Management of patients with severe asthma: results from a survey among allergists and clinical immunologists of the Central Italy Inter-Regional Section of SIAAIC.

BACKGROUND: Asthma, and severe asthma in particular, is often managed within a specialized field with allergists and clinical immunologists playing a leading role. In this respect, the National Scientific Society SIAAIC (Società Italiana di Allergologia, Asma ed Immunologia Clinica), structured in Regional and Inter-Regional sections, interviewed a large number of specialists involved in the management of this respiratory disease. METHODS: A survey entitled "Management of patients with asthma and severe asthma" based on 17 questions was conducted through the SIAAIC newsletter in 2019 thanks to the collaboration between GlaxoSmithKline S.p.A. and the Inter-Regional Section of SIAAIC of Central Italy. RESULTS: Fifty-nine allergists and clinical immunologists participated to the survey, and 40 of them completed the entire questionnaire. Almost all of the specialists (88%) reported that asthma control was achieved in above 50% of their patients, even if only one third (32%) actually used validated clinical tools such as asthma control test (ACT). Poor adherence to inhaled therapy was recognized as the main cause of asthma control failure by 60% of respondents, and 2-5 min on average is dedicated to the patient inhaler technique training by two-thirds of the experts (65%). Maintenance and as-needed therapy (SMART/MART) is considered an appropriate approach in only a minority of the patients (25%) by one half of the respondents (52%). A high number of exacerbations despite the maximum inhalation therapy were recognized as highly suspicious of severe asthma. Patients eligible for biological therapies are 3-5% of the patients, and almost all the responders (95%) agreed that patients affected by severe asthma need to be managed in specialized centers with dedicated settings. Biological drugs are generally prescribed after 3-6 months from the initial access to the center, and once started, the follow-up is initially programmed monthly, and then every 3-6 months after the first year of treatment (96% of responders). After phenotyping and severity assessment, comorbidities (urticaria, chronic rhinosinusitis with or without nasal polyps, vasculitis, etc.) are the drivers of choice among the different biological drugs. In the management of severe asthma, general practitioners (GPs) should play a central role in selecting patients and referring them to specialized centers while Scientific Societies should train GPs to appropriately recognize difficult asthma and promote public disease awareness campaigns. CONCLUSIONS: This survey which collects the point of view of allergists and clinical immunologists from Central Italy highlights that asthma control is still not measured with validated instruments. There is a general consensus that severe asthma should be managed only in dedicated centers and to this aim it is essential to encourage patient selection from a primary care setting and develop disease awareness campaigns for patients.

Migrants and allergy: a new view of the atopic march.

Summary: Atopy is the result of the influence of environmental factors on genetically predisposed individuals. Migration flows represent an interesting model to study the possible reciprocal roles of genes and environment. In this review the following issues influencing the development of allergic sensitization and/or atopic disorders in migrants will be rooted out: 1) ethnicity, genetic polymorphisms and risk of atopy; 2) double faceted effects of parasitic infestations; 3) biodiversity loss and industrial progress. Moreover, an extensive revision of the literature about the relationship between the migratory status and allergy development is provided.

Identification of T-cell epitopes from benzylpenicillin conjugated to human serum albumin and implication in penicillin allergy.

BACKGROUND: There is in vitro evidence that T cells from allergic patients react to benzylpenicillin-human serum albumin (BP-HSA) bioconjugates. Our group has recently shown the existence of naïve CD4+ T cells recognizing BP-HSA in healthy donors. However, BP-haptenated peptides from HSA participating in the immunization of allergic patients have never been identified. The purpose of the present study is to identify immunodominant BP-haptenated peptides from HSA involved in immunization of patients to BP and to refine the frequency calculation of naïve CD4+ T cells recognizing BP. METHODS: Co-cultures were established with CD4+ T cells from non-allergic donors and mature autologous dendritic cells (DCs) loaded with BP-HSA or BP-haptenated peptides from HSA. The CD4+ T-cell response specific for BP-HSA or for individual BP-haptenated peptides was measured using an interferon-γ (IFN-γ) ELISpot assay. The frequency of BP-specific CD4+ T cells was then calculated using the Poisson distribution. BP-HSA and BP-haptenated peptides recognition by allergic patients was evaluated on peripheral blood mononuclear cells (PBMCs) using a lymphocyte transformation test (LTT). RESULTS: Results showed that BP-HSA and BP-haptenated peptides were recognized by naïve T cells from 15/16 and 13/14 tested healthy donors, respectively. Most donors responded to 3 peptides with BP covalently bound on lysines 159, 212, and 525. Two of these benzylpenicilloylated peptides (lysines 159 and 525) were also found to induce PBMCs proliferation in patients with allergic reaction to penicillins. CONCLUSION: This study identifies and characterizes for the first time the BP-haptenated peptides from HSA involved in the immunization of patients to penicillins.

Anti-infliximab IgE and non-IgE antibodies and induction of infusion-related severe anaphylactic reactions.

BACKGROUND: Infliximab is a chimeric monoclonal antibody against TNF-alpha useful in the treatment of many chronic inflammatory diseases. Severe anaphylaxis has been reported during therapy, although the exact mechanism has not been fully defined. The reactions have been related to the infliximab immunogenicity and development of specific antibodies. AIMS OF THE STUDY: Evaluation of the development of IgE and non-IgE antibodies to infliximab and their relationship with infusion reaction. METHODS: Seventy-one patients (11 reactives, 11 therapeutically nonresponders, and 49 unreactive therapeutically responders) and 20 non-infliximab-exposed control subjects (ten rheumatoid arthritis, five spondyloarthropathies, five vasculitis) were evaluated for the presence of IgE (ImmunoCAP assay), IgM, and non-isotype-specific (ELISA assays) anti-infliximab antibodies. Sera were obtained at baseline and during the course of treatment, before each infliximab infusion. RESULTS: Eleven out of 71 patients had a hypersensitivity reaction to infliximab. Non-isotype-specific anti-infliximab antibodies were detected in eight reactive and two nonresponder patients. Three patients with severe reactions displayed anti-infliximab IgE antibodies and positive skin testing. Detectable levels of anti-infliximab IgM antibodies were shown in three additional IgE- and skin testing-negative patients. IgE and IgM antibodies to infliximab were not detectable in the two nonresponder patients. Antibodies developed before the 2nd and the 3rd infusion, and their appearance was strictly related to the timing of the reaction. CONCLUSIONS: This report indicates that in some patients with infliximab-related severe reactions, IgE or IgM antibodies against infliximab were detectable. The majority of reactions could be predicted by the appearance of anti-infliximab antibodies.

Influence of total serum IgE levels on the in vitro detection of beta-lactams-specific IgE antibodies.

BACKGROUND: Allergic reactions to beta-lactams are a frequent cause of adverse drug reactions; the diagnosis is based on history, clinical examination, skin testing (prick and intradermal) and demonstration of serum-specific IgE antibodies (Abs). OBJECTIVE: We compared the diagnostic performance of the Phadia CAP system for the detection of IgE to beta-lactams carried out using the new test with cut-off limits of 0.10 kUA/L and the old test with cut-off limits of 0.35 kUA/L for positive results; subsequently, we analysed the effect of total serum IgE values and of atopic phenotype on the diagnostic performance of the tests. METHODS: The study comprised a total of 34 patients with a history of immediate adverse reactions to beta-lactams, which were confirmed by positive skin testing, and 115 control subjects with tolerance to beta-lactams over the last year. The Phadia CAP System was used for the determination of serum total and specific IgE Abs towards penicilloyl G (c1), penicilloyl V (c2), ampicilloyl (c5) and amoxicilloyl (c6). The overall diagnostic performance was assessed as a diagnostic odds ratio (DOR). RESULTS: The new test showed a higher sensitivity (85% vs. 44%) than the old test and a lower specificity (54% vs. 80%) but the overall diagnostic performance was poor (DOR 6.78 vs. 3.16, P = 0.333) in both tests. The total IgE value influences the DOR of both tests; DOR was better for values under 200 kU/L [DOR = 66; 95% confidence interval (CI): 11.3-384.1] or 500 kU/L (DOR = 45.7; 95% CI: 5.3-394.4) for the new and old tests, respectively. CONCLUSIONS: The reduction in the positive cut off value has not significantly improved the overall diagnostic performance of the beta-lactams-specific IgE assay. Because of the influence of serum total IgE on the detection of beta-lactam-specific IgE Abs, the combination of both tests is mandatory in the in vitro diagnostic approach of beta-lactam allergy.

Safety and tolerability of infliximab therapy: suggestions and criticisms based on wide clinical experience.

Infliximab, an IgG1 monoclonal chimeric antibody against tumor necrosis factor (TNF)-alpha, represents the main biological drug employed for the treatment of several immuno-mediated inflammatory disorders. Infliximab infusion can be complicated by clinically heterogeneous adverse reactions, potentially interfering with the course of treatment. We analysed the adverse events recorded in 49 patients affected by different chronic inflammatory disorders (rheumatoid arthritis, seronegative spondyloarthritis, Behçet's disease, Wegener's granulomatosis, Churg-Strauss syndrome, Cogan's disease) who were receiving a total of 709 infliximab infusions, in order to correlate the development of infliximab reactions and their features to some potential risk factors. We displayed a lower frequency of infusion reactions (1.5 percent; 11 out of 709 infusions) than those previously reported. However, patients suffering from rheumatoid arthritis and/or patients who underwent re-treatment after a long period, showed a higher prevalence of infliximab-related reactions. In conclusion, in our experience infliximab treatment is rarely complicated by adverse reactions which are, more importantly, almost always mild. Some good clinical practices, such as the low rate of infusion, pre-treatment with anti-histamine and prednisone in all patients, chronic immunosuppressive therapy and avoidance of long intervals between infusions may represent a combined useful strategy to reduce the frequency of infliximab reactions and to increase safety.

High intracytoplasmatic levels of Il-4 and Il-5 in a patient with Gleichs syndrome: case report.

Gleichs syndrome is characterized by recurrent localized angioedema, hypereosinophilia, elevated levels of IgM, rapid weight gain, itchy urticaria and fever. Little is known about the pathogenesis of this disease. Increased serum levels for IL5, IL6 and C5a have been reported before and during clinical exacerbations. In order to better understand the role of the T cells in Gleichs syndrome we analyzed the intracellular cytokine expression in CD3+ cells of a patient affected by the disease. As hypereosinophilia was documented, we asked whether IL-4 and IL-5 levels were increased, and the intracytoplasmatic expression of these Th2-cytokines was determined. The percentage of T lymphocytes (CD3-gated cells) of both CD8- and CD8+ phenotype expressing different cytokines showed an unusually high percentage of Th2-related cytokine (IL-4, IL-5 and IL-13) expressing T lymphocytes. The two new variants (myeloproliferative and lymphoproliferative) seem to account for hypereosinophilia in patients with hypereosinophilic syndrome (HES). In the lymphroliferative variant, the presence of a clonal CD3-CD4+ Th2 like lymphocyte secreting IL-4 and IL-5 in peripheral blood, may explain the hypereosinophilia and the hyper-IgE. In our study we show that the patient had a lymphoproliferative variant and her T cell had a Th2 type phenotype. Moreover, we suggest that Th2 lymphocytes may play a role in the pathogenesis of Gleichs syndrome. Further studies are needed to evaluate the possibility that a polyclonal aspecific activation of Th2 type cells can lead to hypereosinophilia, IgE production and the other manifestations typical of Gleichs syndrome.

Reversal of human allergen-specific CRTH2+ T(H)2 cells by IL-12 or the PS-DSP30 oligodeoxynucleotide.

BACKGROUND: The chemoattractant receptor homologous molecule expressed on T(H)2 cells (CRTH2) is a receptor for prostaglandin D(2), which among human T cells is selectively expressed by T(H)2 and type 2 cytotoxic effectors. OBJECTIVE: Our purpose was to assess whether the cytokine production profile of T(H)2 effectors could be reversed by exploiting their selective expression of CRTH2. METHODS: CRTH2(+) T cells were purified from the blood of allergic subjects, stimulated with the specific allergen in the absence or presence of IL-12, and assessed by flow cytometry at the single-cell level for their ability to produce IL-4 and/or IFN-gamma after antigen or polyclonal stimulation. RESULTS: Both IL-12 and the PS-DSP30 oligodeoxynucleotide enabled CRTH2(+) allergen-stimulated T(H)2 cells to produce IFN-gamma. This change in the profile of cytokine production by T(H)2 cells from allergic subjects was related to the upregulation of IL-12 receptor beta2 chain and was associated with the loss of CRTH2. CONCLUSIONS: These data demonstrate that the cytokine production pattern of fully differentiated T(H)2 effectors can be changed to a less polarized profile, thus providing the physiologic basis for new immunotherapeutic strategies in allergic disorders.

Genetic and environmental factors contributing to the onset of allergic disorders.

Evidence has been accumulated to suggest that allergen-reactive Th2 cells play a triggering role in the activation and/or recruitment of IgE antibody-producing B cells, mast cells and eosinophils, the cellular triad involved in allergic inflammation. Recently, chemokines and chemokine receptors involved in such Th2-type response have been also defined. Th2 cells represent the polarized arm of the effector-specific responses that contribute to the protection against gastrointestinal nematodes and act as regulatory cells for chronic and/or excessive Th1-mediated responses. Th2 cells are generated from precursor naive Th cells when they encounter the specific antigen in an IL-4-containing microenvironment. The question of how these Th2 cells are selected in atopic patients is also unclear. Both the nature of the T cell receptor signalling provided by the allergen peptide ligand and a disregulation of IL-4 production likely concur to determine the Th2 profile of allergen-specific Th cells, but the genetic unbalanced IL-4 production is certainly overwhelming. Some gene products selectively expressed in Th2 cells or selectively controlling the expression of IL-4 have recently been described. These findings allow to suggest that the upregulation of genes controlling IL-4 expression and/or abnormalities of regulatory mechanisms of Th2 development and/or function may be responsible for Th2 responses against allergens in atopic people. The increasing prevalence of allergy in developed countries suggests that environmental factors acting either before or after birth also contribute to regulate the development of Th2 cells and/or their function. The reduction of infectious diseases in early life due to increasing vaccinations, antimicrobial treatments as well as changed lifestyle are certainly important in influencing the individual outcome in the Th response to ubiquitous allergens. Moreover, the recent evidence that bacterial DNA or oligodeoxynucleotides containing unmethylated 'CpG motifs' promote the development of Th1 cells via the production of immunomodulatory cytokines (namely IL-12, IL-18 and IFNs) by professional antigen-presenting cells confirms previous epidemiological data. The new insight into the pathophysiology of T cell responses in atopic diseases provides exciting opportunities for the development of novel immunotherapeutic strategies.

Phosphorothioate oligodeoxynucleotides promote the in vitro development of human allergen-specific CD4+ T cells into Th1 effectors.

DNA vaccination is an effective approach in inducing the switch of murine immune responses from a Th2 to a Th1 profile of cytokine production that has been related to the activity of unmethylated CpG motifs present in bacterial, but not mammalian, DNA. We report here that some synthetic phosphorothioate, but not phosphodiester, oligodeoxynucleotides (ODNs) were able to induce B cell proliferation and to shift the in vitro differentiation of Dermatophagoides pteronyssinus group 1-specific human CD4+ T cells from atopic donors into Th cell effectors showing a prevalent Th1, instead of Th2, cytokine profile. This latter effect was completely blocked by the neutralization of IL-12 and IFN (alpha and gamma) in bulk culture, suggesting that the Th1-inducing activity of phosphorothioate ODNs was mediated by their ability to stimulate the production of these cytokines by monocytes, dendritic, and NK cells. Cytosine methylation abolished the Th1-inducing activity of ODNs; however, CpG dinucleotide-containing ODNs exhibited the Th1-shifting effect independently of the presence or the absence of CpG motifs (5'-pur-pur-CpG-pyr-pyr-3'). Moreover, the inversion of CpG to GpC resulted only in a partial reduction of this activity, suggesting that the motif responsible for the Th1-skewing effect in humans is at least partially different from that previously defined in mice. These results support the concept that the injection of allergens mixed to, or conjugated with, appropriate ODNs may provide a novel allergen-specific immunotherapeutic regimen for the treatment of allergic disorders.

Highly Th2-skewed cytokine profile of beta-lactam-specific T cells from nonatopic subjects with adverse drug reactions.

A positive lymphocyte transformation test to beta-lactams (beta-L) was found in 12 of 29 subjects with adverse drug reaction (ADR) to beta-L, irrespective of either the type of clinical manifestation or the presence of specific serum IgE. Short-term T cell lines specific for penicillin G, amoxicillin, and ampicillin could be generated only from subjects with ADR (eight with positive and one with negative lymphocyte transformation test), while streptokinase and Dermatophagoides pteronyssinus group 1 (Der p 1)-specific T cells were obtained from all these subjects, from 7 atopic Der p-sensitive donors without history of ADR and 17 healthy nonatopic donors. Streptokinase-specific T cells from all subjects showed intracellular expression of IFN-gamma with poor or no IL-4, whereas Der p 1-specific T cells exhibited IFN-gamma but low or no IL-4 expression in nonatopics, and remarkable IL-4 expression in atopic donors. By contrast, all penicillin G-, ampicillin-, and amoxicillin-specific short-term T cell lines showed high intracellular expression of IL-4, IL-5, and IL-13, but poor or no expression of IFN-gamma, thus exhibiting a clear-cut Th2 profile. Accordingly, most penicillin G-specific T cell clones derived from two subjects with ADR released high concentrations of IL-4 alone or IL-4 and IFN-gamma. These data suggest that cytokines produced by Th2 cells play an important role in all beta-L-induced ADR, even when late clinical manifestations occur and an IgE-mediated mechanism is apparently indemonstrable.

Influence of both TCR repertoire and severity of the atopic status on the cytokine secretion profile of Parietaria officinalis-specific T cells.

Peripheral blood mononuclear cells (PBMC) from both nonatopic and Parietaria officinalis-sensitive donors proliferated in response to the allergen Par o 1 and developed into Par o 1-specific T cell lines and clones, which also showed reactivity for Par o 1-derived peptides. Virtually all Par o 1-specific T cell lines and large numbers of Par o 1-specific T cell clones proliferated in response to two Par o 1 nonapeptides (p92 and p96), which probably contain immunodominant epitopes of the Par o 1 allergen. Both p92- and p96-specific T cell clones showed the ability to produce IFN-gamma, but p92-specific T cell clones produced significantly lower amounts of IL-4 and IL-5 than p96-specific T cell clones, indicating that distinct epitopes, able to elicit functionally different T helper cell responses, may coexist in Par o 1. However, p92-specific T cell clones derived from atopic subjects with high IgE serum levels (high IgE producers) secreted significantly higher amounts of IL-4 and IL-5 than corresponding T cell clones generated from nonatopic subjects or patients with low IgE serum levels (low IgE producers), whereas p96-specific T cell clones secreted high IL-4 and IL-5 concentrations irrespective of whether they derived from high or low IgE producers. The addition of IL-4 and anti-IL-12 mAb to bulk culture significantly up-regulated the development of p92-specific T cells into IL-4-producing cells, whereas the addition of IL-12 and anti-IL-4 mAb shifted the differentiation of p96-specific T cells towards IFN-gamma-producing cells. Taken together, these results suggest that the cytokine profile of allergen-specific T cells is influenced by both the T cell receptor repertoire and the severity of atopic status and can be modulated, at least in vitro, by stimulation with the specific peptide in the presence, or after removal, of appropriate cytokines.

An update on human Th1 and Th2 cells.

The existence of functionally polarized human T cell responses based on their profile of cytokine secretion in both the CD4+ T helper (Th) and the CD8+ T cytotoxic cell subset has been established. Human Th1 and Th2 cells not only produce a different set of cytokines but also exhibit distinct functional properties and preferential expression of some activation markers, such as LAG-3 and CD30, respectively. Several factors are involved in the Th cell differentiation into the polarized Th1 or Th2 pathway. They include the cytokine profile of 'natural immunity' evoked by different offending agents, the nature of the peptide ligand, as well as the activity of some costimulatory molecules and microenvironmentally secreted hormones, in the context of different host genetic backgrounds. Polarized Th1-type and Th2-type responses play different roles in protection, Th1 being effective in the defense against intracellular pathogens and Th2 against intestinal nematodes. Moreover, they are responsible for different types of immunopathological reactions. Th1 responses predominate in organ-specific autoimmune disorders, acute allograft rejection, unexplained recurrent abortions, and in some chronic inflammatory disorders of unknown etiology. In contrast, Th2 responses predominate in Omenn's syndrome, transplantation tolerance, chronic graft versus host disease, systemic sclerosis; moreover allergen-reactive Th2 cells are involved in the triggering of atopic disorders.

In vivo CD30 expression in human diseases with predominant activation of Th2-like T cells.

CD30 is a member of the tumor necrosis factor (TNF) receptor family, originally described as a marker for Hodgkin and Reed-Sternberg cells in Hodgkin's disease, which has been found to be preferentially expressed by T cells producing Th2-type cytokines. The presence of CD30 expression was assessed by both immunohistochemistry and reverse transcriptase-polymerase chain reaction in the target organs of patients with Th1- or Th2-dominated disorders. CD30 expression was found in neither the gut of patients with Crohn's disease nor in the gastric antrum of Helicobacter pylori-infected patients, where there was high interferon-gamma (IFN-gamma) expression. In contrast, high CD30 expression in the apparent absence of IFN-gamma expression was observed in the skin of patients with systemic sclerosis or chronic graft versus host disease (GVHD), which can be considered Th2-dominated disorders. Moreover, high levels of soluble CD30 were found in the serum of both systemic sclerosis and GVHD patients but not in the serum of patients suffering from multiple sclerosis, a Th1-dominated disorder. Thus, CD30 expression appears to be preferentially associated with Th2-type responses not only in vitro but also in vivo.

Type 1 T-helper cell predominance and interleukin-12 expression in the gut of patients with Crohn's disease.

Crohn's disease (CD) is a chronic bowel inflammatory disorder in which the pathogenic role of immune alterations has been suggested, but the immunologic mechanisms responsible for the inflammatory reaction are still poorly understood. We investigated the profile of cytokine secretion by T-cell clones generated from gut tissue specimens of four patients with active CD, five patients with ulcerative colitis, and four patients with noninflammatory gut disorders (NIGDs). The great majority of CD4+ T-cell clones generated from the gut of patients with CD produced high levels of interferon-gamma (IFN-gamma) but low or undetectable amounts of interleukin-4 (IL-4), whereas substantial proportions of CD4+ T-cell clones derived from the gut of patients with either ulcerative colitis or NIGDs produced IL-4 in addition to IFN-gamma. The immunohistochemical analysis revealed high numbers of activated CD4+ T cells showing IFN-gamma but not IL-4 reactivity, as well as substantial proportions of IL-12-containing macrophages, in the intestinal lamina propria and muscularis propria of patients with CD, whereas these cells were very rare or undetectable in patients with NIGDs. Culturing T cells from gut biopsy specimens of a patient with CD in the presence of a neutralizing anti-IL-12 antibody down-regulated the development of IFN-gamma-producing CD4+ T cells. These findings suggest that a critical event in the initiation of bowel inflammatory lesions in CD may involve up-regulation of IL-12 production, resulting in conditions that maximally promote type 1 T-helper immune responses.

T cells and cytokines in Crohn's disease.

Recent findings indicate that activated T lymphocytes, showing restricted T-cell receptor repertoire and a Th1-like profile of cytokine production, are responsible for macrophage activation and release of inflammatory cytokines, toxic oxygen metabolites and nitric oxide, which initiate and maintain the transmural intestinal inflammation in Crohn's disease. A critical event in the promotion of Th1-type response at gut level may involve up-regulation of IL-12 production and the breakdown of tolerance against the intestinal flora.