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

Phenotypic and genomic assessment of the potential threat of human spaceflight-relevant Staphylococcus capitis isolates under stress conditions

Previous studies have reported that spaceflight specific conditions such as microgravity lead to changes in bacterial physiology and resistance behavior including increased expression of virulence factors, enhanced biofilm formation and decreased susceptibility to antibiotics. To assess if spaceflight induced physiological changes can manifest in human-associated bacteria, we compared three spaceflight relevant Staphylococcus capitis isolates (DSM 111179, ISS; DSM 31028, clean room; DSM 113836; artificial gravity bedrest study) with the type strain (DSM 20326T). We tested the three strains regarding growth, colony morphology, metabolism, fatty acid and polar lipid pattern, biofilm formation, susceptibility to antibiotics and survival in different stress conditions such as treatment with hydrogen peroxide, exposure to desiccation, and irradiation with X-rays and UV-C. Moreover, we sequenced, assembled, and analyzed the genomes of all four strains. Potential genetic determinants for phenotypic differences were investigated by comparative genomics. We found that all four strains show similar metabolic patterns and the same susceptibility to antibiotics. All four strains were considered resistant to fosfomycin. Physiological differences were mainly observed compared to the type strain and minor differences among the other three strains. The ISS isolate and the bedrest study isolate exhibit a strong delayed yellow pigmentation, which is absent in the other two strains. Pigments were extracted and analyzed by UV/Vis spectroscopy showing characteristic carotenoid spectra. The ISS isolate showed the highest growth rate as well as weighted average melting temperature (WAMT) of fatty acids (41.8°C) of all strains. The clean room isolate showed strongest biofilm formation and a high tolerance to desiccation. In general, all strains survived desiccation better in absence of oxygen. There were no differences among the strains regarding radiation tolerance. Phenotypic and genomic differences among the strains observed in this study are not inevitably indicating an increased virulence of the spaceflight isolate. However, the increased growth rate, higher WAMT and colony pigmentation of the spaceflight isolate are relevant phenotypes that require further research within the human spaceflight context. We conclude that combining genetic analysis with classical microbiological methods allows the detailed assessment of the potential threat of bacteria in highly regulated and extreme environments such as spaceflight environments

Helminth antigens differentially modulate the activation of CD4(+) and CD8(+) T lymphocytes of convalescent COVID-19 patients in vitro

BACKGROUND: The coronavirus disease 2019 (COVID-19) is a respiratory disease caused by SARS-CoV-2, a recently discovered strain of coronavirus. The virus has spread rapidly, causing millions of death worldwide. Contrary to the predictions, prevalence and mortality due to COVID-19 have remained moderate on the African continent. Several factors, including age, genetics, vaccines, and co-infections, might impact the course of the pandemic in Africa. Helminths are highly endemic in Sub-Saharan Africa and are renowned for their ability to evade, skew, and suppress human immune responses through various immune-modulatory mechanisms. Such effects will likely impact SARS-CoV-2 transmission and disease progression. METHODS: Here, we analyzed in vitro the impact of antigen extracts from three major helminth parasites, including Onchocerca volvulus, Brugia malayi, and Ascaris lumbricoides, on the immune reactivity to SARS-CoV-2 peptides in COVID-19 patients. Activation of CD4+ and CD8+ T cells was investigated using flow cytometry to monitor the expression of CD137 (4-1BB) and CD69. Cytokine expression, including IL-6, IL-10, IFN-γ, and TNFα, was measured by Luminex in cell culture supernatants. RESULTS: We observed that helminth antigens significantly reduced the frequency of SARS-CoV-2-reactive CD4+ T helper cells. In contrast, the expression of SARS-CoV-2-reactive CD8+ T cells was not affected and even significantly increased when PBMCs from COVID-19 patients living in Benin, an endemic helminth country, were used. In addition, stimulation with helminth antigens was associated with increased IL-10 and a reduction of IFNγ and TNFα. CONCLUSION: Our data offer a plausible explanation for the moderate incidence of COVID-19 in Africa and support the hypothesis that helper T cell-mediated immune responses to SARS-CoV-2 are mitigated in the presence of helminth antigens, while virus-specific cytotoxic T cell responses are maintained

Synergistic effects of TLR ligands with poke weed lectin.

<p><b>A: Effect of TLR2 blockage on human B cell proliferation in response to PWM.</b> CD19+ human peripheral blood B cells were labelled with CFSE, preincubated with anti-TLR2 mAb or the corresponding isotype control (murine IgG_1κ), and stimulated with Pam₃CSK₄ or PWM (10 µg/ml) ± SpA (5 µg/ml) for 5 days. Proliferation was assessed by CFSE dilution. The gate denotes the percentage of live gated proliferating B cells as depicted. One representative experiment of n = 3 is shown. <b>B: Synergistic effects of PWM and immunostimulatory DNA.</b> Human B cells were stained with CFSE for assessment of B cell proliferation. Stimulation was performed with CpG ODN 2006 (PTO), DNAse- or mock (reaction buffer only)-treated PWM, 2006 GC (PTO) or CpG 2006 (PO) and combinations thereof as indicated. The graphs show the results from one representative experiment from n≥3. The percentage of proliferating B cells is indicated in the graphs.</p

In vitro activity of mecillinam and nitroxoline against Neisseria gonorrhoeae - re-purposing old antibiotics in the multi-drug resistance era

In 2018, the European Centre for Disease Prevention and Control reported the first cases of extensively drug-resistant Neisseria gonorrhoeae infections in Europe. Seeking new options for antimicrobial therapy we investigated the susceptibility of N. gonorrhoeae to nitroxoline (NIT) and mecillinam (MCM), both of which are currently only indicated to treat uncomplicated urinary tract infections. Clinical N. gonorrhoeae isolates with non-susceptibility to penicillin from two German medical centres were included (n = 27). Most isolates were also non-susceptible to a range of other anti-gonococcal antimicrobials (cefotaxime, ciprofloxacin, azithromycin, tetracycline). All isolates were further characterized by multi-locus sequence typing. MICs of penicillin and cefotaxime were determined by agar gradient diffusion. Production of penicillinase was tested by cefinase disk test. Susceptibility of MCM was investigated by agar dilution, NIT by agar dilution and disk diffusion. Penicillin MICs ranged from 0.125 to 64 mg l(-1) and MICs of cefotaxime ranged from 128 mg l(-1) whereas MICs of NIT ranged from 0.125 to 2 mg l(-1). NIT disk diffusion (median zone diameter 32 mm) correlated well with results from agar dilution. We demonstrated excellent in vitro activity of NIT against clinical N. gonorrhoeae isolates with non-susceptibility to standard anti-gonococcal antibiotics. MCM activity was unsatisfactory. Correlation of agar dilution and disk diffusion in NIT susceptibility testing is an important aspect with potential clinical implications

Contribution of the lectin component.

<p><b>A:</b> Human B cell proliferation in response to PWM (Lot. B and C) in the presence or absence of N,N-di-acetylchitobiose or N,N,N-tri-acetylchitotriose was assessed by ³H-thymidine incorporation given in counts per minute (cpm). The mean values ± SEM from one representative experiment performed in triplicates of n = 2 is shown. <b>B:</b> Human CD19+ B cells were left unstimulated or stimulated with 0.25 µM GpC PTO ODN (GC) and/or 10 µg/ml of PWM, 5 µg/ml SpA or 10 µg/ml anti-Ig (aIg) for 72 hours. Proliferation was quantified by ³H-thymidine incorporation. The diagram shows the means from n = 4 experiments ± SEM. The values obtained were normalized to GC = 1 (710±280 cpm = mean ± SEM). <b>C:</b> Human CD19+ B cells were stained with CFSE and stimulated with highly purified PWM (Lot. D) or <i>Lycopersicon esculentum</i> lectin (LEA) with or without GpC PTO ODN (GC). Proliferation was quantified by CFSE dilution on day 4. The percentage of proliferating cells is provided in each dot plot. The results from two independent donors of n = 4 are shown.</p

Contribution of TLR ligands to B cell activation.

<p><b>A: Stimulation of human B cells with TLR ligands.</b> CFSE-labelled CD19+ human B cells were stimulated with or without TLR ligands (TLR2 ligand MALP-2, TLR4 ligand LPS (0.1 and 1 µg/ml) or TLR9 ligand CpG ODN 2006 (CpG)) and/or BCR stimuli (5 µg/ml SpA or anti-Ig (aIg)). After 5 days cells were harvested, stained with anti-CD20 and analyzed by flow cytometry. The graphs depict cell survival (percentage of live gated cells; upper right angle) and cell proliferation (CFSE dilution; % proliferating cells of live gated cells (left) where indicated). The experiment shown is representative of n≥3 experiments. <b>B–D: Assessment of TLR2 activity in PWM preparations.</b> HEK293 cells were transfected with pTLR2 or lipofectamine alone (Lf). <b>B:</b> non-transfected and pTLR2-transfected HEK293 cells were stained for TLR2 expression with anti-TLR2 mAb or the respective isotype control as indicated. <b>C:</b> HEK293 cells transfected with pTLR2 or Lf only were stimulated with Pam₃CSK₄ (P3) or PWM (10 µg/ml). After 24 hours cellular supernatants were collected and analyzed for IL-8 secretion. One representative experiment of n≥3 experiments is shown. <b>D:</b> HEK293 cells transfected with pTLR2 were pretreated with anti-TLR2 mAb or the isotype control before stimulation with Pam₃CSK₄ (P3) or PWM (10 µg/ml). IL-8 was quantified in the 24 hour supernatants. <b>E: 16S rDNA PCR.</b> DNA isolation and PCR amplification of bacterial 16S ribosomal DNA from DNA from <i>E. coli</i> (EC), a negative clinical specimen (NC), a positive clinical sample (CS) and the PWM preparation (PW) or the water control (H₂O), with an expected PCR fragment size of approximately 900 bp.</p

TLR-dependency of Poke weed mitogen (PWM)-induced cell stimulation.

<p><b>A: B cell proliferation.</b> Human CD19+ peripheral blood B cells were stained with CFSE and stimulated with 1 or 10 µg/ml PWM in the presence or absence of 5 µg/ml <i>Staphylococcus aureus</i> protein A (SpA) or 5 µg/ml anti-human Ig F(ab′)₂ fragments (aIg) as B cell receptor stimuli, with SpA or aIg alone, or left unstimulated. After 5 days B cells were harvested, stained with anti-CD20 and live gated cells were analyzed for CFSE dilution. The percentages of live gated cells (upper right corner) and proliferating cells (left) are indicated in each diagram. The diagrams depict the results from one representative experiment of n = 3. <b>B: TNF-induction.</b> Human PBMC were stimulated for four hours with or without PWM, TLR2 ligands FSL-1R (FSL) and Pam₃CSK₄ (P3), phosphorothioate-modified DNA ODN CpG 2006 (CpG) and 2006 GC (GC) and LPS in the presence of Brefeldin A (BfA) or with LPS in the absence of BfA. Subsequently, intracellular staining was performed with an anti-human TNF mAb and TNF expression was analyzed by flow cytometry. The results show a summary of the data obtained in n = 4 experiments. Mean values of anti-TNF mean fluorescence intensities are provided ± SEM. <b>C: Antagonization with Polymyxin B.</b> CFSE-stained human CD19+ B cells were stimulated with PWM in the presence and absence of polymyxin B. Proliferation was assessed by flow cytometric analysis of CFSE dilution on day 5. The results obtained in two representative donors of n = 3 are shown. <b>D: MyD88-dependency of B cell stimulation.</b> B220+ B cells were isolated from the spleens of MyD88^−/− mice and their wild type counterparts. B cells were stimulated with CpG, P3, PWM and PMA/Ionomycin (P/I) and harvested after 72 hours and a 18 hour pulse with ³H-thymidine. The diagram shows the average values in counts per minute (cpm) of n = 4 experiments ± SEM. <b>E: TLR-dependency.</b> Wild type, TLR2−/− and TLR9−/− B cells were isolated from murine spleen with anti-CD19 microbeads, labelled with CFSE and stimulated with TLR2 ligands Pam₃CSK₄ (P3) and FSL-1R (FSL), PWM (10 µg/ml), TLR9 ligand CpG ODN 1668 or 1668 GC control ODN. After 4 days B cell proliferation (CFSE dilution) was quantified by flow cytometry. The diagram depicts the mean fluorescence intensity (MFI) for CFSE in live gated cells as mean value ± SEM from n = 4 experiments. Note that low MFI corresponds to strong proliferation while high MFI values reveal absence of proliferation.</p

Detection of TLR ligands in PWM preparations.

<p><b>A:</b> Human B cells were stimulated with different lots of PWM. Proliferation rates were quantified by 3H-thymidine incorporation (cpm = counts per minute). The diagram shows the mean values ± SEM from n = 3 experiments. <b>B:</b> LAL-assay was performed to quantify the LPS content in PWM Lot. A–D. The diagram depicts the mean values ± standard deviation obtained by testing in quadruplicates. <b>C:</b> TLR2 activity was assessed by measuring IL-8 concentrations in the supernatants of pTLR2-transfected or non-transfected HEK293 cells stimulated with different lots of PWM (left) or Pam₃CSK₄ (right) at the concentrations indicated. One representative experiment performed in triplicates of n = 2 is shown.</p