Risk assessment model of occupational exposure to nanomaterials.

Recent development of nanotechnology allows precise assessment of the risks workers are exposed to. To assess the risks, a specific risk assessment model has been developed with reference to workplaces where nanoparticles are present. This model allows identification and quantification of the health hazards for workers. The use of nanomaterials presents some uncertainties regarding effective level of danger, and risk assessment takes this into consideration with the help of an appropriate index. An evaluation algorithm considers both normal work conditions and abnormal/emergency situations. Evaluation outcome consists of several risk levels with several subdivisions. For each single level, it is possible to develop specific behavioral guidelines. The present evaluation model has been implemented in research laboratories. The results are middle to high-level risks.

A hybrid expressing genetically engineered major allergens of the Parietaria pollen as a tool for specific allergy vaccination.

BACKGROUND: Allergy is an immunological disorder affecting about 25% of the population living in the industrialized countries. Specific immunotherapy is the only treatment with a long-lasting relief of allergic symptoms and able to reduce the risk of developing new allergic sensitizations and inhibiting the development of clinical asthma in children treated for allergic rhinitis. METHODS: By means of DNA recombinant technology, we were able to design a head to tail dimer expressing disulphide bond variants of the major allergen of the Parietaria pollen. IgE binding activity was studied by Western blot, ELISA inhibition assays and the skin prick test. T cell recognition was studied by peripheral blood mononuclear cell proliferation. The immunogenicity of the hybrid was studied in a mouse model of sensitization. RESULTS: In vitro and in vivo analysis showed that the disruption of specific cysteine residues in both allergens caused a strong reduction in IgE binding activity of the PjEDcys hybrid. In addition,we were able to show that a reduction in the IgE epitope content profoundly reduced the anaphylactic activity of the hybrid (from 100 to 1,000 times less than wild-type allergens) without interfering with the T cell recognition. Sera from BALB/c mice immunized with the hybrid were able to bind the natural Parietaria allergens and to inhibit the binding of human IgE to wild-type Par j 1 and Par j 2 allergens up to 90%. CONCLUSION: Our results demonstrate that hybrid-expressing disulphide bond variants of the major allergens of the Parietaria pollen displayed reduced allergenicity and maintained T cell reactivity for induction of protective antibodies.

Mast cell production of TNF-alpha induced by substance P evidence for a modulatory role of substance P-antagonists.

Unregulated increasing of Tumor necrosis factor-alpha (TNF-alpha) could be pathogenic in inflammatory diseases. The aim of this study was to investigate the anti-inflammatory role of the Substance P-antagonists (SPAs) through the inhibition of histamine release (HR) and TNF-alpha production from mast cell. Rat peritoneal mast cells (PMC) stimulated with Substance P (SP), in the presence of SPAs or not, were analyzed for HR and TNF-alpha protein production. Competitive Polymerase Chain Reaction, with an internal standard competing with target cDNA for the same primers, was used to determine the TNF-alpha mRNA expression. We show that the increase of either HR and TNF-alpha levels in peritoneal (PMC) after induction with SP was inhibited by pre-incubation with SPA or with the Peptide 101 (P101), while the [D-Pro2, D-Phe7, D-Trp9]-SP (dSP) had no effect. Neuraminidase treatment suggests that dSP, as well as SP, interacts with sialic acid residues on the cell surface. Moreover, SPA and P101 also inhibit the release of histamine and TNF-alpha induced by dSP suggesting that a receptor-independent mechanism is involved. These data could be useful to better understand the mechanisms involved in the mast cell activation and TNF-alpha production in the inflammatory diseases where SP is involved.

Histamine and tumor necrosis factor-alpha production from purified rat brain mast cells mediated by substance P.

The effect of cytokines and neuropeptides on neuroimmune functions has not been completely elucidated and recent evidence suggests an important role for these molecules linking the neuroimmune system and inflammatory events. The aim of this study was to analyse the effect of substance P (SP) on a pure population of hypothalamic brain mast cell (BMC). A pure population of BMC challenged with 10(-8) M SP gave 78% histamine release (HR) and secreted 600 pg/ml of tumor necrosis factor-alpha (TNF-alpha) as determined by ELISA. The production of TNF-alpha mRNA, measured by a competitive RT-PCR, was 14 times higher than that in unstimulated cells. The secretion of histamine and TNF-alpha from BMC after stimulation with SP supports the hypothesis that these mediators could induce an initial response in neuroinflammatory diseases.

Use of antagonist peptides to inhibit in vitro T cell responses to Par j1, the major allergen of Parietaria judaica pollen.

Antigenic peptides with substituted side chains inhibit immune responses to a number of recall Ags from infectious agents in vitro. Here we show that the same strategy can be applied to peptides derived from a pollen protein, the major allergen of Parietaria judaica(Par j1), a plant responsible for most allergenic sensitization in the southern Mediterranean area. Three T cell lines responding to Par j1 protein were used to identify a stimulatory peptide. Two different monosubstituted altered peptide ligands (APL) were identified that bound to the HLA-DR of the responders, did not stimulate the T cell lines on their own, and decreased the response to subsaturating amounts of the unmodified stimulatory peptide. Most important, these APL were able to inhibit the response of these cell lines to intact Par j1 protein. A third monosubstituted peptide bound to the HLA-DR but did not show inhibitory activity. The two APL had a lower affinity than the unsubstituted peptide for the HLA-DR. The last two observations make MHC blockade an unlikely explanation for the observed effect. These results indicate the action of a specific peptide-mediated antagonism that may be useful in controlling the T cell component of an allergic response.

Evidence that brain mast cells can modulate neuroinflammatory responses by tumour necrosis factor-alpha production.

Tumour necrosis factor-alpha (TNF-alpha) levels in mammalian brain increase during neuroinflammatory diseases. We used the competitive polymerase chain reaction (PCR) to quantify the amount of TNF-alpha in stimulated and unstimulated brain mast cells (BMC). A cDNA fragment shortened by a deletion of 56 bp was used as an internal TNF-alpha-specific standard. The immunological stimulus resulted in enhanced TNF-alpha mRNA expression and increased release of histamine and TNF-alpha. This is the first time that BMC showing functional FCepsilonRI-bound IgE receptors have been purified. Our results support the hypothesis that BMC mediators might induce an initial response in neuroinflammatory diseases.

Substance P selectively activates TNF-alpha mRNA in rat uterine immune cells: a neuroimmune link.

Substance P (SP) is a neuropeptide which influences the interaction between the nervous and immune systems. It is an important modulator of cytokines, including tumour necrosis factor-alpha (TNF-alpha) whose role during the reproductive processes has been established. We have investigated the effects of SP on TNF-alpha mRNA expression in macrophages and mast cells (MC) isolated from rat peritoneum and uterus. Cell supernatants were analysed for their histamine content as a measure of stimulation. SP alone increased TNF-alpha expression in peritoneal MC but not in peritoneal macrophages. The addition of SP resulted in a six-fold enhancement of TNF-alpha expression in uterine MC whereas no stimulation was observed in macrophages as determined by competitive polymerase chain reaction (PCR).

Inhibitory effect of neuraminidase on SP-induced histamine release and TNF-alpha mRNA in rat mast cells: evidence of a receptor-independent mechanism.

The neuropeptide substance P (SP) is a mediator of neuro-inflammation and can play a role by induction of histamine release (HR) and TNF-alpha. However, its effect on the heterogeneous response of mast cells (MC) has not been completely studied. We have established that the SR can induce 25% of HR in highly purified rat uterine MC at diestrous but not at proestrous phases of the reproductive cycle and 88% of HR in peritoneal mast cells (PMC). We also found 2.2 fold increase in TNF-alpha mRNA at diestrous, in SP stimulated uterine MC versus control and 2.7 fold increase in PMC; RT and competitive PCR were used to amplify the TNF-alpha mRNA. We have thereafter investigated the mechanism whereby the binding of SP to sialic acid on the MC membrane, could trigger secretion of histamine and induction of TNF-alpha mRNA. The neuraminidase pretreatment (0.1 U/ml) inhibited SP-stimulated HR from either uterine MC and PMC (98% and 50%, respectively) and totally inhibited SP-stimulated TNF-alpha mRNA levels. The neuraminidase effect was not toxic, since it was not observed in IgE mediated HR and TNF-alpha mRNA levels. In conclusion, the inhibitory effect of the neuraminidase on the SP-mediated increase of histamine and TNF-alpha mRNA, suggests that the SP-sialic acid interaction could have a role in the MC heterogeneous response.

A factor secreted by human embryo stimulates cytokine release by uterine mast cell.

The existence of a biochemical network of embryo-maternal communication implies that various secreted molecules constitute a signal-response mechanism, important for the process of embryo implantation in mammals. Here we report the purification of a protein with an apparent molecular weight of 136 kDa, responsible for a 2000-fold increase in embryo-derived histamine-releasing factor (EHRF) activity. This protein, purified from medium from the in-vitro culture of 2-8-cell human embryos, by means of affinity chromatography, was capable of binding immunoglobulin (Ig)E as demonstrated by immunoblotting and enzyme-linked immunosorbent assays. We found EHRF was capable of inducing release of histamine and cytokines in vitro from rat uterine tissue, collected on day 4 of pregnancy (preimplantation stage of embryo development). When EHRF was used as a secretagogue, granulocyte macrophage-colony stimulating factor (GM-CSF) release increased from 3 to 55 pg/g (P < 0.01) and tumour necrosis factor-alpha (TNF-alpha) release increased from 0 to 2.1 ng/g (P < 0.01), as detected by enzyme-linked immunosorbent assay. A simple method was used to purify uterine mast cells using an IgE-Sepharose affinity chromatography column and the purity (90%) was checked with Dynabeads coupled to specific rat IgE antibody. When purified mast cells were stimulated with EHRF in the same way as the uterine explants, a similar pattern of GM-CSF and TNF-alpha release was obtained. We also describe the reverse transcription-polymerase chain reaction (RT-PCR) of GM-CSF and TNF-alpha mRNA from purified uterine mast cells. On day 4 of pregnancy only the mRNA of TNF-alpha was found and this increased after stimulation with the EHRF. In conclusion, the data presented suggest that uterine mast cells isolated during the preimplantation stage release cytokines in vitro following interaction with an embryo factor.

Effect of substance P on uterine mast cell cytokine release during the reproductive cycle.

There is increasing evidence that neuropeptides, steroid hormones and inflammatory cytokines influence the immune response during the reproductive cycle. In the present study, we focus on the effects of neuropeptide Substance P (SP) during the pre-implantation stage of embryo development (day 4 of pregnancy), at pro-estrus and di-estrus (two phases with different hormonal states). We found heterogeneous responses to SP and anti-IgE by the rat uterine mast cells (MCs), as detected by ELISA. In fact, MCs purified from uteri on day 4 of pregnancy released histamine, granulocyte macrophage-colony stimulating factor (GM-CSF) and tumor necrosis factor-alpha (TNF-alpha) in response to anti-IgE, but not to SP. When pre-incubated with SP, the release to anti-IgE was significantly enhanced compared to anti-IgE alone. Exposure of SP to antibodies to SP, prior to pre-incubation with MCs, negated the SP effect on IgE-mediated release. At the pro-estrus phase SP showed similar behavior as on day 4 of pregnancy, whereas at the di-estrus phase SP alone was capable of inducing release of histamine and cytokines from purified uterine MCs. Moreover, non-quantitative RT-PCR analysis of the TNF-alpha mRNA level suggested an SP stimulation at the di-estrus phase, but neither on day 4 of pregnancy nor at the pro-estrus phase. Taken together, these data strongly suggest that SP can modulate IgE-mediated uterine MC release of histamine and inflammatory cytokines in different ways, depending on the phase of the reproductive cycle.

Oestradiol enhances in vitro the histamine release induced by embryonic histamine-releasing factor (EHRF) from uterine mast cells.

The relationship between maternal hormones and factors secreted by the implanting embryo is still controversial. We have analysed the in-vitro effect of oestradiol and human embryo-derived histamine-releasing factor (EHRF) on histamine release from rat uterine mast cells. Rat uterine mast cells which were preincubated with oestradiol and then challenged with human EHRF gave histamine release values two- to threefold higher than those without preincubation. The enhancement observed was time- and temperature-dependent. A similar enhancement was obtained with human sensitized basophils but not with rat peritoneal mast cells. Oestradiol, used as a direct challenge, did not induce any histamine release from either rat uterine or peritoneal mast cells, or from human sensitized basophils. Oestradiol preincubation also enhanced the histamine release induced by anti-IgE but did not enhance the histamine release induced by substance P or compound 48/80, two secretagogues that are not mediated by IgE. Moreover, uterine fragments derived from rats at various oestrus phases, with different amounts of endogenous oestrogen, were challenged in vitro with EHRF. The release of histamine by mast cells was higher at the proestrus and preimplantation phases than at dioestrus. All these findings suggest that the interaction of oestradiol with rat uterine mast cells was capable of enhancing in vitro the histamine releasing effect of EHRF.

Modulation of rat peritoneal mast cell and human basophil histamine release by estrogens.

This study was undertaken to investigate the effect of estrogens on the histamine release mediated by IgE in rat peritoneal mast cells (PMC) and in sensitized human basophils. The estrogens were found to enhance the histamine release of either rat PMC and sensitized human basophils upon stimulation with anti-IgE. The enhancement was estrogens dose-dependent reaching the maximum value of 23% for rat PMC and 41% for sensitized human basophils stimulated with anti-IgE upon preincubation with 10(-8) M estrogens. Moreover, when purified PMC were used, the enhancing effect was still detected, suggesting a direct interaction between estrogens and mast cells. The enhancing effect took place quite rapidly reaching plateau levels in about 60 min. Basophils preincubated at 4 instead of 37 degrees C did not give any appreciable enhancement, suggesting that it was temperature-dependent and that the effect observed was not due to cytotoxicity. Incubation of PMC or human basophils with estrogens alone, without challenge with anti-IgE, did not give any detectable histamine release. The enhancement of histamine release by estrogens is probably mediated by IgE molecules present on the cell membrane, since this effect was not observed on challenge with substance P or compound 48/80, two segretagogues known to induce histamine release not via IgE.

Immunosuppressive effect of early pregnancy factor on early expression of cell surface membrane IgG.

We have adopted a new assay to investigate the influence of early pregnancy factor (EPF) on the modulation of lymphocyte activity. Lymphocytes were attached to the plastic surfaces of microplates in serum-free medium in the presence of Sepharose-Con A. After 2-3 days incubation with EPF, and ELISA assay was used to detect the expression of surface membrane IgG (smIgG); this was done in the same microplates used for the culture, thus avoiding cell manipulation. Using only a few picograms of EPF a significant inhibition (in the range 26-40%) was obtained. The variation in the inhibition observed was mainly due to the different sources of lymphocytes used. Unrelated proteins and hormones, tested at the same concentration as EPF, did not show any inhibitory activity. Using the F(ab)2 fragment of anti-human IgG instead of the whole molecule the same levels of inhibition were obtained, suggesting that the observed inhibition by EPF was not due to a non-specific interaction between the anti-human IgG and the Fc receptors on the cell. Such inhibitory activity detected in vitro by this method provides additional support for a suppressive role for EPF during pregnancy.

Binding of human EPF to receptors on PMBC as a first signal of pregnancy immunomodulation.

The interaction between human early pregnancy factor (EPF) and a specific receptor present on human peripheral blood mononuclear cells (PMBC) has been assessed. EPF was radioiodiated by the lactoperoxidase method to a high specific activity, and the [125I]EPF obtained bound in a specific and saturable manner to the receptors on PBMC. Saturation of the binding sites occurred at 10(-9) M. There were 6600 specific binding sites per cell in cells partially purified from pregnant women and fewer in those from non-pregnant women. Unlabelled EPF was capable of competing for binding sites with [125I]EPF, while the binding of [125I]EPF was not inhibited by human chorionic gonadotrophin. A new assay was used that permits the culture of PBMC and the detection of the surface IgG expression in the same microplate. With this method the influence in vitro of human EPF on lymphocyte expression was tested. The results demonstrated a specific inhibition of surface IgG expression of PBMC using a very low concentration of EPF (10 pg/ml).

Immunochemical characterization of antigens of Parietaria judaica pollen. Identification of allergens by means of crossed radio immunoelectrophoresis.

A crude extract of Parietaria judaica pollen was obtained by means of extraction, centrifugation and dialysis, and studied by means of quantitative immunoelectrophoresis. Crossed immunoelectrophoresis, using a high-titer purified rabbit antibody fraction, showed that the pollen extract contained at least 26 antigens of which 18 moved towards the anode, 6 moved towards the cathode and 1 moved both towards the anode and the cathode. The allergens in the extract were identified by means of crossed radio immunoelectrophoresis. Nine of the 26 antigens were able to bind specific human IgE to their corresponding immunoprecipitates, and 4 of these antigens can be classified as major allergens. The apparent molecular weights of 17 antigens were determined by a combination of size exclusion chromatography and immunochemical analysis. Ten antigens had Kav values corresponding to molecular weights in the range of 10-40 kilodaltons, 1 antigen possessed an apparent molecular weight of less than 10 kilodaltons, and six antigens had molecular weights above 40 kilodaltons. Most of the antigens, as analysed by column isoelectric focusing, had pI values between 4 and 7. Crossed line immunoelectrophoresis using an extract of Parietaria officinalis shows that the antigens of this extract exhibit a high degree of identity with those of P. judaica.

Allergens of Parietaria judaica pollen--I. Purification and characterization of a hapten and a low molecular weight allergenic peptide.

A low mol. wt allergen (Pj-2) and a hapten (Pj-H3) were purified from Parietaria judaica pollen by means of long-term aqueous extraction, dialysis and gel filtrations. The yield of the Pj-2 allergen was 0.94% (w/v) of the total protein present in the aqueous extract of the pollen, while its allergenic activity was about 60% of the total dialyzable activity, as verified by skin prick tests, ELISA- and RAST-inhibition experiments. The homogeneity of this allergen was demonstrated by one single sharp peak on HPLC, one single band on PAGE-SDS and by one single arc on IEF. Its mol. wt, estimated by HPLC and amino acid composition, was 10,400. The amino acid analysis showed 73 amino acid residues, and lysine was predominant, with 20 residues. The hapten Pj-H3 was 0.2% (w/v) of the total protein found in the pollen aqueous extract. It was inactive in skin prick tests even at a protein concn of 2 mg/ml, while it was capable of inhibiting by 60% in ELISA- and RAST-inhibition experiments, suggesting an immunochemical relationship with both IgE and allergens specific to P. judaica. The homogeneity was demonstrated by one single sharp peak on HPLC and one single band on PAGE-SDS. The amino acid analysis showed 10 amino acid residues, with no specific traits, and the mol. wt determined by gel filtration and amino acid composition was 1000. An immunochemical relation between the allergen and the hapten was also suggested by the results of an ELISA-inhibition test, and by the ability of the hapten to partially inhibit the precipitin line between rabbit antibodies to whole P. judaica pollen extract and the Pj-2 allergen. The allergen and the hapten described above, purified at homogeneity and in an antigenically active state, both provide adequate material for further structural and immunological characterizations.

Modulation of lymphocyte response by hormones.

The present study was carried out to investigate the influence of inhibitor factors on the immunological response of the mother. Using a FIVET program (in vitro fertilization and embryo-transfer) we were able to detect in vitro the modulation of lymphocyte response by hormones, utilizing subjects receiving hormone therapy. A new assay has been used to study the antibody response. The lymphocytes were adhered during the culture to the plastic surface of microplates, and the modulation of the expression of the surface IgG was detected with an E.L.I.S.A. assay in the same culture plate. Many hormones were tested in vitro using this assay. The addition to the lymphocyte cultures of hCG and oestradiol give a variable effect. This effect is concentration dependent and whilst in all instances the higher dosages were inhibitory, in same subjects the lower dosages had a stimulatory effect, but if the subjects were treated in vivo with hCG, then an inhibitory effect was observed in vitro even using very low concentrations of hormones. This could suggest that these hormones have an inhibitor effect on the lymphocyte response, inhibition that was enhanced when the lymphocytes were "primed" in vivo.

Circulating immune complexes in patients with usual interstitial pulmonary fibrosis: partial characterization and relationship with Thermoactinomyces vulgaris.

Sixty-six sera were analysed by solid-phase conglutinin binding assay, to detect the levels of circulating immune complexes (CIC), and by enzyme-linked immunosorbent assay (ELISA) to show a correlation with antibodies to Thermoactinomyces vulgaris. Sixty per cent of patients with usual interstitial pulmonary fibrosis (UIP), were positive for CIC; and T. vulgaris antibodies were detected in 60% of the same patients. In comparison, there was a low frequency of positive results in bronchitis patients (5% for CIC and 35% for T. vulgaris), and in normal blood donors (0% for CIC and 30% for T. vulgaris). Furthermore 31% of patients with lung cancer were found positive for CIC, but not for T. vulgaris. Immune complexes purified on Protein A-Sepharose and by sucrose density gradient from patients with UIP, showed a sedimentation coefficient higher than 19 S. The purified material was found to contain IgG and IgM as antibodies. Binding of immune complexes, purified by sedimentation on sucrose gradient, to conglutinin was inhibited by the presence of T. vulgaris antigen; thus suggesting that this antigen might be present in the complexes.

Purification of herpes simplex virus tumor associated antigen from human kidney carcinoma.

In the present study, we attempted to purify herpes simplex virus (HSV) tumor associated antigen(s) (TAA) extracted from human kidney carcinoma. Trypsinized human tumor cells were sonicated for 9 minutes and clarified at 100,000 x g for 1 hour; the supernate yielded 70% of detectable TAA as determined by means of quantitative absorption with specific antisera. The supernate used as source of soluble HSV-TAA was concentrated and the pellet was resuspended in 0.02 M tris, pH 7.2, and purified by means of filtration on Sephadex G-100 followed by chromatography on DEAE Sephadex A-50 and then affinity chromatography on concanavalin A (Con A) sepharose. The TAA bound to Con A sepharose was eluted by 0.5 M of alpha-CH3D-mannoside (alpha-MM) and behaved as a glycoprotein. The molecular weight determined on SDS-PAGE was about 70,000 daltons in relation to standard marker proteins. This antigen reacted in complement fixing tests with hyperimmune guinea pig sera as well as with certain human squamous cancer sera. As a control we used a human kidney carcinoma which showed no complement fixing activity in any of the procedural steps, and as control sera, guinea pig sera prepared by inoculation of uninfected guinea pig cells.