses was ascertained on the Structured Clinical Interview for DSM-IV-TR. Severity of symptoms in patients was assessed on the Positive and Negative Syndrome Scale (PANSS) and a previously validated 5-factor PANSS structure was applied in the subsequent analyses. RESULTS: The FEP sample comprised of 28 (65.1%) individuals with schizophrenia, 12 (27.9%) with schizophreniform disorder and 3 (7%) with schizoaffective disorder. HP gene expression level was noted to be significantly higher in patients than controls at all three time points: recruitment (P=0.049), 1-month follow up (P=0.002) and 3-month follow up (P=0.005). PANSS positive, depression, and excitement symptom factors showed significant associations with HP (P=0.002), A1T (P=0.016) and A2M (P=0.034), respectively. These findings remained significant after controlling for age, gender, smoking status and accumulated chlorpromazine dosage. CONCLUSION: The current study provides information on HP, A1T and A2M gene expression profiles in FEP patients and their associations with psychopathology. This provides support for the hypothesis that inflammation is related to schizophrenia and further encourages studies on immune-inflammatory markers to understand the relationship between inflammation and schizophrenia.

Detection of immunogenic parasite and host-specific proteins in the sera of active and chronic individuals infected with Toxoplasma gondii.

Toxoplasma gondii infection in pregnant women may result in abortion and foetal abnormalities, and may be life-threatening in immunocompromised hosts. To identify the potential infection markers of this disease, 2-DE and Western blot methods were employed to study the parasite circulating antigens and host-specific proteins in the sera of T. gondii-infected individuals. The comparisons were made between serum protein profiles of infected (n=31) and normal (n=10) subjects. Antigenic proteins were identified by immunoblotting using pooled sera and monoclonal anti-human IgM-HRP. Selected protein spots were characterised using mass spectrometry. Prominent differences were observed when serum samples of T. gondii-infected individuals and normal controls were compared. A significant up-regulation of host-specific proteins, α(2)-HS glycoprotein and α(1)-B glycoprotein, was also observed in the silver-stained gels of both active and chronic infections. However, only α(2)-HS glycoprotein and α(1)-B glycoprotein in the active infection showed immunoreactivity in Western blots. In addition, three spots of T. gondii proteins were detected, namely (i) hypothetical protein chrXII: 3984434-3 TGME 49, (ii) dual specificity protein phosphatase, catalytic domain TGME 49 and (iii) NADPH-cytochrome p450 reductase TGME 49. Thus, 2-DE approach followed by Western blotting has enabled the identification of five potential infection markers for the diagnosis of toxoplasmosis: three are parasite-specific proteins and two are host-specific proteins.

Effects of resynchronization programs on pregnancy per artificial insemination, progesterone, and pregnancy-associated glycoproteins in plasma of lactating dairy cows.

Objectives were to develop a timed artificial insemination (TAI) resynchronization program to improve pregnancy per AI and to evaluate responses of circulating progesterone and pregnancy-associated glycoproteins in lactating cows. Cows (n=1,578) were presynchronized with 2 injections of PGF2alpha, given 14 d apart starting on d 45+/-3 postpartum, followed by Ovsynch [2 injections of GnRH 7 d before and 56 h after injection of PGF2alpha, TAI 16 h after second injection (d 0)]. The Resynch-treated cows received an intravaginal progesterone insert from d 18 to 25, GnRH on d 25, and pregnancy diagnosis on d 32, and nonpregnant cows received PGF2alpha., GnRH 56 h later, and TAI 16 h later (d 35). The control cows were diagnosed for pregnancy on d 32 and nonpregnant cows received GnRH, PGF2alpha 39 d after TAI, GnRH 56 h later, and TAI 16 h later (d 42). Pregnancy was reconfirmed on d 60 after AI. Ovarian structures were examined in a subset of cows at the time of GnRH and PGF2alpha injections. Blood samples for analyses of progesterone and pregnancy-associated glycoproteins were collected every 2 d from d 18 to 30 in 100 cows, and collection continued weekly to d 60 for pregnant cows (n=43). Preenrollment pregnancies per AI on d 32 did not differ for cows subsequently treated as Resynch (45.8%, n=814) and control (45.9%, n=764), and pregnancy losses on d 60 were 6.7 and 4.0%, respectively. Resynchronized service pregnancy per AI (36%, n=441; 39.5%, n=412) and pregnancy losses (6.3 and 6.7%) did not differ for Resynch and control treatments, respectively. Days open for pregnant cows after 2 TAI were less for the Resynch treatment than for the control treatment (96.2+/-0.82 vs. 99.5+/-0.83 d). Cows in the Resynch treatment had more large follicles at the time of GnRH. The number of corpora lutea did not differ between treatments at the time of PGF2alpha. Plasma progesterone for pregnant cows was greater for Resynch cows than for control cows (18-60 d; 6.6 vs. 5.3 ng/mL), and plasma concentrations of progesterone on d 18 were greater for pregnant cows than for nonpregnant cows (5.3 vs. 4.3 ng/mL). Plasma pregnancy-associated glycoproteins during pregnancy were lower for cows in the Resynch treatment compared with control cows on d 39 (2.8 vs. 4.1 ng/mL) and 46 (1.3 vs. 3.0 ng/mL). Cows pregnant on d 32 that lost pregnancy by d 60 (n=7) had lower plasma concentrations of pregnancy-associated glycoproteins on d 30 than cows that maintained pregnancy (n=36; 2.9 vs. 5.0 ng/mL). Pregnancy-associated glycoproteins on d 30 (>0.33 ng/mL) were predictive of a positive d 32 pregnancy diagnosis (sensitivity=100%; specificity=90.6%). In conclusion, Resynch and control protocols had comparable pregnancy per AI for first and second TAI services, but pregnancy occurred 3.2 d earlier in the Resynch group because inseminations in the Resynch treatment began 7 d before those in the control treatment. Administration of an intravaginal progesterone insert, or GnRH, or both increased progesterone during pregnancy. Dynamics of pregnancy-associated glycoproteins were indicative of pregnancy status and pregnancy loss.

Pregnancy - associated glycoproteins as a new diagnostic tool in cattle reproduction.

Pregnancy-associated glycoproteins (PAGs) are powerful pregnancy markers in domestic cattle. These proteins are expressed in mono- and binucleate trophoblast cells from the first days of gestation until calving. Different molecules were identified as being expressed at various stages of pregnancy. However, up to date, their functions and activities during pregnancy were not yet established. Specific RIA tests were developed (classic and alternative RIA) and used to measure the concentration of these glycoproteins in blood during gestation and the postpartum period in cattle. In maternal blood, PAGs rise to detectable levels from days 24 to 28 after fertilization. A recent study indicated that PAGs can also be detected in milk samples. However, concentrations in milk are much lower when compared to those of plasma.

[The level of pregnancy-associated alpha2-glycoprotein and the hormonal background during different options of hormonal replacement therapy in the menopausal syndrome].

The authors studied the concentration of pregnancy-associated alpha2-glycoprotein (PA alpha2GP), a sensitive marker of estrogen-dependent tumors, and the association of its level with the serum content of a number of hormones: follicle-stimulating hormone, luteinizing hormone, estradiol (E-2), dehydroepiandrosterone sulfate (DEAS-S), and testosterone in females receiving the groups of drugs containing: 1) estradiol valerate; 2) 17beta-estradiol, and 3) tibolone. The type of the active ingredient of a drug and the duration of its administration were shown to differently affect both the concentration of hypothalamopituitary hormones and steroid sex hormones and the level of PA alpha2GP). The latter increased significantly in Group 1 and insignificantly in Group 2 and did not differ from the normal values in Group 3, at the same time the concentration of E-2 elevated in Groups 1 and 2, rather than in Group 3; the level of DEAS-S increased in Groups 2 and 3 irrespective of the duration of use. Moreover, there were elevated levels of testosterone in Group 3 and those of DEAS-S in Group 1 only when the drugs were administered for 3-6 months. A number of correlations were found in the levels of PA alpha2GP with those of steroid hormones. The authors consider that individual monitoring of the level of PA alpha2GP in the females who need hormonal therapy in menopause provides a useful guide to choosing a drug, monitoring its use efficiency, and preventing malignant proliferation in proper time.

[Alpha-2-macroglobulin ligands and their biotransport mechanisms].

Alpha-2-macroglobulin (MG) is a high-molecular weight glycoprotein that possesses a wide range of regulatory functions. Earlier it has been shown that covalent binding of MG with proteinases results in conformational transformation of MG, which enables MG to transport some additional types of cytokines linked by noncovalent interactions. The results of our study have demonstrated that the range of proteins, with the ability for additional binding with transformed MG is variable and comprises IgG, IgA, IgM, albumin, both types of lipoprotein chain, plasmin, some cytokines and even pregnancy associated alpha-2-glycoprotein (structured MG homolog). The major ligands are found to be albumin, IgG, plasmin and, to a lesser degree, lipoproteins. MG interactions with both acidic and low-alkaline proteinases contribute to neutralization of total charge of the formed complex at neutral pH, typical for internal fluids of the organism, and that the addition of low-density lipoprotein receptor-related protein (LRP) increases the amount of electroneutral complexes at pH 7.4. We suppose, that this mechanism enables the transformed MG (or may be its complex with other regulatory proteins) to rapidly precipitate rapidly on cellular surface and then, after binding with LRP and secondary neutralization of the total charge under physiological pH conditions, to pass through cellular membrane and to realize its own regulatory functions.