Mimosa pudica has been used for decades in SE-Asia to treat inflammation. However mimosine, a well-known toxic molecule, was found invariably in the natural plant extracts. By plant cell culture metabolic engineering, we were able to inhibit intracellularly mimosine expression. It was under HPLC detection level i.e 5 ng per gr dry weight (DW) of cell extract (E13) compared to 2160 ng per gr DW in natural leaves extract (E11). (i) RAW264.7 murine macrophages were incubated with 50 μg DW/ml of E13 or E11, or 1μM Dexamethasone for 1 H and were stimulated with LPS for 24 H. Quantifications of NO, IL-6 and TNFα were performed by Griess, ELISA and Luminex techniques respectively. Similarly to Dexa, E13 and E11 inhibit NO and IL-6. TNFα expression was reduced by E13 and Dexa, in contrast it was increased by E11. (ii) In a Th2 induced model, NHEK cells were incubated with 0, 0.75 and 1.5 mg DW/ml of WO1 a similarly prepared E13 extract for 1 H and were stimulated by (poly I:C + TNFα + IL-4 + IL-13) for 24 H. mRNA quantification of AMP, cytokines and chemokines by RT-qPCR has shown: a dose dependent increase of S100A7 , RNASE7, DEF304A and a significant decrease of IL-1β and CXCL-8. Finally, TSLP and TLR-3 both known to be involved in pruritus were highly down-regulated. In conclusion, mimosine free plant cells culture extracts exhibit anti-inflammatory activities and counteract Th2 responses. This high added value eco extracts could be potentially incorporated in topic formulations for treating inflammatory skin diseases.

Respiratory syncytial virus (RSV) is an important cause of severe upper and lower respiratory disease in infants and in the elderly. There are 2 main RSV subtypes A and B. A recombinant vaccine was designed based on the central domain of the RSV-A attachment G protein which we had previously named G2Na (aa130-230). Here we evaluated immunogenicity, persistence of antibody (Ab) response and protective efficacy induced in rodents by: (i) G2Na fused to DT (Diphtheria toxin) fragments in cotton rats. DT fusion did not potentiate neutralizing Ab responses against RSV-A or cross-reactivity to RSV-B. (ii) G2Nb (aa130-230 of the RSV-B G protein) either fused to, or admixed with G2Na. G2Nb did not induce RSV-B-reactive Ab responses. (iii) G2Na at low doses. Two injections of 3 µg G2Na in Alum were sufficient to induce protective immune responses in mouse lungs, preventing RSV-A and greatly reducing RSV-B infections. In cotton rats, G2Na-induced RSV-reactive Ab and protective immunity against RSV-A challenge that persisted for at least 24 weeks. (iv) injecting RSV primed mice with a single dose of G2Na/Alum or G2Na/PLGA [poly(D,L-lactide-co-glycolide]. Despite the presence of pre-existing RSV-specific Abs, these formulations effectively boosted anti-RSV Ab titres and increased Ab titres persisted for at least 21 weeks. Affinity maturation of these Abs increased from day 28 to day 148. These data indicate that G2Na has potential as a component of an RSV vaccine formulation.

What's known on the subject? and What does the study add? Pervasive inflammatory infiltrates, mainly composed of chronically activated T cells and monocytes/macrophages, have been observed in benign prostatic hyperplasia (BPH). Permixon®, a hexanic lipidosterolic extract of Serenoa repens (hexanic LSESr) used to treat urinary dysfunction in BPH patients, has anti‐inflammatory activities. This paper provides new insights into the anti‐inflammatory properties of Permixon®. We report that hexanic LSESr inhibits early steps of leukocyte infiltration in vitro by downregulating MCP‐1/CCL2 and VCAM‐1 expression. OBJECTIVE To investigate the mechanisms by which hexanic lipidosterolic extract of Serenoa repens (hexanic LSESr) may prevent leukocyte infiltration in benign prostatic hyperplasia by studying its impact on monocyte chemoattractant protein 1/chemokine (C‐C motif) ligand 2 (MCP‐1/CCL2) and vascular cell adhesion molecule 1 (VCAM‐1) expression in vitro . MATERIALS AND METHODS After pretreatment with hexanic LSESr, human prostate (epithelial and myofibroblastic) cells and vascular endothelial cells were stimulated with proinflammatory cytokines. MCP‐1/CCL2 and VCAM‐1 mRNA expression was quantified by real‐time PCR. ELISA kits were used to determine MCP‐1/CCL2 levels in culture supernatants and VCAM‐1 expression in living cells. RESULTS Hexanic LSESr reduced MCP‐1/CCL2 mRNA levels in both epithelial (BPH‐1) and myofibroblastic (WPMY‐1) prostate cell lines. Hexanic LSESr downregulated MCP1/CCL2 secretion by WPMY‐1 cells in a concentration‐dependent manner, more efficiently than Serenoa repens extracts obtained by supercritical carbon dioxide extraction. Hexanic LSESr inhibited tumour‐necrosis‐factor‐α‐induced MCP‐1/CCL2 secretion by the human vascular endothelial cell line EAhy.926, as well as surface VCAM‐1 protein expression, in a concentration‐dependent manner. CONCLUSIONS Hexanic LSESr impedes key steps of monocyte and T cell attraction and adherence by inhibiting MCP‐1/CCL2 and VCAM‐1 expression by human prostate and vascular cells in an inflammatory environment. These findings provide new insights into the anti‐inflammatory effects of the hexanic lipidosterolic extract of Serenoa repens , Permixon®, in benign prostatic hyperplasia.

The limited response rate of cancer patients treated with dendritic cell (DC)-based vaccines indicates that vast improvements remain necessary. In many murine tumour models it has been demonstrated that the use of innate triggers (e.g. TLR triggers) in the maturation of DC results in higher efficacy. However, as few of these innate triggers are generated clinical grade, there remains a great necessity to fill the gap between fundamental mouse studies and a clinical trial in humans. In the present study we used a TLR2/4-agonist (FMKp which is available clinical grade) in combination with IFN-gamma (FI-cocktail) in the maturation of elutriated monocyte-derived DC and compared it with the most used DC in current clinical trials (TNF-alpha/PGE-2, i.e. TP-cocktail). In addition to the assessment of CD4(+) T cell polarizing capacity, we compared the quantity and intrinsic quality of induced CD8(+) T cells of 2 different DC maturation protocols with all cells from the same donor. Besides differences in the cytokine profile, which could be coupled to increased Th1 and Th17 polarization, we demonstrate in this study that FMKp/IFN-gamma matured DC are twice as effective in inducing cytotoxic T cells against known tumor antigens. Both DCs induced phenotypically equivalent effector memory CD8+ T cells that did not show a significant difference in their intrinsic capacity to kill tumor cells. These findings point to the therapeutic applicability of FI-DC as superior inducers of functional antigen-specific T cells. Their increased chemokine secretion is suggestive of a mechanism by which these DC may compensate for the limited migration observed for all ex vivo cultured DC when applied in patients.

Human respiratory syncytial virus (hRSV) is one of the most common causes of respiratory infection in infants and the elderly. Previous attempts to vaccinate children against RSV failed and the induction of an aberrant Th2-type immune response was shown to induce severe to fatal pulmonary disease characterised in part by eosinophilia. BBG2Na is a promising human RSV subunit vaccine candidate which successfully passed phase II clinical trials in adults in association with Adju-Phos®. However, this formulation is not the most suitable for use in children since aluminium salts are known to induce a Th2-based immune response. In this study, we describe a potent and safe adjuvant formulation for BBG2Na in dimethyldioctadecylammonium bromide (DDA) that induces a mixed Th1/Th2 immune response in BALB/c mice. Furthermore, BBG2Na showed the same protective efficacy against RSV challenge when formulated either in DDA or in alum in mice and cotton rats.

Polysaccharides (PSs) derived from Streptococcus pneumoniae include more than 90 serotypes and differ greatly in their immunogenicity. In addition, immunization with PSs does not induce high affinity antibody production and no memory B-cells are generated. Coupling PSs to carrier proteins has been reported to induce B-cell maturation and to install a B-cell memory. As an alternative carrier protein, the outer membrane protein A (OmpA) derived from Klebsiella pneumoniae has been coupled to various PSs. We evaluated the immunogenicity of two PS conjugates, using PS derived from S. pneumoniae types 14 and 19. In this report, we show that anti-PS IgG responses are generated after the conjugation of PSs to P40. In addition, the humoral response generated is able to protect mice from a bacterial challenge. Our results indicate that P40 could be included in the development of new PS conjugate vaccines.

FMKp, the membrane fraction of Klebsiella pneumoniae, is the major component of a ribosomal immunostimulant that has been used in humans for more than 20 years. The aim of this study was to determine the cytokine profile induced by FMKp in human cells. The data presented show that FMKp induces the production of inflammatory cytokines and large amounts of interleukin (IL)-12 by monocytes. Moreover, human peripheral blood mononuclear cells (PBMC) stimulated FMKp-synthesized interferon (IFN)-gamma, a potent down regulator of immunoglobulin (Ig)E production. We therefore tested whether FMKp regulates IgE production. In vitro FMKp inhibited IL-4-induced IgE synthesis by PBMCs from normal donors without affecting the production of IgG. Moreover, the efficacy of FMKp was comparable to that of CpG oligonucleotides. In contrast, the addition of keyhole limpet hemocyanin, a carrier protein used as a negative control, did not alter IgE synthesis. The effect of FMKp was partially inhibited by polymyxin B, thereby suggesting that molecules other than lipopolysaccharide were involved in the inhibition of IgE synthesis. When tested on IgE synthesis in vivo, in BALB/c mice sensitized with ovalbumin, FMKp inhibited both total and specific IgE production. All together, these results show that FMKp induced a type-1 cytokine profile able to inhibit IgE production in vitro and in vivo, suggesting that FMKp may be a novel strategy for the treatment of IgE-related allergic diseases.

Respiratory syncytial virus (RSV) is responsible for severe low respiratory tract infections in young infants and the elderly. To investigate whether BBG2Na, a recombinant subunit vaccine comprising aa 130-230 of the RSV G protein, induced protective Abs in subjects over 60 years during phase II clinical trial, pre- and postimmunization sera of individuals immunized with BBG2Na or placebo were transferred into SCID mice before RSV challenge. These sera dose-dependently reduced lung RSV titers. However at some points of serial dilutions, postimmunization sera of BBG2Na-immunized subjects only were significantly more efficient than the corresponding preimmunization sera, in agreement with the induction of an increased Ab response against multiple epitopes on RSV-A G protein. Thus, BBG2Na is immunogenic in the elderly and confers passive protection in mice after serum transfer. To our knowledge, this is the first description of protective Abs induced by a subunit vaccine in human.

We have developed and validated a process-specific immunoligand assay based on the Threshold™ system for the quantification of residual host cell proteins (HCPs) in a recombinant subunit vaccine candidate against the human respiratory syncytial virus (hRSV). The industrial process of this vaccine produced in Escherichia coli, involved five chromatography steps for the production of clinical-grade batches. The clearance of non-product-related protein throughout the purification process was documented by the evaluation of the HCP content in the chromatographic fractions at each step of the downstream processing. The assay had a detection limit of 0.5 ng/ml of HCP equivalent to 10 parts per million (ppm). The quantification limit was 1.3 ng/ml of HCP, giving a sensitivity range of the assay of 10 to 30 ppm. To our knowledge, this is the first sensitive HCP assay reported for a vaccine.