Decolorization of Synthetic Textile Dyes by Fungal Endophytes Isolated from the Leaves of Philippine Mangrove (Avicennia marina)

Textile dyes in wastewater can be harmful pollutants when released into the environment without treatment. Biodegradation of textile dye effluents by different microbes, including fungi, has become popular as an alternative to physicochemical methods. The mangrove Avicennia marina is known to harbor endophytic fungi which have the potential to carry out dye degradation. Therefore, this study assessed the ability to decolorize synthetic dyes of endophytic fungi isolated from the leaves of A. marina. Of the nine fungal endophytes, Aspergillus niger, Syncephalastrum racemosum and Penicillium citrinum exhibited the highest mycelial growths in solid media, while all endophytes adsorbed Congo red. Through liquid decolorization assay, four isolates decolorized Congo red at greater than 89% decolorization rates. P. citrinum (55.45%), Mycelia sterilia (85.19%), A. flavus (44.91%) showed the highest decolorization rates of Methylene blue, Malachite green and Rhodamine B, respectively. The ligninolytic enzymes produced by the endophytic fungi, laccase exhibited the highest activity with values higher than the positive control

Dengue Virus Activates Polyreactive, Natural IgG B Cells after Primary and Secondary Infection

BACKGROUND: Dengue virus is transmitted by mosquitoes and has four serotypes. Cross-protection to other serotypes lasting for a few months is observed following infection with one serotype. There is evidence that low-affinity T and/or B cells from primary infections contribute to the severe syndromes often associated with secondary dengue infections. such pronounced immune-mediated enhancement suggests a dengue-specific pattern of immune cell activation. This study investigates the acute and early convalescent B cell response leading to the generation of cross-reactive and neutralizing antibodies following dengue infection. METHODOLOGY/PRINCIPAL FINDINGS: We assayed blood samples taken from dengue patients with primary or secondary infection during acute disease and convalescence and compared them to samples from patients presenting with non-dengue related fever. Dengue induced massive early plasmablast formation, which correlated with the appearance of polyclonal, cross-reactive IgG for both primary and secondary infection. Surprisingly, the contribution of IgG to the neutralizing titer 4-7 days after fever onset was more than 50% even after primary infection. CONCLUSIONS/SIGNIFICANCE: Poly-reactive and virus serotype cross-reactive IgG are an important component of the innate response in humans during both primary and secondary dengue infection, and "innate specificities" seem to constitute part of the adaptive response in dengue. While of potential importance for protection during secondary infection, cross-reactive B cells will also compete with highly neutralizing B cells and possibly interfere with their development

Rapid induction of cross-reactive IgG antibodies after dengue infection.

<p>Longitudinal plasma samples of dengue patients were tested by ELISA for dengue-specific IgM (A) and IgG antibodies (B). A and B) Four plasma dilutions from 1∶200 to 1∶25'000 were measured to exclude non-specific binding. For the combined illustration of all samples that were analysed the OD450 of one dilution (1∶5000 for IgG and 1∶1000 for IgM) was divided by the OD450 of a standard serum that was included on each plate. Time points: 1 = 1–3 days after fever, 2 = 4–7 days after fever, 3 = 15–25 days after fever. The same patient samples were analyzed for DENV1–4. Binding intensity correlated for all four serotypes, i.e. high binding to DENV1 would also apply to DENV2, 3 and 4. Primary versus secondary infection status was confirmed with the commercially available Panbio ELISA kit (Inverness Medical, Australia). The experiment was repeated for selected samples, confirming the original results. C) Concentrations of BAFF were measured in the plasma of dengue and flu patients at the indicated time points. D) moDCs were infected with DENV, heat-inactivated (HI) DENV, polyI:C or medium and BAFF was measured in the supernatant at different time points after infection. Data are presented as % of medium control and are the means±SEM of two independent experiments, done in triplicates. TSV01∶medium compared to polyIC∶medium is significantly different (p>0.05, Two-way ANOVA). The source of BAFF in DENV-infected patients is therefore unlikely to be DCs.</p

Polyclonal B cell activation after dengue infection.

<p>Fresh blood samples of dengue and control fever patients were analyzed by flow cytometry at 1–3, 4–7 and 15–25 days after onset of fever. A) CD19⁺CD20⁻CD27⁺CD138⁻ plasmablasts and CD27⁺CD138⁺ plasma cells (B) as % of lymphocytes. squares: other febrile illness (OFI), triangles: primary or secondary dengue infection. Each symbol represents one patient, lines indicate the mean. A Mann-Whitney test was used for statistical analysis. C) Gating strategy for plasmacells and plasmablasts. One secondary dengue patient with high plasma blast formation is shown. Blood was taken 3, 4 and 20 days after onset of fever.</p

Broad specificity of dengue-induced antibodies.

<p>A) Total concentration of IgM, IgA and IgG in the plasma of dengue- and control OFI patients 1–3 days, day 4–7 and day 15–25 after onset of fever. Means±SD, n = 7–10 for dengue-negative and n = 9–16 for dengue-positive patients. A student's t test to compare dengue-positive with fever control samples was performed. B) Polio virus-specific antibodies in paired plasma samples from dengue (grey boxes) and fever control patients (white boxes) 1–3 days of fever, day 4–7 and day 15–25 after onset of fever. Data are combined from three individual experiments, n = 18 for dengue, n = 21 for OFI. A Two-Way Repeated Measures ANOVA test with Bonferroni Post-Hoc test showed a significant difference between dengue and OFI at day 15–25.</p

Contribution of IgM and IgG to virus neutralization.

<p>A) Plasma samples of primary patients 2 and 3 (same patients as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029430#pone-0029430-g003" target="_blank">Fig. 3</a>) collected during acute disease and early convalescence were treated with 0.1 M 2-Mercaptoethanol (2-ME) or medium before serial dilution and incubation with virus. 1∶200 to 1∶437'400 diluted plasma samples were incubated with a constant amount of virus and the percentage of infected cells for each plasma dilution was determined by flow cytometry. B) Plasma samples from two primary patients were IgG-depleted and analyzed as in (A). Lower panels show ELISA results to confirm IgG-depletion. 50% neutralizing titers NT50 are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029430#pone-0029430-t003" target="_blank">table 3</a>. C) IgM and IgG binding to DENV3 (ELISA; ODsample/ODstandard of plasma samples) were correlated with % plasmacells amongst lymphocytes at day 4–7 after fever onset (refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029430#pone-0029430-g001" target="_blank">Fig. 1</a>). Each symbol represents one patient, n = 9 (6 secondary and 3 primary infections). SR: Spearman R. The correlation with IgG is significant, p = 0.03.</p

No serotype-specific selection into the memory B cell pool.

<p>A) Representative wells of the ELISPOT coated with anti-Ig, whole virus particles (E-specific B cells) or NS1 protein. Spots were detected with an anti-IgG antibody. B–D) cells were re-stimulated for six days before incubation on ELISPOT plates for the detection of memory B cells. B) Frequency of E-specific amongst total IgG-ASC in dengue patients 15–25 days after onset of fever. All patients had a DENV2 infection. Memory cells cross-reacting to two or more dengue serotypes but not necessarily binding to DENV2 were detected for most secondary patients, whereas the frequency of dengue-specific memory cells was usually below the detection limit for primary patients. C) Frequency of E-specific ASCs in healthy donors with dengue-specific IgG antibodies. D) Frequency of NS1-specific ASCs in healthy donors with or without dengue-specific IgG antibodies. B–D) Each data point in the x axes represents one donor, and values are the means of duplicates.</p

Absolute numbers of plasmablasts and plasma cells.

a)<p>p value comparing OFI with 1° dengue.</p>b)<p>p value comparing OFI with 2° dengue (Mann Whitney test). Longitudial samples were analyzed. Not all patients were available for the second and third time points.</p

Patient cohort.

a)<p>not all experiments were done with all patient samples. The numbers of patients for each figure are indicated in the figure legend.</p>b)<p>nine patients had a confirmed influenza infection and were analyzed as a separate group for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029430#pone-0029430-g001" target="_blank">Figure 1</a>.</p>c)<p>time point: 4–7days after onset of fever.</p

Cross-binding and cross-neutralizing IgG.

<p>Longitudinal plasma samples of three patients with primary DENV2 infection (A and C) and three patients with secondary DENV2 infection (B) were analyzed at the following time points after onset of fever: 1–3 days, 4–7 days and 15–25 days. Plasma samples were diluted three-fold over a range of 1∶200 to 1∶437'400 and analyzed by ELISA (A and B) and in a neutralization assay (C) for all four serotypes. A) ELISA with plasma from patients with primary infection with DENV2. B) ELISA with plasma from patients with primary infection with DENV2. Cross-binding to all four serotypes was observed after primary infection and was more pronounced after secondary infection. C) For the neutralization assay 1∶200 to 1∶437'400 diluted plasma samples were incubated with a constant amount of virus and the percentage of infected cells for each plasma dilution was determined by flow cytometry. EC50 values after curve fit are shown in the upper left of each graph. All curves fit with an R²>0.95 except for DENV4 of patient 3, where the R² is 0.78. The results of both assays are representative of at least two separate experiments for all patients and time points.</p