Maternal allergy influences the proliferation of neonatal T cells expressing CCR4, CXCR5 or CD103.

BACKGROUND: Elevated proliferative response to allergen in cord blood mononuclear cells (CBMCs) is related to subsequent allergy development of the neonate and has been suggested as a screening marker for high allergy risk. OBJECTIVE: To characterize the proliferating cells in CBMCs from a neonatal group influenced by maternal allergy compared with a control group without known allergic heredity. METHODS: CBMCs were stimulated with bovine beta-lactoglobulin (beta-LG) and proliferation was analysed by radioactive thymidine incorporation and expressed both as the traditional stimulation index (SI) and SI corrected by eliminating non-specific proliferation. After beta-LG combined with endotoxin stimulation, cellular expression of IL-4 and IFN-gamma mRNA was determined by quantitative RT-PCR and adhesion as well as chemokine receptors were analysed by three-colour flow cytometry in proliferating T cells (CD3+ Ki-67+). RESULTS: The percentage of CCR4+ cells correlated weakly with concurrent IL-4 expression (r(S)=0.5, P<0.05), while CXCR3 correlated strongly with IFN-gamma expression (r(S)=0.83, P<0.001). In the allergy risk group, the percentage of proliferating T cells expressing CCR4 or integrin alphaE (CD103) was significantly reduced compared with the control group, while CXCR5 and the corrected SI were relatively increased (CCR4: P=0.01; integrin alphaE: P=0.03; CXCR5: P=0.04; SI: P=0.04). CONCLUSION: Our results implied delayed maturation of immune functions involved in cellular migration, cell-cell interaction and immunoregulatory functions in neonates with hereditary allergy risk. The alterations observed in this subject group suggested that the corrected SI as well as proliferation of CCR4+, CXCR5+ or CD103+ T cells in allergen-stimulated CBMCs might serve as early screening markers for allergy risk.

Impaired interleukin (IL)-4-associated generation of CCR4-expressing T cells in neonates with hereditary allergy risk.

Reduced microbial exposure in early life may contribute to the increase of atopic diseases in 'westernized' societies but the underlying mechanisms remain elusive. The objective of this study was to examine how exposure to bacterial lipopolysaccharide (LPS) during early antigen encounter might influence the maturation of neonatal lymphoid cells, and to define possible differences in this respect between neonates with high risk of allergy due to a family history (FH(+)) and controls with no apparent hereditary risk (FH(-)). Cord blood mononuclear cells from the FH(+) or FH(-) group were stimulated with pure LPS or beta-lactoglobulin (beta-LG) in the presence of LPS. T cell expression of chemokine receptors CCR4 and CXCR3 was determined by flow cytometry and reverse transcription-polymerase chain reaction (RT-PCR). Cellular expression of interleukin (IL)-4 was analysed by quantitative RT-PCR, whereas interferon (IFN)-gamma was analysed by both quantitative RT-PCR and immunoassay. Stimulation with LPS, or beta-LG together with LPS, induced up-regulation of CCR4 (P < 0.05) and CXCR3 (P < 0.05). For CCR4, such up-regulation was related to the level of IL-4 produced by the same T cells (r(S) = 0.49, P = 0.03), while CXCR3 expression was negatively correlated with the IL-4 levels (r(S) = -0.56, P = 0.02). Compared with the FH(-) group, the FH(+) group showed a significantly lower capacity for generation of CCR4(+) T cells (mean percentage of total T cells: FH(+), 2.42%versus FH(-), 5.74%; P < 0.01), whereas induction of CXCR3 and IFN-gamma did not differ significantly between the two groups. When the immune system in early life encounters antigen together with LPS, the T cell potential for compartmentalized interaction with other immune cells might be increased by elevated CCR4- and CXCR3-expression levels. In neonates at hereditary allergy risk, this putative homeostatic mechanism could theoretically be jeopardized due to decreased up-regulation of CCR4. Conversely, Th1 responses to antigen in the presence of LPS did not appear to be reduced compared with controls.

DNA damage induced by the drinking water mutagen 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX) in mammalian cells in vitro and in mice.

3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX) formed during chlorination of water containing natural organic substances, is a very potent bacterial mutagen. Recently, tumours at multiple sites were reported in rats given MX-containing drinking water. We have investigated the genotoxicity of MX in mammalian cells exposed in vitro and in vivo using alkaline filter elution to detect DNA single-strand breaks and/or alkali-labile sites (SSBs). Concentrations as high as 100 and 300 microM MX were required to induce detectable levels of SSBs in the HL-60 cells. If MX treatment was carried out in the presence of DNA repair inhibitors (AraC plus hydroxyurea), the sensitivity of the assay to detect MX-induced SSBs was increased by a factor of 100. The presence of serum proteins during exposure resulted in a minor reduction of the MX-induced DNA damage in HL-60 cells at the lowest MX concentrations. In primary cultures of testicular cells as well as in resting human peripheral blood mononuclear cells (PBMC), a slightly increased level of SSBs was observed at MX-concentrations above 30 microM, this effect was not further increased by repair inhibitors. In LLC-PK1 renal proximal tubular epithelial cells and in growth stimulated human peripheral PBMC, increased SSBs were detected at MX concentrations as low as low as 3-10 microM and higher using repair inhibitors, and at 10 times higher concentrations without repair inhibitors. No dose dependent DNA damage was detected in the liver, kidney, spleen and colon of male B6C3F1 mice administrated high doses of MX (40 and 80 mg kg-1). Moderately increased and dose dependent SSBs were detected in the liver and kidney in the presence of DNA repair inhibitors during MX treatment, but no such increase was observed in the spleen and colon.

D-dimer specific monoclonal antibodies react with fibrinogen aggregates.

Human fibrinogen exposed to 46.5 degrees C was subjected to gel permeation chromatography. The protein eluted in two distinct peaks. The first peak appeared in the void volume containing soluble fibrinogen aggregates, while the other peak represented monomeric fibrinogen. In contrast to the monomeric peak material, the aggregate fraction reacted with a panel of monoclonal antibodies specific for fragment D-dimer using an ELISA system. Edman degradation showed that both the aggregate and the monomeric fractions were devoid of soluble fibrin, and immunoblots of SDS-PAG electrophoretic profiles disclosed no sign of stabilized high molecular weight derivatives. We have previously shown that the aggregate fraction of similarly treated fibrinogen, in contrast to the monomeric fraction, stimulates the t-PA catalyzed conversion of plasminogen to plasmin and concomitantly exposes the sequences Aalpha-(148-160) and gamma-(312-324) involved in t-PA stimulation. Our present and previous findings suggest that soluble fibrinogen aggregates possess a fibrin-like structure, and that fibrin or fibrinogen polymer formation is a prerequisite for the enhancing effect on t-PA-mediated plasminogen to plasmin conversion which is seen even with the polymers in the soluble state.

Aggregated, conformationally changed fibrinogen exposes the stimulating sites for t-PA-catalysed plasminogen activation.

The present paper shows that conformationally changed fibrinogen can expose the sites A alpha-(148-160) and gamma-(312-324) involved in stimulation of the tissue-type plasminogen activator (t-PA)-catalysed plasminogen activation. The exposure of the stimulating sites was determined by ELISA using mABs directed to these sites, and was shown to coincide with stimulation of t-PA-catalysed plasminogen activation as assessed in an assay using a chromogenic substrate for plasmin. Gel permeation chromatography of fibrinogen conformationally changed by heat (46.5 degrees C for 25 min) demonstrated the presence of both aggregated and monomeric fibrinogen. The aggregated fibrinogen, but not the monomeric fibrinogen, has exposed the epitopes A alpha-(148-160) and gamma-(312-324) involved in t-PA-stimulation. Fibrinogen subjected to heat in the presence of 3 mM of the tetrapeptide GPRP neither aggregates nor exposes the rate-enhancing sites. Thus, aggregation and exposure of t-PA-stimulating sites in fibrinogen seem to be related phenomena, and it is tempting to believe that the exposure of stimulating sites is a consequence of the conformational changes that occur during aggregation, or self-association. Fibrin monomers kept in a monomeric state by a final GPRP concentration of 3 mM do not expose the epitopes A alpha-(148-160) and gamma-(312-324) involved in t-PA-stimulation, whereas dilution of GPRP to a concentration that is not longer anti-polymerizing, results in exposure of these sites. Consequently, the exposure of t-PA-stimulating sites in fibrin as well is due to the conformational changes that occur during self-association.

Role of serum in the morphological transformation of Syrian hamster embryo cells: Characterization and partial purification of protein factors in foetal bovine serum.

The expression of transformed colony morphology in Syrian hamster embryo (SHE) cells, and thus the results obtained in the SHE cell transformation assay, is dependent on the source of the foetal bovine serum (FBS) used. The purpose of this study was to characterize the factors in FBS that are necessary for the expression of transformed morphology. The factors were of protein nature (precipitated by ammonium sulfate and non-dialysable), sensitive to heating and thiol reagents, but resistant to acid and solvent treatment. The active factor(s) were found to bind to a number of protein purification media for ion exchange or affinity purification, and it was initially difficult to reconstitute the biological activity from eluted fractions. This loss of activity was not caused by the separation of more than one necessary factor, but by the factors being highly hydrophobic and negatively charged, and therefore strongly bound to the column material. The active factors could be eluted from Affigel Blue in 50% ethylene glycol, but not in 4m NaCl. The bioactive protein fraction could be further fractionated by gel permeation chromatography on Biogel P-60 in 1 m acetic acid, and cation exchange chromatography on MonoS with 20% acetonitrile added to the buffers. Isoelectric focusing on a Rotofor cell indicated two peaks of transforming activity, one with isoelectric point at about pH 8.5, and one at pH 9.5. The finding of two peaks of biological activity is supported by reversed phase chromatography studies. Bioactivity of two fractions from isoelectric focusing with pI around 8.5 and 9.5 were eluted at propanol concentrations of 20 and 27%, respectively. In the present studies, we were unable to identify the factors with transformation supporting activity, probably because of the high content of protein/peptides with similar biochemical properties in FBS. In further studies we will seek to demonstrate whether previously isolated growth factors, or signalling substances, with similar biochemical properties support the expression of the morphologically transformed phenotype in SHE cells.

Stimulating effect on tissue-type plasminogen activator--a new and sensitive indicator of denatured fibrinogen.

A high clottability and a short thrombin clotting time have routinely been considered as evidence of genuineness of the fibrinogen molecule. Since denatured fibrinogen stimulates the t-PA-catalysed conversion of plasminogen to plasmin, it was of interest to study the sensitivity of t-PA-stimulation as evidence of fibrinogen denaturation. Therefore, fibrinogen was intentionally exposed to various denaturating conditions (freeze-drying, heating, EDTA, alkali), and the clottability, the thrombin clotting time and the t-PA-stimulating effect were recorded. We found that the clottability was a poor indicator of fibrinogen denaturation, whereas the t-PA-stimulating effect could detect even mild fibrinogen denaturation. The thrombin clotting time was shortened after freeze-drying or heating at 47 degrees C, in spite of what might have been expected. Thus, denaturation is not necessarily accompanied by a prolonged clotting time. In some instances therefore, the t-PA-stimulation is an even more sensitive and reliable indicator of fibrinogen denaturation than is the thrombin clotting time. Consequently, this parameter should be combined with the thrombin clotting time to characterise preparations of fibrinogen.

Fibrinogen present in EDTA--anticoagulated plasma stimulates the tissue-type plasminogen activator-catalysed conversion of plasminogen to plasmin.

The presence of soluble fibrin in plasma is an early and sensitive indicator of activation of the coagulation system. Quantitative spectrophotometric assays for soluble fibrin can be based on the principle that soluble fibrin stimulates the tissue-type plasminogen activator-catalysed conversion of plasminogen to plasmin. It was previously shown that treatment of purified fibrinogen by EDTA, which removes the three tightly bound Ca2+ ions, results in exposure of tissue-type plasminogen activator-catalytic sites similar to those unveiled by thrombin. Since EDTA is a common anticoagulant, it was of interest to study the effect of EDTA on a test based on plasminogen activation. It is concluded that the determination of soluble fibrin in EDTA-anticoagulated plasma from healthy individuals gives a false positive indication of the presence of soluble fibrin. This was true irrespective of whether the test was performed at pH 7.4, 7.8 or 8.5. The most probable explanation is that tissue-type plasminogen activator-stimulating sites are exposed in fibrinogen by EDTA. Therefore, EDTA-plasma is unsuitable for assaying soluble fibrin with tests based on the tissue-type plasminogen activator-mediated conversion of plasminogen to plasmin.

Freeze-dried fibrinogen or fibrinogen in EDTA stimulate the tissue-type plasminogen activator-catalysed conversion of plasminogen to plasmin.

Both soluble and insoluble fibrin stimulate the tissue-type plasminogen activator-catalysed conversion of plasminogen to plasmin. Whether fibrinogen can exert a similar effect has been a controversial issue. The present investigation shows that while fibrinogen purified by beta-alanine precipitation does not stimulate the tissue-type plasminogen activator-catalysed plasminogen activation, fibrinogen which has been either lyophilized or stripped of bound Ca2+ ions by EDTA chelation, stimulates this reaction. The data indicate that such procedures alter the molecular conformation of fibrinogen, and expose stimulatory sites which are hidden in the native fibrinogen molecule. These results may explain previous findings concerning the capacity of fibrinogen as a stimulator of the tissue-type plasminogen activator-catalysed plasminogen activation. Since even slight alteration of the molecular structure of fibrinogen leads to an increase in the tissue-type plasminogen activator stimulation, the authors suggest that this can be used to test if the fibrinogen is in a native state.

The ubiquity of the highly phosphorylated nuclear protein P1.

The present work shows that antibodies raised in rabbits against rat liver P1 confirmed the presence of P1 in lung, kidney, brain heart, muscle, intestine and thymus in rats. The antiserum reacted with P1 from human and monkey but not from bovine, pig and mouse P1 in spite of there being a close relationship in amino acid composition, electrophoretic properties and peptide mapping. Proteolytic digestion of rat P1 showed that only some of the peptides produced reacted with the antiserum, suggesting that conformational determinants may be dominating compared to sequential determinants in P1, or that only minor parts of P1 which exhibit sequential variation between species are immunoreactive.