631 rankl knock out mesenchymal stromal cells have an unexpected osteogenic differentiation defect which is improved by a rankl expressing lentiviral vector

Osteoclast-poor RANKL-dependent Autosomal Recessive Osteopetrosis (ARO) is a rare bone disease characterized by an increase in bone density due to the failure of bone resorption by impaired osteoclast formation. Hematopoietic stem cell transplantation is not an effective therapy for this ARO form, since in bone RANKL is produced mainly by cells of mesenchymal origin. Therefore Mesenchymal Stromal Cells (MSC) transplantation together with a gene-therapy strategy to correct RANKL defect in MSC could represent a possible effective therapy. Of note, whether also MSC, besides the osteoclasts, are affected by RANKL deficiency is unknown. To verify this, we established and characterized bone marrow derived MSC (BM-MSC) lines from the Rankl−/− (KO) mouse model, which recapitulates the human disease, and from wild type (WT) mice. No differences were found between KO and WT MSC in terms of morphology, immunophenotype and proliferation capacity. However, KO MSC displayed a reduced clonogenic potential with a decrease in stemness genes expression. KO MSC were able to normally differentiate towards the adipogenic and chondrogenic lineages, while showed a significantly impaired osteogenic differentiation capacity compared to WT MSC, as demonstrated by reduced Alizarin Red staining (ARS) and expression of osteogenic genes. To confirm that this alteration was due to the lack of functional RANKL, we developed a third generation lentiviral vector expressing human soluble RANKL (hsRL) for the genetic correction of KO MSC. We first investigated lentiviral transduction in 293T cells to optimize transduction efficiency at different multiplicity of infection (MOI) ranging from 1 to 100. hsRL production increased proportionally to the MOI and was stable over time. However, the higher the MOI the higher the cytotoxicity observed. Based on these data, we performed a lentiviral hsRL transduction in KO MSC at 20 and 50 MOI, to define the optimal transduction conditions. After transduction 99.5% of MSC were GFP+. While in Rankl−/− control cells the cytokine was not detected, in corrected cells hsRL production and secretion was measurable and comparable to sRL levels in WT mouse. KO MSC stably expressing hsRL showed an improved osteogenic differentiation capacity compared to untransduced KO MSC, as demonstrated by increased ARS and expression of osteogenic genes. Moreover, the expression of RANK receptor in both MSC suggested an autocrine role of sRL as possible mechanism. Our data suggest that restoration of RANKL production in lentiviral-transduced KO MSC might not only allow osteoclast differentiation in Rankl−/− mice upon transplantation, but also improve the osteogenic differentiation defect of KO MSC

Setting of Methods for Analysis of Mucosal Antibodies in Seminal and Vaginal Fluids of HIV Seropositive Subjects from Cambodian and Italian Cohorts

International audienceBACKGROUND: Genital mucosae play a key role in protection from STD and HIV infection, due to their involvement in both horizontal and vertical disease transmission. High variability of published observations concerning IgA isolation and quantification underlies the strong requirement of specific methods able to maximize investigation on HIV-specific IgA. METHODOLOGY: Genital fluids from 109 subjects, including male and female cohorts from Italy and Cambodia, were collected, aliquoted and processed with different techniques, to assess optimal conditions maximizing mucosal antibody recovery. Three sampling techniques, up to sixteen preservation conditions, six ELISA methods and four purifications protocols were compared. PRINCIPAL FINDINGS: The optimal method here described took advantage of Weck-Cel sampling of female mucosal fluids. Immediate processing of genital fluids, with the addition of antibiotics and EDTA, improved recovery of vaginal IgA, while the triple addition of EDTA, antibiotics and protease inhibitors provided the highest amount of seminal IgA. Due to low amount of IgA in mucosal fluids, a high sensitive sandwich ELISA assay was set; sensitivity was enhanced by milk-based overcoating buffer and by a two-step biotin-streptavidin signal amplification. Indeed, commercial antisera to detect human immunoglobulins showed weak cross-reactivity to different antibody types. Three-step affinity purification provided reproducible immunoglobulin recovery from genital specimens, while conventional immuno-affinity IgA purification was found poorly manageable. Affinity columns were suitable to isolate mucosal IgA, which are ten-fold less concentrated than IgG in genital specimens, and provided effective separation of IgA monomers, dimers, and J-chains. Jacalin-bound resin successfully separated IgA1 from IgA2 subfraction. CONCLUSIONS/SIGNIFICANCE: Specific, effective and reliable methods to study local immunity are key items in understanding host mucosal response. The sequence of methods here described is effective and reliable in analysing humoral local responses, and may provide a solid advance to identify and measure the effective mucosal responses to HIV

U-CHANGE Project: a multidimensional consensus on how clinicians, patients and caregivers may approach together the new urothelial cancer scenario

IntroductionAdvanced urothelial carcinoma remains aggressive and very hard to cure, while new treatments will pose a challenge for clinicians and healthcare funding policymakers alike. The U-CHANGE Project aimed to redesign the current model of care for advanced urothelial carcinoma patients to identify limitations (“as is” scenario) and recommend future actions (“to be” scenario).MethodsTwenty-three subject-matter experts, divided into three groups, analyzed the two scenarios as part of a multidimensional consensus process, developing statements for specific domains of the disease, and a simplified Delphi methodology was used to establish consensus among the experts.ResultsRecommended actions included increasing awareness of the disease, increased training of healthcare professionals, improvement of screening strategies and care pathways, increased support for patients and caregivers and relevant recommendations from molecular tumor boards when comprehensive genomic profiling has to be provided for appropriate patient selection to ad hoc targeted therapies.DiscussionWhile the innovative new targeted agents have the potential to significantly alter the clinical approach to this highly aggressive disease, the U-CHANGE Project experience shows that the use of these new agents will require a radical shift in the entire model of care, implementing sustainable changes which anticipate the benefits of future treatments, capable of targeting the right patient with the right agent at different stages of the disease

Two Amino Acid Substitutions within the First External Loop of CCR5 Induce Human Immunodeficiency Virus-Blocking Antibodies in Mice and Chickens▿ †

Antibodies to the first loop (ECL1) of CCR5 have been identified in human immunodeficiency virus (HIV)-exposed uninfected individuals (ESN) and in HIV-positive nonprogressing subjects. Thus, these antibodies may confer resistance against HIV infection. To define which amino acids are involved in antibody binding to CCR5, we performed a peptide-scanning assay and studied the immunogenicity of peptides in animal models. A panel of synthetic peptides spanning the CCR5-ECL1 region and displaying glycine or alanine substitutions was assayed for antibody binding with a pool of natural anti-CCR5 antibodies. We used mice and chickens to study the immunogenicity of mutagenized peptide. Structural characterization by nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics simulations were performed to better understand the structural and conformational features of the mutagenized peptide. Amino acid substitutions in positions Ala95 and Ala96 (A95-A96) increased antibody-peptide binding compared to that of the wild-type peptide (Asp95-Phe96). The Ala95-96 peptide was shown to induce, in mice and chickens, antibodies displaying biological activity at very low concentrations. Strikingly, chicken antibodies to the Ala95-96 peptide specifically recognize human CCR5 molecules, downregulate receptors from lymphocytes, inhibit CCR5-dependent chemotaxis, and prevent infection by several R5 viruses, displaying 50% inhibitory concentrations of less than 3 ng/ml. NMR spectroscopy and molecular dynamics simulations proved the high flexibility of isolated epitopes and suggested that A95-A96 substitutions determine a slightly higher tendency to generate helical conformations combined with a lower steric hindrance of the side chains in the peptides. These findings may be relevant to the induction of strong and efficient HIV-blocking antibodies

Induction of HIV-blocking anti-CCR5 IgA in Peyers's patches without histopathological alterations.

<p>UNLABELLED: The chemokine receptor CCR5 is essential for HIV infection and is thus a potential target for vaccine development. However, because CCR5 is a host protein, generation of anti-CCR5 antibodies requires the breaking of immune tolerance and thus carries the risk of autoimmune responses. In this study, performed in mice, we compared 3 different immunogens representing surface domains of murine CCR5, 4 different adjuvants, and 13 different immunization protocols, with the goal of eliciting HIV-blocking activity without inducing autoimmune dysfunction. In all cases the CCR5 sequences were presented as fusions to the Flock House virus (FHV) capsid precursor protein. We found that systemic immunization and mucosal boosting elicited CCR5-specific antibodies and achieved consistent priming in Peyer's patches, where most cells showed a phenotype corresponding to activated B cells and secreted high levels of IgA, representing up to one-third of the total HIV-blocking activity. Histopathological analysis revealed mild to moderate chronic inflammation in some tissues but failed in reporting signs of autoimmune dysfunction associated with immunizations. Antisera against immunogens representing the N terminus and extracellular loops 1 and 2 (Nter1 and ECL1 and ECL2) of CCR5 were generated. All showed specific anti-HIV activity, which was stronger in the anti-ECL1 and -ECL2 sera than in the anti-Nter sera. ECL1 and ECL2 antisera induced nearly complete long-lasting CCR5 downregulation of the receptor, and especially, their IgG-depleted fractions prevented HIV infection in neutralization and transcytosis assays. In conclusion, the ECL1 and ECL2 domains could offer a promising path to achieve significant anti-HIV activity in vivo.</p> <p>IMPORTANCE: The study was the first to adopt a systematic strategy to compare the immunogenicities of all extracellular domains of the CCR5 molecule and to set optimal conditions leading to generation of specific antibodies in the mouse model. There were several relevant findings, which could be translated into human trials. (i) Prime (systemic) and boost (mucosal) immunization is the best protocol to induce anti-self antibodies with the expected properties. (ii) Aluminum is the best adjuvant in mice and thus can be easily used in nonhuman primates (NHP) and humans. (iii) The Flock House virus (FHV) system represents a valid delivery system, as the structure is well known and is not pathogenic for humans, and it is possible to introduce constrained regions able to elicit antibodies that recognize conformational epitopes. (iv) The best CCR5 vaccine candidate should include either extracellular loop 1 or 2 (ECL1 or ECL2), but not N terminus domains.</p

A Nonparametric Procedure for Defining a New Humoral Immunologic Profile in a Pilot Study on HIV Infected Patients

<div><p>This work aims at identifying a set of humoral immunologic parameters that improve prediction of the activation process in HIV patients. Starting from the well-known impact of humoral immunity in HIV infection, there is still a lack of knowledge in defining the role of the modulation of functional activity and titers of serum antibodies from early stage of infection to the development of AIDS. We propose an integrated approach that combines humoral and clinical parameters in defining the host immunity, implementing algorithms associated with virus control. A number of humoral parameters were simultaneously evaluated in a whole range of serum samples from HIV-positive patients. This issue has been afforded accounting for estimation problems typically related to “feasibility” studies where small sample size in each group and large number of parameters are jointly estimated. We used nonparametric statistical procedures to identify biomarkers in our study which included 42 subjects stratified on five different stages of HIV infection, i.e., Elite Controllers (EC), Long Term Non Progressors (LTNP), HAART, AIDS and Acute Infection (AI). The main goal of the paper is to illustrate a novel profiling method for helping to design a further confirmatory study. A set of seventeen different HIV-specific blood humoral factors were analyzed in all subjects, i.e. IgG and IgA to gp120IIIB, to gp120Bal, to whole gp41, to P1 and T20 gp41 epitopes of the MPER-HR2 region, to QARILAV gp41 epitope of the HR1 region and to CCR5; neutralization activity against five different virus strains and ADCC were also evaluated. Patients were selected on the basis of CD4 cell counts, HIV/RNA and clinical status. The Classification and Regression Trees (CART) approach has been used to uncover specific patterns of humoral parameters in different stages of HIV disease. Virus neutralization of primary virus strains and antibodies to gp41 were required to classify patients, suggesting that clinical profiles strongly rely on functional activity against HIV.</p> </div

Immunization with HIV-1 gp41 Subunit Virosomes Induces Mucosal Antibodies Protecting Nonhuman Primates against Vaginal SHIV Challenges

SummaryHuman immunodeficiency virus (HIV)-1 is mainly transmitted mucosally during sexual intercourse. We therefore evaluated the protective efficacy of a vaccine active at mucosal sites. Macaca mulatta monkeys were immunized via both the intramuscular and intranasal routes with an HIV-1 vaccine made of gp41-subunit antigens grafted on virosomes, a safe delivery carrier approved in humans with self-adjuvant properties. Six months after 13 vaginal challenges with simian-HIV (SHIV)-SF162P3, four out of five vaccinated animals remained virus-negative, and the fifth was only transiently infected. None of the five animals seroconverted to p27gag-SIV. In contrast, all 6 placebo-vaccinated animals became infected and seroconverted. All protected animals showed gp41-specific vaginal IgAs with HIV-1 transcytosis-blocking properties and vaginal IgGs with neutralizing and/or antibody-dependent cellular-cytotoxicity activities. In contrast, plasma IgGs totally lacked virus-neutralizing activity. The protection observed challenges the paradigm whereby circulating antiviral antibodies are required for protection against HIV-1 infection and may serve in designing a human vaccine against HIV-1-AIDS