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ABSTRACTIntroduction: Allergen-specific immunotherapy (AIT) is indicated for patients with allergic asthma and/or allergic rhinitis, and can be implemented by either subcutaneous injection (SCIT) or sublingual administration (SLIT). AIT reduces asthma symptoms, lowers the use of pharmacologic controller therapy, and decreases the need for rescue medications. SLIT appears to be safer than SCIT, but SCIT seems to be more efficacious and acts earlier in allergic asthmatic children.Areas covered: This review looks at the pathobiology of allergic asthma as well as the role of regulatory T and B cells in allergen tolerance. It also reviews the immunological mechanisms underlying the clinical effects induced by AIT in allergic asthmatic children.Expert opinion: AIT is very effective in allergic asthmatic children, who can significantly benefit from this particular type of immunotherapy in order to achieve a better control of their disease. AIT is also capable of modifying the natural history of allergic asthma. Furthermore, AIT can potentially represent a valuable therapeutic tool within the context of precision medicine, as recombinant allergen technology might allow the creation of targeted extracts able to be effective against specific proteins to which individual asthmatic children are allergic, thus helping to implement a personalized approach to treatment.

This study aims to evaluate the concentration of -defensin human neutrophil peptide-1 (HNP-1; a peptide released by neutrophils) in salivary fluid among postmenopausal women and to compare bone mineral density among postmenopausal women with different concentrations of this peptide.

Efficient mesoporous proteolytic networks: N‐(2‐Aminoethyl)‐3‐aminopropyl‐derivatized mesoporous silica particles, with a 4.4 nm pore diameter, efficiently promote a comfortable entrapment of trypsin into mesoporous networks, thus providing very fast and effective proteolytic activity. The rapid digestion and highly confident protein identification (100 % sequence coverage in just 1 min) offers a practical and convenient alternative to conventional in‐solution digestion.

Elevated Nuclear Factor κB (NF-κB) levels have been reported in multiple myeloma cells derived from patients relapsing after chemotherapy. In the search of an in vitro a model with molecular features similar to relapsing lesions, we focused our attention on an IL-6 autocrine human myeloma cell line (U266), characterized by apoptosis resistance due to up-regulation of two constitutive signaling pathways: NF-κB and STAT-3. NF-κB activity was inhibited with proteasome inhibitory agents, such as PS-341 and Withaferin A, with an IKK inhibitor (Wedelolactone) or with the adenoviral vector HD IκBαmut-IRES-EGFP encoding a mutant IκBα protein, resistant to proteasomal degradation. We observed that the NF-κB intracellular dislocation at the beginning of the treatment affected therapeutic effectiveness of PS-341, Withaferin A and Wedelolactone; interestingly, the adenoviral vector was highly effective in inducing apopotosis even with NF-κB being predominantly nuclear at the time of infection. We also observed that U266 treated with the Interleukin-6 antagonist Sant7 exhibited reduced STAT3 activity and preferential cytoplasmic NF-κB location; moreover they became capable of undergoing apoptosis mainly from the G1 phase. Adenoviral vector treated U266 have NF-κB localized completely in the cytoplasm and also showed down-regulation of nuclear phospho STAT-3. Finally, combined targeting of NF-κB and STAT3 signaling pathways was the most effective treatment in inducing apoptosis. These findings suggest that combined NF-κB κB and STAT3 targeting warrants further investigations in other apoptosis resistant MM cell lines as well as in suitable MM animal models.

Somatic mutation (E17K) that constitutively activates the protein kinase AKT1 has been found in human cancer patients. We determined the role of the E17K mutation of AKT1 in lung cancer, through sequencing of AKT1 exon 4 in 105 resected, clinically annotated non-small cell lung cancer specimens. We detected a missense mutations G->A transition at nucleotide 49 (that results in the E17K substitution) in two squamous cell carcinoma (2/36) but not in adenocarcinoma (0/53). The activity of the endogenous kinase carrying the E17K mutation immunoprecipitated by tumour tissue was significantly higher compared with the wild-type kinase immunoprecipitated by the adjacent normal tissue as determined both by in vitro kinase assay using a consensus peptide as substrate and by in vivo analysis of the phosphorylation status of AKT1 itself (pT308, pS473) or of known downstream substrates such as GSK3 (pS9/S22) and p27 (T198). Immunostaining or immunoblot analysis on membrane-enriched extracts indicated that the enhanced membrane localization exhibited by the endogenous E17K-AKT1 may account for the observed increased activity of mutant E17K kinase in comparison with the wild-type AKT1 from adjacent normal tissue. In conclusion, this is the first report of AKT1 mutation in lung cancer. Our data provide evidence that, although AKT1 mutations are apparently rare in lung cancer (1.9%), the oncogenic properties of E17K-AKT1 may contribute to the development of a fraction of lung carcinoma with squamous histotype (5.5%).

The bone marrow micro-environment produces a number of different survival factors that are important for the malignant growth and drug resistance of multiple myeloma (MM) cells. One of the main factors reported to be essential for survival and growth of MM cells in some experimental systems is IL-6. Therefore, the development and testing of substances that interfere with IL-6 or IL-6 receptor (IL-6R) function might have therapeutic value for the treatment of MM. We analyzed the effect of the IL-6R antagonist SANT-7 on growth and survival of the IL-6–dependent MM cell lines INA-6 and XG-1 as well as primary MM cells from 7 patients co-cultured with bone marrow stromal cells (BMSCs). In particular, we were interested in whether SANT-7 enhances the growth-inhibitory effects of dexamethasone (Dex) and all-trans-retinoic acid (ATRA). None of the drugs when tested as a single substance, including SANT-7, induced major growth inhibition if MM cells were co-cultured with primary human BMSCs. However, when Dex and ATRA were given in combination with SANT-7, strong growth inhibition was achieved in cell lines and primary MM cells. This effect was due to cell-cycle arrest and induction of apoptosis. © 2001 Wiley-Liss, Inc.

Helper-dependent (HD) adenoviral (Ad) vectors, in which all viral coding sequences are deleted, have been generated. We show here that intravenous delivery of a mouse EPO (mEPO) expression cassette cloned in an HD vector in immunocompetent mice is effective and long lasting, but not permanent. A precise dose–response relationship between the dose of injected virus and stable EPO serum levels was observed, together with a 100-fold increase in gene expression per infectious particle when compared with a first-generation Ad vector bearing the same cassette. As a direct consequence, therapeutic increases in hematocrit that lasted more than 6 months were achieved with minute amounts of virus, which caused no detectable production of neutralizing antibodies. Intravenous readministration of the HD-mEPO vector in the same mice was as effective as in naive animals without any need for prior immunosuppression. Finally, HD-mEPO injection in subtotally nephrectomized rats improved the anemic status induced by surgery. HD Ad vectors are thus excellent tools for EPO gene therapy.

Seroepidemiology and polymerase chain reaction studies have strongly suggested that human herpesvirus type 8 (HHV-8) is associated with Kaposi's sarcoma, Castleman's disease, and body cavity-based lymphoma. The genome of HHV-8 harbors a viral analogue of the interleukin-6 (IL-6) gene. The amino acid sequence of the viral IL-6 (vIL-6) protein is 24.7% identical to human IL-6 (hIL-6). IL-6 as a B-cell growth and differentiation factor is known to play an essential role in the pathophysiology of B-cell tumors. Thus, it seems possible that virus-encoded IL-6 contributes to malignant growth of HHV-8–positive B-cell lymphatic tumors. We have tested a preparation of HHV-8–derived IL-6 for the ability to promote the proliferation of the human myeloma cell line INA-6, which is strictly dependent on exogenous IL-6 for growth and survival. Viral IL-6 significantly induced DNA synthesis of INA-6 cells, but required much more protein on a weight basis when compared with hIL-6 for maximal proliferation. The proliferative effect of vIL-6 was almost completely inhibited by a combination of anti–IL-6 receptor (IL-6R) and anti-gp130 antibodies or IL-6R superantagonist Sant7 and anti-gp130 antibodies. This report demonstrates that vIL-6 has proliferative activity on human cells and that the IL-6R and gp130 are involved in vIL-6 signaling in the myeloma cell line INA-6.

Interleukin-6 (IL-6) is the major growth factor for myeloma cells and is believed to participate in the pathogenesis of chronic autoimmune diseases and postmenopausal osteoporosis. IL-6 has been recently shown to possess three topo-logically distinct receptor binding sites: site 1 for binding to the subunit specific chain IL-6Rα and sites 2 and 3 for the interaction with two subunits of the signaling chain gp130. We have generated a set of IL-6 variants that behave as potent cytokine receptor super-antagonists carrying substitutions that abolish interaction with gp130 at either site 2 alone (site 2 antagonist) or at both sites 2 and 3 (site 2 + 3 antagonist). In addition, substitutions have been introduced in site 1 that lead to variable increases in binding for IL-6Rα up to 70-fold. IL-6 super-antagonists inhibit wild-type cytokine activity with efficacy proportional to the increase in receptor binding on a variety of human cell lines of different origin, and the most potent molecules display full antagonism at low molar excess to wild-type IL-6. When tested on a representative set of IL-6–dependent human myeloma cell lines, although site 2 super-antagonists were in general quite effective, only the site 2 + 3 antagonist Sant7 showed antagonism on the full spectrum of cells tested. In conclusion, IL-6 super-antagonists are a useful tool for the study of myeloma in vitro and might constitute, in particular Sant7, effective IL-6 blocking agents in vivo.

The helical cytokine interleukin-6 (IL-6) assembles a multiprotein receptor complex. The starting event in the activation of intracellular signaling is the binding of the IL-6/IL-6Rα subcomplex to two gp130 chains. The homodimerization of gp130 is triggered by two distinct and independent regions of IL-6 called sites 2 and 3. Several IL-6 antagonists have been obtained that affect signaling, but not IL-6•IL-6Rα subcomplex formation. In this paper, we analyze in detail the impact of these antagonists on gp130 binding and dimerization and show that each signaling variant affects gp130 dimerization in vitroand that biological activity on cells decreases in precise parallel to the decrease in gp130 dimerization in vitro. All IL-6 antagonists can be classified into two groups, mapping at either site 2 or 3 in correspondence to their mode of interaction with gp130. We found that site 3 is a large region, which includes residues at the beginning of helix D spatially flanked by residues in the putative AB loop and located at one extremity of the cytokine 4-helix bundle. Interestingly, in leukemia inhibitory factor, another cytokine that signals through gp130, site 3, is topologically conserved but has evolved to bind leukemia inhibitory factor receptor.

A potent interleukin‐6 (IL‐6) antagonist (Sant 5), which binds tightly to the IL‐6α receptor but has impaired gp130 heterodimerization, has been developed recently by site‐directed mutagenesis of human IL‐6. We report here that Sant 5 inhibits IL‐6‐stimulated osteoclast‐like multinucleated cell (MNC) formation in human marrow cultures but also inhibits the stimulatory effects of IL‐1 or tumor necrosis factor alpha (TNF‐α in these cultures. We further show that a neutralizing antibody to IL‐6 also inhibits the stimulatory effects of IL‐1 or TNF‐α in these cultures. In contrast, Sant 5 had no effect on parathyroid hormone related protein (PTHrP) or 1,25‐dihydroxyvitamin D3stimulated MNC formation in human marrow cultures. Transfection of a human marrow stromal cell line, which normally induces osteoclast formation through production of IL‐6, with the Sant 5 cDNA driven by a cytomegalovirus (CMV) promoter blocked the capacity of these cells to stimulate osteoclast‐like cell formation. These Sant 5 transfected cells and conditioned media from these cells also inhibited the stimulatory effects of the parent cell line on MNC formation. These data suggest that IL‐6 mediates the effects of IL‐1 and TNF on human osteoclast formation, but in contrast to murine systems, does not mediate the effects of PTHrP. These data further demonstrate that stromal cells transfected with the Sant 5 cDNA can constitutively produce high levels of the IL‐6 antagonist and inhibit osteoclast formation in vitro.

Neutralization of cytokine activity by monoclonal antibodies or receptor antagonists is beneficial in the treatment of immune and neoplastic diseases, but the necessity for continuous parenteral delivery of these anticytokine agents poses considerable practical limitations. A viable alternative is to induce a neutralizing antibody response. Using transgenic mice with high circulating levels of human interleukin-6 (hlL-6), we show that injection of the hlL-6 receptor antagonist Santi (an IL-6 variant with seven amino-acid substitutions) induces a strong anti-hlL-6 antibody response. The elicited antibodies bind circulating hlL-6 with very high affinity, totally masking it, and neutralize hlL-6 bioactivity both in vitro and in vivo

A potent interleukin‐6 (IL‐6) antagonist (Sant 5), which binds tightly to the IL‐6α receptor but has impaired gp130 heterodimerization, has been developed recently by site‐directed mutagenesis of human IL‐6. We report here that Sant 5 inhibits IL‐6‐stimulated osteoclast‐like multinucleated cell (MNC) formation in human marrow cultures but also inhibits the stimulatory effects of IL‐1 or tumor necrosis factor alpha (TNF‐α in these cultures. We further show that a neutralizing antibody to IL‐6 also inhibits the stimulatory effects of IL‐1 or TNF‐α in these cultures. In contrast, Sant 5 had no effect on parathyroid hormone related protein (PTHrP) or 1,25‐dihydroxyvitamin D3stimulated MNC formation in human marrow cultures. Transfection of a human marrow stromal cell line, which normally induces osteoclast formation through production of IL‐6, with the Sant 5 cDNA driven by a cytomegalovirus (CMV) promoter blocked the capacity of these cells to stimulate osteoclast‐like cell formation. These Sant 5 transfected cells and conditioned media from these cells also inhibited the stimulatory effects of the parent cell line on MNC formation. These data suggest that IL‐6 mediates the effects of IL‐1 and TNF on human osteoclast formation, but in contrast to murine systems, does not mediate the effects of PTHrP. These data further demonstrate that stromal cells transfected with the Sant 5 cDNA can constitutively produce high levels of the IL‐6 antagonist and inhibit osteoclast formation in vitro.

The ribosomal RNA genes ofTriturus vulgaris meridionalis are clustered at variable and often multiple chromosomal loci. The rDNA repeats exhibit only a limited and discrete length heterogeneity which is accounted for by the non-transcribed spacer (NTS). Interestingly, sequences homologous to the NTS are clustered outside the ribosomal loci. Clones containing such ‘non ribosomal sequences’ have been isolated from a genomic library ofT. v. meridionalis and analyzed by restriction mapping. These sequences appear to consist mostly of repetitive Bam HI fragments ranging from 500 bp to 1000 bp. The Bam HI fragments are internally repetitious and highly homologous to each other.

No relevant conflicts of interest to declare.

Abstract 2825

Genomlc Southern blots showed there are only 14 to 20 copies of U3 snRNA genes per somatic genome in Xenopus laevis, unlike the highly repetitive, tandem arrangement of other snRNA genes in this organism. Sequencing of two U3 snRNA genes from λ clones of a genomlc library revealed striking similarity upstream, but much more divergence downstream. Consensus motifs common to other U snRNA genes were also found: a distal sequence element (DSE, octamer motif at - 222 to - 215), a proximal sequence element (PSE, at - 62 to - 52) and a 3′ Box (15 or 16 bp downstream of the U3 genes). The DSE of mammals also has an Inverted CCAAT motif specific for U3 snRNA genes, and we find this Is conserved in the amphibian U3 snRNA genes. The Xenopus inverted CCAAT motif is exactly one helical turn further upstream of the octamer motif than its mammalian counterpart, suggesting interaction of putative transcription factors bound to these motifs. Mutation of the inverted CCAAT motif and part of an adjacent Sp1 site greatly depresses transcription of the mutant U3 snRNA gene in Xenopus oocytes, implying a role in transcriptional efficiency. Electrophoretic mobility shift assays implicate transcription factor binding to this region.