Serum Fractions Research Articles

Implications for the offspring of circulating factors involved in beta cell adaptation in pregnancy

Several studies have shown an increase in beta cell mass during pregnancy. Somatolactogenic hormones are known to stimulate the proliferation of existing beta cells in rodents whereas the mechanism in humans is still unclear. We hypothesize that in addition to somatolactogenic hormones there are other circulating factors involved in beta cell adaptation to pregnancy. This study aimed at screening for potential pregnancy-associated circulating beta cell growth factors. Serum samples from nonpregnant and pregnant women. The effect of serum from pregnant women on the proliferation of rat beta cells was studied using [3H]thymidine incorporation and 5-ethynyl-2'-deoxyuridine proliferation assays. In addition, serum from pregnant and nonpregnant women was fractionated by gel filtration and high performance liquid chromatography. The fractionated serum was screened for mitogenic activity in INS-1E cells. Proteins and peptides in mitogenic active serum fractions were identified by amino acid sequencing and mass spectrometry. Presence of circulating beta cell proliferating factors. Late gestational pregnancy serum significantly increased proliferation of rat beta cells compared with early pregnancy and nonpregnancy. The mitogenic active serum fractions contained proteins and peptides derived from kininogen-1, fibrinogen-α, α1-antitrypsin, apolipoprotein-A1, placental lactogen, angiotensinogen and serum albumin. Pregnancy serum is able to stimulate proliferation of rat beta cells. We have identified several circulating factors that may contribute to beta cell adaptation to pregnancy. Further studies are needed to elucidate their possible role in glucose homeostasis in the mother and her offspring.

Feasibility and quality development of biomaterials in the pretest studies of the German National Cohort

The German National Cohort (GNC) is designed to address research questions concerning a wide range of possible causes of major chronic diseases (e.g. cancer, diabetes, infectious, allergic, neurologic and cardiovascular diseases) as well as to identify risk factors and prognostic biomarkers for early diagnosis and prevention of these diseases. The collection of biomaterials in combination with extensive information from questionnaires and medical examinations represents one of the central study components. In two pretest studies of the German National Cohort conducted between 2011 and 2013, a range of biomaterials from a defined number of participants was collected. Ten study centres were involved in pretest1 and 18study centres were involved in pretest2. Standard operation procedures (SOP) were developed and evaluated to minimize pre-analytical artefacts during biosample collection. Within the pretest studies different aspects concerning feasibility of sample collection/preparation [pretest1 (a)] and quality control of biomarkers and proteome analyses were investigated [pretest 1 (b), (c)]. Additionally, recruitment of study participants for specific projects and examination procedures of all study centres in a defined time period according to common standards as well as transportation and decentralized storage of biological samples were tested (pretest2). These analyses will serve as the basis for the biomaterial collection in the main study of the GNC starting in 2014. Participants, randomly chosen from the population (n = 1000 subjects recruited at ten study sites in pretest1) were asked to donate blood, urine, saliva and stool samples. Additionally, nasal and oropharyngeal swabs were collected at the study sites and nasal swabs were collected by the participants at home. SOPs for sample collection, preparation, storage and transportation were developed and adopted for pretest2. In pretest2, 18study sites (n = 599subjects) collected biomaterials mostly identical to pretest 1. Biomarker analyses to test the quality of the biomaterials were performed. In pretest1 and 2, it was feasible to collect all biomaterials from nearly all invited participants without major problems. The mean response rate of the subjects was 95 %. As one important result we found for example that after blood draw the cellular fraction should be separated from the plasma and serum fractions during the first hour with no significant variation for up to 6h at 4 ℃ for all analysed biomarkers. Moreover, quality control of samples using a proteomics approach showed no significant clustering of proteins according to different storage conditions. All developed SOPs were validated for use in the main study after some adaptation and modification. Additionally, electronic and paper documentation sheets were developed and tested to record time stamps, volumes, freezing times, and aliquot numbers of the collected biomaterials. The collection of the biomaterials was feasible without major problems at all participating study sites. However, the processing times were in some cases too long. To avoid pre-analytical artefacts in sample collection, appropriate standardisation among the study sites is necessary. To achieve this, blood and urine collection will have to be adapted to specific conditions of usage of liquid handling robots, which will be available at all participating study centres in the main study of the GNC. Strict compliance with the SOPs, thorough training of the staff and accurate documentation are mandatory to obtain high sample quality for later analyses. The so obtained biomaterials represent a valuable resource for research on infectious and other common complex diseases in the GNC.

Transient elevation of serum 5-HIAA by dietary serotonin and distribution of 5-HIAA in serum protein fractions.

Dietary serotonin increases urinary secretion of 5-HIAA. A falsely elevated 5-HIAA may lead to incorrect suspicion of a neuroendocrine tumour. Therefore, we determined the effect and duration of dietary serotonin on serum 5-HIAA concentration. We also studied the distribution of 5-HIAA in serum fractions. We used serum samples from healthy volunteers (31 women and four men). All test subjects avoided serotonin-containing foods for three days before sample collection. They then ate either pineapple, banana, kiwi fruit, tomato or walnuts and additional blood samples were taken after 2, 4, 6, 24, 48 and 72 h. To study the distribution of 5-HIAA in serum, samples from a healthy individual, a test person who had ingested walnuts, and from a neuroendocrine tumour patient were fractionated by gel filtration chromatography. The fractions were analysed for 5-HIAA. Serum 5-HIAA concentration increased significantly (P ≤ 0.001) within 2 h after ingestion of serotonin-containing food. After 2 h, 5-HIAA concentration started to decrease and reached the baseline concentration within 24 h. A calculated half-life of 5-HIAA in circulation was 1.3 h. In fractionated serum, 5-HIAA was found not only in free form but also in the albumin and α2-globulin fractions. The increase of serum 5-HIAA caused by dietary serotonin is significant but transient. Therefore, serotonin-containing foods should be avoided for one day before blood sampling. In serum, 5-HIAA is free and apparently bound to albumin. Minor amounts were also found in the α2-globulin fraction. Our liquid chromatography tandem mass spectrometry assay measures free 5-HIAA in serum.

Natural rubber with nanomatrix of non-rubber components observed by focused ion beam-scanning electron microscopy

Naturally occurring nanomatrix structure formed in natural rubber was observed with a field emission scanning electron microscope equipped with a focused ion beam (FIB-SEM), and it was related to viscoelastic properties of the rubber. Film specimen used for the FIB-SEM observation was prepared from serum fraction of natural rubber latex, in which serum rubber contained about 15 w/w% proteins. Morphology of the film specimen was observed by FIB-SEM, after pinning the nanomatrix structure with glutaraldehyde followed by staining the serum rubber with OsO4. Three-dimensional image of the nanomatrix structure was successfully reconstructed in mesoscale without defects and voids, in which rubber particles of 200 nm in diameter were dispersed in the matrix of non-rubber components such as the proteins and phospholipids of several 10 nm in thickness. Storage modulus at plateau region of the serum rubber was about 500 times as high as that of deproteinized natural rubber, suggesting that the nanomatrix of the non-rubber components was completely continuous.

Extraction of perfluorinated alkyl acids from human serum for determination of the combined xenoestrogenic transactivity: A method development

Humans are exposed to perfluorinated alkyl acids (PFAAs) through food, drinking water, consumer products, dust, etc. The human metabolism and excretion of the long-chain PFAAs is slow with half-lives up to 8.8years. Studies suggest that the PFAAs are potential endocrine-disrupting compounds that might affect human health.We developed a method for extraction of PFAAs from human serum with simultaneous removal of endogenous sex hormones. The developed method includes solid phase extraction, liquid/liquid extraction, HPLC fractionation and weak anion exchange. The method was validated by extraction of seven persistent PFAAs spiked to human male serum obtaining mean recoveries between 49.6% and 78.6%. Using an estrogen receptor (ER) transactivation luciferase reporter gene assay, analysis of the extracted PFAA serum fraction from three pregnant women showed the ER-active endogenous hormones were removed.The developed method was further documented by extraction of the PFAAs from the serum of 18 Danish pregnant women. The PFAA fraction from three of the 18 samples significantly induced the ER-transactivity. Upon co-exposure with the natural ER-ligand 17β-estradiol (E2), 17 of the 18 PFAA fractions caused a significant further increase of the E2 induced ER-transactivity. In conclusion, we developed a method to extract PFAAs from human serum, and the method documentation suggested that PFAAs at the levels found in human serum can transactivate the ER.

Novel HIV-1 miRNAs stimulate TNFα release in human macrophages via TLR8 signaling pathway.

PurposeTo determine whether HIV-1 produces microRNAs and elucidate whether these miRNAs can induce inflammatory response in macrophages (independent of the conventional miRNA function in RNA interference) leading to chronic immune activation.MethodsUsing sensitive quantitative Real Time RT-PCR and sequencing, we detected novel HIV-derived miRNAs in the sera of HIV+ persons, and associated with exosomes. Release of TNFα by macrophages challenged with HIV miRNAs was measured by ELISA.ResultsHIV infection of primary alveolar macrophages produced elevated levels of viral microRNAs vmiR88, vmiR99 and vmiR-TAR in cell extracts and in exosome preparations from conditioned medium. Furthermore, these miRNAs were also detected in exosome fraction of sera from HIV-infected persons. Importantly, vmiR88 and vmiR99 (but not vmiR-TAR) stimulated human macrophage TNFα release, which is dependent on macrophage TLR8 expression. These data support a potential role for HIV-derived vmiRNAs released from infected macrophages as contributing to chronic immune activation in HIV-infected persons, and may represent a novel therapeutic target to limit AIDS pathogenesis.ConclusionNovel HIV vmiR88 and vmiR99 are present in the systemic circulation of HIV+ persons and could exhibit biological function (independent of gene silencing) as ligands for TLR8 signaling that promote macrophage TNFα release, and may contribute to chronic immune activation. Targeting novel HIV-derived miRNAs may represent a therapeutic strategy to limit chronic immune activation and AIDS progression.

Effect of Ethanolic Extract of Silkworm on Reactive Oxygen Species Formation in vivo

The purpose of this study is to investigate the effects of silkworm extracts (SWE) on reactive oxygen species formation in mice (C57BL/6). Mice were administrated intraperitoneally with SWE (20 mg/kg/day) for 14 days. All animals were sacrificed 24 hours after the last SWE treatment and then extracted the blood and brain tissue in mouse. The researcher measured several parameters related to reactive oxygen species formation, malondialdehyde (MDA) and hydrogen peroxide (H2O2) contents in serum, whole brain, cerebral cortex and cerebellum. The results showed that MDA content of pre-SWE treatment was decreased significantly in serum, mitochondrial and cytosolic fraction of whole brain and cerebellum (P<0.01). The H2O2 content of pre-SWE treatment was decreased significantly in mitochondrial fraction of whole brain, cerebral cortex and cerebellum (P<0.01). These results suggest that SWE plays an important role for inhibition of oxidative damage of cells as well as antioxidant effect, aging delay and cells protected from irradiation. 

Proteomic Profiling of Invasive Ductal Carcinoma (IDC) using Magnetic Beads‐based Serum Fractionation and MALDI‐TOF MS

To reveal the serum proteomic profiling of intraductal carcinoma (IDC) patients in China, establish a serum proteome fractionation technique for choosing magnetic beads for proteomic analysis in breast cancer research; and identify differentially expressed peptides (m/z; P < 0.0001) as potential biomarkers of early IDCs. We used two different kinds of magnetic beads (magnetic bead-based weak cation exchange chromatography (MB-WCX) and immobilized metal ion affinity chromatography (MB-IMAC-Cu)) to analyze 32 patients with early stage (stages I-II) IDC and 32 healthy control serum samples for proteomic profiling by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis. The mass spectra, analyzed using ClinProTools software, distinguished between IDC patients and healthy individuals based on k-nearest neighbor genetic algorithm. The serum samples purified in the MB-WCX group provided better proteomic patterns than the MB-IMAC-Cu group. The samples purified by MB-WCX had better average peak numbers, higher peak intensities, and better capturing ability of low abundance proteins or peptides in serum samples. In addition, the MB-WCX and MB-IMAC-Cu purification methods, followed MALDI-TOF MS identification and use of ClinProTools software accurately distinguished patients with early stage IDC from healthy individuals. Serum proteomic profiling by MALDI-TOF MS is a novel potential tool for the clinical diagnosis of patients with IDC in China.

MicroRNA-122 regulates hypoxia-inducible factor-1 and vimentin in hepatocytes and correlates with fibrosis in diet-induced steatohepatitis.

miR-122 is the most abundant miRNA in the liver particularly in hepatocytes where it targets cholesterol metabolism. Steatosis, a key component of non-alcoholic fatty liver disease, is regulated by hypoxia-inducible factor-1α (HIF-1α). Here, we hypothesized that reduced miR-122 has a pathogenic role in steatohepatitis. miR-122 and its target genes were evaluated in mouse livers and/or isolated hepatocytes after methionine-choline-deficient (MCD) or methionine-choline-supplemented (MCS) diet. Liver and hepatocyte miR-122 expression was significantly decreased in steatohepatitis. A maximum reduction in miR-122 occurred at the fibrosis stage (8 weeks of MCD diet). MAP3K3, a miR-122 target gene, was induced at all stages of non-alcoholic steatohepatitis (NASH; 3-8 weeks) only at the mRNA level. Increased NF-κB activation was found in MCD diet-fed mice and MAP3K3 regulated the NF-κB DNA binding in naive hepatocytes. HIF-1α mRNA and DNA binding and expression of the HIF-1α target gene, profibrotic lysyl oxidase, was increased in advanced steatohepatitis (8 weeks). In addition, increase in vimentin and Sirius red staining (liver fibrosis) was found at 8 weeks of MCD diet. Using miR-122 overexpression and inhibition approaches, we confirmed that HIF-1α, vimentin and MAP3K3 are novel miR-122 targets in hepatocytes. We report transcriptional repression of miR-122 in NASH. Decreased liver miR-122 was associated with elevated circulating miR-122 in both exosome-rich and protein-rich serum fractions. Our novel data suggest that decreased liver miR-122 contributes to upregulation of modulators of tissue remodelling (HIF-1α, vimentin and MAP3K3) and might play a role in NASH-induced liver fibrosis.

Copper subtype of Alzheimer's disease (AD): Meta-analyses, genetic studies and predictive value of non-ceruloplasmim copper in mild cognitive impairment conversion to full AD

Alzheimer's disease (AD) is the most common form of dementia. A myriad of complex factors contribute to AD, promoting the deposition in plaques of amyloid-beta (Aβ), which is the main constituent of this pathognomonic sign of AD at autopsy brain inspection. Aβ toxicity is related to oxidative stress, which results in synaptic loss in specific brain areas, eventually leading to cognitive decline. Metal, and especially copper, dyshomeostasis is a key factor in these processes. Recent studies have demonstrated that the serum fraction of copper that is not bound to ceruloplasmin (Non-Cp copper, also known as ‘free’ or labile copper) increases in a percentage of AD patients and mild cognitive impairment (MCI) subjects; this is considered a precursor of AD. Non-Cp copper is the exchangeable fraction of low molecular weight copper in serum. It is distinguished from the copper structurally bound to the ceruloplasmin protein, a master protein of iron metabolism. Non-Cp copper levels are higher than normal reference values (range 0–1.6μmol/L) in about 50% of amnestic MCI subjects and 60% of AD patients, typifying them in a subset of AD. Meta-analyses, genetic studies and a prognostic study evaluating the predictive value of Non-Cp copper in MCI conversion to full AD demonstrate the existence of this copper phenotype of AD.

Humoral immune response in Oreochromis niloticus: modified direct agglutination test

Understanding the functional mechanisms of the humoral and cellular immune responses among teleost broadens the possibilities for intervention and improvement of vaccination methods. The aim of this study was to evaluate the dynamics of the humoral immune response, hematological biochemical changes, and electrophoretic profile of proteins in Oreochromis niloticus, when submitted to antigen stimulation using sheep erythrocytes in a single dose and with booster doses. Seventy O. niloticus were distributed in two groups (n = 35): one received a single dose and the other received two doses of antigen. Evaluations were made at the following predetermined times: zero (pre-immune, i.e., before inoculation with the first injection of sheep erythrocytes, negative control) and on 7th, 14th, 21st, and 28th days after the first immunization to evaluate the hemogram, biochemistry, and electrophoresis of proteins and direct hemagglutination. Twenty-eight days after antigen inoculation, it was observed that there had been an increase in the serum antibody titer and fractions gamma globulins after second antigen stimulation. The results suggest that teleost fish have the capacity to produce antibodies from a single stimulation, and a second antibody production wave after second stimulation.

Inhibitor(s) of the classical complement pathway in mouse serum limit the utility of mice as experimental models of neuromyelitis optica

Neuromyelitis optica (NMO) is an inflammatory demyelinating disease of the central nervous system in which anti-aquaporin-4 (AQP4) autoantibodies (AQP4-IgG) cause damage to astrocytes by complement-dependent cytotoxicity (CDC). Various approaches have been attempted to produce NMO lesions in rodents, some involving genetically modified mice with altered immune cell function. Here, we found that mouse serum strongly inhibits complement from multiple species, preventing AQP4-IgG-dependent CDC. Effects of mouse serum on complement activation were tested in CDC assays in which AQP4-expressing cells were incubated with AQP4-IgG and complement from different species. Biochemical assays and mass spectrometry were used to characterize complement inhibitor(s) in mouse serum. Sera from different strains of mice produced almost no AQP4-IgG-dependent CDC compared with human, rat and guinea pig sera. Remarkably, addition of mouse serum prevented AQP4-IgG-dependent CDC caused by human, rat or guinea pig serum, with 50% inhibition at <5% mouse serum. Hemolysis assays indicated that the inhibitor(s) in mouse serum target the classical and not the alternative complement pathway. We found that the complement inhibitor(s) in mouse serum were contained in a serum fraction purified with protein-A resin; however, the inhibitor was not IgG as determined using serum from IgG-deficient mice. Mass spectrometry on the protein A-purified fraction produced several inhibitor candidates. The low intrinsic complement activity of mouse serum and the presence of complement inhibitor(s) limit the utility of mouse models to study disorders, such as NMO, involving the classical complement pathway.

Electrochemical immunosensor for the analysis of the breast cancer biomarker HER2 ECD

Human epidermal growth factor receptor 2 (HER2) is a breast cancer biomarker that plays a major role in promoting breast cancer cell proliferation and malignant growth. The extracellular domain (ECD) of HER2 can be shed into the blood stream and its concentration is measurable in the serum fraction of blood. In this work an electrochemical immunosensor for the analysis of HER2 ECD in human serum samples was developed. To achieve this goal a screen-printed carbon electrode, modified with gold nanoparticles, was used as transducer surface. A sandwich immunoassay, using two monoclonal antibodies, was employed and the detection of the antibody–antigen interaction was performed through the analysis of an enzymatic reaction product by linear sweep voltammetry. Using the optimized experimental conditions the calibration curve (ip vs. log[HER2 ECD]) was established between 15 and 100ng/mL and a limit of detection (LOD) of 4.4ng/mL was achieved. These results indicate that the developed immunosensor could be a promising tool in breast cancer diagnostics, patient follow-up and monitoring of metastatic breast cancer since it allows quantification in a useful concentration range and has an LOD below the established cut-off value (15ng/mL).

Investigating the effects of food matrix and food components on bioaccessibility of pomegranate (Punica granatum) phenolics and anthocyanins using an in-vitro gastrointestinal digestion model

Effects of food matrix and individual food components on potential bioaccessibility of pomegranate were investigated by means of simulating in vitro gastrointestinal (GI) digestion. The foodstuffs (sunflower oil, skim milk, cooked lean meat, bread, skim yogurt, probiotic yogurt, apple, lemon, honey, soy milk, cream, and soybean) and the food components (gluten, casein, isolated meat protein, lactose, fructose, galactose, glucose, salt, ascorbic acid, starch, cooked starch, tocopherol, linoleic acid, cellulose, citric acid and pectin) were codigested with pomegranate in model systems to better understand matrix effects. Total phenolic content (TPC) and total anthocyanin content (TAC) were determined by spectrophotometric methods and major phenolics/anthocyanins were analyzed by RP-HPLC/PDA detection both before and after in vitro GI digestion at post gastric (PG), dialyzable (IN) and non-dialyzable (OUT) fractions. Phenolics of pomegranate were found to be stable during gastric conditions (115%), with 25% loss in pancreatic digestion, available (14%) in IN. Although preserved (89%) in PG, anthocyanins were lost in pancreatic digestion (38%), but still available (12%) in IN. Milk, bread, yogurt, probiotic yogurt, lactose, starch, cellulose, salt, citric acid or tocopherol codigestion with pomegranate decreased TPC for all fractions. Proteins affected losses in PG and OUT fractions. Carbohydrates such as starch, lactose, glucose and pectin appeared to affect the loss of phenolics and exerted 2-fold decreases in serum fraction (IN). For TAC, only meat, soymilk or cream codigestion with pomegranate resulted in IN losses. Proteins did not significantly affect TAC in IN, but were inhibitory in PG. However, carbohydrates and fatty acids significantly increased TAC in IN. Generally cyanidins were found to be more stable in food matrices and pancreatic conditions than other anthocyanins.

Dysfunctional lipoproteins from young smokers exacerbate cellular senescence and atherogenesis with smaller particle size and severe oxidation and glycation.

Until now, there has been limited information on the effects of smoking on atherogenesis and senescence in the context of lipoprotein parameters, particularly in young smokers who have smoked fewer than 10 cigarettes per day for 3 years. In this study, lipoprotein profiles and functions were compared between smoker (n = 21) and control groups (n = 20). In the smoking group, ferric ion reduction abilities of serum and high-density lipoprotein (HDL) fractions were significantly reduced, and low-density lipoprotein (LDL) was severely oxidized. All lipoprotein particles from the smoker group showed higher advanced glycated end products with more triglyceride (TG) content compared with the control group. Lipoproteins from smokers showed faster agarose gel electromobility as well as greater smear band intensity in SDS-PAGE due to oxidation and glycation. LDL from smokers was more sensitive to oxidation and promoted foam cell forma-tion in macrophages. Gel filtration column chromatography revealed that the protein and cholesterol peaks of VLDL and LDL were elevated in the smoker group, whereas those of HDL were reduced. Human dermal fibroblast cells from the smoker group showed severe senescence following treatment with HDL2 and HDL3. Although HDL from young smokers showed impaired antioxidant ability, smaller particle size, and increased TG content, cholesteryl ester transfer protein activities were greatly enhanced in the serum and HDL fractions of the smoker group. In conclusion, smoking can cause production of dysfunctional lipoproteins having a smaller particle size that exacerbate senescence and atherogenic progress due to oxidation and glycation.

13C Enrichment of the CO2 in Breast Milk and in the Breath Is Rapidly Modified by Changes in the 13C Content of the Diet

Background: C4 plants (e.g. corn and sugar cane) have greater ¹³C enrichment than C3 plants (e.g. wheat and sugar beet). Objective: To assess whether ¹³C enrichment of CO₂ in the breath and breast milk of women on diets based on C3 and C4 foods changes from one diet to the other. Materials and Methods: Six breast-feeding women were studied at 5-6 months postpartum. They ate a controlled C4 diet on days 1 and 2 followed by a C3 diet on days 3 and 4. Diet duplicates, breast milk on days 2 and 4 and hourly breath samples were collected over 4 days. ¹³C enrichment was measured by isotope-ratio mass spectrometry. Values of δ¹³C were calculated from the international PDBV standard (δ¹³C_PDBV). Differences between means were compared by paired t test or t test for repeated measurements. Results: δ¹³C_PDBV values were significantly higher in the C4 diet than in the C3 diet composites (p < 0.01). In breath CO₂, the δ¹³C_PDBV value was greater on days 1 and 2 (range -15.4 to -13.2, respectively) and declined on days 3 and 4 (range -20.0 to -21.8, respectively, p < 0.01). The lipid and milk serum fractions of breast milk had significantly higher δ¹³C_PDBV on the C3 diet than on the C4 diet (p < 0.01). Conclusions: Subjects eating a C4 diet have a higher δ¹³C_PDBV value in the breath and breast milk fractions, which diminish rapidly on a C3 diet. Further studies focusing on individual nutrients are warranted.

Estudio electroforético de las proteínas totales y fraccionadas en nativos de la selva

La sangre ha sido desde tiempo inmemorial materia de estudio de los investigadores ávidos de desentrañar los secretos del cuerpo humano y sus sistemas. Considerada por sí misma como un tejido, expresa fidedignamente muchas veces el estado fisiológico del individuo. Las sustancias proteicas constituyentes del suero sanguíneo, representan una de las más importantes partes del tejido hemático. A su estudio y comprensión se ha contribuido con una infinidad de trabajos y experiencias investigatorias cuya enumeración no es necesaria. Fue quizá William Hewson (20), el primero en separar una fracción sérica al aislar por medios biológicos el fibrinógeno plasmático en 1771. Desde esa fecha fueron ganándose lentamente una serie de conocimientos, pero podemos decir que data de tan sólo 20 años a esta parte el enorme adelanto alcanzado en esta rama del conocimiento. A ello han contribuido poderosamente: el fraccionamiento por las sales neutras, la electroforesis y la ultracentrifugación.

Antibody against immunoglobulin E contained in blood components as causative factor for anaphylactic transfusion reactions

Determining the mechanism underlying the development of transfusion reactions is important in transfusion therapy. Two bags of fresh-frozen plasma obtained from a donor (index donor) were implicated in two cases of anaphylactic transfusion reactions. The serum prepared from the index donor plasma transfused into the second patient (Patient 2) was evaluated using cord blood-derived mast cells (CBMCs) incubated with Patient 2 plasma. The component in the serum required for the degranulation was determined and quantified by chromatography in combination with degranulation assay, Western blot analysis, and enzyme-linked immunosorbent assay. The component in the plasma required for CBMC sensitization was determined using human immunoglobulin (Ig)E or normal plasma in place of Patient 2 plasma in the assay. Sera collected from the index donor between 2001 and 2008 were examined for the CBMC degranulation factor. The donor serum activated CBMCs incubated with Patient 2 plasma. The IgG fraction of the donor serum induced degranulation of CBMCs sensitized with IgE or plasma containing a normal IgE concentration. The IgG anti-IgE at a concentration higher than 2200 ng/mL, which showed CBMC degranulation activity, was detected in the donor sera for at least 7 years. Transfusion of a high concentration of the anti-IgE in the donor plasma was suggested to induce mast cell degranulation in the patients leading to the development of anaphylactic transfusion reactions. Antibodies existing in not only the patient circulation but also the transfused blood might cause transfusion-induced anaphylaxis.

High density lipoproteins improve insulin sensitivity in high-fat diet-fed mice by suppressing hepatic inflammation

Obesity-induced liver inflammation can drive insulin resistance. HDL has anti-inflammatory properties, so we hypothesized that low levels of HDL would perpetuate inflammatory responses in the liver and that HDL treatment would suppress liver inflammation and insulin resistance. The aim of this study was to investigate the effects of lipid-free apoAI on hepatic inflammation and insulin resistance in mice. We also investigated apoAI as a component of reconstituted HDLs (rHDLs) in hepatocytes to confirm results we observed in vivo. To test our hypothesis, C57BL/6 mice were fed a high-fat diet (HFD) for 16 weeks and administered either saline or lipid-free apoAI. Injections of lipid-free apoAI twice a week for 2 or 4 weeks with lipid-free apoAI resulted in: i) improved insulin sensitivity associated with decreased systemic and hepatic inflammation; ii) suppression of hepatic mRNA expression for key transcriptional regulators of lipogenic gene expression; and iii) suppression of nuclear factor κB (NF-κB) activation. Human hepatoma HuH-7 cells exposed to rHDLs showed suppressed TNFα-induced NF-κB activation, correlating with decreased NF-κB target gene expression. We conclude that apoAI suppresses liver inflammation in HFD mice and improves insulin resistance via a mechanism that involves a downregulation of NF-κB activation.

Extracellular Vesicles Modulate Host-Microbe Responses by Altering TLR2 Activity and Phagocytosis

Oral delivery of Gram positive bacteria, often derived from the genera Lactobacillus or Bifidobacterium, can modulate immune function. Although the exact mechanisms remain unclear, immunomodulatory effects may be elicited through the direct interaction of these bacteria with the intestinal epithelium or resident dendritic cell (DC) populations. We analyzed the immune activation properties of Lactobacilli and Bifidobacterium species and made the surprising observation that cellular responses in vitro were differentially influenced by the presence of serum, specifically the extracellular vesicle (EV) fraction. In contrast to the tested Lactobacilli species, tested Bifidobacterium species induce TLR2/6 activity which is inhibited by the presence of EVs. Using specific TLR ligands, EVs were found to enhance cellular TLR2/1 and TLR4 responses while TLR2/6 responses were suppressed. No effect could be observed on cellular TLR5 responses. We determined that EVs play a role in bacterial aggregation, suggesting that EVs interact with bacterial surfaces. EVs were found to slightly enhance DC phagocytosis of Bifidobacterium breve whereas phagocytosis of Lactobacillus rhamnosus was virtually absent upon serum EV depletion. DC uptake of a non-microbial substance (dextran) was not affected by the different serum fractions suggesting that EVs do not interfere with DC phagocytic capacity but rather modify the DC-microbe interaction. Depending on the microbe, combined effects of EVs on TLR activity and phagocytosis result in a differential proinflammatory DC cytokine release. Overall, these data suggest that EVs play a yet unrecognized role in host-microbe responses, not by interfering in recipient cellular responses but via attachment to, or scavenging of, microbe-associated molecular patterns. EVs can be found in any tissue or bodily fluid, therefore insights into EV-microbe interactions are important in understanding the mechanism of action of potential probiotics and gut immune homeostasis.