Pooled Human Serum Research Articles

An immunoprecipitation coupled with fluorescent Western blot analysis for the characterization of a model secondary serum cardiac troponin I reference material

Background Cardiac troponin I (cTnI) is considered the ‘gold standard’ cardiac biomarker. However, the result comparability of commercial cTnI immunoassays is still lacking despite the availability of NIST Standard Reference Material, SRM 2921 (human cardiac troponin). To facilitate the standardization of the cTnI immunoassays, a secondary reference material consisting of a panel of three cTnI-positive human serum pools is proposed by the IFCC Working Group on Standardization of Troponin I. The objective of this study is to develop measurement procedures for the characterization of the future secondary reference material using a pooled cTnI-positive serum sample as a development model. Methods We used magnetic beads coupled with 6 different anti-cTnI monoclonal antibodies that bind specifically to different amino acid sequence regions of the cTnI molecule to immunoprecipitate cTnI proteins from the pooled cTnI-positive serum sample followed by sensitive detection using a fluorescent Western blot. Results The degradation of cTnI in the pooled sample was detected and the concentration of cTnI was determined. Conclusion We demonstrated the utility of this measurement procedure in support of the development of the proposed secondary cTnI-positive, serum-based reference material.

Multiplexed Evaluation of a Cell-Based Assay for the Detection of Antidrug Neutralizing Antibodies to Panitumumab in Human Serum Using Automated Fluorescent Microscopy

The method described here employs a high-content cell-based assay format for the detection of neutralizing antibodies (NAbs) to panitumumab, a fully human monoclonal antagonistic antibody to the human epidermal growth factor (EGF) receptor in human serum (screening assay). A specificity assay was also developed and qualified to confirm that the neutralizing activity was attributable to the presence of NAbs and not due to serum interference (serum interference assay). The ArrayScan IV HCS reader was used for the measurement of tyrosine phosphorylation of the epidermal growth factor receptor (EGFR) and STAT-1 redistribution between the cytoplasm and nucleus in the human epidermoid carcinoma cell line A431. Assay conditions were developed by (1) optimizing the response of the A431 cells to recombinant human EGF in pooled human serum, (2) evaluating the ability of panitumumab to inhibit the EGF response, and (3) assessing the assay's sensitivity for detecting a positive control affinity purified rabbit polyclonal anti-panitumumab antibody. Panitumumab dose-dependently inhibited 4 ng/mL EGF, and the positive control antibody showed a dose-dependent neutralization of 50 ng/mL panitumumab. The experiments indicated that in comparison to STAT-1 translocation, EGFR phosphorylation was the optimal endpoint for the screening and serum interference assays. Assay cut points were derived for the screening and serum interference assays by obtaining normalized ratios of mean fluorescence intensity values obtained with EGFR phosphorylation by testing sera from healthy human donor sera. The assay sensitivity was determined to be 0.125 microg/mL for the positive control antibody.

Use of a Single-Chain Antibody Library for Ovarian Cancer Biomarker Discovery

The discovery of novel early detection biomarkers of disease could offer one of the best approaches to decrease the morbidity and mortality of ovarian and other cancers. We report on the use of a single-chain variable fragment antibody library for screening ovarian serum to find novel biomarkers for the detection of cancer. We alternately panned the library with ovarian cancer and disease-free control sera to make a sublibrary of antibodies that bind proteins differentially expressed in cancer. This sublibrary was printed on antibody microarrays that were incubated with labeled serum from multiple sets of cancer patients and controls. The antibodies that performed best at discriminating disease status were selected, and their cognate antigens were identified using a functional protein microarray. Overexpression of some of these antigens was observed in cancer serum, tumor proximal fluid, and cancer tissue via dot blot and immunohistochemical staining. Thus, our use of recombinant antibody microarrays for unbiased discovery found targets for ovarian cancer detection in multiple sample sets, supporting their further study for disease diagnosis.

Liquid chromatographic analysis of the anticancer alkaloid luotonin A and some new derivatives in human serum samples

The quantitation of the natural cytotoxic and anti-inflammatory alkaloid luotonin A and five recently synthesized derivatives is described, constituting the first report of a HPLC method for the analysis of these compounds in human serum samples. The conditions for the chromatographic separation were optimized and the method was validated for the analysis of these compounds in biological samples according to international guidelines. An RP-HPLC method with fluorimetric detection and a C(18) stationary phase was applied. Different ACN/water mobile phases were assayed, including 0-4% of a mobile phase modifier such as tetrahydrofuran, dioxane or tert-butyl methyl ether. Isocratic and gradient elution conditions are compared. The influence of pH on the efficiency and resolution of the separation was also considered. The developed method was applied to the determination of luotonins in pooled human serum samples by gradient elution RP-HPLC using a simple cleanup procedure. The proposed chromatographic method exhibits satisfactory analytical figures of merit, with LOD from 1.0 x 10(-10) to 2.0 x 10(-10) M, intraday and interday precision below 6% except for the concentration level closest to LOD, and good agreement between experimental and theoretical concentrations. Therefore, the developed method is suitable, reliable, rapid, and simple.

Consistency of creatinine results in Shanghai and the preliminary clinical application of estimated glomerular filtration rate

Objective To explore the consistency of the clinical serum creatinine results in Shanghai district and investigate the population distribution of apparently healthy people, modified MDRD formula adapted to Chinese population was used to estimate glomerular filtration rate and its distribution for further assessment of the clinical applicability of the eGFR.Methods A fresh pooled human serum sample with given IFCC's creatinine level from c.f.a.s.(calibration for automatic system) ,was used to calibrate the creatinine detecting systems of each participating hospital laboratory before every examination. Fourteen hospital laboratories successively conducted 6 experiments, and the test results were almost identical.They tested and studied the creatinine values of 6 837 ( male 3 289, female 2 132, children and teenagers 1 416)apparently healthy individuals, age from 0 to 99 years old, and estimated eGFR value of these apparently healthy individuals according to the documented eGFR formula [ eGFR = 175 × (Scr) -1.154 × (age) -0.203 ×0.742 (female) × 0.827 ] which was applied especially to Shanghai people.Results Before calibration,the inter-laboratory CVs of creatinine results varied from 3.1％ -9.1％, and after calibration, CVs decreased to less than 5％.A good consistency of the creatinine results was established among all these hospitals.The result of population distribution study of creatinine for men was 63.0-102.8 μmol/L,for women was 45.0-76.0 μmol/L, and for children and teenagers was 0-1 year old 11.0-77.0 μmol/L, 2 years old 15.5-33.3 μmol/L,3-5 years old 19.0-42.0 μmol/L, 6-19 years old 41.4-62.0 μmol/L.The Cr value were different between the male and femal [ male: ( 82.1 ± 10.9 ) μmol/L, femal: ( 59.4 ± 8.4 ) μmol/L, t =94.3 ,P 0.05 ].The difference of Cr and eGFR could not be eliminated between the groups divided by every 10 years(x2Cr =2 601 ,P <0.01 ;x2eGFR2= 1 105 ,P <0.01 ＝.Conclusions The pooled patients' sera could be used as calibrator for harmonizing of creatinine results among the laboratories. The reference rang of Cr should be differentiated by age and sex.Although eGFR can decrease the difference of sex, it cannot eliminate the difference of age. Key words: Glomerular filtration rate; Creatinine; Sex distribution; Age distribution

Bovine Serum–Based Bilirubin Calibrators Are Inappropriate for Some Diazo Methods

In 2003, the College of American Pathologists (CAP)1 included in the Neonatal Bilirubin Surveys a human serum–based sample enriched solely with unconjugated bilirubin (UBIL). This sample, shipped frozen in a cold pack, resembled closely a clinical sample from a healthy neonate and was commutable, as expected. The mean of all results submitted to CAP (CAP All Data) for bilirubin (199.4 mg/L) was close to that of the reference method (194.4 mg/L)(1). In subsequent years, the bias has increased(2). Most recently (2009, neonatal bilirubin NB-01), the difference between the mean value for CAP All Data and the reference method value was 9 mg/L, and the differences for the Olympus, Ortho Vitros, and Siemens ADVIA assays were closer to 25 mg/L. Because ditaurobilirubin (DTB), which is present in most commercial calibrators, and bovine serum, which is used instead of human serum for preparing calibrators, have been shown to interfere with the measurement of bilirubin(3), we investigated the effects of DTB and bovine serum on the measurement of total bilirubin by diazo methods. UBIL and DTB were purchased from Lee Biosolutions. Human serum pools (nos. 1 and 2) were obtained from volunteers from the laboratory personnel of the Children’s Hospital of Wisconsin. Bovine sera and donor calf serum were purchased from Sigma–Aldrich and MP Biomedicals. Fresh bovine serum was obtained from a local abattoir in Milwaukee, Wisconsin. Stock solutions of UBIL and DTB were prepared in all of the above-described protein matrices. The concentrations of UBIL (210 mg/L) …

Application of five frozen human-pooled serum samples assigned by the International Federation of Clinical Chemistry and Laboratory Medicine reference procedure in a traceability investigation of γ-glutamyltransferase catalytic concentration measurements in China

The in vitro directive of the European Union requires traceability to the international recommended reference procedures. The application of the reference procedures is necessary in order to evaluate the accuracy of gamma-glutamyltransferase (GGT) assays of routine measurement systems in China. Five frozen patient-pooled serum samples were assigned values by the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) reference procedure in order to evaluate the traceability of the results of GGT catalytic activity from six homogeneous systems. One of the serum samples was used to calibrate seven non-homogeneous systems. All of the homogeneous systems, except the Dade system (Dade Bering Inc, IL, USA), achieved traceability within the measurement range. The Roche and Hitachi systems were better than the other systems. After calibration, the variance of the non-homogeneous systems decreased dramatically from between 14.50% and 25.23% to between 1.25% and 3.09% and the bias decreased from between -11.4% and -4.1% to between 0.5% and 3.5%. Manufacturers in China should ensure that their calibration systems correspond to the IFCC reference procedures. Fresh frozen pooled patient serum assigned by reference laboratories can be used to calibrate non-homogeneous systems in order to achieve traceability.

Candida dubliniensis screening using the germ tube test in clinical yeast isolates and prevalence of C. dubliniensis in Korea

The aim of this study was to screen for C. dubliniensis using the germ tube test with human pooled serum (HPS) in clinical isolates and investigate the prevalence of C. dubliniensis in Korea. Among 1,854 yeast strains isolated, 1,404 strains of C. albicans (on the basis of positive results of the germ tube test) and 192 germ tube-negative yeast strains were examined. All 1,596 clinical isolates were examined using the germ tube test with HPS, the differential temperature, and NaCl tolerance test. Only 81 isolates that did not grow at 45 degrees C nor on Sabouraud 6.5% NaCl broth were selected and tested using the VITEK 2 ID-YST system and the multiplex-PCR assay for the study. The two strains, C. dubliniensis ATCC MYA-646 and KCTC 17427 failed to produce germ tubes in HPS but produced them in fresh rabbit serum (FRS) and fetal bovine serum (FBS). No C. dubliniensis was found in this study population. The results of this study suggest that the germ tube test with HPS in combination with FRS or FBS can be used for discriminating between C. albicans and C. dubliniensis strains and that the prevalence of C. dubliniensis appears to be extremely low in Korea.

Stability and mixing compatibility of dog epithelia and dog dander allergens

Little information or data are available concerning the stability and compatibility of dog epithelia and dog dander allergens. To determine the immunochemical reactivities of commercial, nonstandardized dog epithelia and dog dander extracts after exposures to various temperatures or after mixing with high-protease fungal and cockroach extracts at concentrations recommended for maintenance immunotherapy (IT) injections. Quantitative enzyme-linked immunosorbent assay and qualitative (immunoblot) analyses were performed to compare specific compositional changes with total or individual allergen activities. Assays for dog allergens Can f 1 and Can f 3 (albumin) used specific mouse or rabbit antibodies. Multiallergen enzyme-linked immunosorbent assay inhibition and immunoblot methods were conducted using a human serum pool with high levels of IgE to dog allergens. Dog allergen recoveries ranged from 22% to 134% after short exposures to moderate or extreme temperatures and from 28% to 118% after mixing with fungal or insect extracts and storage for up to 15 months at 2 degrees C to 8 degrees C. Recoveries in dog dander extracts varied up to 2.5-fold with different test methods. Immunoblots revealed partial degradation of dog albumin molecules to discrete fragments that retained antibody-binding activities. In most cases, recoveries improved at elevated glycerin concentrations. Dog allergens in epithelia and dander extracts exhibited favorable temperature stabilities. Compatibilities with fungal or insect extracts may be compromised or at risk in some combinations. These data support current IT practice parameter recommendations of separating high-protease extracts from other products if possible; they also demonstrate that dog extracts possess allergen stabilities suitable for many IT formulations.

Identification of Continuous Human B-Cell Epitopes in the Envelope Glycoprotein of Dengue Virus Type 3 (DENV-3)

BackgroundDengue virus infection is a growing global public health concern in tropical and subtropical regions of the world. Dengue vaccine development has been hampered by concerns that cross-reactive immunological memory elicited by a candidate vaccine could increase the risk of development of more severe clinical forms. One possible strategy to reduce risks associated with a dengue vaccine is the development of a vaccine composed of selected critical epitopes of each of the serotypes.Methodology/Principal FindingsSynthetic peptides were used to identify B-cell epitopes in the envelope (E) glycoprotein of dengue virus type 3 (DENV-3). Eleven linear, immunodominant epitopes distributed in five regions at amino acid (aa) positions: 51–65, 71–90, 131–170, 196–210 and 246–260 were identified by employing an enzyme- linked immunosorbent assay (ELISA), using a pool of human sera from dengue type 3 infected individuals. Peptides 11 (aa51–65), 27 and 28 (aa131–150) also reacted with dengue 1 (DENV-1) and dengue 2 (DENV-2) patient sera as analyzed through the ROC curves generated for each peptide by ELISA and might have serotype specific diagnostic potential. Mice immunized against each one of the five immunogenic regions showed epitopes 51–65, 131–170, 196–210 and 246–260 elicited the highest antibody response and epitopes131–170, 196–210 and 246–260, elicited IFN-γ production and T CD4+ cell response, as evaluated by ELISA and ELISPOT assays respectively.Conclusions/Significance Our study identified several useful immunodominant IgG-specific epitopes on the envelope of DENV-3. They are important tools for understanding the mechanisms involved in antibody dependent enhancement and immunity. If proven protective and safe, in conjunction with others well-documented epitopes, they might be included into a candidate epitope-based vaccine.

Effect of iron on cytolysin a expression in Salmonella enterica serovar Typhi

Previously, a novel protein ClyA (Cytolysin A) has been identified in Escherichia coli K-12, Salmonella enterica serovars Typhi and Paratyphi A and Shigella. Salmonella spp. synthesize substantial amounts of ClyA upon infection of the human host, although the mechanism by which ClyA is induced in vivo is unclear. Since environmental signals could control the expression of virulence determinants, ClyA expression in S. Typhi Ty2 was tested by Western blotting in the presence of normal pooled human serum (NPS). The level of ClyA expression increased in the presence of NPS in a concentration-dependent manner. RPMI 1640 medium similarly induced ClyA expression. ClyA expression was inversely proportional to the concentration of iron in RPMI medium. Therefore, we speculated that iron inhibited the expression of ClyA in S. Typhi Ty2, and free iron depletion may be one of the causes of S. Typhi-mediated induction of ClyA in vivo. Transcription from a clyA-lacZ fusion gene decreased as iron concentration increased, but not as significantly as the ClyA protein expression. It is concluded that the regulatory effect of iron on clyA expression is mainly at translational level.

Proteomic Expression Profiling and Identification of Serum Proteins Using Immobilized Trypsin Beads with MALDI-TOF/TOF

MALDI-TOF mass spectrometry is a widely used technique for serum protein expression profiling and biomarker discovery. Many profiling strategies typically employ chemical affinity beads or surfaces to decrease sample complexity of dynamic fluids such as serum or plasma. However, many of the proteins captured on a particular surface or bead are not resolved in the lower mass ranges where time-of-flight mass spectrometers are most effective. Thus, a majority of reported protein expression profiling studies primarily interrogate the native low molecular mass constituents of the target sample. We report an expression profiling workflow that utilizes immobilized trypsin paramagnetic beads following an initial affinity bead fractionation step, thereby reducing large mass proteins to peptides that are better suited to analysis and sequencing determinations. Our bead-based trypsin approach resulted in more efficient digestion of complex serum protein extracts at short incubation times. This method was reproducible and readily adaptable to robotic sample handling and may be combined in tandem with other bead fractionation surfaces. When weak cationic and weak anionic bead surfaces were used, experimental conditions were optimized for tandem combinations of these beads with the immobilized trypsin step to produce an efficient serum fractionation strategy. A proof-of-concept pilot experiment using pooled human serum samples demonstrating reproducibility is presented, along with the sequence determination of selected tryptic peptides of serum proteins.

Superiority of nitric acid for deproteinization in the determination of trace lithium in serum by graphite furnace atomic absorption spectrometry

Physiological level of trace lithium in human serum was determined by graphite furnace atomic absorption spectrometry (GFAAS). 3.5% HNO 3 (v/v) was employed as a protein precipitant for sample treatment and at the same time verified as a very effective chemical modifier to eliminate the interference of chloride. The analytical conditions for lithium determination in serum were investigated and the optimal pyrolysis and atomization temperatures were 800 °C and 2700 °C. The accuracy and precision of the method were tested by determining lithium in a RANDOX HN1530 assayed human multi-sera and a pooled human serum. The result was in good agreement with the target value and CV of the pooled human serum was 4.74% ( n = 10). The characteristic mass, the limit of detection (LOD) of the proposed method were 0.8 pg and 0.01 μmol/L, respectively. Median ± S.E.M. of serum lithium in 220 Chinese people was 0.25 ± 0.02 μmol/L.

The role of complement opsonization in interactions between F. tularensis subsp. novicida and human neutrophils

The remarkable infectiousness of Francisella tularensis suggests that the bacterium efficiently evades innate immune responses that typically protect the host during its continuous exposure to environmental and commensal microbes. In our studies of the innate immune response to F. tularensis, we have observed that, unlike the live vaccine strain (LVS) of F. tularensis subsp. holarctica, F. tularensis subsp. novicida U112 opsonized in pooled human serum activated the NADPH oxidase when incubated with human neutrophils. Given previous observations that F. tularensis fixes relatively small quantities of complement component C3 during incubation in human serum and the importance of C3 to neutrophil phagocytosis, we hypothesized that F. tularensis subsp. novicida may fix C3 in human serum more readily than would LVS. We now report that F. tularensis subsp. novicida fixed approximately six-fold more C3 than did LVS when incubated in 50% pooled human serum and that this complement opsonization was antibody-mediated. Furthermore, antibody-mediated C3 deposition enhanced bacterial uptake and was indispensable for the neutrophil oxidative response to F. tularensis subsp. novicida. Taken together, our results reveal important differences between these two strains of F. tularensis and may, in part, explain the low virulence of F. tularensis subsp. novicida for humans.

Serologic Assay To Quantify Human Immunoglobulin G Antibodies to the Staphylococcus aureus Iron Surface Determinant B Antigen

A direct binding Luminex assay has been developed and validated for the detection of human immunoglobulin G (IgG) antibodies to the Staphylococcus aureus iron surface determinant B protein (IsdB) in serum following natural infection or immunization with investigational Saccharomyces cerevisiae-derived IsdB-based vaccines. To ensure that IsdB-specific IgG antibodies are measured following immunization with S. cerevisiae-derived IsdB, an Escherichia coli-produced IsdB antigen is used in the assay. The IsdB antigen is covalently conjugated to maleimide microspheres via an engineered carboxy-terminal cysteine residue. Antibody titers are determined in a direct binding format, where the phycoerythrin-labeled monoclonal antibody (HP6043) specific for IgG1 to IgG4 binds to human serum IgG antibodies. Fluorescent signal emitted from bound HP6043 is directly proportional to an individual's antibody levels. A pooled human reference serum from vaccinees with high titers to IsdB is used to generate a 12-point standard curve. The correlation of mean fluorescent intensity (MFI) units to microg/ml of IsdB-specific IgG is made by interpolating the MFI data through a four-parameter curve-fitting algorithm. The assay is sensitive to 1.06 microg/ml with a dynamic range of 2.1 to 10,625 microg/ml. The overall specificity of the assay is >96% and the linearity (parallelism) of the assay is -4% per 10-fold dilution. The total precision of the assay was 16.6% relative standard deviation across three different IsdB antigen lots, three different microsphere lots, two secondary antibody lots, and three different operators. The assay has proven useful for evaluating the immune response following the administration of different dosages and formulations of investigational IsdB-based vaccines.

Serum bactericidal activity against Helicobacter pylori in patients with hypogammaglobulinaemia

The two major primary antibody deficiency disorders are X-linked hypogammaglobulinaemia (XLA) and common variable immunodeficiency (CVID). CVID patients have an elevated risk for gastric cancer and extra-nodal marginal zone lymphoma. Both diseases are associated with Helicobacter pylori infection. We investigated whether antibody deficiency leads to defective serum bactericidal activity against H. pylori. We also investigated the correlation with immunoglobulin (Ig)M levels and observed the terminal complement complex (TCC) activity. Sera of 13 CVID patients (four H. pylori positive), one patient with hyper-IgM syndrome, one patient with Good syndrome (both H. pylori positive), five XLA patients, four H. pylori seropositive controls, four H. pylori seronegative controls and a sample of pooled human serum (PHS) were incubated in vitro with bacterial suspensions of H. pylori for 30 min. After 72 h of culture, colony-forming units were counted. TCC formation was measured by enzyme-linked immunosorbent assay. We found that normal human serum is bactericidal for H. pylori, whereas heat-inactivated serum shows hardly any killing of H. pylori. Serum (1%) of hypogammaglobulinaemia patients has a decreased bactericidal activity against H. pylori. Helicobacter pylori-positive (HP(+)) normal individuals show more than 90% killing of H. pylori, whereas CVID patients show 35% killing (P = 0.007) and XLA patients only 19% (P = 0.003). Serum (1%) of HP(+) volunteers showed significantly better killing compared with serum of H. pylori-negative (HP(-)) volunteers (P = 0.034). No correlation between (substituted) IgG levels and serum bactericidal activity was found, but a weak correlation between total serum IgM and serum bactericidal activity was found. In conclusion, serum bactericidal activity against H. pylori is decreased in patients with hypogammaglobulinaemia. Heat treatment of the serum abolished the bactericidal capacity, indicating that complement activity is essential for the bactericidal effect.

Evaluation of acetaminophen recovery in human serum

Background Acetaminophen toxicity can lead to hepatic and renal damage. Early and accurate measurement of acetaminophen is critical for the management of patients. The Royal College of Pathologists Quality Assurance Program (RCPA-QAP) end of cycle report for acetaminophen shows a significant negative bias (18%) when compared to the median of all values and methods. This may have serious implications for patient samples if the true values are underestimated by the same extent. The aim of this study is to evaluate whether this bias is real. Methods A series of samples was prepared using an acetaminophen spiked human serum pool diluted with non-spiked matching serum across the stated measurement range and measured on the Roche Modular PPE by enzymatic assay. Recoveries were calculated and the values at the decision points for hepatic and renal toxicity were evaluated (200–1300 µmol/L). Results Ten levels of acetaminophen were examined. The bias across the concentration range was Conclusion The negative bias evident from EQAP measurements is absent when tested in human serum pools. This indicates that acetaminophen is accurately measured in the Modular PPE and is fit for purpose for monitoring of acetaminophen toxicity.

Selection of CD271+ cells and human AB serum allows a Large expansion of mesenchymal stromal cells from human bone marrow

Mesenchymal stromal cells (MSC) are promising candidates for cell therapy and tissue engineering and may be used to treat acute graft-versus-host disease (GvHD). However, major obstacles for their clinical use are the required cell dose and the biosafety and potential immunogenicity of fetal bovine serum (FBS), which is a crucial supplement of all media currently used for the culture of MSC. In this study MSC were successfully expanded after selection of CD271 cells from human bone marrow (BM) mononuclear cells in medium supplemented with 10% pooled allogeneic human serum. We isolated MSC from 10 healthy donor BM by plastic adherence and immunomagnetic selection of the CD271(+) fraction and expanded MSC in medium supplemented with pooled human allogeneic serum and animal serum. We isolated a homogeneous multipotent population by CD271(+) selection with a proliferation rate that was higher than MSC isolated by plastic adherence, 6.8+/-1.57 compared with 2.07+/-1.40 logs. Similar to cells generated in animal serum medium, MSC from allogeneic human serum were positive for mesenchymal markers and negative for hematopoietic markers; moreover they expressed embryonic stem cell genes. A normal karyotype and differentiation capacity into adipogenic, osteogenic and chondrogenic lineages and neurosphere-like structures were preserved throughout long-term culture. Expansion of MSC is both feasible and large with a CD271-selected population in medium supplemented with 10% pooled allogeneic human serum, without loss of multipotent differentiation capacity or karyotype alterations.

Human Mesenchymal Stem Cells Exert Immunosuppressive Capacities by Induction of Regulatory T Cells

It is widely accepted that Mesenchymal Stem Cells (MSCs) exhibit immunosuppressive capacities in vitro and in vivo. In an effort to understand the mechanism of suppression, co-cultures of MSCs with several types of immune cells have been studied. MSCs inhibit B- and T cell proliferation and inhibit the generation, maturation and function of dendritic cells. In the present study, we evaluated the effect of MSCs on the expansion and function of regulatory T cells (T regs). Human bone-marrow derived MSCs were cultured and expanded in low-glucose DMEM containing 10% FCS and frozen until used in experiments. Freshly isolated, MACS selected human CD4+CD25+ T regs were cultured in IMDM, supplemented with 10% pooled human serum and 300 units/ml IL-2, in the presence or absence of irradiated MSCs (60 Gy) (ratio MSCs: T regs = 1:5). Results are derived from 3 to 15 independent experiments. In the presence of IL-2 and MSCs, the percentage of FOXP3+ CD4+CD25+ T cells increased from 26.8% ±2.2 (no MSCs) to 42.3% ±2.4 (with MSCs) over a period of 5 days, representing a 1.6 (±0.1)-fold induction. Moreover, a distinct CD4+CD25+ population with high FOXP3 expression appeared after 5 days of culture in the presence of MSCs (23.3% ±2.5 in the presence of MSCs versus 7.2% ±1.0 in the absence of MSCs). This CD4+CD25+FOXP3hi population was not observed after co-culturing MSCs and CD4+CD25− T cells (1.7% ±0.6). To show that the MSC-induced T regs were functionally suppressive, freshly isolated CFSE-labeled CD4+ T cells were stimulated with PHA (0.8 μg/ml). After 3 days, about 40% of the T cells had undergone one or more cell divisions as measured by CFSE dilution. Addition of MSC-induced T regs (in a 1:1 ratio) resulted in a 50% reduction of the proliferation of CFSE-labeled T cells (down to 15–20% of cells undergoing one or more cell divisions). Control CD4+CD25+ T cells cultured in the absence of MSCs did not suppress T cell proliferation. These results indicate that MSC-induced CD4+CD25+ FOXP3hi cells exert regulatory function. To study whether the induction of T regs by MSCs was dependent on cell-cell contact, co-culture experiments were performed in transwells where MSCs were physically separated from T cells. CD4+CD25+ T cells co-cultured with MSCs in transwells showed a 1.4 (±0.1)-fold increase in the percentage of FOXP3hi cells, in comparison with T cells cultured in the absence of MSCs. Similarly, direct co-cultures of MSCs and T cells resulted in a 1.8 (±0.2)-fold increase in the percentage of FOXP3hi T regs. In addition, medium derived from co-cultures of MSCs and CD4+CD25+ T cells and added to freshly isolated CD4+CD25+ T cells resulted in a 2.6 (±0.6) fold increase in the percentage of CD4+CD25+FOXP3hi T regs, implicating that FOXP3hi induction by MSCs was mediated by soluble factors. Since T Cell Receptor-stimulated naïve T cells (as well as natural T regs, generated in the thymus) require both IL-2 and TGF-β to become induced T regs, we analyzed the involvement of TGF-β. Addition of the pharmacological inhibitor of the TGF-β receptor (SB431542) only marginally reduced FOXP3 induction in the presence of MSCs, suggesting that MSC-mediated expression of high FOXP3 levels requires alternate or additional cytokines. In conclusion, we show thatMSCs promote the induction of CD4+CD25+ T cells that express high levels of FOXP3 andthese MSC-induced T regs suppress proliferation of PHA-stimulated CD4+ T cells.These effects are mediated by soluble factors produced during the co-culture of MSCs and T cells. The cytokines involved are presently unknown, but likely do not involve TGF-β.

Exogenous Immunoglobulin Downregulates T Cell Receptor Signaling and Cytokine Production

BACKGROUND: Immune globulin, a polyvalent solution of pooled human serum immunoglobulin, when administered intravenously (IGIV) is used as replacement therapy in patients with hypogammaglobulinemia. In addition, IGIV has immunosuppressive properties and is FDA-approved to treat idiopathic thrombocytopenic purpura; it has also been used to treat several other autoimmune diseases including autoimmune hemolytic anemia and Kawasaki Disease. The mechanisms of action underlying IGIVs immunomodulatory effects are poorly understood, although a few reports have shown IGIV can inhibit T cell activation-induced upregulation of CD38 and CD69 in vitro and variably affect cytokine secretion by Th1 and Th2 cells. We hypothesized that the immunosuppressive effects of IGIV might be lymphocyte subset specific and mediated through T cell antigen receptor signaling interference, thus modulating Th1/Th2 cytokine production.METHODS: Mature (CD45RO) and naïve (CD45RA) lymphocytes from adult and umbilical cord blood were cultured for 8 days in the presence of IGIV (Gamunex™, 0–2.0 mg/mL). T, B, and natural killer (NK) lymphocyte subsets were then quantified by flow cytometry (FACS) over a time course. Additionally, the integrity of the T cell antigen receptor signaling pathway was assessed by FACS, quantifying expression of CD25 and CD69 following anti-CD3 cross-linking. Finally, multiplexed microsphere-based immunoassays were used to quantify cytokine secretion from IGIV-treated cells.RESULTS: IGIV, in a dose-dependent manner, reduced lymphocyte proliferation up to 3-fold after 6 days in culture in both cord and adult blood. IGIV also decreased activation-induced upregulation of CD25 and CD69, with the highest concentrations of IGIV leading to a 70% reduction in cells with an activated phenotype. This inhibitory effect was sustained over a 7-day period in both CD4 and CD8 T cell subsets and was independent of maturation markers (CD45RA/CD45RO). The percentage and absolute number of NK cells varied with IGIV treatment, but a marked increase was observed by day 6. IGIV also selectively reduced the absolute number and percentage of CD45RO+ T cells in cord blood over a time course. Direct cell surface Ig binding was not detected on T cell subsets. IGIV, in a dose-dependent manner, significantly decreased Th2 cytokine secretion, specifically IL-5, IL-10, and IL-13. This effect was more pronounced in cord blood lymphocytes but also was observed in adult blood lymphocytes. IGIV treatment also decreased Th1 cytokine secretion, particularly IL-2 and IFN-γ.CONCLUSIONS: IGIV inhibits T cell proliferation and activation as well as Th1 and Th2 cytokine secretion in a dose- and time-dependent manner. These effects may be responsible for the immunophenotypic changes observed—specifically a reduction in mature T cells and expansion of NK cells. While it is not entirely clear how IGIV is mediating its effects, the inhibition of Th1 and Th2 cytokine production and reduction of T cell activation signals in vitro may explain its role in blocking autoimmune responses in the clinical setting. Further studies are required to identify more precisely the molecular target and mechanism of action of IGIV in vivo.