Surface plasmon resonance-based immunoassay for human C-reactive protein.

A rapid and highly-sensitive surface plasmon resonance (SPR)-based immunoassay (IA) has been developed and validated for detecting human C-reactive protein (CRP), a specific biomarker for inflammatory and metabolic disorders, and infections. The 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC)-activated protein A/G (Pr A/G) was diluted in 1% (v/v) 3-aminopropyltriethoxysilane (APTES), dispensed on a KOH-treated gold (Au)-coated SPR chip, and incubated for 30 min. The Pr A/G functionalized Au SPR chip was then bound to anti-human CRP capture antibody (Ab), blocked with bovine serum albumin, and subsequently used for the detection of CRP. The highly-simplified oriented Ab immobilization strategy enabled the leach-proof binding of capture Ab in 5-fold shorter time than conventional procedures. The developed IA detected 1.2-80 ng mL(-1) of CRP with a limit of detection (LOD) and a limit of quantification (LOQ) of 1.2 ng mL(-1) and 4.6 ng mL(-1), respectively. It detected CRP spiked in diluted human whole blood, serum and plasma as well as the CRP levels in the ethylenediaminetetraacetic acid (EDTA) plasma samples of patients with the same precision as the clinically-accredited analyzer-based IA and conventional CRP sandwich ELISA. The Ab-bound SPR chips stored at 4 °C retained their functional activity for 10 weeks, resulting in significant reduction in the overall analysis time.

Surface plasmon resonance-based immunoassay for human fetuin A.

This article describes a highly-sensitive surface plasmon resonance (SPR)-based immunoassay (IA) for human fetuin A (HFA), a specific biomarker for atherosclerosis and hepatocellular carcinoma. The assay is based on a novel immobilization procedure that simply involves the dilution of an anti-HFA capture antibody (Ab) in 1% (v/v) 3-aminopropyltriethoxysilane (APTES), followed by its dispensing on a KOH-treated gold (Au)-coated SPR chip and incubation for 30 min. The developed SPR IA detected 0.3-20 ng mL(-1) of HFA with a limit of detection and sensitivity of 0.7 ng mL(-1) and 1 ng mL(-1), respectively. The highly-simplified Ab immobilization procedure is also 5-fold more rapid than conventional procedures. It leads to the leach-proof binding of the capture Ab, which means that the developed SPR IA is highly cost-effective, as the Ab-bound SPR chip could be reused for many repeated HFA IAs after regeneration with 10 mM glycine-HCl, pH 2.0. The Ab-bound SPR chip, stored at 4 °C, lost only 18% of its original activity after 4 months. For the detection of HFA spiked in diluted human whole blood and plasma, the results obtained by the developed SPR IA agreed well with the commercial HFA sandwich ELISA.

The (-765 G→C) promoter variant of the COX-2/PTGS2 gene is associated with a lower risk for end-stage hip and knee osteoarthritis.

BACKGROUND: Functional polymorphisms in genes of proinflammatory signalling cascades may contribute to the genetic risk of osteoarthritis (OA). OBJECTIVE: To examine a possible association between end-stage OA of the hip and knee joint and a known single nucleotide polymorphism (SNP) of the COX-2 gene promoter. METHODS: The SNP -765 G→C (rs20417) of the COX-2 gene promoter was genotyped by pyrosequencing in 531 (320 women/211 men) patients with OA from the Ulm Osteoarthritis Study and 400 (200 women/200 men) regional controls from the south-west of Germany. RESULTS: In the whole study population the C allele was associated with a lower risk (per allele OR 0.57; 95% CI 0.43 to 0.75, p<0.0001) and the G allele with a higher risk for end-stage OA. Analysis of subgroups confirmed this result for primary, bilateral, hip and knee OA. CONCLUSION: The promoter polymorphism rs20417 of the COX-2 gene contributes to the genetic risk for end-stage hip and knee OA.

DY determinants, possibly associated with novel class II molecules, stimulate autoreactive CD4+ T cells with suppressive activity.

A set of T cell clones (TCC) isolated from HLA-DR-, Dw-, DQ-matched allogeneic MLCs was found to proliferate autonomously when stimulated with cells carrying a wide range of class I or II specificities. This apparently unrestricted proliferation was relatively weak, and only low levels of IL-2 were present in the supernatants of stimulated cells. Autologous as well as allogeneic PBMC and B lymphoblastoid cell lines (B-LCL) were capable of stimulating such clones, which were also restimulated by suppressive, but not by helper, TCC. Moreover, such clones displayed the unusual property of autostimulation. mAb inhibition experiments suggested that class II- or class II-restricted antigens were involved in stimulation. Thus, certain "broad" mAbs (TU39, SG520) reacting with multiple locus products inhibited activation of these reagents, but none of those reacting more specifically with DR (TU34, TU37, L243, Q2/70, SG157), DQ (TU22, SPV-L3, Leu 10), or DP (B7/21), or mixtures of these mAbs, were able to do so. Evidence from sequential immunoprecipitation experiments suggested that mAb TU39 bound class II-like molecules other than DR, DQ, and DP on TCC and B-LCL, and it is therefore proposed that such putative novel class II-like molecules may carry the stimulating determinants for these autoreactive clones. DY-reactive clones lacked helper activity for B cells but mediated potent suppressive activity on T cell proliferative responses that was not restricted by the HLA type of the responding cells. Suppressive activity was induced in normal PBMC by such clones, as well as by independent suppressive clones, which was also inhibited only by mAb TU39. These findings lead to the proposal that DY-reactive autostimulatory cells may constitute a self-maintaining suppressive circuit, the level of activity of which would be regulated primarily by the availability of IL-2 in the microenvironment.

Generation of mature CD3+ and T cell receptor (TCR) + T cells from a leukemic analogue of the putative human stem cell by T cell conditioned medium containing IL-3, IL-2, and GM-CSF.

Malignant cells of a patient with acute leukemia expressed hematopoietic stem cell antigens such as CD34 and HLA-class II but lacked lineage specific differentiation markers. The leukemic blasts differentiated into mature T cells within 14 days in the presence of a T cell conditioned medium or with a mixture of highly purified interleukin-2 (IL-2) plus recombinant interleukin-3 (IL-3) and recombinant granulocyte/macrophage colony-stimulating factor (GM-CSF). Phenotypically, the maturing cells acquired the T cell-specific differentiation antigens CD2, CD3, and CD8, whereas immature differentiation antigens such as CD34 and Leu19 as well as HLA-class II and the IL-2 receptor CD25 were concomitantly down-regulated within 14 days of in vitro culture. This in vitro maturation involved two to three synchronized cell divisions. Beyond 10 days of culture the leukemic cells produced mRNA specific for the T cell receptor beta and alpha chain, but at no time transcription of T cell receptor gamma chain-specific message was detectable. To our knowledge, these data represent the first in vitro model demonstrating the differentiation of phenotypically mature T cells from immature leukemic cells induced by the combined activities of IL-2 plus IL-3 and GM-CSF.

Acute leukemia coexpressing myeloid, B- and T-lineage associated markers: multiparameter analysis of criteria defining lineage commitment and maturational stage in a case of undifferentiated leukemia.

Coexpression of myeloid, B-, and T-lineage associated markers was found in a patient with morphologically and cytochemically undifferentiated acute leukemia. Surface marker analysis using two-color immunofluorescence staining characterized blast cells to express CD34, CD38, CD117, and class II antigens, coexpressing TdT, CD4, CD7, CD13, CD19, and CD33. Cytoplasmic expression of myeloperoxidase, CD3, and CD22 could not be demonstrated. Monosomy for chromosome 7 was found by cytogenetic analysis. The absence of clonal rearrangements of immunoglobulin or T-cell receptor genes was shown by Southern blot analysis. Using a 3H-thymidine incorporation assay, DNA synthesis of leukemic blasts could be stimulated by IL-3, IL-6 and G-CSF in vitro. The present case did not offer specific criteria of lineage commitment. Corresponding to an equivalent counterpart in normal hematopoiesis, the involved cell population may reflect an early, most immature developmental stage within a multipotent progenitor cell compartment.

Rapid and sensitive mRNA phenotyping for interleukins (IL-1 to IL-6) and colony-stimulating factors (G-CSF, M-CSF, and GM-CSF) by reverse transcription and subsequent polymerase chain reaction.

Oligonucleotide primer pairs specific for interleukins (IL)-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-5, and IL-6, as well as for granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNA/cDNA were synthesized in order to detect cytokine transcripts by reverse transcription and subsequent polymerase chain reaction (RT/PCR). Analysis of RNA preparations of the human bladder carcinoma cell line 5637 by this methodology reveals expression of mRNAs for IL-1 alpha, IL-1 beta, IL-6, G-CSF, M-CSF, and for GM-CSF, whereas mRNAs for IL-2, IL-3, IL-4, and IL-5 are not detectable. These results are in agreement with data obtained by classical methods. Thus, for the cytokines IL-2, IL-3, IL-4, and IL-5, it was not possible to detect a phenomenon described as 'illegitimate transcription,' defined as the low level transcription of any gene in any cell type. This finding is of importance for the applicability of mRNA phenotyping employing RT/PCR for the determination of mRNA expression patterns. For M-CSF mRNA detection, two oligonucleotide primer pairs had to be used to distinguish between the alpha-(pcCSF17) and beta-splicing forms and to overcome the problem of non-amplification of a larger fragment in the presence of a competing smaller one, defined here as 'incomplete positivity.' For G-CSF, IL-4, IL-2, and IL-5, RT/PCR reveals two fragments. Restriction enzyme analysis of the additional fragments suggests that they may arise from alternative splicing events. For G-CSF and IL-4, exons 3 and 2 seem to be spliced out, respectively. The additional fragments for IL-2 and IL-5 RT/PCR have not yet been further characterized, but the size of the fragments makes it seem probable that exons 2 and 3 are spliced out for IL-2 and IL-5, respectively. The biological role of these alternative mRNAs has yet to be determined.

Identification of an alternatively spliced transcript of human interleukin-4 lacking the sequence encoded by exon 2.

The expression of interleukin-4 (IL-4) mRNA of human peripheral blood mononuclear cells stimulated with phytohemagglutinin (PHA) for 15 hours was analyzed by reverse transcription and subsequent polymerase chain reaction (RT/PCR). These analyses revealed an additional smaller fragment that hybridizes with an IL-4 cDNA probe and an oligonucleotide that is specific for a fragment lacking the sequence encoded by exon 2. Sequencing of this fragment demonstrates that it is generated from an alternatively spliced transcript of the IL-4 gene with the sequence encoded by exon 2 being skipped. Skipping of exon 2 does not result in a frame shift but would delete part of the mature protein (48 bp coding for amino acid residues 22 to 37), including Cys24 but not a region directly involved in receptor binding. Differential splicing of other exons or exon combinations has not been observed. The data suggest that the alternatively spliced transcript is not generated by a splicing or PCR error and is not detectable solely because of the high sensitivity of RT/PCR, but in contrast, argue for a physiological role of the transcript and its potentially encoded protein.

Base-free Knoevenagel condensation catalyzed by copper metal surfaces.

For the first time Knoevenagel condensation has been catalyzed by elemental copper with unexpected activity and excellent isolated yields. Inexpensive, widely available copper powder was used to catalyze the condensation of cyanoacetate and benzaldehyde under mild conditions. To ensure general applicability, a wide variety of different substrates was successfully reacted.

Stable dispersions of azide functionalized ferromagnetic metal nanoparticles.

Ferromagnetic nanoparticles are covalently modified in order to enhance the dispersion stability as well as the antifouling properties. Insertion of an azide moiety allows "click"-reaction of a relevant tag molecule. This and the high saturation magnetization of the presented nanocomposite offer a promising platform for magnetic biosensors.

Cloning, sequencing and functional studies of the gene encoding human GTP cyclohydrolase I.

We have identified a genomic clone containing the 5' regulatory region of the gene GTP-CH encoding human GTP cyclohydrolase I. The transcription start point (tsp) was mapped by 5'-rapid amplification of cDNA ends (5'-RACE). The 2.6-kb region upstream from the tsp showed promoter activity when ligated upstream from a reporter gene. The truncation of approximately 2 kb of the promoter did not change expression activity, while a further removal of 243 bp halved the activity. The promoter contains CCAAT and TATA boxes. The GC-rich region close to the tsp, which contains several putative Sp1-responsive elements, is required for maximum promoter activity. Interferon-gamma treatment of B-cells transfected with reporter constructs had no influence on the expression activity.

Semliki Forest virus vectors: efficient vehicles for in vitro and in vivo gene delivery.

Rapidly generated high-titer Semliki Forest virus (SFV) vectors can infect numerous mammalian cell lines and primary cell cultures, and result in high levels of transgene expression. SFV-based expression of transmembrane receptors has been characterized by specific ligand-binding activity and functional responses. Adaptation of the SFV technology for mammalian suspension cultures has allowed the production of hundreds of milligrams of recombinant receptor for purification and structural studies. The same SFV stock solutions used for the infection of mammalian cells in culture have also been successfully applied for efficient transgene expression in organotypic hippocampal slices, as well as in vivo in rodent brain.

Expression of the antioxidant stress protein heme oxygenase-1 (HO-1) in human leukocytes.

Inducible heme oxygenase (HO-1) is an antioxidant stress protein, that is mainly induced by reactive oxygen species (ROS), cytokines and hyperthermia. By using flow cytometry the present investigation demonstrated a rise in the cytoplasmic expression of HO-1 in lympho- (L), mono- (M) and granulocytes (G) of 9 endurance-trained male subjects after a half marathon run. The expression was more pronounced in M (median: 98.3% HO-1 positive cells/4.31 mfc) and G (94.8%/1.93 mfc) than in L (80.1%/1.51 mfc) when measured 3 h post-exercise. Additionally the exercise protocol caused a rise in the plasma levels of myeloperoxidase, TNF alpha and interleukin-8 (IL-8), indicating an inflammatory response. We could detect a correlation between IL-8 and HO-1, directly after exercise, that was apparent in G (r = 0.67, p < .05) and L (r = 0.80, p < .05), but did not reach significance in M (r = 0.65, p = 0.06). An additional detection of HO-1 at rest in 12 untrained subjects showed a higher baseline expression of HO-1 compared to the athletes. The regulatory pathways leading to an increased expression of HO-1 after endurance exercise are not completely clear, but a causal involvement of a cytokine-mediated generation of ROS must be discussed. We supposed that the down-regulation of the baseline expression of HO-1 in athletes reflects an adaptional mechanism to regular exercise training.

Generation of CD4-positive suppressor T cells from mixed lymphocyte cultures in the presence of interleukin 2 receptor antibody TU69. An in vitro model for transplantation tolerance induction.

The specificity of a novel monoclonal antibody (moAB), TU69, directed to the interleukin 2 receptor (IL-2R) was verified by sequential immunoprecipitation with anti-Tac. TU69 cross-competed with anti-Tac in binding analyses. When TU69 was added during the sensitization of normal peripheral blood mononuclear cells (PBMC) to allogeneic HLA-class I or -class II mismatched stimulator PBMC, alloproliferative responses and specific cytotoxicity were no longer detectable and the generation of natural killer (NK)-like effector cells was partially inhibited. Remarkably, however, the generation of CD4+ nonspecific suppressor T cells in such mixed lymphocyte cultures (MLC) was not inhibited--but, in contrast, was strongly enhanced in the presence of TU69. These suppressor cells inhibited unrelated allospecific responses in vitro to background levels even at a ratio of 50:1 responder:irradiated suppressor T cell lines. Such a potent experimental suppressor system suggests a possible application of TU69 for in vivo tolerance induction after transplantation, by down-regulating allospecific effector cells and allowing the generation of tolerance to graft antigens.

High frequency of graft-versus-host-like syndromes following syngeneic bone marrow transplantation.

Occurrence of acute graft-versus-host reactivity-like (GvHR) syndromes has been shown in at least 3 and possibly in 4 further cases of 9 patients with bone marrow transplants from identical twin donors. The diagnosis of GvHR-like syndromes is based on clinical, immunologic, and histologic features indistinguishable from those observed in graft-versus-host disease (GvHD) grades I-III of patients receiving allogeneic major histocompatibility complex (MHC) matched bone marrow transplants. Induction of GvHR-like symptoms appeared to be correlated with reactivated viral infections after bone marrow transplantation (BMT) or, like in animal models, was due to specific conditioning therapy with cyclophosphamide. The high incidence of acute GvHR-like syndromes in the first months after syngeneic BMT suggests inability of the immune system to discriminate appropriately self from nonself antigens during a normal tolerance induction period after grafting.

Dissection of suppressor cell generation in vitro.

Suppressor cells (SC) that nonspecifically inhibited lymphoproliferative (LP) responses were found after culturing peripheral blood mononuclear cells: with suppressor T-cell clones, in mixed lymphocyte cultures (MLC), and with recombinant interleukin 2 (IL-2), but were not found after culture in medium alone. A monoclonal anti-IL-2 receptor (R) antibody (MoAb), TU69, which blocked LP responses of IL 2-dependent T-cell lines, also blocked SC induction by T-cell clones, but completely failed to inhibit SC generation in MLC or with IL-2. This suggests that the IL-2R epitope defined by TU69 was not involved in SC induction in the latter systems. MoAb against HLA-DQ (TU22), -DR (TU34, SG157), -DP (B7/21), or DR and DP (TU43, 58), all of which were able to block stimulation of appropriately specific clones, did not block SC induction in any of the three systems studied. In contrast, the broadly reactive moAb TU39, which binds at least DR and DP but also has additional reactivity for determinants tentatively designated "DY," blocked SC induction by T-cell clones and in MLC. Finally, an anti-HLA class I MoAb, W6/32.HL, greatly decreased SC generation in MLC, but not with rIL-2 or T-cell clones. Thus, the induction of nonspecific SC was dissected into three pathways involving: class I and TU39-defined but not DR, DQ, or DP determinants (in MLC) which was independent of the IL-2R epitope bound by TU69; only TU39-defined determinants (with T-cell clones), which were IL 2R dependent; and, neither class I, class II nor TU39-defined determinants (induction by rIL-2), which was also TU69+ IL-2R independent.

Strong lymphoproliferative suppressive function of PLT clones specific for SB-like antigens.

From a total of 37 different priming combinations between donors matched for HLA-A,B, and/or Dw/DR, but mismatched for SB, antigens, T cell clones strongly restimulated with concordance for SB specificities were isolated from only two. Most of the alloproliferative (PLT) clones obtained were restimulated by determinants not correlated with any currently known HLA product. Nonetheless, their stimulation was inhibited by a monoclonal antibody TU 39, which preferentially blocks stimulation by SB-, rather than by Dw/DR-associated determinants. Despite having an OKT4+, OKT-, Leu8- phenotype, and secreting Interleukin-2 after contact with stimulatory cells, these clones strongly suppressed proliferative responses of cloned PLT reagents as well as unprimed lymphocytes in mixed leukocyte cultures. They may thus represent a novel type of immunoregulatory T cell, stimulated by SB-related antigens, which despite their "helper/inducer" phenotype are able directly to suppress lymphoproliferative responses.

Alloproliferative human T cell clones primed and cultured in vitro lose proliferative and gain suppressive activity with age.

Alloreactive human T lymphocyte clones were found to lose antigen-stimulated proliferative capacity and their ability to secrete interleukin 2 (IL 2) after a critical period in tissue culture. Instead, they gained the previously absent capacity to suppress lymphoproliferative (LP) responses in the presence or absence of exogenous IL 2. Such "ex-PLT" suppressive clones continued to grow perfectly well, retaining IL 2 and filler cell dependency, apparently normal karyotypes, and their OKT4+, OKT8- phenotypes. At least two suppressive mechanisms were demonstrated: (1) the "induction" of suppressive effectors in normal peripheral lymphocytes, and (2) a direct suppressive activity on lymphocyte proliferation shown by their ability to inhibit restimulation of cloned lymphocytes lacking suppressor cell precursors. The consistent "differentiation" from IL 2-secreting "helper" status to nonspecific suppressive status may represent a novel immunoregulatory phase in the long-term differentiation of normal human T cells.

Transforming growth factor beta is a growth-inhibitory cytokine of B cell lymphoma in SIV-infected macaques.

Cytokine dysregulation is accepted as one of the pivotal factors in the pathogenesis of B cell lymphomas in HIV-positive patients. So far no data exist on inhibitory cytokines in the regulatory network of HIV-associated B-NHL. Simian immunodeficiency virus (SIV)-infected macaques are a well-established in vivo model of HIV infection in humans. We used this model for the identification of TGF-beta as a growth-inhibitory cytokine of SIV-associated B cell lymphomas. Fifty-seven rhesus macaques were infected with SIVmac. Nine animals developed B cell lymphomas: eight with high-grade lymphomas of the immunoblastic, centroblastic, and "Burkitt-like" type, and one with the centroblastic/centrocytic type according to the Kiel classification. Six of seven analyzed lymphomas were infected with the macaque EBV, herpes virus macaca mulatta (HVMM). The lymphomas and the SIV-associated B cell lymphoma cell line H50 were positive for transcription of the TGF-beta gene. Protein expression and secretion of the active cytokine were proved by immunohistochemistry and ELISA. H50 transcribed the TGF-beta type I and type II receptor (R I/II), betaglycan, and endoglin. Furthermore, all primary lymphoma samples tested were positive for receptor type I/II transcription and protein expression. TGF-beta induced reduction of cell viability by 67% (range, 50-84% and enhanced apoptosis by 69% (range, 33-111%) compared with the control. TGF-beta activity was blocked by a specific anti-TGF-beta antibody. Thus, TGF-beta fulfilled the criteria of a negative autocrine inhibitor in H50. These data identify TGF-beta as a promising candidate as an inhibitory factor in the regulatory network of HIV-associated lymphomagenesis.

Persistence of decreased T-helper cell function in industrial workers 20 years after exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

In experimentally exposed animals 2,3,7,8-tetrachlorodibenzo-n-dioxin (TCDD) causes severe immunosuppression. However, the overall susceptibility of humans for the different pathological effects of TCDD has remained unclear. We examined the long-term effects of TCDD in 11 industrial workers who were exposed to high doses of TCDD for several years 20 years ago. Current TCDD body burdens were still at least 10 times higher (between 43 and 874 pg/g blood far) in these exposed persons than in the average German population. To evaluate possible TCDD-induced changes in the percentage of different lymphocyte subsets, we determined a large panel of lymphocyte subsets in the blood by flow cytometric analysis. Immunocompetence of T-and B-lymphocytes was tested by nitrogen (phytohemagglutinin, pokeweed mitogen)- induced lymphoproliferation assays and by assays using sensitive mixed-lymphocyte cultures. No significant differences could be detected between the individuals tested and controls for surface marker distribution or mitogen-induced lymphoproliferation TCDD-exposed subjects showed a reduced response to human lymphocyte antigen-allogeneic lymphocytes and interleukin-2-boosted proliferation. Responder cells of the dioxin-exposed persons proliferated less in response to irradiated stimulator cells (p < or = 0.05), and the third-party mixed lymphocyte reaction against unirradiated stimulator cells revealed suppressive activity in the responder cell fraction compared to the controls (p < or = 0.01). Furthermore, the capacity of a pool of T cells isolated from TCDD-exposed subjects to proliferate upon interleukin-2 stimulation was significantly diminished (p < or = 0.05). TCDD has a long-term immunosuppressive-effect on T-helper cell function, which is mediated more likely by a reduced functionality of individual cells rather than by a reduction in absolute cell numbers in the peripheral blood.