Allergen immunotherapy on the way to product-based evaluation - a WAO statement

Allergen immunotherapy (AIT) is widely used in clinical practice for patients with moderate to severe allergic rhinitis due to inhalant allergens and may be delivered via subcutaneous (SCIT) and sublingual routes (SLIT). However, the quality of evidence for individual AIT products is very heterogeneous, and extensions of overall conclusions ("class effects") on the efficacy and disease-modifying effects to all AIT products are unjustified. In contrast, each product needs to be evaluated individually, based on available study results, to justify efficacy and specific claims on sustained and disease modifying effects per allergen and targeted patient group (children vs. adults, allergic rhinitis vs. asthma). WAO intends to support the current development to evidence-based AIT, which ultimately will lead to a more efficacious treatment of allergic patients and the appropriate recognition of AIT

Short-term antigen presentation and single clonal burst limit the magnitude of the CD8(+) T cell responses to malaria liver stages.

Malaria sporozoites induce swift activation of antigen-specific CD8(+) T cells that inhibit the intracellular development of liver-stage parasites. The length of time of functional in vivo antigen presentation, estimated by monitoring the activation of antigen-specific CD8(+) T cells, is of short duration, with maximum T cell activation occurring within the first 8 h after immunization and lasting approximately 48 h. Although the magnitude of the CD8(+) T cell response closely correlates with the number of parasites used for immunization, increasing the time of antigen presentation by daily immunizations does not enhance the magnitude of this response. Thus, once a primary clonal burst is established, the CD8(+) T cell response becomes refractory or unresponsive to further antigenic stimulation. These findings strongly suggest that the most efficient strategy for the induction of primary CD8(+) T cell responses is the delivery of a maximal amount of antigen in a single dose, thereby ensuring a clonal burst that involves the largest number of precursors to become memory cells

Vaccination with nanoparticles combined with micro-adjuvants protects against cancer.

Induction of strong T cell responses, in particular cytotoxic T cells, is a key for the generation of efficacious therapeutic cancer vaccines which yet, remains a major challenge for the vaccine developing world. Here we demonstrate that it is possible to harness the physiological properties of the lymphatic system to optimize the induction of a protective T cell response. Indeed, the lymphatic system sharply distinguishes between nanoscale and microscale particles. The former reaches the fenestrated lymphatic system via diffusion, while the latter either need to be transported by dendritic cells or form a local depot. Our previously developed cucumber-mosaic virus-derived nanoparticles termed (CuMV <sub>TT</sub> -VLPs) incorporating a universal Tetanus toxoid epitope TT830-843 were assessed for their draining kinetics using stereomicroscopic imaging. A nano-vaccine has been generated by coupling p33 epitope as a model antigen to CuMV <sub>TT</sub> -VLPs using bio-orthogonal Cu-free click chemistry. The CuMV <sub>TT</sub> -p33 nano-sized vaccine has been next formulated with the micron-sized microcrystalline tyrosine (MCT) adjuvant and the formed depot effect was studied using confocal microscopy and trafficking experiments. The immunogenicity of the nanoparticles combined with the micron-sized adjuvant was next assessed in an aggressive transplanted murine melanoma model. The obtained results were compared to other commonly used adjuvants such as B type CpGs and Alum. Our results showed that CuMV <sub>TT</sub> -VLPs can efficiently and rapidly drain into the lymphatic system due to their nano-size of ~ 30 nm. However, formulating the nanoparticles with the micron-sized MCT adjuvant of ~ 5 μM resulted in a local depot for the nanoparticles and a longer exposure time for the immune system. The preclinical nano-vaccine CuMV <sub>TT</sub> -p33 formulated with the micron-sized MCT adjuvant has enhanced the specific T cell response in the stringent B16F10p33 murine melanoma model. Furthermore, the micron-sized MCT adjuvant was as potent as B type CpGs and clearly superior to the commonly used Alum adjuvant when total CD8 <sup>+</sup> , specific p33 T cell response or tumour protection were assessed. The combination of nano- and micro-particles may optimally harness the physiological properties of the lymphatic system. Since the nanoparticles are well defined virus-like particles and the micron-sized adjuvant MCT has been used for decades in allergen-specific desensitization, this approach may readily be translated to the clinic

IL-2 Mediates CD4+ T Cell Help in the Breakdown of Memory-Like CD8+ T Cell Tolerance under Lymphopenic Conditions

Background: Lymphopenia results in the proliferation and differentiation of naïve T cells into memory-like cells in the apparent absence of antigenic stimulation. This response, at least in part due to a greater availability of cytokines, is thought to promote anti-self responses. Although potentially autoreactive memory-like CD8 + T cells generated in a lymphopenic environment are subject to the mechanisms of peripheral tolerance, they can induce autoimmunity in the presence of antigen-specific memory-like CD4 + T helper cells. Methodology/Principal Findings: Here, we studied the mechanisms underlying CD4 help under lymphopenic conditions in transgenic mice expressing a model antigen in the beta cells of the pancreas. Surprisingly, we found that the self-reactivity mediated by the cooperation of memory-like CD8 + and CD4 + T cells was not abrogated by CD40L blockade. In contrast, treatment with anti-IL-2 antibodies inhibited the onset of autoimmunity. IL-2 neutralization prevented the CD4-mediated differentiation of memory-like CD8 + T cells into pathogenic effectors in response to self-antigen cross-presentation. Furthermore, in the absence of helper cells, induction of IL-2 signaling by an IL-2 immune complex was sufficient to promote memory-like CD8 + T cell self-reactivity. Conclusions/Significance: IL-2 mediates the cooperation of memory-like CD4 + and CD8 + T cells in the breakdown of crosstolerance, resulting in effector cytotoxic T lymphocyte differentiation and the induction of autoimmune disease

Fibroblast cell-based therapy prevents induction of alopecia areata in an experimental model

YesAlopecia areata (AA) is an autoimmune hair loss disease with infiltration of proinflammatory cells into hair follicles. Current therapeutic regimens are unsatisfactory mainly because of the potential for side effects and/or limited efficacy. Here we report that cultured, transduced fibroblasts, which express the immunomodulatory molecule indoleamine 2,3-dioxygenase (IDO), can be applied to prevent hair loss in an experimental AA model. A single intraperitoneal (IP) injection of IDO-expressing primary dermal fibroblasts was given to C3H/HeJ mice at the time of AA induction. While 60–70% of mice that received either control fibroblasts or vehicle injections developed extensive AA, none of the IDO-expressing fibroblast-treated mice showed new hair loss up to 20 weeks post injection. IDO cell therapy significantly reduced infiltration of CD4+ and CD8+ T cells into hair follicles and resulted in decreased expression of TNF-α, IFN-γ and IL-17 in the skin. Skin draining lymph nodes of IDO fibroblast-treated mice were significantly smaller, with more CD4+ CD25+ FoxP3+ regulatory T cells and fewer Th17 cells than those of control fibroblast and vehicle-injected mice. These findings indicate that IP injected IDO-expressing dermal fibroblasts can control inflammation and thereby prevent AA hair loss.Canadian Institutes of Health Researches (Funding Reference Number: 134214 and 136945)

Degradation of millimolar concentration of the herbicide dalapon (2,2-Dichloropropionic Acid) by rhizobium Sp isolated from soil

The herbicide Dalapon is widely used in agricultural areas and is persistent in ground water. A Rhizobium sp. was able to grow at 0.2 mM 2,2-dichloropropionic acid (2,2DCP), which was 100-fold lower than the concentration of the substrate routinely used. Apparently, no new dehalogenases are required to allow growth on this low concentration of 2,2DCP as judged by electrophoretic mobility of dehalogenase proteins in native-PAGE analysis and protein separation by anion-exchange column chromatography. The kinetic analysis suggested that the known dehalogenases were able to act efficiently on low concentrations of haloalkanoic acids. The amount of each dehalogenase, from cells grown on low substrate concentration was different compared to that seen at 20 mM 2,2DCP due to complex regulatory controls, which respond to the growth environment

Naphthalene Complexes. V. Arene Exchange Reactions in Naphthalenechromium Complexes

Di-?6-naphthalenechromium(0) (1) reacts at 150°C with benzene to yield (?6-naphthalene)(?6-benzene)chromium(0) (3) in 76% yield. In the presence of THF, 1 undergoes Lewis base catalyzed arene exchange at 80°C. Reactions of 1 with substituted arenes yield the mixed sandwich complexes 4 and 6–10 (arene = 1,4-C6H4Me2, 1,3,5-C6H3Me3, C6Me6, 1,4-C6H4(OMe)2, 1,4-C6H4F2 and 1,4-C10H6Me2). In all but one case (with 1,4-dimethylnaphthalene) exchange of a single naphthalene ligand is observed. In marked contrast to the lability of 1, dimesitylenechromium(0) (5) is inert to arene displacement in benzene up to 240°C. The molecular structure of 3 has been determined by X-ray crystallography. The crystal data are as follows: a 7.784(1), b 13.411(2), c 22.772(5) Å, Z = 8, space group Pbca. The structure was refined to a Rw value of 0.043. The naphthalene ligand in 3 is nearly planar and parallel to the approximately eclipsed benzene ring. Metal atom-ring distances are 1.631(9) and 1.611(4) Å for naphthalene and benzene, respectively. Catalyzed and uncatalyzed naphthalene exchanges in the sandwich complex are compared to the analogous reactions with the Cr(CO)3 complex 2. Naphthalene exchange in 2 in benzene is 103 to 104 times faster than arene exchange in other arenetricarbonylchromium compounds. The mild conditions for Lewis base catalyzed naphthalene exchange make 2 a good precursor of other arenetricarbonylchromium compounds. Examples include the Cr(CO)3 complexes of styrene, benzocyclobutene, 1-ethoxybenzocyclobutene, 1,8-dimethoxy-9,10-dihydroanthracene and 1,4-dimethylnaphthalene

Profiling of proteolytic activities secreted by cancer cells using phage display substrate technology

Although the cellular steps required for metastasis are similar for all cancer cells, proteases involved in this process and their expression levels vary greatly between different cancer types. Thus, the identification of these proteolytic activities represents a crucial issue in the understanding of cancer development. Until now, phage display substrate technology has been successfully employed for the characterization of purified proteases but was never used with a mix of proteases. In the present work, we report an easy protocol to identify multiple proteolytic activities secreted by cancer cells. We selected substrates from a phage display library of high diversity using secreted media of three established prostate cancer cell lines (DU-145, LNCaP and PC-3) with variable degrees of invasive capability. Some of these selected peptide substrates were hydrolyzed by the secreted proteins of all three prostatic cancer cell lines, demonstrating similarities in their proteolytic activities. On the other hand, a few substrates were cancer cell specific, indicating differences in the phenotypes of protease expression in prostate cancer. This work reports for the first time the selection of substrates from a mix of proteases using phage display technology and opens a new avenue for the direct identification of proteolytic activities for tumor extracts