Trastuzumab and lapatinib modulation of HER2 tyrosine/threonine phosphorylation and cell signaling.

Cell surface transmembrane signaling receptors EGFR, HER3, and HER4 are activated by ligand-binding-mediated dimerization and phosphorylation. In contrast, HER2 amplification promotes signaling by increasing homo/heterodimerization and ligand binding. Trastuzumab or lapatinib therapy of HER2 amplicon-positive breast cancer cells induces growth inhibition and intracellular growth pathway signaling modulation. The mechanism(s) by which trastuzumab, an IgG1 humanized antibody, induces modification of cell signaling upon binding to an extracellular determinant on a ligand-less "receptor" membrane protein remains unexplained. Using immune detection methodology comprised of antibodies detecting three distinct domains of HER and five tyrosine/threonine phosphorylation sites, the effects of trastuzumab and lapatinib were defined during steady state growth inhibition. Here, we show that lapatinib markedly reduces HER2 tyrosine phosphorylation, while in contrast, no change in tyrosine phosphate levels is detected during trastuzumab-mediated cell growth inhibition. As trastuzumab treatment does not change either the steady state HER2 protein levels or HER2 mRNA, these findings argue against an antibody-dependent alteration in internalization kinetics. We further show a sequenced relationship between lapatinib-induced blockage of phosphorylation (6-8 h) and induction of delayed cell death (5-6 days), while trastuzumab-treated cells showed no evidence of cell death up to 9 days. Taken together, these results demonstrate that inhibition of HER2 phosphorylation by lapatinib is sufficient to induce apoptosis while trastuzumab binding to the extracellular HER2 domain may function by sterically modulating the detection of phosphate moieties by cytoplasmic signal transducers. This investigation also detected a 20 kD protein, which is down-regulated by lapatinib, further demonstrating the complexity of this signal transduction system.

Glove powder's carrying capacity for latex protein: analysis using the ASTM ELISA test.

Glove donning powders carry latex proteins and disperse them into the workplace environment. We have used the ASTM D6499 ELISA to quantify the amount of latex antigen bound to and carried by glove powders. We could differentiate between a small amount of protein actually bound to the powders and a larger amount carried by the powder. Enhanced binding of a major allergen, Hev b 5, to the starch powders was demonstrated by Western blot. The D6499 ELISA is able to measure total latex antigen, soluble and powder bound, simultaneously without the need to centrifuge the samples.

Latex protein: a hidden "food" allergen?

Avoidance of latex allergens is the primary method to prevent adverse reactions. Natural rubber latex is found in many different products in both the health care industry and in modern society, and consequently results in unexpected exposures of sensitized individuals. The use of latex gloves by food handlers provides one potential route for inadvertent exposure to latex allergens. In this study we have used two immunological methods to determine whether latex proteins are transferred to foods following contact with latex gloves. Direct transfer of latex protein to cheese was visualized using a modified immunoblot method. Sliced cheese was touched with a gloved finger. A nitrocellulose membrane was applied to lift the potential fingerprints and a rabbit anti-latex antiserum was used to visualize the transfer of any latex finger-prints. After handling lettuce with gloves, transferred protein was recovered by extracting the lettuce and quantified using an inhibition ELISA for latex proteins. Fingerprints of latex protein were readily detectable on cheese after contact with powdered latex gloves, but not with vinyl gloves. Furthermore, powdered latex glove use resulted in measurable amounts of latex protein on lettuce with an exposure-dependent increase in the latex protein levels. Lettuce alone or lettuce handled with vinyl gloves was negative for latex protein. The use of latex gloves by food handlers is the source of an indirect food additive in the form of latex proteins. It is recommended that food handlers avoid the use of latex gloves to eliminate inadvertent exposure of latex-sensitive individuals.

Cloning and characterization of a latex allergen (Hev b 7): homology to patatin, a plant PLA2.

We previously identified a 46-kD protein allergen in latex as having amino acid sequence homology to the patatin gene family. The objective of this study was to characterize this protein by molecular techniques. RNA was isolated from the latex or leaf material from Hevea brasiliensis and from potato tubers. Specific polymerase chain reaction (PCR) primers were designed from the amino acid sequence and reverse transcriptase (RT)-PCR amplified a specific product from latex RNA that was subsequently cloned and sequenced. This product was 1493 bp in length with an 1167 bp open reading frame. The deduced amino acid sequence encodes for a 389 aa protein, pI 4.82 with 43% homology to tobacco patatin. Northern analysis of potato, Hevea leaf, and latex RNA demonstrated the message to be most abundant in latex, weakly present in Hevea leaf, but no hybridization occurred with potato RNA. Patatin has lipid acyl-transferase and PLA2-like activity, suggesting it plays a role as a defence-related protein. Other defence-related proteins in latex such as hevein, glucanase, and hevamine are also allergens. Increased production of defence-related proteins as a result of increased tapping of the rubber trees to meet the demand for latex may explain the increased allergenicity of latex.

IgE epitope analysis of the hevein preprotein; a major latex allergen.

We have previously identified the hevein preprotein as a common allergen for latex allergic healthcare workers. The B cell epitopes in the hevein protein that are recognized by IgE of latex-allergic individuals have not been identified. In this study, we examined the hevein preprotein using epitope mapping. Overlapping synthetic peptides of 10 amino acids (two aa overlap) were synthesized on a derivatized cellulose membrane using Fmoc chemistry. The peptide spots were probed with pooled sera from 10 latex-allergic patients, and the IgE-reactive peptides identified with anti-IgE MoAbs. We identified six B cell epitopes within the full length hevein preprotein which bound IgE from latex-allergic patients. Two were located in the N-terminal 5-kD hevein domain and four were observed in the 14-kD C-domain. A broad epitope was located between the N-terminal amino acids 13-24. This epitope had nearly complete homology to wheat germ agglutinin (WGA). Immunological cross-reactivity to WGA was confirmed by Western blot analysis with purified WGA, and this reactivity could be inhibited by latex proteins or WGA. Of the five remaining epitopes, four had homologies to other proteins in the pathogenesis-related family of plant proteins (PR-4). The data demonstrate that hevein has multiple IgE epitopes. The significant homology of these epitopes to a broad family of plant defence proteins further explains the increased prevalence of food allergies in latex-allergic individuals.

Measurement of natural rubber proteins in latex glove extracts: comparison of the methods.

BACKGROUND: Health care workers and individuals with frequent contact with latex are at risk for latex protein allergy. OBJECTIVE: The purpose of this study was to compare several established methods for measuring protein in extracts from latex-containing medical devices. METHODS: Extracts from latex gloves were analyzed for natural rubber proteins using a modified Lowry assay and two different immunochemical assays. The immunochemical methods were competitive inhibition assays that employed either immune rabbit serum or human serum with antibodies directed against natural rubber proteins. RESULTS: Seventy extracts representing five different brands of gloves from four manufacturers were analyzed. A good linear correlation (R = 0.88) was found between the immunoassay methods. Correlation to the modified Lowry method was not possible because many of the samples were below the limit of detection for the Lowry assay. Reference extracts and antisera were further characterized by Western blot analysis. The data demonstrate that the proteins recognized by rabbit antisera and the proteins recognized by human IgE are similar. The greatest difference in the immunochemical assays appears to be the relative binding of the antibody sources to high and low molecular weight natural rubber proteins in the reference extracts. CONCLUSIONS: The immunochemical assays are specific for latex proteins and provide a more sensitive and biologically relevant method for determining protein levels in latex medical products.

Mycoplasma hyorhinis molecules that induce tumor necrosis factor alpha secretion by human monocytes.

Mycoplasma hyorhinis has been shown to induce the secretion of tumor necrosis factor alpha (TNF-alpha) from monocytes. To identify the molecules responsible for this activity, we separated sonicated M. hyorhinis lysate material by centrifugation at 100,000 x g into soluble (S) and particulate (P) fractions. The fractions were assayed for TNF-alpha-inducing activity by the L929 bioassay. Both the soluble and particulate fractions were able to induce TNF-alpha in roughly equal amounts. The optimum dose for both fractions was 1 micrograms/ml. Proteinase K treatment of either fraction eliminated the activity, suggesting that a protein component is involved in induction. Phase partitioning into Triton X-114 aqueous (A) and detergent (D) phases showed that the soluble fraction was composed of 80% aqueous-phase proteins, while the particulate fraction was > 75% detergent-phase proteins. All four fractions (SA, SD, PA, and PD) were able to induce TNF-alpha release. Treatment with NaIO4 to remove carbohydrate reduced the inducing activity of the SA phase by 80%, whereas that of the other fractions was unaffected by this treatment. The M(r)S of the inducing activity were determined by the monocyte Western (immunoblot) technique. The SA phase activity was associated with a single periodate-sensitive peak of 69 to 75 kDa. The two detergent phases had similar profiles of inducing activity, containing four peaks of activity. These peaks corresponded to 48 to 52, 43 to 45, 39 to 40, and 31 to 32 kDa. The PA fraction also contained four peaks of activity, 69 to 75, 55 to 57, 48 to 52, and 39 to 40 kDa. Thus, both a protein and glycan moiety from M. hyorhinis are capable of inducing TNF-alpha release from human monocytes.

Identification of a 46-kD latex protein allergen in health care workers.

Latex allergy is an occupational hazard for health care workers. Extractable latex proteins are known to be allergenic, but most latex allergens have not been specifically identified. The purpose of this study was to characterize the IgE response of latex-allergic patients to latex proteins and to identify common protein allergens. Serum was obtained from 40 individuals who were skin test-positive to latex; 85% were health care workers. Western blots for IgE reactivity were performed using both ammoniated (AL) and non-ammoniated (NAL) latex proteins and IgE-reactive NAL proteins were analysed by microsequence analysis. The patients were grouped according to common patterns of reactivity. Pattern 1, the most common pattern of reactivity (9/40 patients) recognized two protein bands in both NAL and AL at 46 and 110 kD. A second, heterogeneous pattern of reactivity (pattern 2) recognized a diffuse pattern of polypeptides in the AL preparation. The n-terminal amino acid sequences for allergens at 14, 18, 29, 46 and 110 kD were determined. Sequence analysis identified the 14-kD and 18-kD allergens as the hevein proprotein. The 46-kD and 110-kD had identical sequences which were unique from known latex proteins. We conclude that multiple latex proteins are allergens with hevein preprotein and a previously unidentified 46/110-kD protein being commonly recognized in health care workers.

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.

Flash sterilization and instrument tape--an experimental study.

A letter appearing in the AORN Journal questioned whether flash sterilization is appropriate for instruments coded with color identification tape. The reply stated that porous, colored tape required a longer time to penetrate and sterilize the area beneath, and thus if it should peel off, the zone beneath might not be sterile. This conclusion was, as far as we can determine, reached by intuitive reasoning and not by experimental evidence. Therefore, the following experimental approach was undertaken to test the hypothesis. Spores on discs were placed between the color-code tape and a metal instrument. Exposure to heat (135 degrees C) and time (3 min.) was in a gravity displacement sterilizer. We then determined whether spore kill has been achieved. The test organism was B. stearothermophilus. None of the discs that were in contact with the instruments while being sterilized showed any growth. Thus, it appears that sterility can be achieved on the instrument surfaces that are beneath color-code tape in three minutes.

Differentiation-inducing cytokine P48 exists in a membrane-associated form.

P48 is a recently described 48-kDa differentiation-inducing cytokine isolated from the culture medium of the human leukemia line Reh. P48 induces differentiation and cytolytic activity in the promyelocytic cell line HL-60, and stimulates the release of TNF-alpha and IL-1 from peripheral blood monocytes. In further studies designed to examine the biosynthesis and function of P48, surface immunofluorescence flow cytometry analysis as well as 125I surface labeling and immunoprecipitation, revealed the presence of P48 on the surface of Reh cells. Triton X-114-treated Reh cells were partitioned into detergent and aqueous phases and separated by SDS-PAGE. Western blot analysis revealed that P48 partitioned exclusively into the detergent phase, suggesting an integral membrane association. Reh cells fixed with paraformaldehyde, but not K562 or P815, were able to stimulate the release of TNF-alpha from peripheral blood monocytes in a manner similar to that of secreted P48. Isolated plasma membranes from Reh cells could also stimulate TNF-alpha release. This TNF-alpha-releasing activity could be removed from detergent solubilized Reh membranes by immunoaffinity chromatography on an anti-P48 column. This study suggests that, in addition to being secreted into the culture medium, P48 is expressed on the surface of Reh cells in a biologically active form. The membrane form of P48 may be 1) a final maturation step before secretion or 2) a cell membrane-associated form that may be analogous to the membrane forms of TNF-alpha and IL-1.

Synthetic peptides to identify antigenic determinants on Epstein-Barr virus gp350/220.

We synthesized three peptides, MA1 - Thr19-Val28(+Tyr) -, MA2 - Ser807-Ala816-, and MA3-Ser718-Glu729(+Tyr) from the sequence of Epstein-Barr virus gp350/220 and immunized rabbits with these peptides. Rabbit antisera to the peptides had antipeptide radioimmunoassay titers of 1:400 for anti-MA1, 1:200 for anti-MA2, and 1:1600 for anti-MA3. The anti-MA1 serum recognized gp350/220 in Western blotting to SDS-electrophoresed proteins from 12-O-tetradecanoylphorbol-13-acetate- and n-butyrate-treated B95-8 cells, but anti-MA2 and MA3 sera did not. None of the sera reacted with gp350/220 by membrane or cytoplasmic immunofluorescence or by immunoprecipitation of Triton X-100 solubilized proteins.

Replication of an RK2 miniplasmid derivative in vitro by a DNA/membrane complex extracted from Escherichia coli: involvement of the dnaA but not dnaK host proteins and association of these and plasmid-encoded proteins with the inner membrane.

A DNA/membrane complex extracted from a miniplasmid derivative of the broad host range plasmid RK2 cultured in Escherichia coli capable of synthesizing new plasmid supercoiled DNA in vitro was treated with antibodies that were made against or reacted with the dnaA and dnaK host-encoded proteins, respectively. Anti-dnaA protein antibody inhibited total plasmid DNA synthesis significantly and the synthesis of supercoil plasmid DNA almost completely. In contrast, anti-dnaK protein antibody and nonimmune serum had little or no effect on total plasmid DNA synthesis. Both proteins were found to be present in the inner but not outer membrane fraction of E. coli. A variety of miniplasmid-encoded proteins which had previously been found in the DNA/membrane complex have also been localized to the inner but not outer membrane fraction. These include an essential initiation protein of 32 kDa (and an overlapping protein of 43 kDa coded for by the same gene), as well as a 30-kDa protein that may be linked to incompatibility functions. Various extraction methods were used to distinguish between the associated and the integral nature of the plasmid-encoded proteins. The results demonstrated that the essential replication proteins (32 and 43 kDa) as well as the 30-kDa protein was tightly bound to the inner membrane. Computer analysis of the amino acid sequence of the 32 (and 43)-kDa protein revealed a hydrophobic region that is only half that normally required to span the membrane. Other interactions are discussed with respect to attaching this protein to the membrane.