Suggestions for the assessment of the allergenic potential of genetically modified organisms

The prevalence of allergic diseases has been increasing continuously and, accordingly, there is a great desire to evaluate the allergenic potential of components in our daily environment (e.g., food). Although there is almost no scientific evidence that genetically modified organisms (GMOs) exhibit increased allergenicity compared with the corresponding wild type significant concerns have been raised regarding this matter. In principle, it is possible that the allergenic potential of GMOs may be increased due to the introduction of potential foreign allergens, to potentially upregulated expression of allergenic components caused by the modification of the wild type organism or to different means of exposure. According to the current practice, the proteins to be introduced into a GMO are evaluated for their physiochemical properties, sequence homology with known allergens and occasionally regarding their allergenic activity. We discuss why these current rules and procedures cannot predict or exclude the allergenicity of a given GMO with certainty. As an alternative we suggest to improve the current evaluation by an experimental comparison of the wild-type organism with the whole GMO regarding their potential to elicit reactions in allergic individuals and to induce de novo sensitizations. We also recommend that the suggested assessment procedures be equally applied to GMOs as well as to natural cultivars in order to establish effective measures for allergy prevention.     

A2 adenosine receptors inhibit calcium influx through L-type calcium channels in rod photoreceptors of the salamander retina.

Presynaptic inhibition is a major mechanism for regulating synaptic transmission in the CNS and adenosine inhibits Ca(2+) currents (I(Ca)) to reduce transmitter release at several synapses. Rod photoreceptors possess L-type Ca(2+) channels that regulate the release of L-glutamate. In the retina, adenosine is released in the dark when L-glutamate release is maximal. We tested whether adenosine inhibits I(Ca) and intracellular Ca(2+) increases in rod photoreceptors in retinal slice and isolated cell preparations. Adenosine inhibited both I(Ca) and the [Ca(2+)]i increase evoked by depolarization in a dose-dependent manner with approximately 25% inhibition at 50 microM. An A2-selective agonist, (N(6)-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)-ethyl]adenosine) (DPMA), but not the A1- or A3-selective agonists, (R)-N(6)-(1-methyl-2-phenylethyl)adenosine and N(6)-2-(4-aminophenyl)ethyladenosine, also inhibited I(Ca) and depolarization-induced [Ca(2+)]i increases. An inhibitor of protein kinase A (PKA), Rp-cAMPS, blocked the effects of DPMA on both I(Ca) and the depolarization-evoked [Ca(2+)]i increase in rods. The results suggest that activation of A2 receptors stimulates PKA to inhibit L-type Ca(2+) channels in rods resulting in a decreased Ca(2+) influx that should suppress glutamate release.     

Papillary carcinoma of the thyroid: hepatocyte growth factor (HGF) stimulates tumor cells to release chemokines active in recruiting dendritic cells

Tissue distribution of dendritic cells was investigated in eight cases of papillary carcinoma of the thyroid using immunohistochemistry. Most dendritic cells had an immature phenotype (CD1a++, CD11c+, CD40+, CD86-, HLA-DR-) and were located at the invasion edge of the tumor. This pattern of distribution was profoundly different from that of CD68+ macrophages, which were evenly distributed throughout the tumor. The ability of tumor cells to release chemotactic factors active on dendritic cells was investigated in primary cultures of the same cases of papillary carcinoma, and was compared to that of the corresponding normal thyroid cells obtained from the tumor-free contralateral lobe. Chemotactic activity of culture supernatants was tested against dendritic cells in a chemotaxis chamber. It was found that papillary carcinoma cells were active in releasing chemotactic activity, that hepatocyte growth factor (HGF; 100 ng/ml) or interleukin (IL)-1beta (10(3) U/ml) induced a fourfold increase in the amount of chemotactic activity released, and that normal thyroid cells obtained from the same patients were as effective as tumor cells. Characterization of chemokines at RNA level revealed that unstimulated cells contain large amounts of IL-8 and monocyte chemotactic protein (MCP)-1 RNAs, and that stimulation with HGF or IL-1beta induced RNAs for regulated upon activation normal T expressed and secreted (RANTES), macrophage inflammatory protein (MIP)-3alpha, interferon-gamma-inducible protein 10 (IP-10), and, to a lesser extent, MIP-1alpha and MIP-1beta. The possibility that HGF/Met interaction has a biological role in vivo was investigated in serial sections of six tumors immunostained for CD1a+, Met protein, and HGF. It was found that all six tumors were intensely and diffusely positive for Met protein, that HGF staining was present in tumor cells of the advancing edge, and that HGF+/Met+ tumor cell nests were infiltrated by CD1a+ dendritic cells. The foregoing observations are consistent with the possibility that HGF stimulation of Met+ tumor cells is one of the molecular mechanisms involved in the recruitment of dendritic cells.     

Molecular epidemiology of macrolide resistance in neonatal bloodstream isolates of group B streptococci

Pulsed-field gel electrophoresis (PFGE) was performed on 122 neonatal bloodstream isolates of group B streptococci (GBS) to further examine the relationship between macrolide resistance and serotype V GBS (GBS-V). Over one-third (35%) of macrolide-resistant GBS belonged to a single PFGE subtype of GBS-V, which was also the most common GBS-V subtype noted in previous Centers for Disease Control and Prevention surveillance studies. Erm methylase (ermA and ermB) was the most common resistance mechanism detected, present in 12 of 20 macrolide-resistant GBS.     

Relationship between poliovirus and echovirus 6 antigens II. Antibody Patterns in sera from patients with poliovirus infections

Summary Sera from 40 patients infected with poliovirus 1, 2 or 3 were surveyed for antibody activity against antigens of these viruses and echovirus 6 by immunodiffusion. Antibody activity common to the virion (N) antigens of two or three poliovirus types were encountered in a few sera; cross absorption tests confirmed the findings. The same sera also contained antibodies directed to one single type of N antigen, this was, however, not that of the infecting virus. Antibodies to echovirus 6 N was found only in one serum.Antibodies to empty capsid antigens (H) of polioviruses and echovirus 6 were demonstrable in the majority of sera; the occurrence of common antibodies to the H antigens of echovirus 6 and poliovirus H antigens was evident.The antibody patterns of different sera varied. Even early during the course of the disease heterologous anti H activity was associated with 7 S type antibodies, whereas the early homotypic anti N activity was present in the 19 S serum fraction. The data indicate that the heterologous antibody responses seem to represent booster responses from earlier experience with similar antigens.Antibodies of the 19 S and 7 S type showed precipitating activity whereas CF activity was found with certainty only in the 7 S fractions of sera.     

Detection of Campylobacter spp. in chicken fecal samples by real-time PCR

A real-time PCR assay for detecting thermophilic Campylobacter spp. directly in chicken feces has been developed. DNA was isolated from fecal material by using magnetic beads followed by PCR with a prealiquoted PCR mixture, which had been stored at -18 degrees C. Campylobacter could be detected in less than 4 h, with a detection limit of 100 to 150 CFU/ml, in a fecal suspension. A bacterial internal control was added before DNA extraction to control both DNA isolation and the presence of PCR inhibitors in the samples. The assay was performed on 111 swab samples from a Danish surveillance program and compared to conventional culturing using selective enrichment. There was no statistically significant difference in performance between real-time PCR and culture by selective enrichment, and the diagnostic specificity was 0.96 with an agreement of 0.92. Therefore, the assay should be useful for screening poultry flocks for the presence of Campylobacter.     

Construction and characterization of affibody-Fc chimeras produced in Escherichia coli

Affibody-Fc chimeras were constructed by genetic fusion between different affibody affinity proteins with prescribed specificities and an Fc fragment derived from human IgG. Using affibody ligands previously selected for binding to respiratory syncytial virus (RSV) surface protein G and Thermus aquaticus (Taq) DNA polymerase, respectively, affibody-Fc fusion proteins showing spontaneous Fc fragment-mediated homodimerization via disulfide bridges were produced in Escherichia coli and affinity purified on protein A Sepharose from bacterial periplasms at yields ranging between 1 and 6 mg/l culture. Further characterization of the chimeras using biosensor technology showed that the affibody moieties have retained high selectivities for their respective targets after fusion to the Fc fragment. Avidity effects in the target binding were observed for the affibody-Fc chimeras compared to monovalent affibody fusion proteins, indicating that both affibody moieties in the chimeras were accessible and contributed in the binding. Fusion of a head-to-tail dimeric affibody moiety to the Fc fragment resulted in tetravalent affibody constructs which showed even more pronounced avidity effects. In addition, the Fc moiety of the chimeras was demonstrated to be specifically recognized by anti-human IgG antibody enzyme conjugates. One application for this class of "artificial antibodies" was demonstrated in a western blotting experiment in which one of the anti-RSV surface protein G affibody-Fc chimeras was demonstrated to be useful for specific detection of the target protein in a complex background consisting of a total E. coli lysate. The results show that through the replacement of the Fab portion of an antibody for an alternative binding domain based on a less complicated structure, chimeric proteins compatible with bacterial production routes containing both antigen recognition domains and Fc domains can be constructed. Such "artificial antibodies" should be interesting alternatives to, for example, whole antibodies or scFv-Fc fusions as detection devices and in diagnostic or therapeutic applications.     

T- and B-Cell Immune Responses of Patients Who Had Undergone Colectomies to Oral Administration of Salmonella enterica Serovar Typhi Ty21a Vaccine

The capacity of an oral live attenuated Salmonella enterica serovar Typhi Ty21a vaccine to induce immune responses in patients who had undergone colectomies because of ulcerative colitis was evaluated, and these responses were compared with those of healthy volunteers. Purified CD4⁺ and CD8⁺ T cells from peripheral blood were stimulated in vitro by using the heat-killed Ty21a vaccine strain, and the proliferation and gamma interferon (IFN-γ) production were measured before and 7 or 8 days after vaccination. Salmonella-specific immunoglobulin A (IgA) and IgG antibody responses in serum along with IgA antibody responses in ileostomy fluids from the patients who had undergone colectomies were also evaluated. Three doses of vaccine given 2 days apart failed to induce proliferative T-cell responses in all the six patients who had undergone colectomies, and increases in IFN-γ production were found only among the CD8⁺ cells from three of the patients. In contrast, both proliferative responses and increased IFN-γ production were observed among CD4⁺ and CD8⁺ T cells from 3 and 6 of 10 healthy volunteers, respectively. Salmonella-specific IgA and/or IgG antibody responses in serum were observed for five (56%) of nine patients who had undergone colectomies and in 15 (88%) of 17 healthy volunteers. In ileostomy fluids, significant anti-Salmonella IgA antibody titer increases were detected in six (67%) of nine patients who had undergone colectomies. The impaired T- and B-cell immune responses found after vaccination in the circulation of patients who have undergone colectomies may be explained by a diminished colonization of the Ty21a vaccine strain due to the lack of a terminal ileum and colon.     

The in vitro transformation of thymocytes and lymphocytes from humans, rabbits and guinea-pigs and from thymomas

The response of human, rabbit and guinea-pig thymocytes in culture to blastogenic stimulants such as phytohaemagglutinin (PHA) and staphylococcal filtrate (SF) was usually considerably less than that seen with equivalent numbers of peripheral blood and lymph node lymphocytes.