Surgical glove powders bind latex antigens.

Latex surgical gloves have recently been identified as a potential source of allergens. Much of the current information suggests that the soluble proteins in latex may cause significant reactions in sensitive individuals. The starch powders used as a lubricant on some latex gloves have also been identified as potential allergens in some patients. In this study, we determined these powders to act as potential carriers of latex allergens. We have produced a polyclonal antiserum to be used as a reagent to study latex proteins. By Western blot analysis, we identified a significant interaction between latex proteins and starch powders. The binding of latex proteins to starch particles results in a glove particle that may have an increased potential to act as an allergen. The latex protein-starch particles represent a potential mechanism for exposure and sensitization of health care workers to latex allergens. Elimination of these particles from the operating room should reduce the route of sensitization and the potential for adverse reactions to latex.     

A 48-kilodalton Mycoplasma fermentans membrane protein induces cytokine secretion by human monocytes.

Mycoplasma fermentans is one of several Mycoplasma species that have been reported to stimulate tumor necrosis factor (TNF) secretion from monocytes. This activity has been associated primarily with the mycoplasma membrane fraction. In this article, we have characterized a membrane protein that stimulates TNF and interleukin 1 beta secretion. The TNF-releasing activity partitioned into the Triton X-114 detergent phase, suggesting that the molecules is hydrophobic. The secretion of TNF is elevated in the presence of serum, which suggests that a serum component may play a role in the interaction between this mycoplasma protein and monocytes. Treatment of monocytes with monoclonal anti-CD14 antibody had no effect on the levels of TNF-releasing activity. By using the monocyte Western blot (immunoblot) technique, we have determined the molecular mass of the active molecule to be 48 kDa. This molecule appears to be distinct from the recently described family of variable lipoproteins of M. fermentans. Mycoplasma particulate material treated with proteinase K lost all inducing activity, whereas lipoprotein lipase-treated samples retained some level of activity.     

The latex allergen hev B 5 is an antigen with repetitive murine B-cell epitopes

BACKGROUND: Hev b 5 is a potent latex allergen. In this study, we characterize the linear B-cell epitopes for three monoclonal antibodies (mAbs) to Hev b 5. METHODS: The mAbs included 2 IgG1 (6A10, 3G3) and 1 IgG2b (6F6) isotypes. We used SPOTscan analysis with overlapping octapeptides to identify the binding regions for the antibodies and then methionine substitution analysis to further define the critical amino acids (aa) in each epitope. Site-directed mutagenesis was used to selectively eliminate the IgG binding for each epitope and single and multiple mutations were expressed as recombinant GST fusion proteins. Antibody recognition of the mutant proteins was determined by inhibition ELISA. RESULTS: All three mAbs recognized the same aa sequence by SPOTs analysis with slight variations, and this epitope was repeated 3 times in the Hev b 5 sequence; APETEK (63-68), PAEGEK (120-125), and PAEEEK (126-131). Sequential methionine substitution by SPOTsalogue identified K68, E122, and K131 as critical aa in each epitope to change by site-directed mutagenesis. Inhibition ELISA with the mutant proteins indicated that epitope 126-131 was the dominant epitope, but mutation of epitope 120-125 was also required to eliminate mAb reactivity to Hev b 5. The antibodies did not appear to recognize the epitope 63-68 in the recombinant fusion protein. CONCLUSIONS: We identified an immunodominant B-cell epitope in Hev b 5 that is repeated 3 times within the sequence, making Hev b 5 multivalent. Well-characterized monoclonals recognizing repeated epitopes would be a good choice for immunodetection of Hev b 5 in glove extracts where individual epitopes could get altered by the manufacturing process.     

Hev b 8, the Hevea brasiliensis latex profilin, is a cross-reactive allergen of latex, plant foods and pollen

BACKGROUND: Plant profilins are important pan-allergens. They are responsible for a significant percentage of pollen-related allergies. Limited information is available about their involvement in the latex-fruit syndrome and the cross-reactivities between latex and pollen. We aimed to clone and express the Hevea brasiliensis latex profilin to investigate its allergological significance and serological cross-reactivities to profilins from plant foods and pollens. METHODS: A DNA complementary to messenger RNA (cDNA) coding for the Hevea latex profilin, Hev b 8, was amplified by polymerase chain reaction from latex RNA. Recombinant (r)Hev b 8 was produced in Escherichia coli and used to screen sera from 50 latex- allergic health care workers (HCWs) with well-documented histories of food and pollen allergy and 34 latex-allergic spina bifida (SB) patients. The cross-reactivity of natural Hev b 8 and rHev b 8 with other plant profilins was determined by ELISA inhibition assays. A three-dimensional homology model of Hev b 8 was constructed based on known profilin structures. RESULTS: The cDNA of Hev b 8 encoded a protein of 131 amino acids with a predicted molecular mass of 14 kD. Twelve of the 50 HCWs and 2 of the 34 SB patients were sensitized to Hev b 8. All Hev b 8-sensitized patients showed allergic symptoms to pollen or plant foods. Cross-reactivities between profilins of latex, pollen and plant food were illustrated by their ability to inhibit IgE binding to rHev b 8. Homology modeling of Hev b 8 yielded a structure highly similar to Bet v 2, the birch pollen profilin, with the most distinct differences located at the N-terminus. CONCLUSIONS: We conclude that primary sensitization to latex profilin in the majority of cases takes place via pollen or food profilins. Additionally, pollinosis and food-allergic patients with profilin-specific IgE can be at risk of developing latex allergy.     

Synergistic induction of interleukin-1 by endotoxin and toxic shock syndrome toxin-1 using rat macrophages.

We studied interleukin-1 (IL-1) secretion by rat peritoneal exudate macrophages stimulated with purified toxic shock syndrome toxin-1 (TSST-1). TSST-1 was observed to be a more potent inducer of IL-1 than was endotoxin. The induction of IL-1 secretion by TSST-1 was not blocked by polymyxin B but could be blocked by monoclonal antibodies directed against TSST-1. Synergistic induction of IL-1 was observed when the cells were stimulated with TSST-1 and endotoxin. The sequence of addition was found to be important for the synergistic response. Enhanced IL-1 production was observed only when macrophages were exposed to endotoxin before or simultaneously with TSST-1. Prior exposure of macrophages to TSST-1 had no enhancing effect on endotoxin-induced IL-1 secretion. We conclude that stimulation of the macrophage by endotoxin enhances the responsiveness of the cells to TSST-1 and may thereby play a role in the pathogenesis of toxic shock syndrome.     

Modulation of rat lymphocyte transformation by plasma fibronectin

Purified rat plasma fibronectin (Fn) was studied for its ability to influence nonspecific lymphocyte transformation to various mitogens. Fn added to lymph node cells (LNC) stimulated with phytohemagglutinin (PHA), concanavalin A (Con A), or bacterial lipopolysaccharide (LPS) resulted in a noncytotoxic dose-dependent inhibition of the blastogenic response. Effective inhibition (greater than 50%) of LNC responses to PHA and LPS occurred with concentrations of Fn that ranged from 10-50 micrograms/culture. The proliferative response to Con A was less affected by the presence of Fn: 50% inhibition was observed only with the highest concentration of Fn studied. Fn at low concentrations was more effective than Fn-depleted plasma in inhibiting lymphocyte responses to PHA. Optimal expression of the regulatory activity of Fn occurs during and following the peak proliferative period for both PHA and LPS responses. In order to evoke maximum inhibition it is necessary for Fn to be present within 12 hours following stimulation of LNC with mitogen. Inhibition does not appear to be the result of Fn-mitogen complexes which reduce the amount of mitogen available for stimulation, since increasing concentrations of either PHA or LPS did not significantly reduce the inhibitory effect. Furthermore, inhibition was not influenced by the concentration of fetal calf serum present in the culture medium. Thus, Fn may function as an important nonspecific immunoregulatory factor at inflammatory sites and in areas of tissue repair.     

ELISA inhibition assay for the quantitation of antigenic protein in natural rubber latex

Evaluating allergenicity of natural rubber latex (NRL) products is essential for the successful reduction of the consumer's exposure to potentially allergenic NRL proteins. We have developed an ELISA Inhibition method for the quantitation of extractable proteins from NRL products which has good sensitivity and specificity. The method utilizes rabbit anti-NRL protein serum as a detection mechanism. The source of NRL proteins for immunization and as a reference protein in the assay is ammoniated raw latex (AL). By comparison with the Western blot analysis of the rabbit sera, it appears that the ELISA detects most of the latex proteins present in extracts. To investigate, further, this assumption, we compared the ELISA Inhibition test with two other methods for total protein measurement. We also compared the values generated by the ELISA Inhibition test with other immunological methods for quantitation of antigenic and allergenic proteins. Comparisons were performed with 15 extracts from randomly selected surgical and examination gloves. The samples were coded separately for each test to insure the objectivity of evaluation. The antigenic protein values obtained by the ELISA Inhibition test correlated best with the HPLC amino acid analysis (correlation coefficient (cc) = 0.79) and with the LEAP assay (cc = 0.97). The antigenic protein levels obtained by the ELISA test were 3-10 times lower than those obtained by the HPLC analysis. A lesser correlation was observed with the Modified Lowry assay (cc = 0.45), which is likely due to chemical interference bias in the Lowry method. Our findings suggest that the antigenic proteins measured by the ELISA Inhibition test described here more closely represent the measure of the total protein content in the extracts. It is important to note that the relative values obtained by this method are lower than the values obtained by other total protein methods, possibly due to a large number of small peptides present in NRL products, that would only be measured by the chemical method.     

Prevalence of allergic sensitization to indoor fungi in West Virginia

Exposure to indoor fungi is of growing concern in residential and occupational environments in the United States. The purpose of this study was to determine the prevalence of sensitization to common indoor fungal species in an atopic population. We evaluated 102 patients (73 female and 29 male patients) for immunoglobulin E (IgE) reactivity to a panel of skin-prick test (SPT) reagents used for routine allergy testing. Patients also were tested for six additional fungi that are common indoor contaminants. All patients had symptoms consistent with allergic rhinitis or asthma. The presence of specific IgE against the fungal species was determined using immunoblotting. Of the 102 eligible patients, 68% had at least one positive skin test. The most prevalent positive SPTs were to dust mites, cats, vernal grass, and short ragweed. Overall, 21/102 (21%) patients with asthma or allergic rhinitis were skin test positive to at least one fungal extract. Of the patients with a positive SPT to fungi, 12/21 (58%) showed sensitivity to one or more of the newly tested species; most notably Trichoderma viride (8%), Chaetomium globosum (7%), Paecilomyces variotii (7%), and Acremonium strictum (6%). Immunoblotting revealed specific IgE against a number of protein bands belonging to these fungal species. The prevalence of fungal sensitization was common, particularly for indoor fungal contaminants that are not routinely included in SPT panels. Cross-reactivity with other fungi may partially explain our results; however, skin testing for these indoor fungi may provide useful diagnostic information.