Poke Weed Mitogen Requires Toll-Like Receptor Ligands for Proliferative Activity in Human and Murine B Lymphocytes

Poke weed mitogen (PWM), a lectin purified from Phytolacca americana is frequently used as a B cell-specific stimulus to trigger proliferation and immunoglobulin secretion. In the present study we investigated the mechanisms underlying the B cell stimulatory capacity of PWM. Strikingly, we observed that highly purified PWM preparations failed to induce B cell proliferation. By contrast, commercially available PWM preparations with B cell activity contained Toll-like receptor (TLR) ligands such as TLR2-active lipoproteins, lipopolysaccharide and DNA of bacterial origin. We show that these microbial substances contribute to the stimulatory activity of PWM. Additional experimental data highlight the capacity of PWM to enable B cell activation by immunostimulatory DNA. Based on these findings we propose that the lectin sensitizes B cells for TLR stimulation as described for B cell receptor ligation and that B cell mitogenicity of PWM preparations results from synergistic activity of the poke weed lectin and microbial TLR ligands present in the PWM preparations

Driving change in dtap batch release testing

The complexity of vaccine manufacturing has raised the need to drive standardization and quality control requirements as well as batch release of vaccines. The purpose of release testing is to ensure that efficacy and safety of the vaccine product are maintained in all batches. Classical testing includes challenge experiments in animals that provide proof of vaccine potency and identify subpotent vaccines. However, novel concepts such as “consistency testing” question the continued need for in vivo experiments and propose to implement rigorous QC for lot-to-lot consistency testing with other methods at an earlier stage. Please click Download on the upper right corner to see the full abstract

A 5-year look-back at the notification and management of vaccine supply shortages in Germany

Background: Unavailability of vaccines endangers the overall goal to protect individuals and whole populations against infections. Methods: The German notification system includes the publication of vaccine supply shortages reported by marketing authorisation holders (MAH), information on the availability of alternative vaccine products, guidance for physicians providing vaccinations and an unavailability reporting tool to monitor regional distribution issues. Aim: This study provides a retrospective analysis of supply issues and measures in the context of European and global vaccine supply constraints. Results: between October 2015 and December 2020, the 250 notifications concerned all types of vaccines (54 products). Most shortages were caused by increased demand associated with immigration in Germany in 2015 and 2016, new or extended vaccine recommendations, increased awareness, or changes in global immunisation programmes. Shortages of a duration up to 30 days were mitigated using existing storage capacities. Longer shortages, triggered by high demand on a national level, were mitigated using alternative products and re-allocation; in a few cases, vaccines were imported. However, for long lasting supply shortages associated with increased global demand, often occurring in combination with manufacturing issues, few compensatory mechanisms were available. Nevertheless, only few critical incidents were identified: (i) shortage of hexavalent vaccines endangering neonatal immunisation programmes in 2015;(ii) distribution issues with influenza vaccines in 2018; and (iii) unmet demand for pneumococcal and influenza vaccines during the coronavirus disease (COVID)-19 pandemic. Conclusion: Vaccine product shortages in Germany resemble those present in neighbouring EU states and often reflect increased global demand not matched by manufacturing capacities.Peer Reviewe

Impact of rifaximin on the frequency and characteristics of spontaneous bacterial peritonitis in patients with liver cirrhosis and ascites

BACKGROUND: Rifaximin is a non-absorbable antibiotic used to prevent relapses of hepatic encephalopathy which may also be a candidate for prophylaxis of spontaneous bacterial peritonitis (SBP). AIM: To detect the impact of rifaximin on the occurrence and characteristics of SBP. METHODS: We prospectively studied all hospitalized patients that underwent a diagnostic paracentesis in our department from March 2012 to April 2013 for SBP and recorded all clinical data including type of SBP prophylaxis, prior use of rifaximin, concomitant complications of cirrhosis, as well as laboratory results and bacteriological findings. Patients were divided into the following three groups: no antibiotic prophylaxis, prophylaxis with rifaximin or with systemically absorbed antibiotic prophylaxis. RESULTS: Our study cohort comprised 152 patients with advanced liver cirrhosis, 32 of whom developed SBP during the study period. As expected, our study groups differed regarding a history of hepatic encephalopathy and SBP before inclusion into the study. None of the 17 patients on systemic antibiotic prophylaxis developed SBP while 8/27 patients on rifaximin and 24/108 without prophylaxis had SBP (p = 0.02 and p = 0.04 versus systemic antibiotics, respectively). In general, episodes of SBP were similar for patients treated with rifaximin and those without any prophylaxis. However, Escherichia coli and enterococci were dominant in the ascites of patients without any prophylaxis, while mostly klebsiella species were recovered from the ascites samples in the rifaximin group. CONCLUSION: Rifaximin pretreatment did not lead to a reduction of SBP occurrence in hospitalized patients with advanced liver disease. However, the bacterial species causing SBP were changed by rifaximin

Revisiting the B-cell compartment in mouse and humans: more than one B-cell subset exists in the marginal zone and beyond.

International audienceABSTRACT: The immunological roles of B-cells are being revealed as increasingly complex by functions that are largely beyond their commitment to differentiate into plasma cells and produce antibodies, the key molecular protagonists of innate immunity, and also by their compartmentalisation, a more recently acknowledged property of this immune cell category. For decades, B-cells have been recognised by their expression of an immunoglobulin that serves the function of an antigen receptor, which mediates intracellular signalling assisted by companion molecules. As such, B-cells were considered simple in their functioning compared to the other major type of immune cell, the T-lymphocytes, which comprise conventional T-lymphocyte subsets with seminal roles in homeostasis and pathology, and non-conventional T-lymphocyte subsets for which increasing knowledge is accumulating. Since the discovery that the B-cell family included two distinct categories - the non-conventional, or extrafollicular, B1 cells, that have mainly been characterised in the mouse; and the conventional, or lymph node type, B2 cells - plus the detailed description of the main B-cell regulator, FcγRIIb, and the function of CD40+ antigen presenting cells as committed/memory B-cells, progress in B-cell physiology has been slower than in other areas of immunology. Cellular and molecular tools have enabled the revival of innate immunity by allowing almost all aspects of cellular immunology to be re-visited. As such, B-cells were found to express "Pathogen Recognition Receptors" such as TLRs, and use them in concert with B-cell signalling during innate and adaptive immunity. An era of B-cell phenotypic and functional analysis thus began that encompassed the study of B-cell microanatomy principally in the lymph nodes, spleen and mucosae. The novel discovery of the differential localisation of B-cells with distinct phenotypes and functions revealed the compartmentalisation of B-cells. This review thus aims to describe novel findings regarding the B-cell compartments found in the mouse as a model organism, and in human physiology and pathology. It must be emphasised that some differences are noticeable between the mouse and human systems, thus increasing the complexity of B-cell compartmentalisation. Special attention will be given to the (lymph node and spleen) marginal zones, which represent major crossroads for B-cell types and functions and a challenge for understanding better the role of B-cell specificities in innate and adaptive immunology

Plant virus particles carrying tumour antigen activate TLR7 and induce high levels of protective antibody

Induction of potent antibody is the goal of many vaccines targeted against infections or cancer. Modern vaccine designs that use virus-like particles (VLP) have shown efficacy for prophylactic vaccination against virus-associated cancer in the clinic. Here we used plant viral particles (PVP), which are structurally analogous to VLP, coupled to a weak idiotypic (Id) tumour antigen, as a conjugate vaccine to induce antibody against a murine B-cell malignancy. The Id-PVP vaccine incorporates a natural adjuvant, the viral ssRNA, which acts via TLR7. It induced potent protective anti-Id antibody responses in an in vivo mouse model, superior to the "gold standard" Id vaccine, with prevalence of the IgG2a isotype. Combination with alum further increased antibody levels and maintained the IgG2a bias. Engagement of TLR7 in vivo was followed by secretion of IFN-? by plasmacytoid dendritic cells and by activation of splenic CD11chi conventional dendritic cells. The latter was apparent from up-regulation of co-stimulatory molecules and from secretion of a wide range of inflammatory cytokines and chemokines including the Th1-governing cytokine IL-12, in keeping with the IgG2a antibody isotype distribution. PVP conjugates are a novel cancer vaccine design, offering an attractive molecular form, similar to VLP, and providing T-cell help. In contrast to VLP, they also incorporate a safe "in-built" ssRNA adjuvant

T cell-independent, TLR-induced IL-12p70 production in primary human monocytes

IL-12p70 is a key cytokine for the induction of Th1 immune responses. IL-12p70 production in myeloid cells is thought to be strictly controlled by T cell help. In this work we demonstrate that primary human monocytes can produce IL-12p70 in the absence of T cell help. We show that human monocytes express TLR4 and TLR8 but lack TLR3 and TLR7 even after preincubation with type I IFN. Simultaneous stimulation of TLR4 and TLR8 induced IL-12p70 in primary human monocytes. IL-12p70 production in peripheral blood myeloid dendritic cells required combined stimulation of TLR7/8 ligands together with TLR4 or with TLR3 ligands. In the presence of T cell-derived IL-4, but not IFN-gamma, stimulation with TLR7/8 ligands was sufficient to stimulate IL-12p70 production. In monocytes, type I IFN was required but not sufficient to costimulate IL-12p70 induction by TLR8 ligation. Furthermore, TLR8 ligation inhibited LPS-induced IL-10 in monocytes, and LPS alone gained the ability to stimulate IL-12p70 in monocytes when the IL-10 receptor was blocked. Together, these results demonstrate that monocytes are licensed to synthesize IL-12p70 through type I IFN provided via the Toll/IL-1R domain-containing adaptor inducing IFN-beta pathway and the inhibition of IL-10, both provided by combined stimulation with TLR4 and TLR8 ligands, triggering a potent Th1 response before T cell help is established

TLR1/2, TLR7, and TLR9 Signals Directly Activate Human Peripheral Blood Naive and Memory B Cell Subsets to Produce Cytokines, Chemokines, and Hematopoietic Growth Factors

Recently, it has been reported that using multiple signals, murine and human B cells secrete several cytokines with pro-inflammatory and immunoregulatory properties. We present the first comprehensive analysis of 24 cytokines, chemokines, and hematopoietic growth factors production by purified human peripheral blood B cells (CD19+), and naive (CD19+CD27-) and memory (CD19+CD27+) B cells in response to direct and exclusive signaling provided by toll-like receptor (TLR) ligands Pam3CSK (TLR1/TLR2), Imiquimod (TLR7), and GpG-ODN2006 (TLR9). All three TLR ligands stimulated B cells (CD19+) to produce cytokines IL-1α, IL-1β, IL-6, TNF-α, IL-13, and IL-10, and chemokines MIP-1α, MIP-1β, MCP-1, IP-10, and IL-8. However, GM-CSF and G-CSF production was predominantly induced by TLR2 agonist. Most cytokines/chemokines/hematopoietic growth factors were predominantly or exclusively produced by memory B cells, and in general, TLR2 signal was more powerful than signal provided viaTLR7 and TLR9. No significant secretion of eotaxin, IFN-α, IFN-γ, IL-2, IL-3, IL-4, IL-5, IL-7, IL-15, IL-17, IL-12p40, IL-12p70, and TNF-β (lymphotoxin) was observed. These data demonstrate that human B cells can be directly activated viaTLR1/TLR2, TLR7, and TLR9 to induce secretion of cytokines, chemokines, and hematopoietic growth factors and suggest a role of B cells in immune response against microbial pathogenesis and immune homeostasis