Current asthma and respiratory symptoms among pupils in Shanghai, China: influence of building ventilation, nitrogen dioxide, ozone, and formaldehyde in classrooms.

We investigated 10 naturally ventilated schools in Shanghai, in winter. Pupils (13-14 years) in 30 classes received a questionnaire, 1414 participated (99%). Classroom temperatures were 13-21 degrees C (mean 17 degrees C), relative air humidity was 36-82% (mean 56%). The air exchange rate was 2.9-29.4 ac/h (mean 9.1), because of window opening. Mean CO2 exceeded 1000 ppm in 45% of the classrooms. NO2 levels were 33-85 microg/m3 indoors, and 45-80 microg/m3 outdoors. Ozone were 1-9 microg/m3 indoors and 17-28 microg/m3 outdoors. In total, 8.9% had doctors' diagnosed asthma, 3.1% wheeze, 23.0% daytime breathlessness, 2.4% current asthma, and 2.3% asthma medication. Multiple logistic regression was applied. Observed indoor molds was associated with asthma attacks [odds ratio (OR) = 2.40: P < 0.05]. Indoor temperature was associated with daytime breathlessness (OR = 1.26 for 1 C; P < 0.001), and indoor CO2 with current asthma (OR = 1.18 for 100 ppm; P < 0.01) and asthma medication (OR = 1.15 for 100 ppm; P < 0.05). Indoor NO2 was associated with current asthma (OR = 1.51 for 10 microg/m3; P < 0.01) and asthma medication (OR = 1.45 for 10 microg/m3; P < 0.01). Outdoor NO2 was associated with current asthma (OR = 1.44 for 10 microg/m3; P < 0.05). Indoor and outdoor ozone was negatively associated with daytime breathlessness. In conclusion, asthma symptoms among pupils in Shanghai can be influenced by lack of ventilation and outdoor air pollution from traffic. Practical Implications Most urban schools in Asia are naturally ventilated buildings, often situated in areas with heavy ambient air pollution from industry or traffic. The classes are large, and window opening is the only way to remove indoor pollutants, but this results in increased exposure to outdoor air pollution. There is a clear need to improve the indoor environment in these schools. Building dampness and indoor mold growth should be avoided, and the concept of mechanical ventilation should be introduced. City planning aiming to situate new schools away from roads with heavy traffic should be considered.

Depletion of Vbeta5.2/5.3 T cells with a humanized antibody in patients with multiple sclerosis.

A potentially pathogenic expansion of T cells expressing T cell receptor (TCR) Vbeta5.2/5.3 has been demonstrated in patients with multiple sclerosis (MS). A humanized antibody (ATM-027) directed against these T cells has been developed to further investigate the role of this subpopulation of T cells in MS. The pharmacokinetics/dynamics and safety of ATM-027 (0.3-300 mg intravenously over 30 min) were investigated in 14 patients with MS. The effect of treatment on cytokine expression and autoreactivity to peptides of myelin basic protein (MBP) was also studied. ATM-027 was well tolerated and raised no safety concerns. Clearance of the antibody was low and elimination half-life was approximately 3 weeks. The majority of the target Vbeta5.2/5.3 expressing T cells were depleted for at least 18 months. The small remaining fraction of target cells showed a marked decrease in their TCR expression, which was recovered within 8 months. The numbers of peripheral blood mononuclear cells (PBMCs) with spontaneous expression of IFN-gamma was decreased at 72 h and 8 weeks after treatment, whilst no clear effects on TNF-alpha, IL-4, IL-10, TGF-beta expression were observed. There was also a significant decrease in the number of PBMCs producing IFN-gamma in response to MBP peptide 80-102. We conclude that long-term depletion of T cells expressing defined Vbeta subgroups in MS patients is feasible using selective immunotherapy. The selective depletion of Vbeta5.2/5.3 expressing T cells in this study resulted in a decrease in potentially disease promoting anti-MBP reactivity and pro-inflammatory cytokine production.

Identification of fish species after cooking by SDS-PAGE and urea IEF: a collaborative study.

A collaborative study, to validate the use of SDS-PAGE and urea IEF, for the identification of fish species after cooking has been performed by nine laboratories. By following optimized standard operation procedures, 10 commercially important species (Atlantic salmon, sea trout, rainbow trout, turbot, Alaska pollock, pollack, pink salmon, Arctic char, chum salmon, and New Zealand hake) had to be identified by comparison with 22 reference samples. Some differences in the recoveries of proteins from cooked fish flesh were noted between the urea and the SDS extraction procedures used. Generally, the urea extraction procedure appears to be less efficient than the SDS extraction for protein solubilization. Except for some species belonging to the Salmonidae family (Salmo, Oncorhynchus), both of the analytical techniques tested (urea IEF, SDS-PAGE) enabled identification of the species of the samples to be established. With urea IEF, two laboratories could not differentiate Salmo salar from Salmo trutta. The same difficulties were noted for differentiation between Oncorhynchus gorbuscha and Oncorhynchus keta samples. With SDS-PAGE, three laboratories had some difficulties in identifying the S. trutta samples. However, in the contrast with the previous technique, SDS-PAGE allows the characterization of most of the Oncorhynchus species tested. Only Oncorhynchus mykiss was not clearly recognized by one laboratory. Therefore, SDS-PAGE (Excel gel homogeneous 15%) appears to be better for the identification, after cooking, of fish such as the tuna and salmon species which are characterized by neutral and basic protein bands, and urea IEF (CleanGel) is better for the gadoid species, which are characterized by acid protein bands (parvalbumins). Nevertheless, in contentious cases it is preferable to use both analytical methods.

A standardized method of identification of raw and heat-processed fish by urea isoelectric focusing: a collaborative study.

A urea-isoelectric focusing (urea-IEF) method of identifying fish species in processed fishery products was investigated as an interlaboratory collaborative study. The technique was optimized with respect to (i) protein extraction conditions, composition of the extraction solution (urea and SDS solutions), determination of protein concentrations of the fish extracts (five tested methods); (ii) nature of gel (with carrier ampholytes and Immobilines), conditions of rehydration of commercial dry gels, urea concentration; (iii) staining conditions, Coomassie blue and silver staining. The results of various experiments were compared to select the most appropriate methodology, with respect to the discrimination power of differentiating species with the minimal influence of heat processing, reproducibility, speed, and ease of application. The method recommended meets the requirements of food control and customs laboratories.

Field intercomparison of diffusive samplers for measuring ammonia.

Agricultural production systems are recognised as a major source of atmospheric ammonia. Deposition of ammonia and ammonium may contribute to undesired changes in oligotrophic ecosystems. The continuous measurement of atmospheric ammonia requires expensive and sophisticated techniques and is performed only in a very restrict number of ambient air stations in Europe. Therefore, the application of passive samplers, which have the advantage of being easy to handle and cost-efficient, is useful. In the past the comparability of different passive samplers must be considered as rather scarce. In a joint European project under the leadership of the GSF-Forschungszentrum für Umwelt und Gesundheit, Neuherberg, in 1997 a comparison of different passive ammonia monitoring methods was carried out in a prealpine rural site near Garmisch-Partenkirchen. It was considered valuable to include not only well established systems but also methods still being developed. For the comparative test ten working groups with different methods took part. A wet annular denuder system, which has been developed by the Netherlands Energy Research Foundation for on-line measurement of atmospheric ammonia, served as reference of passive methods. The experiment, which started in June and finished in December, showed that most of the passive samplers fulfil the requirements and can be recommended for further measurements. Additional measurements of meteorological parameters were performed to check the influences of different weather conditions on passive sampling.

IFN-gamma treatment increases insulin binding and MHC class I expression in erythroleukemia cells.

We have investigated if interferon-gamma (IFN-gamma) treatment of human K562 tumor cells, which upregulates the expression of MHC class I antigens (MHC-I), simultaneously would influence insulin binding. Treatment of K562 cells with recombinant human IFN-gamma for 48 h caused a significant increase of insulin binding at 37 degrees C. Recombinant human tumor necrosis factor-alpha (TNF-alpha) alone had no effect but acted synergistically with IFN-gamma, leading to a two-fold increase of insulin binding. No change in affinity, number of binding sites or cell surface expression of insulin receptors (IR) after IFN-gamma treatment could be detected. The increased insulin binding observed at 37 degrees C was not seen at 4 degrees C, suggesting alteration of insulin internalization. The dose-response curve, as well as the time curve, for the increase in insulin binding after IFN-gamma treatment correlated with enhanced cell surface expression of MHC-I antigens. However, the correlation was not absolute. Our results show that IFN-gamma treatment alone or together with TNF-alpha, can alter the insulin binding to K562 cells without changing the expression or affinity of the IR. This correlates with the effect of IFN-gamma on MHC-I expression. These results support the findings that MHC-I molecules associate and interact with the IR at the cell surface.

Engagement of MHC class I proteins on natural killer cells inhibits their killing capacity.

We have studied whether engagement of MHC class I (MHC-I) molecules on natural killer (NK) cells can influence the NK killing activity. Human NK effector cells, enriched by nylon wool passage, were incubated with monoclonal antibodies (MoAb) to MHC-I followed by cross-linking with secondary rabbit anti mouse Ig or streptavidin. Cross linking of MHC-I molecules on NK cells resulted in a clear inhibition of the NK activity against the target cells K562, Molt-4 and U937. The inhibitory effect was selective for MHC-I and was not seen with MoAb to MHC-II or CD56 molecules. The inhibition was not mediated via Fc receptors since F(ab)2 fragments of the MHC-I MoAb W6/32 were as effective as the intact antibody. The best inhibition of NK activity was obtained using biotin-labelled F(ab)2 fragments of W6/32 and streptavidin as a cross-linker, where up to 70% reduction in NK cell activity was observed. Antibody dependent cellular cytotoxicity (ADCC) was also inhibited by cross-linking MHC-I molecules on the effector cells. The results show that antibody mediated cross-linking of MHC-I proteins on NK cells can inhibit their killing capacity. This indicates that MHC-I molecules on NK cells can be involved in the regulation of NK cytotoxicity, perhaps by transmitting inhibitory signals into the NK cell.

Regulation of lymphocyte aggregation and proliferation through adhesion molecule CD54 (ICAM-1).

The effect of two mouse mAb (LB-2 and G1B2) against human CD54 (intercellular adhesion molecule-1, ICAM-1) in lymphocyte aggregation and proliferation systems was investigated. The LB-2 mAb, but not G1B2, inhibited phorbol ester-induced aggregation of B lymphoblastoid cells. In addition, LB-2, but not G1B2, induced aggregation and proliferation of peripheral blood mononuclear cells (PBMC) in cultures containing FCS. The Fab fragment of LB-2 always (10/10 donors) induced proliferation while the intact mAb was active in 3/11 donors. When cultures contained human serum (HS), LB-2 and its Fab fragment induced proliferation in 1/9 and 1/4 donors, respectively. Addition of HS to FCS cultures inhibited proliferation induced by LB-2 Fab, indicating the presence of an inhibitory factor in human serum. Addition of anti-CD18 mAb to cultures stimulated by LB-2 Fab caused partial inhibition of proliferation but did not prevent aggregate formation. A combination of anti-CD18 and anti-CD29 mAb resulted in a nearly complete inhibition of proliferation but did not inhibit aggregate formation. In these experiments it was found that the anti-CD29 mAb 4B4 in itself induced cell aggregation of PBMC and enhanced aggregation induced by the anti-CD3 mAb OKT3. Both LB-2 and G1B2 showed significant inhibition (> 60%) of proliferation when human PBMC were stimulated by the antigen PPD in the presence of HS, but not when stimulated by staphylococcal enterotoxin A (SEA) or IL-2. This study describes two mAb against separate epitopes on CD54 which are differentially involved in cell aggregation or induction of proliferation but are of similar importance in antigen-specific responses. Furthermore, the new finding that the LB-2 mAb or its Fab fragment can induce cell aggregation and proliferation defines a signaling function of CD54 which may work independent of crosslinking or costimulation.

Induction of human hsp60 expression in monocytic cell lines.

Both bacterial and mammalian heat shock proteins (HSP) are recognized by some T cells, and hsp60 recognition has been implicated in rheumatoid arthritis. We have developed a model to study the induction of hsp60 in human monocytic cell lines. An anti-mycobacterial hsp65 mAb (ML30), cross-reacting with human hsp60 was used to screen 21 human tumor cell lines in Western blot analysis. All T cell and B cell lymphomas constitutively expressed hsp60 protein at moderate to high levels, while little or no hsp60 protein was detected in two monocytic leukemia lines. Moderate to high levels of hsp60 mRNA and protein could be induced in the THP-I monocytic leukemia cell line by heat shock, retinoic acid, interferon (IFN)-gamma or tumor necrosis factor (TNF)-alpha treatment, the highest levels obtained with a combination of IFN-gamma/TNF-alpha. This was also seen using two rabbit anti-hsp60 antisera directed against the N-terminal or C-terminal part of the human hsp60 protein. The determinants detected by the ML30 mAb or the two rabbit anti-hsp60 antisera were not cell surface expressed, as measured with immunofluorescence (FACS) analysis on control cultured or cytokine treated cell lines. This could be a useful model for studies related to the induction of hsp60 in human cells.

Human MHC class I antigens are associated with a 90-kDa cell surface protein.

Human MHC class I proteins are expressed on almost all nucleated cells as a heavy chain (about 45 kDa) non-covalently associated with beta 2-microglobulin (12 kDa). In this report we show that MHC class I (MHC-I) proteins can also be associated with a 90-kDa protein in the cell membrane. Surface-radiolabelled cells were treated with dithiobis succinimidyl propionate (DSP) in order to preserve multimer protein complexes during cell lysis. The lysates were immunoprecipitated and analysed by SDS-PAGE and autoradiography. Immunoprecipitation of human MHC-I proteins co-precipitated another protein of about 90 kDa in molecular weight-p90. p90 was coprecipitated from all the MHC-I expressing cells tested: U937, Raji, Molt-4 and IFN-gamma treated K562, but not from untreated, MHC-I negative K562. A 90-kDa protein was also co-precipitated with MHC-I from fresh peripheral blood mononuclear cells (PBMC). Furthermore, p90 was coprecipitated by different MoAbs to the MHC-I heavy chain or beta 2-microglobulin, but not by control antibodies. Two additional co-precipitating proteins at 34 kDa and 28 kDa were seen in MHC-I precipitates from Raji cells. Our results suggest that MHC-I proteins and the 90-kDa protein are associated in the cell membrane, probably by a close but weak, non-covalent interaction. Two additional cell surface proteins at 34 kDa and 28 kDa seem to be MHC-I associated on Raji Burkitt's lymphoma cells.

A monoclonal IgM antibody to a methylcholanthrene-induced tumor. 2. A biologically relevant glycoprotein antigen with a molecular weight of 40-43 kilodaltons.

We have recently described a monoclonal IgM antibody, H17, to a mouse methylcholanthrene-induced tumor with specificity for a common saccharide, namely alpha-N-acetylgalactosamine. Although recognizing a glycosidic determinant most likely to be part of many cell surface glycoproteins, H17 was highly specific for the immunizing tumor. This discriminatory ability was investigated by testing the antibody binding to a panel of normal and transformed mouse and human cells. Cross-linking studies showed that H17 reacts with a glycoprotein with a molecular weight of 40-43 kD, which through blockage during the process of immunization has an impact in vivo on tumor growth. Moreover, the uncommon sensitivity to complement-mediated lysis by this methylcholanthrene-induced tumor was blocked by the H17 monoclonal antibody.

Presence of human 65 kD heat shock protein (hsp) in inflamed joints and subcutaneous nodules of RA patients.

Monoclonal antibodies to the human homologue of the bacterial 65 kD heat shock protein (hsp) were used to investigate the tissue distribution of endogenous hsp 65 in normal versus rheumatoid synovial tissue, in subcutaneous nodules of patients with rheumatoid arthritis (RA) and in several instances of non-rheumatoid inflammation. A strong reactivity of the anti-hsp antibody was found in the cartilage-pannus junction in rheumatoid joints and in rheumatoid nodules, but not in normal joints or in normal or inflamed kidney or liver (irreversible graft rejection, chronic glomerulonephritis or primary biliary cirrhosis). The findings provide a new hypothetical explanation for a role of T cells reactive with the 65 kD hsp in the generation of both articular and extra-articular lesions in chronic rheumatoid arthritis.

Interferon is able to reduce tumor cell susceptibility to human lymphokine-activated killer (LAK) cells.

It is known that IL-2 induces lymphocytes to produce interferon-gamma (IFN-gamma) and this IFN type is particularly efficient in inducing tumor cell resistance to natural killer (NK) cell-mediated lysis. We have investigated the effect of IFN on tumor cell sensitivity to LAK cell-mediated cytotoxicity. Pretreatment of the human K562 leukemia and HHMS melanoma with IFN-gamma and the Daudi lymphoma with IFN-alpha caused a significant reduction in sensitivity to lysis by human LAK cells generated in vitro in the presence of human recombinant IL-2 (100 U/ml). The LAK activity was mediated by cells expressing NK cell markers (CD16,NKH1) as well as by cells with T cell markers (CD3, CD5). IFN-treated K562 cells were protected from lysis mediated by all these populations. Supernatants from LAK cultures containing IFN-gamma were able to induce NK and LAK resistance when used to pretreat K562 overnight. Antibodies to IFN-gamma but not to IFN-alpha were able to neutralize this activity. Taken together, these results indicate that the production of IFN-gamma by LAK cells may be of importance in induction of tumor cell resistance to LAK cell-mediated lysis.

IFN-gamma treatment of K562 cells inhibits natural killer cell triggering and decreases the susceptibility to lysis by cytoplasmic granules from large granular lymphocytes.

Pretreatment of human K562 leukemia cells with rIFN-alpha and rIFN-gamma resulted in decreased susceptibility to lysis by human peripheral blood NK cells. The reduction of NK-susceptibility after IFN treatment was not due to a general effect of IFN on the stability of the cell membrane because the susceptibility of K562 cells to lysis by antibodies plus C, distilled water, or lysolecithin was unaffected. Binding studies with effector cell preparations enriched for NK cells with large granular lymphocyte morphology revealed no difference in binding to control and IFN-gamma-treated target cells. The sensitivity to soluble NK cytotoxic factors was not affected significantly by the IFN treatment. In contrast, the susceptibility of IFN-treated target cells to the cytotoxic activity of purified cytoplasmic granules from a rat large granular lymphocyte tumor was significantly reduced, indicating that the IFN-induced resistance acted at the level of susceptibility to the lytic mechanism of NK cells. However, IFN-alpha was more effective than IFN-gamma in inducing resistance to the cytoplasmic granules although resulting in only a weak resistance in the cell-mediated cytotoxic assay. IFN-gamma but not IFN-alpha caused a reduction in the frequency of effector cells that had reoriented their Golgi apparatus toward their bound target cell. In addition, IFN-gamma treated K562 cells failed to elicit an influx of Ca2+ into effector cells. Taken together, the results suggest that IFN-gamma in addition to an increased resistance to the lytic molecules released by NK cells can also induce changes in the target cells which prevent the triggering and activation of the effector cell.

The effect of T cell growth factor on the generation of cytolytic T cells.

The development of cytotoxic effector cells through primary allogeneic mixed tumor-lymphocyte culture (MTLC) was found to be accompanied by the production of T cell growth factor (TCGF). Addition of supplemental TCGF to MTLC resulted in the generation of significantly greater quantities of effector cells, and these effector cells displayed augmented cytotoxic activity. The TCGF-induced effect could not by duplicated by the addition of fresh medium or a mitogenic concentration of concananvalin A. Although TCGF augmented the proliferation of antigen-nonreactive cells, antigen-reactive cells appeared to be preferentially stimulated by TCGF. Finally, it was shown that depletion of TCGF from MTLC resulted in an impairment of proliferation and differentiation of cytotoxic effector cells. These findings demonstrate that soluble factors are involved in the regulation of in vitro cell-mediated immune responses in an analogous manner to similar factors that have been shown to regulate humoral immune responses. Therefore, the forces affecting TCGF production may modulate the amplitude of a T cell-mediated cytolytic response.

T cell growth factor: parameters of production and a quantitative microassay for activity.

Several soluble factors have recently been associated with the proliferation and differentiation of thymus-derived lymphocytes. One of these factors present in medium conditioned by T cell mitogen-stimulated lymphocytes has the ability to promote the long-term culture of normal and antigen-specific cytotoxic T cells. We report a method to test for this proliferative stimulus in the form of a sensitive microassay based upon the tritiated-thymidine incorporation of continuous murine tumor-specific cytotoxic T cell lines (CTLL). The microassay requires microliter quantitites of sample fluid and is amenable to quantitative analysis. This highly reproducible, quantitative assay for T cell growth factor (TCGF) has allowed investigation as to the kinetics of TCGF generation and has revealed that T lymphocytes are required for its production. Further investigation has supported the notion that this nonspecies-specific factor is actively removed from tissue culture medium by the proliferation of either T cell mitogen-activated lymphocytes or CTLL.

Anti-keyhole limpet hemocyanin antibody in normal unsensitized individuals.

Antibody to keyhole limpet hemocyanin (KLH) has been detected in normal unimmunized subjects by means of a sensitive micropassive hemagglutination technique. Prior sensitization was not detected by either skin testing or lymphocyte transformation. After immunization with KLH there was no correlation between the level of this antibody and subsequently acquired skin test reactivity and lymphocyte transformation.