Performance and quality analysis of membrane cartridges used in long-term operation.

In this study, operation and maintenance performance in two long-term operating Membrane Bioreactor (MBR) wastewater treatment facilities were investigated. One facility in Japan started its operation in 1999 showed that both effluent BOD and n-hexane extracts were less than 5 mg/L, and a cumulative replacement percentage (CR%) of membrane cartridges was 7.8% as of 2008. Another facility in the United Kingdom (UK) started its operation in 1998 showed that average effluent BOD was less than 5 mg/L and more than 5-log removal of faecal coliforms was maintained. The CR% was 6.4% as of 2008. Amongst 95 facilities in Europe, the CR% was 6.4% after 5-year operation. In order to inspect product quality of membrane cartridges after 10-year operation in the UK facility, clean water flow (CWF) rate and pore size distribution were measured. The CWF rate was approximately 100% of that of new membrane cartridge's, and the pore size distribution was well maintained at less than 0.46 microm. A microscopic observation showed some scratches on the membrane surface. However, they did not lead to deteriorate permeate quality. These data suggested that the membrane cartridges can be used as long as 10 years.

Critical requirement for the membrane-proximal cytosolic tyrosine residue for CD28-mediated costimulation in vivo.

The YMNM motif that exists in the CD28 cytoplasmic domain is known as a binding site for phosphatidylinositol 3-kinase and Grb-2 and is considered to be important for CD28-mediated costimulation. To address the role of the YMNM motif in CD28 cosignaling in primary T cells, we generated transgenic mice on a CD28 null background that express a CD28 mutant lacking binding ability to phosphatidylinositol 3-kinase and Grb-2. After anti-CD3 and anti-CD28 Ab stimulation in vitro, the initial proliferative response and IL-2 secretion in CD28 Y189F transgenic T cells were severely compromised, while later responses were intact. In contrast to anti-CD3 and anti-CD28 Ab stimulation, PMA and anti-CD28 Ab stimulation failed to induce IL-2 production from CD28 Y189F transgenic T cells at any time point. Using the graft-vs-host reaction system, we assessed the role of the YMNM motif for CD28-mediated costimulation in vivo and found that CD28 Y189F transgenic spleen cells failed to engraft and could not induce acute graft-vs-host reaction. Together, these results suggest that the membrane-proximal tyrosine of CD28 is required for costimulation in vivo. Furthermore, these results indicate that the results from in vitro assays of CD28-mediated costimulation may not always correlate with T cell activation in vivo.

Antiadhesive function of 130-kd glycoform of CD43 expressed in CD4 T-lymphocyte clones and transfectant cell lines.

Conflicting findings regarding proadhesion and antiadhesion in cell-to-cell interactions were previously reported for CD43. We examined possible differences in the role of the 130-kd glycoform and the 115-kd glycoform of CD43 in cellular adhesion in vitro. We generated a monoclonal antibody (MFT3) that discriminates between helper and nonhelper murine T-cell clones. Characterization of MFT3 with use of biochemical analysis and complementary DNA (cDNA) transfection experiments showed that it is specific for the 130-kd glycoform of CD43. T-cell clones that expressed the 130-kd CD43 glycoform showed decreased homocytic aggregation and decreased adhesion to spleen cells, B-lymphoma cell lines, and fibroblastic cell lines compared with T-cell clones negative for the 130-kd glycoform. Expression of core 2 beta-1, 6-N-acetylglucosaminyltransferase (C2GnT) cDNA together with CD43 cDNA resulted in expression of both the 130-kd CD43 glycoform and the 115-kd CD43 glycoform in fibroblastic cell lines. Using these cell lines, we showed that the 130-kd glycoform but not the 115-kd glycoform of CD43 has an antiadhesive function in cellular interactions. Our findings suggest that the antiadhesive function of CD43 is primarily carried out by the 130-kd glycoform. (Blood. 2000;96:4267-4275)

Analysis of Vbeta4 T cell receptor CDR3 repertoire in BALB/c and (NZB x NZW)F1 mice.

To examine the unique TCR repertoire in auto-immune-prone (NZB x NZW)F1 (B/WF1) mice, we analysed the Vbeta4 CDR3 region of TCRbeta chain in spleens of young (1 month old) and aged (6 month old) BALB/c and B/WF1 mice. Total RNA from spleens was used for cDNA synthesis and TCRVbeta4 PCR products were cloned and sequenced. Young B/WF1 mice showed high frequency (38.5%) of anionic amino acid residues at position beta100 in TCRVbeta4 chain compared to that (19.0%) in young BALB/c mice. Aged BALB/c mice and B/WF1 mice showed increase of frequency (38.1 and 51.9%, respectively) of anionic residues at beta100. These results indicate that Vbeta4-T cells that have anionic residues at beta100 in CDR3 region of TCRbeta chain increase with age in normal mice. Auto-immune prone mice show high frequency of anionic residues at beta100 in TCRVbeta4 chain even at the age of 1 month. These T cells may interact with cationic self-antigen(s) and might contribute to the onset and/or the progression of systemic autoimmunity in concert with other genetic elements in B/WF1 mice.

Effects of transgenic mixed-haplotype MHC class II molecules A alpha d A beta z on autoimmune disease in New Zealand mice.

The MHC haplotype heterozygosity (H-2d/H-2z) acts as one major predisposing genetic element for autoimmune disease resembling systemic lupus erythematosus (SLE) in the F1 hybrid of NZB (H-2d) and NZW (H-2z) mice. To determine a possible role of mixed-haplotype A molecules, we introduced a transgene A beta z into H-2d/H-2d homozygous (NZB x NZW.H-2d)F1 mice, in which, compared with the original H-2d/H-2z heterozygotes, the incidence and titer of IgG anti-DNA antibodies, serum levels of nephritogenic retroviral gp70/anti-gp70 immune complexes (ICs) and the associated incidence of IC-type lupus nephritis were reduced. Evidence for the formation of mixed-haplotype A alpha d A beta z molecules in the transgenic mice was obtained by A beta z molecule expression on the cell surface of splenic B cells and macrophages, thymic epithelial cells and mature B cells in the bone marrow. A alpha d A beta z-restricted T cell clones showed good proliferative responses to spleen cells from the transgenic mice, to an extent much larger than seen in cells from the original (NZB x NZW)F1 mice, suggesting that the expression of functional A alpha d A beta z molecules on cells in transgenic mice is considerably high. Compared with findings in transgene-negative littermates and the original (NZB x NZW)F1 mice, the A beta z transgene to a greater extent promoted serum levels of IgM anti-double-stranded (ds) DNA antibodies and IgM anti-gp70/gp70 ICs in the transgenic mice. None the less, the production of IgG anti-dsDNA antibodies and IgG anti-gp70/gp70 ICs as well as the development of renal disease was markedly suppressed. Possible mechanisms of such effects of the transgene are discussed.

Efficient isolation of mutant antigen presenting cell lines by functional selection using T cell clones.

An efficient method for the isolation of mutant antigen-presenting cell (APC) lines is described. When mixtures of transfectant APC lines TA beta z (that express A beta z/A alpha d MHC class II molecules) and hypothetical variant APC lines TA beta d (that express A beta d/A alpha d class II molecules) were cultured with and selected by autoreactive A beta z/A alpha d-restricted T cell clones, the percentage of TA beta d APC lines increased from less than 1% of the original APC mixtures to almost 100% after several cycles of selection. This increase of hypothetical variant was shown to be due to the formation of aggregates of wild-type TA beta z APC lines with A beta z/A alpha d-restricted autoreactive T cell clones that results in the inhibition of proliferation and probably killing of TA beta z APC lines. Based on this, ethyl methane sulfonate (EMS)-treated TA beta z APC lines or B-B hybridoma APC lines MW4 (that express A beta z/A alpha d and A beta z/A alpha z class II molecules) were cultured with and selected by A beta z/A alpha d-restricted autoreactive T cell clones to obtain mutant APC lines that escaped the recognition by T cell clones. After cloning, about 43% of clones examined lost the ability to stimulate T cell clones with concomitant loss of class II molecule expression. Less than 1% showed loss of stimulatory activity against T cell clones in spite of the expression of normal amounts of class II molecules. Initial analysis revealed that they include APC mutant lines with (1) altered MHC class II sequences, (2) loss of adhesion molecule expression and (3) possible impairment of the peptide loading. The method described here may provide a variety of mutant APC lines that are useful for the analysis of antigen processing and presentation pathways as well as of class II structure for T cell stimulation.

In vivo induction of IgG anti-DNA antibody by autoreactive mixed haplotype A beta z/A alpha d MHC class II molecule-specific CD4+ T-cell clones.

We characterized autoreactive T-cell clones derived from (NZB x NZW)F1 (B/WF1) mice. These autoreactive T-cell clones are shown to be CD4+ by immunofluorescence staining and to belong to Th2 type by cytokine release assay. Specificity analysis revealed the existence of mixed haplotype A beta z/A alpha d major histocompatibility complex (MHC) class II molecule-specific T-cell clones as well as A beta z/A alpha z- or A beta d/A alpha d-specific T-cell clones. Some but not all of the mixed haplotype A beta z/A alpha d-specific autoreactive T-cell clones showed strong activity to induce IgG anti-DNA antibody production upon transfer to young (4-month-old) B/WF1 mice, indicating that T cells with these specificities might be involved in B/WF1 autoimmunity.

Mixed haplotype A beta Z/A alpha d class II molecule in (NZB x NZW)F1 mice detected by T cell clones.

We have tried to demonstrate the existence of a mixed haplotype MHC class II molecule in (NZB x NZW)F1 (B/WF1) mice. When a large panel of keyhole limpet hemocyanin-specific T cell clones derived from B/WF1 mice was analyzed, several clones were shown to be restricted by a F1-specific A beta Z/A alpha d class II molecule. Autoreactive A beta Z/A alpha d-specific T cell clones were also obtained. The ability of the association and expression of A beta Z with A alpha d was confirmed by hybridoma and transfection experiments. Hybridoma cell lines created by fusion of NZW (H-2z) spleen cells with M12.C3 (a A beta d- variant cell line derived from M12.4.1 (H-2d) B lymphoma) cells expressed A beta Z determinants. Transfection of A beta Z genomic DNA to M12.C3 cells resulted in the expression of A beta Z determinants. These hybridoma cell lines and transfectants were able to stimulate A beta Z/A alpha d-specific T cell clones, suggesting the expression of A beta Z/A alpha d molecules on the cell surface. However, attempts to demonstrate the existence of mixed haplotype MHC class II molecules in B/WF1 mice by two-dimensional (nonequilibrium pH gradient gel electrophoresis/SDS-PAGE) gel electrophoresis analysis with the use of anti-class II mAb failed to demonstrate the existence of mixed haplotype A beta Z/A alpha d or A beta d/A alpha z class II molecules in B/WF1 mice. Analysis of mixture of TA beta Z cell and B/WF1 spleen cell lysates immunoprecipitated by anti-A beta Z mAb suggested that the amount of haplotype mixed A beta Z/A alpha d molecules in B/WF1 spleen cells is less than 1/10 that of haplotype matched A beta/A alpha pairs. Our results suggest that, although undetectable by biochemical analysis, small amounts of mixed haplotype A beta Z/A alpha d molecules exist in B/WF1 spleen cells. Also, T cell clones which recognize them exists in B/WF1 mice. Because autoimmune symptoms of B/WF1 mice are shown to be related to heterozygosity at the H-2 region, autoreactive T cell clones which recognize the mixed haplotype A beta Z/A alpha d class II molecule might be involved for the induction of autoimmunity in B/WF1 mice.

Generation of cells with morphological and antigenic properties of microglia from cloned EBV-transformed lymphoid progenitor cells derived from human fetal liver.

Single-cell clones from the Epstein Barr virus transformed lymphoid progenitor-like cell line established from human fetal liver at 8-week gestation, have been derived and characterized. These clones retained immunoglobulin (Ig) and T cell receptor (TCR) genes in their germ line configuration. They expressed HLA-DR and some B lymphoid markers such as CD19, CD20, and in some, the T lymphoid marker, CD2. They did not express surface Igs, CD3, CD4, CD8 or TCRs (alpha/beta, gamma/delta). A sensitive RT-PCR assay revealed that they did not express mRNA for a recombination activating gene-1, which is expressed after commitment to lymphoid cells. These results suggest that the established cloned lines are very early lymphoid progenitors that have not yet been committed to lymphoid cell lineage. In one of the lines, FL8.2.1.4, a marked morphological change that resembled microglia was induced when the cells were cultured in the presence of phorbol myristate acetate (PMA). After 72 hr of culture, 5-10% of FL8.2.1.4 cells developed a microglial morphology when stimulated with 10 to 100 ng/ml PMA. The newly generated cells with microglial morphology expressed HLA-DR and stained with Recinus communis agglutinin-1, which has been reported to bind specifically to brain microglia. In contrast, expression of lymphoid markers on cells with microglia-shaped morphology was remarkably diminished by PMA stimulation. Thus, the early lymphoid progenitor cells have the capacity to differentiate into cells with the morphological and antigenic properties of microglia cells. This system might be useful for further understanding of the characteristics and functions of microglia cells distributed in the central nerve system.

Changes in the lymphocyte response and in the production of IgE regulatory factors in relation to the course of immunotherapy.

In previous studies we described isotype-specific but antigen non-restricted soluble factors produced by human lymphocytes. In subsequent studies we demonstrated changes in the production of these factors during the course of immunotherapy (IT) by testing them on lymphocytes from normal healthy controls and allergic patients. Through these studies we confirmed that there exists a difference in lymphocytes' responsiveness to soluble factors between both groups. In this report, we investigated the effect of soluble factors on lymphocytes from allergic patients without IT (LyG1) and with IT longer than 2 years (LyG4). Peripheral blood samples were collected from healthy controls and allergic patients at different time periods of IT, and bidirectional mixed cultures were performed with the isolated lymphocytes. Supernatants obtained from chromatography were tested on lymphocytes of allergic patients without IT and with IT greater than 2 years to determine their effect on IgE synthesis. Long periods of IT reduce the production of Suppressor Factors (SF) by allergic patients as well as their responsiveness. Long periods of IT increase the responsiveness of lymphocytes of allergic patients to Enhancing Factors (EF) and decrease EF production. We propose a "receptor hypothesis" to explain these events.

Effect of immunotherapy on the production of IgE regulatory soluble factors. The response of normal lymphocytes.

IgE synthesis is controlled not only by antigen-specific mechanism but also by other factors which selectively affect the IgE isotype. Several groups demonstrated that factors released by lymphocytes were involved in this regulation. Our previous investigations were also directed in this field. Immunotherapy improves clinical symptoms in allergic respiratory diseases and produces cellular and humoral changes that affect IgE production. So we decided to investigate if immunotherapy had any influence in the production of soluble factors. Bidirectional mixed cultures were performed with lymphocytes from healthy controls and allergic patients with different time periods of IT, and the supernatants obtained from chromatography were tested to determine their effect on IgE synthesis of normal lymphocytes. IT time periods exerted influence on the production of enhancing factors. Suppressor factors derived from allergic patients had no effect on IgE synthesis of normal lymphocytes.

Lymphocyte response to IgE regulatory factors in allergic patients during the course of immunotherapy.

In previous studies we demonstrated isotype-specific but antigen non-restricted soluble factors produced by human lymphocytes of allergic patients and normal controls. In a subsequent investigation we isolated these soluble factors from allergic patients classified in various groups according to their immunotherapy (IT) time periods and from controls, and we tested them on lymphocytes of healthy controls. Then, we decided to amplify the study to include the effects on allergic patients' lymphocytes. Bidirectional mixed cultures were grown with lymphocytes from healthy controls and allergic patients with different IT time periods and supernatants obtained from chromatography were tested on lymphocytes of twenty-one allergic patients. None of the enhancing factors showed statistical significant effects on IgE synthesis of allergic lymphocytes. There clearly existed a change in the production of suppressor factors during the course of IT, but the long time period of IT did not increase this production.

Alpha 1-adrenoceptor antagonist activity of novel pyrimidine derivatives (SHI437 and IK29) in rabbit aorta and trigone of the bladder.

1. In the rabbit isolated aorta and trigone of the bladder, noradrenaline, phenylephrine and clonidine elicited concentration-dependent contractions, which may be caused through activation of postsynaptic alpha 1-adrenoceptors. 2. SHI437, IK29, prazosin and yohimbine competitively antagonized the contractile responses induced by noradrenaline in the aorta and trigone. The pA2 values of SHI437, IK29, prazosin and yohimbine were 7.35 +/- 0.09, 7.47 +/- 0.10, 8.55 +/- 0.02 and 6.28 +/- 0.05 in the aorta, and 8.07 +/- 0.04, 8.30 +/- 0.03, 8.22 +/- 0.04 and 6.46 +/- 0.04 in the trigone, respectively. 3. SHI437, IK29, prazosin and yohimbine also possessed competitive alpha 2-adrenoceptor blocking properties, judging from their antagonism of the clonidine-induced inhibitory effect on the twitch responses in the electrically stimulated vas deferens of the rat. The pA2 values of SHI437, IK29, prazosin and yohimbine were determined to be 4.76 +/- 0.02, 4.74 +/- 0.02, 5.06 +/- 0.03 and 7.86 +/- 0.04, respectively. 4. SHI437, IK29 and prazosin inhibited the contractile responses elicited by transmural electrical stimulation without affecting the evoked 3H-overflow from the [3H]-noradrenaline-preloaded rabbit aorta. Yohimbine augmented the contractile responses and 3H-overflow. 5. SHI437 and IK29 at a concentration sufficient to inhibit noradrenaline-induced contraction failed to attenuate the contractile responses of aorta to KCl, 5-hydroxytryptamine and prostaglandin F2 alpha, and of the trigone to acetylcholine and histamine. 6. The present results suggest that SHI437 and IK29 are highly selective alpha 1-adrenoceptor antagonists, especially in the trigone of the bladder.