Search

Background: We evaluated the effects of high-protein dairy milk ingestion on changes in body composition, strength, power, and skeletal muscle regulatory markers following 6 weeks of resistance training in trained young males., Methods: Thirty resistance-trained young males (age: 27 ± 3 years; training experience: 15 ± 2 months) were randomly assigned to one of two groups: high-protein dairy milk (both whey and casein) + resistance training (MR; n = 15) or isoenergetic carbohydrate (maltodextrin 9%) + resistance training (PR; n = 15). Milk and placebo were ingested immediately post-exercise (250 mL; 30 g protein) and 30 min before sleep (250 mL; 30 g protein). Before and after 6 weeks of linear periodized resistance training (4 times/week), body composition (bioelectrical impedance), strength, power, and serum levels of skeletal muscle regulatory markers (insulin-like growth factor 1 (IGF-1), growth hormone, testosterone, cortisol, follistatin, myostatin, and follistatin-myostatin ratio) were assessed., Results: The MR group experienced a significantly higher ( p < 0.05) increase in lean mass, strength, and power (upper- and lower-body) than the PR group. Further, IGF-1, growth hormone, testosterone, follistatin, and follistatin-myostatin ratio were significantly increased, while cortisol and myostatin significantly decreased in the MR group than the PR group ( p < 0.05)., Conclusions: The strategic ingestion of high-protein dairy milk (post-exercise and pre-sleep) during 6 weeks of resistance training augmented lean mass, strength, power, and altered serum concentrations of skeletal muscle regulatory markers in trained young males compared to placebo.

Acute pancreatitis (AP) remains a significant clinical challenge. Mitochondrial dysfunction contributes significantly to the pathogenesis of AP. Milk fat globule EGF factor 8 (MFG-E8) is an opsonizing protein, which has many biological functions via binding to αvβ3/5 integrins. Ligand-dependent integrin-FAK activation of STAT3 was reported to be of great importance for maintaining a normal mitochondrial function. However, MFG-E8's role in AP has not been evaluated., Methods: Blood samples were acquired from 69 healthy controls and 134 AP patients. Serum MFG-E8 levels were measured by ELISA. The relationship between serum concentrations of MFG-E8 and disease severity were analyzed. The role of MFG-E8 was evaluated in experimental models of AP., Results: Serum concentrations of MFG-E8 were lower in AP patients than healthy controls. And serum MFG-E8 concentrations were negatively correlated with disease severity in AP patients. In mice, MFG-E8 administration decreased L-arginine-induced pancreatic injury and mortality. MFG-E8's protective effects in experimental AP were associated with improvement in mitochondrial function and reduction in oxidative stress. MFG-E8 knockout mice suffered more severe pancreatic injury and greater mitochondrial damage after l-arginine administration. Mechanistically, MFG-E8 activated the FAK-STAT3 pathway in AP mice. Cilengitide, a specific αvβ3/5 integrin inhibitor, abolished MFG-E8's beneficial effects in AP. PF00562271, a specific FAK inhibitor, blocked MFG-E8-induced STAT3 phosphorylation. APTSTAT3-9R, a specific STAT3 antagonist, also eliminated MFG-E8's beneficial effects under such a condition., Conclusions: MFG-E8 acts as an endogenous protective mediator in the pathogenesis of AP. MFG-E8 administration protects against AP possibly by restoring mitochondrial function via activation of the integrin-FAK-STAT3 signaling pathway. Targeting the action of MFG-E8 may present a potential therapeutic option for AP., (© 2021 The Authors. Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.)

Large (> 1 μm) tumor-derived extracellular vesicles (tdEVs) enriched from the cell fraction of centrifuged whole blood are prognostic in metastatic castration-resistant prostate cancer (mCRPC) patients. However, the highest concentration of tdEVs is expected in the cell-free plasma fraction. In this pilot study, we determine whether mCRPC patients can be discriminated from healthy controls based on detection of tdEVs (< 1μm, EpCAM+) and/or other EVs, in cell-free plasma and/or urine. The presence of marker+ EVs in plasma and urine samples from mCRPC patients (n = 5) and healthy controls (n = 5) was determined by flow cytometry (FCM) and surface plasmon resonance imaging (SPRi) using an antibody panel and lactadherin. For FCM, the concentrations of marker positive (+) particles and EVs (refractive index <1.42) were determined. Only the lactadherin+ particle and EV concentration in plasma measured by FCM differed significantly between patients and controls (p = 0.017). All other markers did not result in signals exceeding the background on both FCM and SPRi, or did not differ significantly between patients and controls. In conclusion, no difference was found between patients and controls based on the detection of tdEVs. For FCM, the measured sample volumes are too small to detect tdEVs. For SPRi, the concentration of tdEVs is probably too low to be detected. Thus, to detect tdEVs in cell-free plasma and/or urine, EV enrichment and/or concentration is required. Furthermore, we recommend testing other markers and/or a combination of markers to discriminate mCRPC patients from healthy controls., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: Frank Coumans is majority shareholder in Exometry, Amsterdam, The Netherlands. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Purpose: To examine whether supplementation with low doses of fish or milk proteins would affect glucose regulation and circulating lipid concentrations in overweight healthy adults., Methods: Ninety-three overweight adults were assigned to receive 2.5 g protein/day from herring (HER), salmon (SAL), cod (COD) or milk (CAS, a casein-whey mixture as positive control) as tablets for 8 weeks., Results: Seventy-seven participants were included in the analyses. HER and SAL did not affect glucose and insulin concentrations. COD significantly reduced within-group changes in 90 and 120 min postprandial glucose concentrations but changes were not different from HER and SAL groups. CAS supplementation significantly reduced the area under the curve for glucose concentrations (- 7%), especially when compared to SAL group, and reduced postprandial insulin c-peptide concentration (- 23%). Reductions in acetoacetate (- 24%) and β-hydroxybutyrate (- 29%) serum concentrations in HER group were more prominent compared to SAL and COD groups, with no differences between fish protein groups for α-hydroxybutyrate. Serum concentrations of α-hydroxybutyrate (- 23%), acetoacetate (- 39%) and β-hydroxybutyrate (- 40%) were significantly reduced within CAS group, and the decreases were significantly more pronounced when compared to SAL group. Serum lipid concentrations were not altered in any of the intervention groups., Conclusion: Findings indicate that 2.5 g/day of proteins from fish or milk may be sufficient to improve glucose regulation in overweight adults. The effects were most pronounced after supplementation with proteins from cod, herring and milk, whereas salmon protein did not affect any of the measurements related to glucose regulation., Clinical Trail Registration: This trial was registered at clinicaltrials.gov as NCT01641055.

Systemic lupus erythematosus (SLE) is characterized by impaired clearance of apoptotic cells. Milk fat globule epidermal growth factor 8 (MFGE8) is a protein that connects αvβ3 integrin on phagocytic macrophages with phosphatidylserine on apoptotic cells. We investigated whether genetic variation of the MFGE8 gene and serum MFGE8 concentration are associated with SLE. Single nucleotide polymorphisms (SNPs) were genotyped and serum concentrations were analyzed. The rs2271715 C allele and rs3743388 G allele showed higher frequency in SLE than in healthy subjects (HSs). Three haplotypes were found among 4 SNPs (rs4945, rs1878327, rs2271715, and rs3743388): AACG, CGCG, and CGTC. CGCG haplotype was significantly more common in SLE than in HSs. rs4945 was associated with the erythrocyte sedimentation rate and rs1878327 was associated with alopecia, C-reactive protein, complement 3, anti-dsDNA antibody, and high disease activity. rs2271715 and rs3743388 were associated with renal disease, cumulative glucocorticoid dose, and cyclophosphamide and mycophenolate mofetil use. Serum MFGE8 concentrations were significantly higher in SLE than in HSs. Furthermore, the levels of MFGE8 were significantly higher in SLE than HSs of the rs2271715 CC genotype. In conclusion, MFGE8 genetic polymorphisms are associated not only with susceptibility to SLE but also with disease activity through modulation of gene expression.

Current serum hepatocellular carcinoma (HCC) biomarkers are insufficient for early diagnosis. We aimed to clarify whether serum MFG-E8 can serve as a diagnostic or prognostic biomarker of HCC. Serum MFG-E8 levels of 282 HCC patients, who underwent primary hepatectomy, were examined by ELISA. We also quantified serum MFG-E8 levels in patients with chronic hepatitis (CH), liver cirrhosis (LC), as well as in healthy volunteers (HVs). Serum MFG-E8 levels were significantly lower in HCC patients than in HVs regardless of the etiology of liver disease (3.6 ± 0.1 vs 5.8 ± 0.2 ng/mL, p < 0.0001), and recovered after treatment of HCC. Serum MFG-E8 levels in CH and LC patients were comparable to those in HVs. Serum MFG-E8 could detect HCCs, even α-fetoprotein (AFP)-negative or des-γ-carboxy prothrombin (DCP)-negative HCCs, in CH and LC patients. Our new HCC prediction model using MFG-E8 and DCP (Logit(p) = 2.619 - 0.809 × serum MFG-E8 + 0.0226 × serum DCP) distinguished HCC patients from CH and LC patients with an area under the curve of 0.923, a sensitivity of 81.1%, and a specificity of 89.8%. Futhermore, low preoperative serum MFG-E8 was an independent predictor of poor overall survival. Thus, serum MFG-E8 could serve as a feasible diagnostic and prognostic biomarker for HCC.

Introduction: Type 1 diabetes is a prothrombotic state strongly linked to vascular complications. The role of microvesicles (MVs) as mediators and potential biomarkers in microangiopathy in type 1 diabetes remains unclear., Materials and Methods: MV levels in plasma samples from 106 patients with type 1 diabetes with microangiopathy, 130 patients without microangiopathy and 100 healthy controls were analysed using flow cytometry. Phosphatidylserine (PS) expression in MVs was assessed by lactadherin, and the ability of MVs to induce thrombin generation was investigated in vitro. Endogenous plasma lactadherin levels were measured using ELISA., Results: Patients with type 1 diabetes had higher MV levels compared to healthy controls, with no significant differences between patients with and without microangiopathy. MV-induced thrombin generation in normal-pooled plasma was blocked by addition of lactadherin. Endogenous lactadherin levels were higher in patients compared to controls, and the highest levels were found in patients with microangiopathy. Plasma lactadherin levels did not correlate with levels of PS positive/negative MVs., Conclusion: Patients with type 1 diabetes with and without microangiopathy have higher levels of circulating MVs than healthy controls, probably reflecting higher cellular activation and turnover. However, we found no associations between clinical microangiopathy and levels of MVs in total or PS-expressing MVs. Plasma levels of lactadherin, which is a glycoprotein important in the clearance of cells and MVs, are increased in patients with type 1 diabetes and correlate with microangiopathy., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Dairy, as a major component of a high protein diet, is a critical dietary source of branched chain amino acids (BCAA), which are biomarkers of health and diseases. While BCAA are known to be key stimulators of protein synthesis, elevated circulatory BCAA is an independent risk factor for type 2 diabetes mellitus. This study examined the impact of altered dairy intake on plasma BCAA and their potential relationship to insulin sensitivity. Healthy adults ( n = 102) were randomized to receive dietary advice to reduce, maintain, or increase habitual dairy intake for 1 month. Food intake was recorded with food frequency questionnaires. Self-reported protein intake from dairy was reported to be reduced (-14.6 ± 3.0 g/day), maintained (-4.0 ± 2.0 g/day) or increased (+13.8 ± 4.1 g/day) according to group allocation. No significant alterations in circulating free amino acids (AA), including BCAA, were measured. Insulin sensitivity, as assessed by homeostatic model assessment-insulin resistance (HOMA-IR), was also unaltered. A significant change in dairy protein intake showed no significant effect on fasting circulatory BCAA and insulin sensitivity in healthy populations.

Background: Increased amino acid availability stimulates muscle protein synthesis (MPS), which is critical for maintaining or increasing muscle mass when combined with training. Previous research suggests that whey protein is superior to soy protein in regard to stimulating MPS and muscle mass. Nevertheless, with respect to a future lack of dietary protein and an increasing need for using eco-friendly protein sources it is of great interest to investigate the quality of alternative protein sources, like insect protein., Objective: Our aim was to compare the postprandial amino acid (AA) availability and AA profile in the blood after ingestion of protein isolate from the lesser mealworm, whey isolate, and soy isolate., Design: Six healthy young men participated in a randomized cross-over study and received three different protein supplementations (25 g of crude protein from whey, soy, insect or placebo (water)) on four separate days. Blood samples were collected at pre, 0 min, 20 min, 40 min, 60 min, 90 min, and 120 min. Physical activity and dietary intake were standardized before each trial, and participants were instructed to be fasting from the night before. AA concentrations in blood samples were determined using ¹H NMR spectroscopy., Results: A significant rise in blood concentration of essential amino acids (EAA), branched-chain amino acids (BCAA) and leucine was detected over the 120 min period for all protein supplements. Nevertheless, the change in AA profile was significantly greater after ingestion of whey than soy and insect protein ( p < 0.05). Area under the curve (AUC) analysis and AA profile revealed comparable AA concentrations for soy and insect protein, whereas whey promoted a ~97% and ~140% greater AUC value than soy and insect protein, respectively. A tendency towards higher AA concentrations beyond the 120 min period was observed for insect protein., Conclusion: We report that ingestion of whey, soy, and insect protein isolate increases blood concentrations of EAA, BCAA, and leucine over a 120 min period (whey > insect = soy). Insect protein induced blood AA concentrations similar to soy protein. However, a tendency towards higher blood AA concentrations at the end of the 120 min period post ingestion was observed for insect protein, which indicates that it can be considered a "slow" digestible protein source.

Background & Aims: Breastfeeding is beneficial for mothers and infants. Underlying mechanisms and biochemical mediators thus need to be investigated to develop and support improved infant nutrition practices promoting the child health. We analysed the relation between maternal breast milk composition and infant metabolism., Methods: 196 pairs of mothers and infants from a European research project (PreventCD) were studied. Maternal milk samples collected at month 1 and month 4 after birth were analysed for macronutrient classes, hormone, and fatty acid (FA) content. Phospholipids, acylcarnitines, and amino acids were measured in serum samples of 4-month old infants. Associations between milk components and infant metabolites were analysed with spearman correlation and linear mixed effect models (LME). P-values were corrected for multiple testing (PLME)., Results: Month 1 milk protein content was strongly associated with infant serum lyso-phosphatidylcholine (LPC) 14:0 (PLME = 0.009). Month 1 milk insulin was associated to infant acetylcarnitine (PLME = 0.01). There were no associations between milk protein content and serum amino acids and milk total fat content and serum polar lipids. Middle- and odd-chain FA% in breast milk at both ages were significantly related to serum LPC and sphingomyelins (SM) species in infant serum (all PLME<0.05), while FA% 20:5n-3 and 22:6n-3 percentages were significantly associated to serum LPC 22:6 (PLME = 1.91×10-4/7.93×10-5) in milk only at month 4. Other polyunsaturated fatty acids and hormones in milk showed only weak associations with infant serum metabolites., Conclusions: Infant serum LPC are influenced by breast milk FA composition and, intriguingly, milk protein content in early but not late lactation. LPC 14:0, previously found positively associated with obesity risk, was the serum metabolite which was the most strongly associated to milk protein content. Thus, LPC 14:0 might be a key metabolite not only reflecting milk protein intake in infants, but also relating high protein content in milk or infant formula to childhood obesity risk., Competing Interests: The authors report no conflict of interest with respect to the study reported.

Atherosclerosis (AS) is an age‑related inflammatory disease. Globule‑epidermal growth factor‑8 (MFG‑E8) and transforming growth factor-β1 (TGF‑β1) are involved in the pathogenesis of AS. However, age‑related changes in circulating levels of MFG‑E8 and TGF‑β1, and their correlation with the severity of AS is not well‑characterized. In this study, we investigated age‑related changes in serum levels of MFG‑E8, TGF‑β1 and examined their association with the severity of AS. Sixty healthy volunteers were divided into young, middle‑age and old‑age groups. In addition, carotid ultrasound examination was performed to assess the intima‑media thickness (IMT) of carotid artery. Sixty‑seven patients with carotid AS and 30 age‑matched healthy persons were divided into IMT increased group, plaque group and control group. Serum levels of MFG‑E8, TGF‑β1, tumor necrosis factor-α (TNF‑α) and intercellular adhesion molecule‑1 were measured in all subjects. A positive association between serum MFG‑E8 levels and age was observed in healthy volunteers, while a significant negative association was observed between TGF‑β1 levels and age. Serum levels of MFG‑E8 and TNF‑α showed a positive correlation while those of TGF‑β1 showed a negative correlation with Crouse scores for carotid artery IMT (P<0.05 for both). Both MFG‑E8 and TGF‑β1 were age‑related inflammatory factors. MFG‑E8 showed a positive correlation, while TGF‑β1 showed a negative correlation with the severity of AS. Our findings suggest that both MFG‑E8 and TGF‑β1 are age‑related inflammatory factors and are related to the degree of AS. In conclusion, both MFG‑E8 and TGF‑β1 may serve as potential markers of the severity of AS.

Cardiac hypertrophy occurs in response to numerous stimuli like neurohumoral stress, pressure overload, infection, and injury, and leads to heart failure. Mfge8 (milk fat globule-EGF factor 8) is a secreted protein involved in various human diseases, but its regulation and function during cardiac hypertrophy remain unexplored. Here, we found that circulating MFGE8 levels declined significantly in failing hearts from patients with dilated cardiomyopathy. Correlation analyses revealed that circulating MFGE8 levels were negatively correlated with the severity of cardiac dysfunction and remodeling in affected patients. Deleting Mfge8 in mice maintained normal heart function at basal level but substantially exacerbated the hypertrophic enlargement of cardiomyocytes, reprogramming of pathological genes, contractile dysfunction, and myocardial fibrosis after aortic banding surgery. In contrast, cardiac-specific Mfge8 overexpression in transgenic mice significantly blunted aortic banding-induced cardiac hypertrophy. Whereas MAPK (mitogen-activated protein kinase) pathways were unaffected in either Mfge8 -knockout or Mfge8 -overexpressing mice, the activated Akt/PKB (protein kinase B)-Gsk-3β (glycogen synthase kinase-3β)/mTOR (mammalian target of rapamycin) pathway after aortic banding was significantly potentiated by Mfge8 deficiency but suppressed by Mfge8 overexpression. Inhibition of Akt with MK-2206 blocked the prohypertrophic effects of Mfge8 deficiency in angiotensin II-treated neonatal rat cardiomyocytes. Finally, administering a recombinant human MFGE8 in mice in vivo alleviated cardiac hypertrophy induced by aortic banding. Our findings indicate that Mfge8 is an endogenous negative regulator of pathological cardiac hypertrophy and may, thus, have potential both as a novel biomarker and as a therapeutic target for treatment of cardiac hypertrophy., (© 2017 American Heart Association, Inc.)

Human milk fat globule-EGF factor 8 (MFG-E8), also known as lactadherin, is a secreted glycoprotein that plays essential roles in the clearance of apoptotic cells and angiogenesis. It has been reported that serum MFG-E8 levels are higher in systemic lupus erythematosus (SLE) patients compared with in healthy controls; however, a previous study reported no correlation between serum MFG-E8 levels and SLE disease activity. The objective of this study was to assess serum MFG-E8 levels and their clinical associations in patients with SLE. Serum MFG-E8 levels in 49 Japanese patients with SLE, eight with cutaneous LE, and 28 healthy controls were examined. Serum MFG-E8 levels in SLE patients were significantly higher than those in cutaneous LE patients and healthy individuals. In addition, serum MFG-E8 levels were positively correlated with the SLE Disease Activity Index score, which reflects the disease activity of SLE. Notably, the frequency of the presence of high-intensity cerebral lesions on MRI in the SLE patients with elevated serum MFG-E8 levels was significantly higher than that in SLE patients with normal serum MFG-E8 levels. These findings suggest that elevated serum MFG-E8 levels may be associated with cerebrovascular diseases or neuropsychiatric SLE in patients with SLE, and that the measurement of serum MFG-E8 levels in SLE patients is useful for risk stratification of cerebrovascular disease or cerebrovascular disease-related neuropsychiatric SLE., (© 2017 Japanese Dermatological Association.)

Objectives: Milk basic protein (MBP), a mixture of proteins isolated from bovine milk, is known to increase bone formation. Ghrelin, a stomach-derived peptide hormone, also has been reported to stimulate osteoblast formation. The aim of this study was to determine whether MBP-induced bone formation is mediated via ghrelin., Methods: MBP was chronically administered to mice in their drinking water for 3 wk, and body weight, water intake, and bone mineral density were measured. Additionally, plasma bone-specific alkaline phosphatase, tartrate-resistant acid phosphatase isoform 5b, and ghrelin concentrations were determined by enzyme-linked immunosorbent assay. To examine the direct effect of MBP on ghrelin secretion, gastric tissue culture and primary mucosal cells were stimulated by MBP., Results: The in vivo study of young, growing mice showed that chronic MBP intake for 3 wk increased the plasma ghrelin concentration and bone mineral density of the hind limb tibia. In vitro studies using minced rat gastric mucosa tissues and primary murine isolated gastric mucosal cells revealed that MBP stimulated ghrelin release in a dose-dependent manner. Moreover, MBP-induced ghrelin secretion was partly inhibited by adrenergic blockers., Conclusions: These findings suggest a novel mechanism by which MBP directly acts on ghrelin secretion. Additionally, the elevated ghrelin level induced by MBP may act as a mediator for bone formation., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Aims/introduction: Milk fat globule-epidermal growth factor 8 (MFG-E8) is the key mediator in anti-inflammatory responses that facilitate phagocytosis of apoptotic cells, and play an essential role in type 2 diabetes and pregnancy, both of which are under a low-grade inflammatory state. However, the action of MFG-E8 in gestational diabetes mellitus (GDM) is unclear. We measured plasma MFG-E8 levels in pregnancy and GDM for the first time, and elucidated possible relationships between its plasma levels and various metabolic parameters., Materials and Methods: Plasma MFG-E8 levels were quantified by enzyme-linked immunosorbent assay in 66 women with GDM, 70 with normal pregnancy (p-NGT) and 44 healthy non-pregnant controls (CON), who were matched for age and body mass index. Inflammatory factors tumor necrosis factor-α (TNF-α) and C-reactive protein levels were measured, oral glucose tolerance test was carried out and β-cell function was evaluated., Results: Plasma MFG-E8 levels were remarkably higher in p-NGT than in CON (P = 0.024), and were further elevated in GDM vs p-NGT (P = 0.016). MFG-E8 concentrations correlated positively with hemoglobin A1c, glucose levels and insulin resistance (homeostasis model assessment for insulin resistance), and correlated inversely with TNF-α and insulin secretion evaluated by disposition indices in pregnancies. Fasting glucose levels, disposition index of first phase insulin secretion and TNF-α were independent predictors of MFG-E8 levels in pregnancies. Logistic regression analyses showed that women in the third tertile of MFG-E8 levels had a markedly elevated risk of GDM., Conclusions: Circulating MFG-E8 levels are dramatically elevated in pregnancy, and are significantly higher in GDM vs p-NGT. MFG-E8 concentrations are significantly associated with TNF-α, fasting glucose levels, homeostasis model assessment for insulin resistance and disposition indices. However, further studies are required to elucidate the regulation mechanism of MFG-E8 during pregnancy and GDM., (© 2016 The Authors. Journal of Diabetes Investigation published by Asian Association for the Study of Diabetes (AASD) and John Wiley & Sons Australia, Ltd.)

Background: Bacterial lipopolysaccharides (LPS) induce cell death and procoagulant tissue factor (TF) in purified cultured human monocytes. Phosphatidylserine (PS)-expression on the outer membrane of monocytes following cell death contributes to increased procoagulant activity. LPS also induce TF on monocytes in whole blood (WB), but it is unknown whether LPS induce cell death. Activated platelets that aggregate with monocytes in non-EDTA anticoagulated WB also express PS and may potentially interfere in viability measurement on monocytes in WB., Methods: Viability of monocytes was estimated in purified monocytes and unlysed citrated WB incubated without and with LPS using two markers of early cell death; Lactadherin (PS-exposure) and Mitoprobe™ DilC (Mitochondrial Membrane Potential, MMP), and one late marker Sytox® Blue (membrane impermeant DNA stain). To study the interfering effect of monocyte-platelet aggregates (MPAs) on estimated monocyte viability, addition of EDTA or anti-CD62P was used to inhibit monocyte-platelet binding., Results: In WB, the median percentage of viable monocytes without and with EDTA was 21% and 93% with PS-exposure and 89% and 99% with MMP, respectively. Addition of EDTA reduced the percentage of MPAs (81%-8.2%). Addition of anti-CD62P also reduced the percentage of MPAs (97-42%) and PS-expression on monocytes (MFI 28.9-3.9). LPS induced death of monocytes after 3 h in purified monocytes, but not in WB., Conclusions: MPAs interfered in monocyte viability measurement in citrated WB with PS-exposure, but not with MMP. LPS induced death of monocytes in purified culture, but not in WB. © 2015 International Clinical Cytometry Society., (© 2015 Clinical Cytometry Society.)

Background: The association between serum milk fat globule-epidermal growth factor 8 (MFG-E8) concentrations and vascular complications in T2DM remains unclear., Methods: A total of 149 patients with T2DM were included. The serum concentrations of MFG-E8, glycosylated hemoglobin (HbA1c), and high-sensitivity C-reactive protein (hs-CRP) were measured., Results: There was no significant difference in serum MFG-E8 concentrations between the T2DM group and the T2DM with subclinical atherosclerosis (AS) group (615.49±143.54 vs. 596.22±79.46ng/ml, P=0.365), while the serum concentrations of MFG-E8 in the T2DM with microvascular complications group (446.70±61.53ng/ml) and the T2DM with subclinical AS and microvascular complications group (200.87±38.86ng/ml) were significantly lower than those in the T2DM group (P=0.000 for both). In addition, hs-CRP and HbAlc concentrations were independently associated with serum MFG-E8 concentrations (P=0.024 and P=0.01, respectively), and low serum MFG-E8 concentrations were significantly associated with an increased risk of microvascular complications in T2DM patients., Conclusions: Serum concentrations of MFG-E8 were negatively associated with the risk of microvascular complications in patients with T2DM. Thus, it might be a potential candidate biomarker for diabetic microvascular complications., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Inflammation is the most important link between obesity and type 2 diabetes (T2D). Although milk fat globule-epidermal growth factor 8 (MFG-E8) is a key mediator in anti-inflammatory responses, its role in obesity and diabetes is not yet completely understood. We aimed to measure MFG-E8 serum levels and to explore the role of MFG-E8 in obesity and T2D. Fasting serum MFG-E8 levels were quantified by enzyme-linked immunosorbent assay for 168 individuals, whose oral glucose tolerance test was conducted, and levels of inflammatory factors, including tumor necrosis factor-α (TNF-α) and C-reactive protein, were measured. The participants were subdivided into 66 newly diagnosed T2D individuals, 44 impaired glucose tolerance (IGT) subjects and 58 healthy controls. Their characteristics were further classified as lean or nonlean for investigation. MFG-E8 levels were significantly higher in T2D subjects than in healthy controls (P = 0.028). Decreased levels of MFG-E8 were found in overweight or obese individuals, compared to those in lean subjects, in both the T2D and IGT groups (P < 0.001). Interestingly, MFG-E8 levels showed a negative correlation with body mass index (BMI) and TNF-α levels in the total population and the T2D subgroup. Further, BMI and TNF-α concentrations were found to be independent predictors of MFG-E8 levels in all subjects. MFG-E8 levels are elevated in T2D but suppressed by increased adipose tissues, thereby allowing inflammatory factors to rise to high levels. MFG-E8 may serve as a potential biomarker for obesity and T2D in the clinical setting. © 2017 IUBMB Life, 69(2):63-71, 2017., (© 2017 International Union of Biochemistry and Molecular Biology.)

Cardiolipin (CL) is an anionic phospholipid located exclusively in the mitochondrial inner membrane. Its presence in blood indicates mitochondrial damage and release from injured cells. Here, we report the detection of CL-exposed brain-derived mitochondrial microparticles (mtMPs) at 17 547 ± 2677/μL in the peripheral blood of mice subjected to fluid percussion injury to the brain. These mtMPs accounted for 55.2% ± 12.6% of all plasma annexin V-binding microparticles found in the acute phase of injury. They were also released from cultured neuronal and glial cells undergoing apoptosis. The mtMPs synergized with platelets to facilitate vascular leakage by disrupting the endothelial barrier. The disrupted endothelial barrier allowed the release of mtMPs into the systemic circulation to promote coagulation in both traumatically injured and mtMP- or CL-injected mice, leading to enhanced fibrinolysis, vascular fibrin deposition, and thrombosis. This mtMP-induced coagulation was mediated by CL transported from the inner to the outer mitochondrial membrane and was blocked by the scavenging molecule lactadherin. The mtMP-bound CL was ∼1600 times as active as purified CL in promoting coagulation. This study uncovered a novel procoagulant activity of CL and CL-exposed mitochondria that may contribute to traumatic brain injury-associated coagulopathy and identified potential pathways to block this activity., (© 2016 by The American Society of Hematology.)

Cortical gray matter loss in schizophrenia remains a great therapeutic difficulty. Each psychotic episode causes irreversible cortical gray matter loss, that causes the patients to never regain their previous state of functioning. Microglial cells are part of the innate immune system and their functions, among others, include phagocytosis and release of neurotrophic factors. They have a key impact on developmental and plasticity-induced removal of neuronal precursors, live-but-stressed neurons and synapses, while also stimulating synaptic growth and development. We hypothesize that microglia are the culprit for the cortical gray matter loss in schizophrenia through abnormal synaptic pruning, phagocytosis of stressed neurons and lacking neurotrophic factor release. Furthermore, we propose a research that could validate the hypotheses using serum samples of first-episode early-onset patients. By measuring the serum levels of milk fat globule-EGF factor 8 (MFG-E8), subcomponent in the classical pathway of complement activation (C1q), brain-derived neurotrophic factor (BDNF), interleukin-6 (IL-6) and interleukin-10 (IL-10), we could gain an insight into the state of microglial activation during various stages of the disease. If this hypothesis is valid, new targeted drugs could be developed in order to reduce the deterioration of cortical gray matter, thereby possibly improving negative symptoms and cognitive deficits., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Milk fat globule-epidermal growth factor 8 (MFG-E8) is an anti-inflammatory glycoprotein that mediates the clearance of apoptotic cells and is implicated in the pathogenesis of autoimmune and inflammatory diseases. Because MFG-E8 also controls bone metabolism, we investigated its role in rheumatoid arthritis (RA), focusing on inflammation and joint destruction. The regulation of MFG-E8 by inflammation was assessed in vitro using osteoblasts, in arthritic mice and in patients with RA. K/BxN serum transfer arthritis (STA) was applied to MFG-E8 knock-out mice to assess its role in the pathogenesis of arthritis. Stimulation of osteoblasts with lipopolysaccharide (LPS) and tumor necrosis factor (TNF)-α downregulated the expression of MFG-E8 by 30% to 35%. MFG-E8-deficient osteoblasts responded to LPS with a stronger production of pro-inflammatory cytokines. In vivo, MFG-E8 mRNA levels were 52% lower in the paws of collagen-induced arthritic (CIA) mice and 24% to 42% lower in the serum of arthritic mice using two different arthritis models (CIA and STA). Similarly, patients with RA (n = 93) had lower serum concentrations of MFG-E8 (-17%) compared with healthy controls (n = 140). In a subgroup of patients who had a moderate to high disease activity (n = 21), serum concentrations of MFG-E8 rose after complete or partial remission had been achieved (+67%). Finally, MFG-E8-deficient mice subjected to STA exhibited a stronger disease burden, an increased number of neutrophils in the joints, and a more extensive local and systemic bone loss. This was accompanied by an increased activation of osteoclasts and a suppression of osteoblast function in MFG-E8-deficient mice. Thus, MFG-E8 is a protective factor in the pathogenesis of RA and subsequent bone loss. Whether MFG-E8 qualifies as a novel biomarker or therapeutic target for the treatment of RA is worth addressing in further studies., (© 2015 American Society for Bone and Mineral Research.)

Milk fat globule epidermal growth factor 8 (MFG-E8) is an opsonin involved in the phagocytosis of apoptotic cells. In patients with chronic obstructive pulmonary disease (COPD), apoptotic cell clearance is defective. However, whether aberrant MFG-E8 expression is involved in this defect is unknown. In this study, we examined the expression of MFG-E8 in COPD patients. MFG-E8, interleukin (IL)-1β and transforming growth factor (TGF)-β levels were measured in the plasma of 96 COPD patients (93 males, 3 females; age range: 62.12±10.39) and 87 age-matched healthy controls (85 males, 2 females; age range: 64.81±10.11 years) using an enzyme-linked immunosorbent assay. Compared with controls, COPD patients had a significantly lower plasma MFG-E8 levels (P<0.01) and significantly higher plasma TGF-β levels (P=0.002), whereas there was no difference in plasma IL-1β levels between the two groups. Moreover, plasma MFG-E8 levels decreased progressively between Global Initiative for Chronic Obstructive Lung Disease (GOLD) I and GOLD IV stage COPD. Multiple regression analysis showed that the forced expiratory volume in 1 s (FEV1 % predicted) and smoking habit were powerful predictors of MFG-E8 in COPD (P<0.01 and P=0.026, respectively). MFG-E8 was positively associated with the FEV1 % predicted and negatively associated with smoking habit. The area under the receiver operating characteristic curve was 0.874 (95% confidence interval: 0.798-0.95; P<0.01). Our findings demonstrated the utility of MFG-E8 as a marker of disease severity in COPD and that cigarette smoke impaired MFG-E8 expression in these patients.

A component of polycomb repressor complex 2, enhancer of zeste homolog 2 (EZH2), plays an important role in tumor malignancy and metastasis, while milk fat globule-epidermal growth factor-factor 8 (MFGE8) plays a key role in tumor progression and prognosis. MicroRNAs (miRs) are also critically involved in various physiological and pathological processes. We here evaluated the relationship between overall survival (OS) in colorectal cancer patients and the expression of onco-miRs and miRs, which may target EZH2 and MFGE8. Plasma and formalin-fixed paraffin-embedded (FFPE) samples were obtained from 71 colorectal cancer patients. The expression levels of miRs complementary to EZH2 and MFGE8 mRNA and cancer malignancies were evaluated. The miRs analyzed were as follows: miR-16, miR-21, miR-26a, miR-34a, miR-98, miR-101-3p, miR-101-5p, miR-124-5p (also known as miR-124*), miR-126-3p, miR-126-5p, miR-210, miR-217, and miR-630. The plasma expression levels of MFGE8 in completely resected patients were significantly lower than those in unresectable patients. Lower miR-26a expression levels were correlated with a higher probability of OS. Higher miR-124-5p expression levels in plasma and FFPE samples were correlated with a higher probability of OS. The transfection of mimic miR-124-5p into WiDr and COLO201 cells inhibited the expression of structural maintenance of chromosomes 4 (SMC4) mRNA. Our results indicate that miR-124-5p may target the tumorigenesis gene, SMC4, which suggests that expression levels of miR-124-5p in plasma and FFPE samples; therefore, the expression of MFGE8, miR-26a, and miR-124-5p in plasma may be used as biomarkers to determine the prognosis of colorectal cancer patients., (© 2014 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Objective: Inflammation is now considered a main pathogenic factor in coronary atherosclerotic heart disease (CHD), and it has a positive correlation with plaque vulnerability. A novel anti-inflammatory factor, milk fat globule-epidermal growth factor 8 (MFG-E8), has been reported as having prominent anti-inflammatory effects in sepsis. However, few studies have reported on the association between MFG-E8 and CHD. In the present study, we aimed to investigate the serum MFG-E8 concentrations in patients with different stages of CHD or without CHD. Then, we studied the associations among MFG-E8, Gensini score, and high-sensitivity C-reactive protein (hs-CRP) in Chinese patients with CHD to illustrate the role of MFG-E8 in CHD., Methods: A total of 176 controls and 295 patients with CHD were selected for this study. To evaluate CHD severity, we calculated the Gensini score for all of the subjects. Serum levels of MFG-E8 were determined by an enzyme-linked immunosorbent assay (ELISA) kit; serum total cholesterol (TC), high density lipoprotein-cholesterol (HDL-c), low density lipoprotein-cholesterol (LDL-c), triglyceride (TG), and hs-CRP were detected by an automatic biochemistry analyzer; and fibrinogen (FIB) was analyzed with an automatic coagulation analyzer., Results: Compared with the controls, the CHD group had a lower level of MFG-E8 (673.20±112.34 ng/mL vs. 134.89±4.74 ng/mL, p<0.001). The level of serum MFG-E8 in the acute myocardial infarction group (118.07±10.10 ng/mL) was significantly less than that in the stable angina group (p=0.025). Further analysis showed that MFG-E8 had a negative association with the Gensini score and the hs-CRP level (r=-0.590, p<0.001; r=-0.105, p=0.022, respectively). In addition, multiple regression analysis of the association between MFG-E8 and the main cardiovascular risk factors in our cases showed that MFG-E8 had a negative association with hs-CRP and a positive association with LDL-c (all p<0.05)., Conclusion: The serum level of MFG-E8 was negatively associated with the severity of coronary artery stenosis and the risk of clinical events. Thus, MFG-E8 has the potential to be a marker of vascular complications., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Objective: Impaired clearance of apoptotic cells is a potential trigger of systemic lupus erythematosus (SLE). Milk fat globule epidermal growth factor 8 (MFG-E8) plays an important role in the clearance of dying cells. Previously, we reported serum MFG-E8 was elevated in some SLE patients. Here we further investigated the prevalence of MFG-E8 in active SLE and other autoimmune diseases and also tried to clarify the characteristics of MFG-E8-positive and -negative SLE., Methods: Serum MFG-E8 was measured in 40 active non-treated SLE patients, 104 disease controls and 104 healthy controls by ELISA. Clinical characteristics and serum cytokine profiles were compared between MFG-E8-positive and MFG-E8-negative SLE patients., Results: Prevalence of MFG-E8 was significantly higher in SLE patients (40%) than in various controls (p < 0.05). MFG-E8 level became negative after treatment, and increased again upon relapse. When compared, MFG-E8-positive SLE patients showed higher immune complex (p = 0.021) and lower complement (p = 0.004 for CH50). In contrast, MFG-E8-negative SLE patients tended to show higher CRP (p = 0.094). There was a positive correlation between MFG-E8 level and immune complex level (r s = 0.49, p = 0.049). TNF-α (p = 0.019), IFN-γ (p = 0.031) and IL-10 (p = 0.013) were significantly higher in MFG-E8-positive SLE., Conclusion: MFG-E8-positive SLE and -negative SLE may have different clinical features, the one with stronger immunological response and the other with stronger inflammatory response, and those two groups may be two distinct subtypes of SLE driven by different mechanisms. Further, MFG-E8 could be used as a biomarker for diagnosis and monitoring of disease activity in certain SLE patients.

Although biomarkers exist for a range of disease diagnostics, a single low-cost platform exhibiting the required sensitivity, a large dynamic-range and multiplexing capability, and zero sample preparation remains in high demand for a variety of clinical applications. The Interferometric Reflectance Imaging Sensor (IRIS) was utilized to digitally detect and size single gold nanoparticles to identify protein biomarkers in unprocessed serum and blood samples. IRIS is a simple, inexpensive, multiplexed, high-throughput, and label-free optical biosensor that was originally used to quantify biomass captured on a surface with moderate sensitivity. Here we demonstrate detection of β-lactoglobulin, a cow's milk whey protein spiked in serum (>10 orders of magnitude) and whole blood (>5 orders of magnitude), at attomolar sensitivity. The clinical utility of IRIS was demonstrated by detecting allergen-specific IgE from microliters of characterized human serum and unprocessed whole blood samples by using secondary antibodies against human IgE labeled with 40 nm gold nanoparticles. To the best of our knowledge, this level of sensitivity over a large dynamic range has not been previously demonstrated. IRIS offers four main advantages compared to existing technologies: it (i) detects proteins from attomolar to nanomolar concentrations in unprocessed biological samples, (ii) unambiguously discriminates nanoparticles tags on a robust and physically large sensor area, (iii) detects protein targets with conjugated very small nanoparticle tags (~40 nm diameter), which minimally affect assay kinetics compared to conventional microparticle tagging methods, and (iv) utilizes components that make the instrument inexpensive, robust, and portable. These features make IRIS an ideal candidate for clinical and diagnostic applications.

Aims: This study was carried out with the purpose of investigating the association between serum lactadherin, monocyte chemoattractant protein 1 (MCP-1), tumor necrosis factor alpha (TNFα) and carotid-femoral pulse wave velocity (PWV) in elderly patients with type 2 diabetes mellitus (T2DM)., Methods: A total of 105 subjects including 27 T2DM patients without vascular complications (DM), 28 T2DM patients with vascular complications (DC), 25 elderly healthy volunteers (older) and 25 younger healthy volunteers (younger) were recruited into the study. Carotid-femoral PWV was measured using an automatic device. Serum lactadherin, MCP-1 and TNFα were determined by enzyme linked immunosorbent assay., Results: PWV and lactadherin, MCP-1 and TNFα were significantly higher in DM and DC groups than those of older and younger groups. PWV and lactadherin were higher in older group than those of younger group. Moreover, lactadherin was significantly correlated with MCP-1, TNFα, PWV, HbA1c and 2 h postprandial blood glucose (P2hBG) (P<0.05). In multivariate regression analysis, the independent determinants of lactadherin were HbA1c, P2hBG and age (P<0.05)., Conclusions: These findings underscore that lactadherin is correlated with poor blood glucose control and diabetic vascular complications., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Background: Atherosclerosis is one of the major complications of type 2 diabetic patients (T2DM), leading to morbidity and mortality. Grape seed procyanidin B2 (GSPB2) has demonstrated protective effect against atherosclerosis, which is believed to be, at least in part, a result of its antioxidative effects. The aim of this study is to identify the target protein of GSPB2 responsible for the protective effect against atherosclerosis in patients with DM., Methods and Results: GSPB2 (30 mg/kg body weight/day) were administrated to db/db mice for 10 weeks. Proteomics of the aorta extracts by iTRAQ analysis was obtained from db/db mice. The results showed that expression of 557 proteins were either up- or down-regulated in the aorta of diabetic mice. Among those proteins, 139 proteins were normalized by GSPB2 to the levels comparable to those in control mice. Among the proteins regulated by GSPB2, the milk fat globule epidermal growth factor-8 (MFG-E8) was found to be increased in serum level in T2DM patients; the serum level of MFG-E8 was positively correlated with carotid-femoral pulse wave velocity (CF-PWV). Inhibition of MFG-E8 by RNA interference significantly suppressed whereas exogenous recombinant MFG-E8 administration exacerbated atherogenesis the db/db mice. To gain more insights into the mechanism of action of MFG-E8, we investigated the effects of MFG-E8 on the signal pathway involving the extracellular signal-regulated kinase (ERK) and monocyte chemoattractant protein-1 (MCP-1). Treatment with recombinant MFG-E8 led to increased whereas inhibition of MFG-E8 to decreased expression of MCP-1 and phosphorylation of ERK1/2., Conclusion: Our data suggests that MFG-E8 plays an important role in atherogenesis in diabetes through both ERK and MCP-1 signaling pathways. GSPB2, a well-studied antioxidant, significantly inhibited the arterial wall changes favoring atherogenesis in db/db mice by down-regulating MFG-E8 expression in aorta and its serum level. Measuring MFG-E8 serum level could be a useful clinical surrogate prognosticating atherogenesis in DM patients.

Background & Aims: In patients with recent hip fracture, reduced serum IGF-I in relation to protein undernutrition is frequent. Elevation of circulating IGF-I in response to a daily oral supplement of 20 g of casein was observed after 6 months. This study determined if the response to casein as compared to whey protein can be observed as early as after one week., Methods: 45 women were randomized after recent hip fracture in 3 groups receiving a preparation of 20 g of casein, an isocaloric supplement of 20 g of whey protein or an isocaloric supplement of 15 g of whey protein combined with 5 g of essential amino acids (a.a.)., Results: A similar significant elevation of serum IGF-I was already observed after 7 days for casein (+37.3 microg/L), whey (+29.4) and for whey+a.a. (+34.3). From day 7-28, no further significant rise in IGF-I was recorded., Conclusion: After one week of protein supplementation, the percent increase of IGF-I was of similar magnitude to that previously observed after 6 months of protein supplementation. It suggests that in hip fracture patients, long-term effects of various protein preparations on IGF-I could be predicted from changes observed as early as 7 days after the onset of supplementation., (Copyright 2009 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.)

We have previously demonstrated that increasing the habitual protein intake widened the gap in nutritional quality between proteins through mechanisms that are not yet fully understood. We hypothesized that the differences in gastrointestinal kinetics between dietary proteins were an important factor affecting their differential response to an increased protein intake. To test this hypothesis, we built a 13-compartment model providing integrative insight into the sequential dynamics of meal nitrogen (Nm) absorption, splanchnic uptake, and metabolism, and subsequent peripheral transfer and deposition. The model was developed from data on postprandial Nm kinetics in certain accessible pools, obtained from subjects having ingested a (15)N-labeled milk or soy protein meal, after adaptation to normal (NP) or high (HP) protein diets. The faster absorption of Nm after soy vs. milk caused its earlier and stronger splanchnic delivery, which favored its local catabolic utilization (up to +30%) and limited its peripheral accretion (down to -20%). Nm absorption was also accelerated after HP vs. NP adaptation, and this kinetic effect accounted for most of the HP-induced increase (up to +20%) in splanchnic Nm catabolic use, and the decrease (down to -25%) in peripheral Nm anabolic utilization. The HP-induced acceleration in Nm absorption was more pronounced with soy than with milk, as were the HP effects on Nm regional metabolism. Our integrative approach identified Nm absorption kinetics, which exert a direct and lasting impact on Nm splanchnic catabolic use and peripheral delivery, as being critical in adaptation to both qualitative and quantitative changes in protein intake.

Background: Enhanced and prolonged postprandial triglyceride responses involve increased cardiovascular disease risk in type 2 diabetes. Dietary fat and carbohydrates profoundly influence postprandial hypertriglyceridemia, whereas little information exists on the effect of proteins., Objective: The objective was to compare the effects of the proteins casein, whey, cod, and gluten on postprandial lipid and incretin responses to a high-fat meal in persons with type 2 diabetes., Design: A crossover study was conducted in 12 patients with type 2 diabetes. Blood samples were collected over 8 h after ingestion of a test meal containing 100 g butter and 45 g carbohydrate in combination with 45 g casein (Cas-meal), whey (Whe-meal), cod (Cod-meal), or gluten (Glu-meal). We measured plasma concentrations of triglycerides, retinyl palmitate (RP), free fatty acids, insulin, glucose, glucagon, glucagon-like peptide 1, and glucose-dependent insulinotropic peptide., Results: The incremental area under the curve for triglyceride was significantly lower after the Whe-meal than after the other meals. The RP response was lower after the Whe-meal than after the Cas-meal and Cod-meal in the chylomicron-rich fraction and higher after the Whe-meal than after Cod- and Glu-meals in the chylomicron-poor fraction. Free fatty acids were most pronouncedly suppressed after the Whe-meal. The glucose response was lower after the Whe-meal than after the other meals, whereas no significant differences were found in insulin, glucagon, glucagon-like peptide 1, and glucose-dependent insulinotropic peptide responses., Conclusion: The data suggest that as a supplement to a fat-rich meal in patients with type 2 diabetes, whey protein seems to outperform other proteins in terms of postprandial lipemia improvement, possibly because of the formation of fewer chylomicrons or increased clearance of chylomicrons. The trial was registered at ClinicalTrials.gov as NCT00817973.

Background & Aims: Dietary protein plays a role in body weight regulation, partly due to its effects on satiety. The objective was to compare the effects of casein-, soy-, whey-, whey without glycomacropeptide (GMP)-, alpha-lactalbumin-, gelatin-, or gelatin with tryptophan (TRP)-protein breakfasts at two concentrations on subsequent satiety and energy intake (EI)., Methods: Twenty-four healthy subjects (mean+/-SEM BMI: 24.8+/-0.5 kg/m(2); age: 25+/-2 years) received a breakfast; a custard with casein, soy, whey, whey-GMP, alpha-lactalbumin, gelatin, or gelatin+TRP as protein source with either 10/55/35 (normal) or 25/55/20 (high) En% protein/carbohydrate/fat in a randomized, single-blind design. At the precedingly determined time point for lunch, 180 min, subjects were offered an ad lib lunch. Appetite profile (Visual Analogue Scales, VAS) and EI were determined., Results: Both at the level of 10 and 25 En% from protein, EI at lunch was approximately 20% lower after an alpha-lactalbumin or gelatin (+TRP) breakfast (2.5+/-0.2 MJ) compared with after a casein, soy, or whey-GMP breakfast (3.2+/-0.3 MJ, p<0.05). Appetite ratings at 180 min differed 15-25 mm (approximately 40%, p<0.05) between types of protein. Differences in EI were a function of differences in appetite ratings (R(2)=0.4, p<0.001)., Conclusions: Different proteins (alpha-lactalbumin, gelatin, gelatin+TRP) that are approximately 40% more satiating than other proteins (casein, soy, whey, whey-GMP) induce a related approximately 20% reduction of subsequent energy intake.

Background: In red blood cells (RBCs) anionic phospholipids, such as phosphatidylserine, are present in the inner leaflet of the membrane bilayer. Exposure of phosphatidylserine occurs during senescence and during long-term storage of RBCs and is considered as the tag for removal from the circulation by macrophages. Lactadherin is a phosphatidylserine-binding glycoprotein secreted by macrophages that promotes the engulfment of phosphatidylserine-expressing apoptotic lymphocytes. This study investigates the role of lactadherin in the phagocytosis of phosphatidylserine-expressing RBCs., Study Design and Methods: Transbilayer movement of phosphatidylserine was induced in RBCs either by storage beyond 30 days or by treatment with calcium ionophore A23187 and N-ethylmaleimide. Phosphatidylserine-expressing RBCs were incubated with phorbol ester-stimulated THP-1, and phagocytosis was determined by measuring the pseudoperoxidase activity of hemoglobin. The in vivo clearance of phosphatidylserine-enriched RBCs was measured in lactadherin-deficient mice and in their littermate controls., Results: Lactadherin promoted phagocytosis of phosphatidylserine-expressing RBCs by macrophages in a concentration-dependent manner. Splenic macrophages from lactadherin-deficient mice had diminished capacity to phagocytose phosphatidylserine-expressing RBCs. The life span of RBCs in lactadherin-deficient mice was similar to wild-type littermate controls in vivo. However, when an excess of phosphatidylserine-expressing RBCs were infused, there was only a mild impairment in the clearance in lactadherin-deficient mice compared to wild-type littermate controls., Conclusion: These results show that clearance of phosphatidylserine-expressing RBCs is not diminished in a significant way in lactadherin-deficient mice under physiologic conditions and suggest the presence of redundant pathways.

Mouse milk fat globule epidermal growth factor 8 (MFG-E8), which is secreted by a subset of activated macrophages, binds to apoptotic cells by recognizing phosphatidylserine and promotes their engulfment. Many apoptotic cells are left unengulfed in the germinal centers of the spleen in MFG-E8(-/-) mice, and these mice develop an autoimmune disease resembling human systemic lupus erythematosus (hSLE). Here, we report that hMFG-E8 bound to phosphatidylserine and an integrin alpha(v)beta(3) complex. Increasing concentrations of MFG-E8 generated a bell-shaped response curve for the efficiency of phagocytosis. That is, in NIH3T3 and MFG-E8(-/-) thioglycollate-elicited peritoneal macrophages that do not express MFG-E8, hMFG-E8 enhanced engulfment at low concentrations but inhibited it at high concentrations. On the other hand, hMFG-E8 dose-dependently inhibited the engulfment of apoptotic cells by MFG-E8(+/+) thioglycollate-elicited peritoneal macrophages, indicating that an excess of MFG-E8 has an inverse effect on the engulfment of apoptotic cells. To investigate the role of MFG-E8 in human disease, we generated two mAb against MFG-E8 and screened human blood samples for MFG-E8 using an ELISA. We found that some childhood-onset and adult SLE patients carried a significant level of MFG-E8 in their blood samples. These results suggested that the aberrant expression of MFG-E8 is involved in the pathoetiology of some cases of hSLE.

Background: Due to a transient age-related low renal capacity for net acid excretion, preterm infants fed formula are at a considerable risk of spontaneously developing incipient late metabolic acidosis, clinically characterized by e.g., disturbed bone mineralization and impaired growth., Aim of the Study: From acid-base data in blood and urine under different diets of modified human milk or preterm formulas is attempted to explore the impact of food mineral (and protein) composition on renal regulation and systemic acid-base balance in preterm infants., Patients and Methods: Data were collected from 48 infants fed their own mother's milk (28 native human milk, 20 enriched with fortifier) and 34 patients on formula (23 on a standard batch, 11 on a modified batch with reduced acid load). Intake of food was measured and acid-base data were determined in blood and timed-urine (8-12 h) samples., Results: Differences in mineral composition of the diets led to considerable differences of daily "alkali-intake", without significant effects on non-respiratory (base excess, BE) and respiratory (PCO(2)) acid-base data in the blood. In contrast, a highly significant proportionality between individual dietary alkali intake and daily renal base (Na(+) + K(+)-Cl(-)) excretion was observed (y = 0.32x-0.70, n = 80, r = 0.77, P < 0.0001), irrespective of the type of the diet., Conclusion: Renal base saving mechanisms are normally effective in preterm infants to compensate for differences in dietary acid-base load. Generally, nutritional acid-base challenges can be judged much earlier and more safely by urinary than by blood acid-base analysis. Taking into account the age specific low capacity for renal NAE, the relatively high nutritional acid load of preterm standard formula should be reduced.

This work elucidates the role of ceruloplasmin (Cp): the main soluble copper containing glycoprotein, in newborn infant copper metabolism. An over 3-fold drop of Cp and copper concentration in samples of skimmed milk from 2 to 5 days of lactation was demonstrated. It has been shown that [125I]Cp of the breast milk is discovered in the blood plasma after its was administered per os to 6-day old rats. In the body, [125I]Cp was specifically bound to cells and lost copper ions. Alternatively, after copper metabolism change to adult type, milk [125I]Cp isn't carried from gastrointestinal tract to the blood. During 8 days, newborn rats were fed with baby formula and we have found that in this case changing of the copper metabolism type takes place earlier in contrast to the litter fed by females. Transcription of the Cp gene activated in liver, Cp and the copper ions concentration in the blood plasma increased proportionally, whereas in liver the copper concentration is decreased. In the brain, changes typical for adult copper metabolism, were not discovered. But in cerebrospinal fluid copper and Cp concentration sharply increased. The role of milk Cp in controlling the copper balance in newborn rats and the tissue-specific mechanism of Cp gene activity regulation are under discussion.

Allergic inflammation is characterized by elevated eosinophil numbers and by the increased production of the cytokines IL-5 and GM-CSF, which control several eosinophil functions, including the suppression of apoptosis. The JAK/STAT pathway is important for several functions in hemopoietic cells, including the suppression of apoptosis. We report in this study that STAT3, STAT5a, and STAT5b are expressed in human eosinophils and that their signaling pathways are active following IL-5 or GM-CSF treatment. However, in airway eosinophils, the phosphorylation of STAT5 by IL-5 is reduced, an event that may be related to the reduced expression of the IL-5Ralpha on airway eosinophils. Furthermore, IL-5 and GM-CSF induced the protein expression of cyclin D3 and the kinase Pim-1, both of which are regulated by STAT-dependent processes in some cell systems. Pim-1 is more abundantly expressed in airway eosinophils than in blood eosinophils. Because Pim-1 reportedly has a role in the modulation of apoptosis, these results suggest that Pim-1 action is linked to the suppression of eosinophil apoptosis by these cytokines. Although cyclin D3 is known to be critical for cell cycle progression, eosinophils are terminally differentiated cells that do not proceed through the cell cycle. Thus, this apparent cytokine regulation of cyclin D3 suggests that there is an alternative role(s) for cyclin D3 in eosinophil biology.

Diverse pathogenic mechanisms elicit different clinical manifestations in cow's milk allergy (CMA). Our aim was to determine the concentration of serum immunoglobulin levels to different cow's milk proteins in patients with CMA and to determine how these values were related to clinical symptoms and prognosis. Fifty children (mean age 10.9 months, range: 1-34 months) with previously confirmed CMA were enrolled in this study. All had various clinical manifestations of CMA, including gastrointestinal, skin, and respiratory symptoms. At the diagnosis of CMA the serum total and the milk-specific immunoglobulin (Ig)E values were measured by enzyme immunoassay and fluoroimmunoassay, respectively, while the relative levels of serum IgA and IgG antibodies against different cow's milk proteins were determined by a sensitive enzyme-linked immunosorbent assay (ELISA). The results were compared to those of 30 non-atopic age-matched control children. On average, after 9.2 months (range 2-31 months) on a milk-free diet, a repeated challenge was performed in 38 children. At the re-challenge, 12 patients had clinical symptoms while the remaining 26 children were symptom-free. The IgG antibody level to bovine serum albumin (BSA) was significantly lower in the patients than in the controls (median: 0.36 vs. 2.94, p < 0.01). There was a close correlation among all individual IgA and IgG antibodies to different cow's milk proteins. The anti-alpha-casein IgG level (of 2.10) in children with a positive reaction at the re-challenge was significantly higher than in those with a negative reaction (0.89) (p < 0.05). The total IgE serum concentration was also significantly higher in those who had symptoms at the re-challenge compared to those who did not have any reaction at this time (22.9 vs. 6.8 kU/l, geometric mean, p < 0.02). There was no association between the clinical manifestations and the IgG and IgA antibody levels to the cow's milk proteins studied, except for the anti-BSA IgA level, which was higher in patients with gastrointestinal symptoms. The serum total IgE and anti-alpha-casein IgG levels could have prognostic values; their increase at the beginning of the disease may indicate the development of tolerance to cow's milk only at a later age and after a longer duration of CMA. However, as there is considerable overlap among the values observed in different groups of patients, there is a limitation of these tests for predicting the prognosis.

Background: The precise role of leucocytes in human milk is still unresolved., Objective: To assist in clarifying the immune mechanisms involved in the development of CMA in suckling infants, we studied the role of immunoregulatory leucocytes and their mediators in human breast milk., Methods: The study population consisted of 43 lactating mothers and their infants, aged 0.25-8.0 months, followed-up prospectively from birth. Of these mothers, 27 had an infant with challenge-proven cow's milk allergy manifested with either skin (n = 23), gastrointestinal (n = 2) or skin and gastrointestinal symptoms (n = 3). Sixteen mothers with a healthy infant served as controls. We evaluated the spontaneous and mitogen-induced tumour necrosis factor-alpha (TNFalpha) and interferon-gamma (IFNgamma) production of human milk leucocytes and isolated peripheral blood lymphocytes in vitro with a commercial ELISA kit., Results: TNFalpha production of breast milk leucocytes was significantly lower in the mothers with a cow's milk-allergic infant, whereas IFNgamma production of these cells was comparable in the two groups., Conclusion: Our results suggest that in the breast milk of mothers having an infant with cow's milk allergy, the number and function of TNFalpha-producing cells is defective. This might lead to a disturbance in the development of oral tolerance and thereby to the development of CMA in suckling infants. These novel results may help in clarifying the etiopathogenesis of CMA.

The whey proteins alpha-lactalbumin and beta-lactoglobulin have been investigated as potential markers of mammary development in sows by measuring their concentrations in plasma. The whey proteins were isolated from porcine milk by gel filtration, ion-exchange and hydrophobic interaction chromatography, characterized by several criteria and used to raise antibodies. Specific radioimmunoassays were set up for porcine alpha-lactalbumin and beta-lactoglobulin and validated for use in porcine blood and milk. Plasma levels of the whey proteins were measured in sows that were pregnant, suckling litters post partum, weaned abruptly at birth or were pregnant but mastectomized. Both whey proteins showed similar patterns in plasma post partum, falling from a maximum 1 d after parturition to values < 0.02% those in milk by day 4-5 post partum in suckling sows and showing a transient peak associated with early involution before declining to very low concentrations in non-suckling sows. alpha-Lactalbumin was first detected in the last week prepartum, rising markedly in the 3 d before parturition, correlated with rising prolactin (r = 0.986) and falling progesterone (r = -0.998). beta-Lactoglobulin rose much earlier from 5 weeks prepartum, at the time when lobulo-alveolar mammary development is occurring, and correlated (r = 0.929) with oestradiol-17 beta. In mastectomized sows, concentrations of whey proteins in plasma were reduced by 90% or more when compared with intact animals, though following a similar pattern. This study shows that whey protein concentrations in plasma vary with physiological state and reflect aspects of the development of the mammary gland. The very different profiles for alpha-lactalbumin and beta-lactoglobulin prepartum indicate that they are differently controlled.

We have previously reported that normal differentiation of PRL-secreting cells in neonatal rats requires a maternal signal specific to the first few days of lactation. Subsequently, we found that milk proteins from this period had a greater capacity than those from later in lactation to stimulate lactotrope differentiation in vitro, suggesting that the maternal signal was a milk-borne peptide. We reasoned in the present study that if this were the case, the signal should be present in the serum of suckling pups, and its level should reflect that in mothers' milk. To test this hypothesis, we first compared serum from 3-day-old pups with that from newborn pups that had not yet suckled. Anterior pituitary cells from 1-day-old pups were cultured for 6 days in the presence of various serum samples (0.01-1.0% by volume). Lactotrope differentiation was assessed using a reverse hemolytic plaque assay for PRL. Serum from 3-day-old pups was more effective (P < 0.01; n = 4) than that from unsuckled newborns at stimulating lactotrope differentiation in vitro (to 3.5 +/- 0.5% and 1.5 +/- 0.5% of all anterior pituitary cells, respectively). Next, we tested whether this increase in serum bioactivity in 3-day-old pups required maternal influences restricted to the first few days of lactation. Newborn pups were placed with foster mothers that had been lactating for 0 or 7 days. When the pups were 3 days old, their serum was tested for lactotrope-differentiating activity. Serum from pups placed with day 0 foster mothers (control) increased the abundance of PRL cells to 2.4 +/- 0.3% of all cells (P < 0.01; n = 3), whereas serum from pups placed with day 7 foster mothers failed to stimulate lactotrope differentiation. In similar experiments, litters placed the day after birth with day 7 foster mothers were separated on day 3 postpartum for 3 h, after which littermates were allowed to suckle for 90 min on different foster mothers on day 3 or 9 of lactation. This brief suckling bout increased (P < 0.01; n = 3) the capacity of serum from pups on day 3 mothers to stimulate PRL cell differentiation (to 3.9 +/- 0.4%) above that of serum from littermates placed with day 9 foster mothers (to 1.8 +/- 0.2%). Finally, the differentiating activities in pup serum and mothers' milk eluted from gel filtration chromatography in identical fractions (4-8 kilodaltons).(ABSTRACT TRUNCATED AT 400 WORDS)

Rabbit whey acidic protein has been purified from whey using an AcA54 column. The purified whey acidic protein had an amino acid composition in agreement with the previously defined cDNA sequence. An antibody against whey acidic protein was raised in guinea pig. This antibody did not crossreact with mouse or cow milk or with rabbit alpha s1-casein and beta-casein. Whey acidic protein concentration was measured in rabbit milk using the antibody with a radioimmunoassay. The concentration of whey acidic protein in rabbit milk was 15 mg/ml, whereas the concentrations of alpha s1-casein and beta-casein were 16 and 45 mg/ml, respectively. The concentration of the three proteins was also evaluated in culture medium of rabbit primary mammary cells. The three proteins were induced by prolactin alone. Glucocorticoids amplified the prolactin effect on whey acidic protein more intensively than on caseins. The three proteins were present in mammary extract from virgin rabbit. The concentration of these proteins was lower at d 8 and 14 of pregnancy, and it was very high at d 25 of pregnancy. Whey acidic protein was undetectable in blood of virgin, weaned, and midpregnant females and of males. Whey acidic protein was present in blood of lactating rabbits, but alpha s1-casein and beta-casein were not detectably present in rabbit blood at the examined physiological states.

Among 76 eczematous children, we realized a RAST test for whole milk and milk specific proteins: lactalbumin, lactoglobulin and casein. One of these RAST tests, at least, was positive for 27 children. Whole milk RAST test is the most frequently positive (77%) but some non specific reactions seem to interfere: only three children are clinically milk sensitive. Among specific proteins, lactalbumin is more frequently positive (59%) than lactoglobulin (51%) and casein (18%). Eczematous children with positive milk RAST tests, if compared to other eczematous children, have currently more mite allergies and positive egg RAST tests. They have a tendency (non significative) to be more frequently breast fed. The positivity of milk RAST tests seems to belong to a polysensitization and infrequently concerns a clinical allergy.

Intramammary leucocytosis was induced by injection of Escherichia coli endotoxin via the teat canal in three lactating Holstein cows. Concentrations of alpha-lactalbumin and casein in blood serum were measured, and somatic cell concentration and yield and composition of milk were determined. Endotoxin injection elicited mean increases of 100-fold in somatic cell concentration and 50% in protein concentration, whereas milk yield declined 5-fold and lactose concentration was halved. Concentrations of alpha-lactalbumin and casein in blood rose from 80 to 1909 and 0 to 1231 ng/ml, respectively. By 96 h postinjection, all variables were approximately equal to those preinjection. In a second study, concentration of alpha-lactalbumin was determined in blood of lactating cows in two herds (n = 332) and related to milk somatic cell count. Concentrations of alpha-lactalbumin in blood were correlated with somatic cell counts (r = .60). Mean concentrations of alpha-lactalbumin increased with increasing cell count even at low somatic cell concentrations (25 to 250 X 10(3)). Concentrations of milk proteins in blood serum apparently reflect competency of the blood-milk barrier and may therefore yield an indirect measure of udder health.