Perspectives in allergen immunotherapy: 2017 and beyond.

The Future of the Allergists and Specific Immunotherapy (FASIT) workshop provides a regular platform for global experts from academia, allergy clinics, regulatory authorities and industry to review developments in the field of allergen immunotherapy (AIT). The most recent meeting, held in February 2017, had two main themes: advances in AIT and hot topics in AIT from the regulatory point of view. The first theme covered opportunities for personalized AIT, advances in adjuvants and delivery systems, and the development of new molecules and future vaccines for AIT. Key topics in the second part of the meeting were the effects of the enactment of European Directive 2001/83 on the availability of allergens for therapy and diagnosis across the EU, the challenges of conducting Phase 3 studies in the field, the future role of allergen exposure chambers in AIT studies and specific considerations in performing AIT studies in the paediatric population. Finally, the group highlighted the forthcoming EAACI guidelines and their particular importance for the standardization of practice in the treatment of allergies. This review presents a comprehensive insight into those panel discussions and highlights unmet needs and also possible solutions to them for the future.

A survey of fumonisins, deoxynivalenol, zearalenone and aflatoxins contamination in corn-based food products in Argentina.

The presence of mycotoxins in corn-based foods available in Argentina was determined in order to make a preliminary exposure assessment. Thirty-eight samples [corn meal ('polenta') and corn flakes] of different local brands were analysed for zearalenone, deoxynivalenol and aflatoxins by TLC and fumonisins (FB1, FB2 and FB3) by HPLC. None of the 38 samples contained any detectable amount of aflatoxins (< 2 micrograms/kg), zearalenone (< 50 micrograms/kg) and deoxynivalenol (< 50 micrograms/kg). By contrast fumonisin contamination was found in 95% of the samples. The highest fumonisin levels were found in corn meal: FB1 (range positives: 60-2860 micrograms/kg; mean positive value: 556 micrograms/kg), FB2 (61-1090 micrograms/kg; 232 micrograms/kg) and FB3 (18-1015 micrograms/kg; 150 micrograms/kg). Low levels of fumonisin B1 were detected in 16/17 corn flakes samples (2-38 micrograms/kg). Total fumonisin levels in corn meal were more than 1000 micrograms/kg in 24% (5/21) of the samples. Although it is not the staple food in Argentina, maize consumption is very important, especially among children. A daily fumonisin intake of 11.3 micrograms/kg of body weight was estimated for child consumers (1-5 years old) based on an average consumption of 200 g of corn meal/day. Calculated at an average rate for all children (consumers or not) the intake estimate was 0.9 microgram/kg of body weight.

IL-1 alpha and TNF-alpha are required for IL-12-induced development of Th1 cells producing high levels of IFN-gamma in BALB/c but not C57BL/6 mice.

The development of Th1- or Th2-type responses determines the type of immune response that is elicited in response to Ag. Responsiveness to IL-12 is critical for the development of Th1-type CD4+ T cells required for cell-mediated immune responses. Addition of IL-12 to primary cultures of CD4+ T cells stimulated with OVA and splenocytes or dendritic cells resulted in the development of a Th1 phenotype with the capacity to secrete high levels of IFN-gamma upon restimulation with splenic APC. The present study shows that using dendritic cells to present Ag upon restimulation reveals a requirement for additional cofactors, including IL-1 alpha and TNF-alpha, which were provided by spleen cells but not dendritic cells. Furthermore, these cofactors are required for optimal IL-12-induced Th1 development in BALB/c but not C57BL/6 mice. This differential requirement for such cofactors in IL-12-driven Th1 development may play a role in genetic predisposition to Th1 or Th2 responses to infectious agents.

[Meningitis due to Salmonella in the neonatal period]. / Meningitis por Salmonella en período neonatal.

INTRODUCTION: Meningitis due to Salmonella is an unusual sign of salmonellosis. Usually Salmonella causes clinical disorders of the digestive tract, but on occasions, especially in babies, may cause focalized infections such as meningitis. Although meningitis due to Salmonella is unusual, it should be remembered because of its gravity, since it has a high morbimortality. It mainly affects neonatal babies and those under 4 months of age. It usually precedes or is accompanied by gastroenteritis and has a rapid clinical course. CLINICAL CASE: We present the case of a neonatal baby girl, 17 days old, who was very irritable, had liquid or semi-liquid faeces and high fever of unknown origin with poor response to antipyretic drugs. On lumbar puncture a cloudy liquid, compatible with bacterial meningitis was obtained. Treatment was therefore started immediately with intravenous ampicillin and cefotaxima. CSF culture grew Salmonella which was resistant to ampicillin but sensitive to cefotaxima. Antibiotic treatment was given for 21 days. There was excellent clinical recovery. After eight months of follow-up no sequelae have been seen and her development, both psychomotor, in height and in weight is normal for her age. CONCLUSION: Meningitis due to Salmonella is an unusual condition. Immediate, suitable treatment is essential to obtain satisfactory recovery.

Contribution of nitric oxide to exercise-induced changes in healthy volunteers: effects of acute exercise and long-term physical training.

Endothelium plays a central role in the regulation of regional blood flow through the release of certain vasoactive substances. We conducted this study to test whether an increase in the production of nitric oxide (NO) metabolites, atrial natriuretic peptide (ANP) and plasma and intraplatelet cyclic guanosine 3':5' monophosphate (cGMP) is involved in the adaptation to chronic exercise in physically trained people and in the vasodilatation induced by acute physical exercise. We studied one group of 10 trained athletes and another group of 10 untrained people. We measured plasma levels of nitrites, nitrates and cGMP and intraplatelet levels of cGMP, as an indicator of intracellular guanylate cyclase activity, and ANP before and after a maximal treadmill test. Resting cardiac rate (CR) and systolic blood pressure (SBP) were lower in the athlete group than in the control group (73.8 +/- 3.6 vs. 92 +/- 5.9; P < 0.02 and 110 +/- 2.58 vs. 118 +/- 3.27; P < 0.02 respectively). SBP did not show differences between groups after the exercise test. Diastolic blood pressure (DBP) at rest was lower in the athlete group (71 +/- 1.79 vs. 80.5 +/- 3.53; P < 0.03) and the decrease after maximal exercise was more pronounced in this group (64 +/- 2.67 vs. 74.5 +/- 3.2; P < 0.02). Basal plasma nitrites were 4.9 +/- 0.8 in the athlete group and 1.9 +/- 0.3 in the control group (P < 0.05). After exercise, test differences between groups remained (P < 0.05). Nitrates were significantly higher in the group of athletes and did not show exercise-related changes. Plasma levels of cGMP and ANP increased in both groups after the treadmill test, with no differences between groups. Among the athletes, cGMP increased from 1.11 +/- 0.1 to 2.6 +/- 0.4 (P < 0.001), whereas in the untrained group plasma cGMP rose from 1.14 +/- 0.09 to 1.86 +/- 0.2 (P < 0.01). There was a significant correlation between the increases in plasma cGMP and the atrial natriuretic peptide in both groups (r = 0.91, P < 0.0002, for athletes; and r= 0.68, P < 0.04, for control group). The intraplatelet concentration of cGMP did not show differences between groups and did not change after exercise. In conclusion, we have found increased basal levels of plasma nitrite and nitrate in trained subjects. Exercise does not produce differences in the increments of these metabolites. Therefore, we speculate the release of nitric oxide is not augmented by exercise in trained athletes.

Role of CD4 epitopes outside the gp120-binding site during entry of human immunodeficiency virus type 1.

CD4 is the primary receptor for human immunodeficiency virus (HIV). The binding site for the surface glycoprotein of HIV type 1 (HIV-1), gp120, has been mapped to the C'-C" region of domain 1 of CD4. Previously, we have shown that a mutant of rat CD4, in which this region was exchanged for that of human CD4, is able to mediate infection of human cells by HIV-1, suggesting that essential interactions between HIV and CD4 are confined to this region. Our observations appeared to conflict with mutagenesis and antibody studies which implicate regions of CD4 outside the gp120-binding site in postbinding events during viral entry. In order to resolve this issue, we have utilized a panel of anti-rat CD4 monoclonal antibodies in conjunction with the rat-human chimeric CD4 to distinguish sequence-specific from steric effects. We find that several antibodies to rat CD4 inhibit HIV infection in cells expressing the chimeric CD4 and that this is probably due to steric hinderance. In addition, we demonstrate that replacement of the rat CDR3-like region with its human homolog does not increase the affinity of the rat-human chimeric CD4 for gp120 or affect the exposure of gp41 following binding to CD4, providing further evidence that this region does not play a crucial role during entry of virus.

CD28/CTLA-4 ligands: the gene encoding CD86 (B70/B7.2) maps to the same region as CD80 (B7/B7.1) gene in human chromosome 3q13-q23.

CD86 (B70/B7.2) is an antigen of the immunoglobulin superfamily expressed on monocytes, dendritic cells and activated B, T, and natural killer cells. CD86 was recently identified as a second ligand for the T cell antigens CD28 and CTLA-4, and plays an important role in the co-stimulation of T cells in a primary immune response. We report here the assignment of the CD86 gene to human chromosome 3 using Southern blot analysis on a panel of hamster x human somatic cell hybrid genomic DNA. Fluorescence hybridization in situ on metaphase chromosomes coupled with GTG banding (G-bands by trypsin using Giemsa staining) confirmed this assignment and localized the CD86 gene to 3q13-q23 region. The CD86 gene is, therefore, located in the proximity of the CD80 (B7/B7.1) gene, the first identified ligand for CD28 and CTLA-4, previously mapped to chromosome 3q13.3-q21. Deletions, inversions and insertions of chromosome 3q21-q26, as well as translocations of 3q21 with other chromosomes have been described in many cases of acute myeloid leukemia (AML), acute non-lymphocytic leukemia (ANLL), chronic myeloid leukemia (CML) and myelodisplastic syndromes (MDS), suggesting that this region contains several genes involved in the leukemic process.

CD80 (B7) and CD86 (B70) provide similar costimulatory signals for T cell proliferation, cytokine production, and generation of CTL.

Signals initiated through both the TCR complex and CD28 are required for optimal activation of T lymphocytes. Recently, it has been demonstrated that CD28 interacts with two different ligands, designated CD80 (B7/B7-1) and CD86 (B70/B7-2). We have produced stable transfectants that express CD80, CD86, or both ligands and have examined their ability to costimulate T cell proliferation, cytokine production, and the generation of CTL. When we used small, resting human peripheral blood T cells as responders, both CD80 and CD86 transfectants efficiently costimulated anti-CD3 mAb-induced proliferation and the secretion of IL-2 and IFN-gamma. Additionally, both CD80 and CD86 transfectants were able to generate functional CTL. The magnitude and kinetics of these responses were similar, which indicates that both ligands provide efficient costimulatory signals. Because many APCs coexpress both CD80 and CD86, we compared the ability of anti-CD80 and anti-CD86 mAbs to inhibit allogeneic MLR stimulated with B lymphoblastoid cell lines and showed that it is necessary to inhibit interactions with both ligands to optimally block CD28-dependent proliferation. Given the limited homology of CD80 and CD86, it was surprising that the binding of CD28-Ig fusion protein to CD80 and that to CD86 transfectants were essentially indistinguishable. Binding of CTLA-4-Ig fusion protein to both transfectants also was quite similar, but was of higher affinity than CD28-Ig binding. Results from these studies indicate that both CD80 and CD86 are potent and similar costimulators of T lymphocytes. Therefore, the role of CD80 and CD86 in an immune response may be determined primarily by their differential expression on APC.

B70 antigen is a second ligand for CTLA-4 and CD28.

The membrane antigen B7/BB1 (refs 1, 2) is expressed on activated B cells, macrophages and dendritic cells, and binds to a counter-receptor, CD28, expressed on T lymphocytes and thymocytes. Interaction between CD28 and B7 results in potent costimulation of T-cell activation initiated through the CD3/T-cell receptor complex. Discrepancies between results with anti-CD28 and anti-B7 antibodies have suggested the existence of a second ligand for CD28 and CTLA-4 (refs 3, 6-8). We have generated a monoclonal antibody, IT2, that reacts with a 70K glycoprotein (B70). B70 complementary DNA was cloned from a B-lymphoblastoid cell line library and encodes a new protein of the immunoglobulin superfamily with limited homology to B7. B70 is expressed on resting monocytes and dendritic cells and on activated, but not resting, T, NK and B lymphocytes. IT2 substantially inhibited the binding of a CTLA4-immunoglobulin fusion protein to human B-lymphoblastoid cell lines and, together with anti-B7 antibody, completely blocked CTLA-4 binding. Further IT2 efficiently inhibited primary allogeneic mixed lymphocyte responses. These findings indicate that B70 is a second ligand for CD28 and CTLA-4 and may play an important role for costimulation of T cells in a primary immune response.

[The acute and chronic effects of 10-mg nitroglycerin patches in stable angina of effort]. / Estudio del efecto agudo y crónico de los parches de 10 mg de nitroglicerina en la angina de esfuerzo estable.

INTRODUCTION AND OBJECTIVES: To assess the anti-ischemic efficacy of nitroglycerin patches (10 mg/day), we studied, by means of serial exercise testing (Bruce protocol), 10 patients with stable effort angina in a randomized, placebo-controlled, cross-over, double-blind essay. METHODS: Patients were exercised 1, 4, 12 and 24 hours after a single patch, and 4 and 12 hours after a 48 hours therapy course. Chronic therapy was assessed after both continuous and intermittent (intermission of 12 hours) patch application. RESULTS: After single patch, time to angina and time to 1 mm ST depression were significantly increased with respect to placebo at 1-hour test (83 +/- 27 s and 119 +/- 39 s, respectively), 4-hour test (100 +/- 34 s and 87 +/- 29 s, respectively) and 12-hour test (46 +/- 15 s and 64 +/- 20 s, respectively). No effect was demonstrated at 24-hour test. After continuous treatment no differences with respect to placebo were found at any test. After intermittent treatment time to angina was prolonged (75 +/- 23 s) only at 4-hour test, and time to 1 mm ST depression at 4-hour test (61 +/- 19 s) and 12-hour test (41 +/- 14 s). CONCLUSIONS: Nitroglycerin patches improve parameters of exercise ischemia for a 12 hours period. Tolerance is developed very early and provokes absolute lack of efficacy. Tolerance can be avoided with intermittent patch application. No treatment schedule shows 24 hours efficacy.

The recognition of chimeras of rat and human CD4 by HIV-1 gp120 and by monoclonal antibodies.

The use of chimeras of rat and human CD4 to probe the HIV-1 gp120 and antibody binding properties of CD4 is reviewed. Short segments of human CD4 sequence were substituted for the equivalent regions of rat CD4 which does not bind gp120, and analysis of the properties of these chimeras established: (i) that residues 33-58 of the NH2-terminal domain of human CD4 encompass the high-affinity gp120 binding site; and (ii) that chimeras containing residues 33-62 mediate HIV-1 infection. The chimera-binding specificities of gp120 and a large panel of anti-CD4 antibodies were also determined. This allowed a critical test of the popular notion that receptor mimics appear at high frequency among antibodies elicited by immunization with receptor ligands and that anti-idiotypic antibodies can be used to identify novel receptors. The data suggest that such mimics appear infrequently, if at all, a result which is consistent with the failure of the anti-idiotype approach to identify new genes encoding receptors with prescribed functions.

Mutational analysis of the CD2/CD58 interaction: the binding site for CD58 lies on one face of the first domain of human CD2.

The adhesion interaction between the immunoglobulin superfamily molecules CD2 and CD58 (lymphocyte function-associated antigen 3) plays an important role in T cell and natural killer cell interaction with various antigen-presenting and target cells. Determination of the solution structure of rat CD2 domain 1 has allowed a model of human CD2 domain 1 to be generated, and a series of mutants based on this model have been made. Residues of domain 1 of human CD2 predicted to be solvent exposed were substituted with the equivalent residues present in the rat CD2 molecule. The ability of these mutants to mediate rosetting with human and sheep erythrocytes was studied. Results show that the binding site of CD2 for both human and sheep CD58 maps to the beta sheet containing beta strands CC'C"F and G. Residues K34 and E36 in beta strand C, R48 and K49 in beta strand C', and K91 and N92 in the loop connecting beta strands F and G are shown to be critical in the interaction. The data support the proposition that the interaction between CD2 and CD58 involves the major beta sheet face of CD2.

A rat CD4 mutant containing the gp120-binding site mediates human immunodeficiency virus type 1 infection.

CD4 is the primary receptor for the human immunodeficiency virus type 1 (HIV-1). Early mutational studies implicated a number of residues of CD4, centered in the region 41-59, in binding to gp120. However, further mutational analyses, together with studies using inhibitory antibodies or CD4-derived peptides, have suggested that other regions of CD4 are also involved in binding or postbinding events during infection. To resolve these ambiguities, we used rat CD4 mutants in which particular regions were replaced with the corresponding sequence of human CD4. We have previously shown that some of these are able to bind HIV-1 gp120, and here we test their ability to act as functional receptors. We find that the presence of human CD4 residues 33-62 is enough to confer efficient receptor function to rat CD4, and we conclude that it is unlikely that regions of CD4 outside this sequence are involved in specific interactions with HIV-1 during either infection or syncytium formation.

Site-specific glycosylation of recombinant rat and human soluble CD4 variants expressed in Chinese hamster ovary cells.

The rat and human forms of the T-cell surface glycoprotein CD4 share a common glycosylation site at the Asn270/271 position but differ with respect to the locations of the second glycosylation sites at Asn159 (rat) and Asn300 (human). The glycosylation of soluble recombinant forms of human and rat CD4 (sCD4) expressed in Chinese hamster ovary cells has been characterized. The most obvious differences between the rat and human sCD4 oligosaccharides were the greater abundance of oligomannose and hybrid oligosaccharides on rat sCD4 and the presence of oligosaccharides carrying a terminal alpha-galactose residue on human sCD4. This is the first report of the occurrence of alpha-galactose residues on a glycoprotein expressed in Chinese hamster ovary cells. Comparison of mutant rat sCD4 molecules with single glycosylation sites and glycopeptides indicated that site-specific and independent processing occurred at each glycosylation site. The glycosylation at the conserved site at Asn270 of rat sCD4 was identical to that seen for the equivalent site in human sCD4, and the oligomannose and hybrid structures were restricted to the nonconserved site at Asn159 in rat sCD4. However, there was more oligosaccharide processing at this site in a truncated form of rat sCD4 consisting of the two NH2-terminal domains. These results indicate that not only the local three-dimensional structure but also domain interactions can influence the processing at individual glycosylation sites.

Antibody and HIV-1 gp120 recognition of CD4 undermines the concept of mimicry between antibodies and receptors.

It has been proposed that antibodies can mimic the binding of a receptor to its ligand and that anti-idiotype antibodies raised against such antibodies can be used to identify the receptor. A large number of antibodies have been raised against CD4, the receptor on T cells for the envelope glycoprotein gp120 of the human immunodeficiency virus, and the site at which gp120 binds to CD4 has been delineated. It has therefore become possible to contrast the fine specificities of a natural ligand (gp120) and antibodies that interact with the receptor at the same site. Here we report that out of a panel of 225 anti-CD4 antibodies, only one showed fine binding specificity that was broadly like that of gp120, but the evidence was against this being an exact mimic. Thus the data indicate that the production of antibody mimics will occur very rarely or not at all and that the anti-idiotype approach is unlikely to be useful. This contention is supported by a review of the results of attempts to use this approach. Taking strict criteria for success, there is no example for which the anti-idiotype approach has led to the discovery of a previously undescribed receptor or other protein of interest.

[The acute and chronic effects of 50 mg of isosorbide 5-mononitrate with delayed action in patients with stable angina of effort]. / Estudio del efecto agudo y crónico de 50 mg de 5-mononitrato de isosorbida de liberación retardada en pacientes con angina de esfuerzo estable.

In 10 patients with stable effort angina and angiographically demonstrated coronary artery disease, serial exercise test were performed in order to assess the efficacy and duration of the anti-ischemic effects of a single dose (50 mg) of a sustained-release preparation of 5-isosorbide mononitrate (5-IMN). The possible presence of a tolerance phenomenon was also sought. The study was randomized, double-blind and placebo-controlled. Four hours after an acute dose of 5-IMN, time for -1 mm ST segment depression significatively increased as compared with basal test and placebo test (367 +/- 92 vs 199 +/- 87 and 250 +/- 78 sec respectively, p less than 0.0004). At the same test, total exercise time also increased from 282 +/- 92 sec (basal) and 323 +/- 91 sec (placebo) to 424 +/- 91 sec (p less than 0.008). At 12 hours test, total exercise time was also significantly increased as compared with basal test and placebo test (354 +/- 109 vs 282 +/- 92 and 291 +/- 90 sec respectively; p less than 0.01). These effects were not present when the patients were tested 24 hours after active drug administration. After daily administration of a single dose of 5-IMN during a 3 week period, 4 and 12 hours test demonstrated a persistent and significant anti-ischemic effect, similar to the acute figures. Thus, an acute dose of 50 mg of a sustained-release preparation of 5-IMN reveals significant anti-ischemic effects which remain 4 and 12 hours after drug administration. Chronic administration of the preparation for 3 weeks (single daily dose) is equally effective, without any evidence of tolerance phenomenon.

The hinge region of the CD8 alpha chain: structure, antigenicity, and utility in expression of immunoglobulin superfamily domains.

The lymphocyte surface CD8 antigen is a heterodimer with each chain containing a single Ig-related domain, a hinge-like sequence, a transmembrane segment, and a short cytoplasmic sequence. A soluble form of the rat CD8 alpha chain was produced by introducing a stop codon into the cDNA at the end of the region encoding the extracellular sequence and expressed in Chinese hamster ovary cells. sCD8 alpha was produced at 20 mg/l, and consisted of monomers, dimers, and higher aggregates. The latter could be minimized, but not eliminated, by removal of one of the two cysteine residues in the hinge region by mutation and by growth in serum-free medium. The positions of the N- and O-linked glycosylation sites and the disulphide bond in the Ig-like domain were determined. The MRC OX-8 antibody was shown to react with a region from the CD8 alpha hinge containing 24 amino acids and the antigenic determinant was sensitive to neuraminidase digestion. A construct encoding the Ig-like domain of rat CD8 alpha without the hinge was not expressed in CHO cells, indicating the importance of the hinge region for expression. It seemed possible that the CD8 alpha hinge might facilitate expression of other Ig-related domains and such expression could be detected using the MRC OX-8 antibody. To test the system cDNA constructs were made with the rat CD8 alpha hinge spliced to the V-like domain of mouse CD8 alpha, to the V alpha and V beta domains of a T lymphocyte antigen receptor, and to one or both of the Ig-like domains of the MRC OX-47 membrane antigen. All these forms were expressed as soluble proteins that were detected with the MRC OX-8 antibody. This method may prove useful for the expression of Ig superfamily domains for raising antibodies and other studies.

Construction of a binding site for human immunodeficiency virus type 1 gp120 in rat CD4.

The human immunodeficiency virus (HIV-1) infects T lymphocytes via an interaction between the virus envelope glycoprotein gp120 and the CD4 antigen of T helper cells. Previous studies demonstrated that mutations in various regions of CD4 domain 1 lead to the loss of gp120 binding. In the present study the gp120 binding site was constructed in rat CD4 by replacing rat with human CD4 sequence. A series of mutants was constructed the best of which bound gp120 with an affinity only twofold less than that of human CD4. The data indicate that the gp120 binding site of human CD4 is constituted by residues 33-58 of domain 1.