Infection with Mansonella perstans Nematodes in Buruli Ulcer Patients, Ghana.

During August 2010-December 2012, we conducted a study of patients in Ghana who had Buruli ulcer, caused by Mycobacterium ulcerans, and found that 23% were co-infected with Mansonella perstans nematodes; 13% of controls also had M. perstans infection. M. perstans co-infection should be considered in the diagnosis and treatment of Buruli ulcer

CD83 Modulates B Cell Function In Vitro: Increased IL-10 and Reduced Ig Secretion by CD83Tg B Cells

The murine transmembrane glycoprotein CD83 is an important regulator for both thymic T cell maturation and peripheral T cell responses. Recently, we reported that CD83 also has a function on B cells: Ubiquitous transgenic (Tg) expression of CD83 interfered with the immunoglobulin (Ig) response to infectious agents and to T cell dependent as well as T cell independent model antigen immunization. Here we compare the function of CD83Tg B cells that overexpress CD83 and CD83 mutant (CD83mu) B cells that display a drastically reduced CD83 expression. Correlating with CD83 expression, the basic as well as the lipopolysaccharide (LPS) induced expression of the activation markers CD86 and MHC-II are significantly increased in CD83Tg B cells and reciprocally decreased in CD83mu B cells. Wild-type B cells rapidly upregulate CD83 within three hours post BCR or TLR engagement by de novo protein synthesis. The forced premature overexpression of CD83 on the CD83Tg B cells results in reduced calcium signaling, reduced Ig secretion and a reciprocally increased IL-10 production upon in vitro activation. This altered phenotype is mediated by CD83 expressed on the B cells themselves, since it is observed in the absence of accessory cells. In line with this finding, purified CD83mu B cells displayed a reduced IL-10 production and slightly increased Ig secretion upon LPS stimulation in vitro. Taken together, our data strongly suggest that CD83 is expressed by B cells upon activation and contributes to the regulation of B cell function

Temporal and spatial analysis of the 2014-2015 Ebola virus outbreak in West Africa

West Africa is currently witnessing the most extensive Ebola virus (EBOV) outbreak so far recorded. Until now, there have been 27,013 reported cases and 11,134 deaths. The origin of the virus is thought to have been a zoonotic transmission from a bat to a two-year-old boy in December 2013 (ref. 2). From this index case the virus was spread by human-to-human contact throughout Guinea, Sierra Leone and Liberia. However, the origin of the particular virus in each country and time of transmission is not known and currently relies on epidemiological analysis, which may be unreliable owing to the difficulties of obtaining patient information. Here we trace the genetic evolution of EBOV in the current outbreak that has resulted in multiple lineages. Deep sequencing of 179 patient samples processed by the European Mobile Laboratory, the first diagnostics unit to be deployed to the epicentre of the outbreak in Guinea, reveals an epidemiological and evolutionary history of the epidemic from March 2014 to January 2015. Analysis of EBOV genome evolution has also benefited from a similar sequencing effort of patient samples from Sierra Leone. Our results confirm that the EBOV from Guinea moved into Sierra Leone, most likely in April or early May. The viruses of the Guinea/Sierra Leone lineage mixed around June/July 2014. Viral sequences covering August, September and October 2014 indicate that this lineage evolved independently within Guinea. These data can be used in conjunction with epidemiological information to test retrospectively the effectiveness of control measures, and provides an unprecedented window into the evolution of an ongoing viral haemorrhagic fever outbreak.status: publishe

Reduced IL-10 secretion by CD83mu B cells.

<p>Untouched B cells were isolated from pooled spleens of two 8–10 week old sex matched C57BL/6 (open bars), CD83Tg (black bars) and CD83mu (dark grey bars) mice. 2×10⁵ B cells were cultured in triplicates in the presence of titrated amounts of LPS as indicated on the x-axis. 6A: Total mouse Ig in the supernatant (dilution 1∶1024) was measured after seven days by ELISA. 6B: IL-10 in the supernatant was measured after 48 h. Presented are the combined results of 3 independent experiments, error bars show SEM. Asterisks indicate a significant difference of the mean (* p<0.05; ** p<0.005 *** p<0.0005) employing students t test.</p

Reduced calcium signaling in CD83Tg B cells.

<p>Untouched B (7AB) or T cells (7CD) were isolated from pooled spleens of two 8–10 week old sex matched C57BL/6 (open bars) and CD83Tg (black bars) mice. Purified B and T cells were loaded with Fura-2-AM and stimulated with anti-ΒCR (Lo-MK) or anti-CD3 (145-2C11) respectively (10 µg/ml each). [Ca²⁺]_i was calculated from the fluorescence intensity ratio at 340 and 380 nm. 7AC: Bars represent the combined results from nine independent measurements, error bars show SEM. Asterisks indicate a significant difference of the mean. 7BD: Shown is a characteristic calcium tracing from a single experiment for B cells (7B) and T cells (7D) obtained from C57BL/6 (solid line) or CD83Tg (dotted line) mice.</p

CD83Tg spleen cells display reduced Ig and increased IL-10 response to LPS stimulation.

<p>3A-D: 2×10⁵ spleen cells derived from sex and age matched C57BL/6 (open squares) or CD83Tg founder 2 (closed squares) were cultured in the presence of LPS (10 µg/ml) or anti-CD3 (145-2C11, 1 µg/ml) or no further stimulus, as indicated on the x-axis. 3AB: Proliferation was estimated after 48h by ³H thymidine incorporation. 3C: Total mouse Ig in the supernatant was measured after seven days by ELISA. Results are presented as relative ELISA units (REU: OD₄₅₀ sample: OD₄₅₀medium control) of a 1∶1024 dilution. 3D: IL-10 in the supernatant was quantified by ELISA after 48 h. Each dot represents the analysis of an individual mouse (mean of five replicates the SEM not shown, being below 10%). The lines indicate the median of all mice analyzed and the asterisks indicate a significant difference of the median (*p<0.05; **p<0.005) employing students t test. 3E: HEL-specific Ig in the serum of six IgHEL Tg mice (open bar) or six IgHEL CD83double Tg mice (black bar) was quantified by ELISA, error bars indicate SEM. 3F: 2×10⁵ spleen cells derived from six IgHEL Tg mice (open bars) or from spleen cells derived from IgHEL CD83 double Tg mice (black bars) were cultivated <i>in vitro</i> for 48 h in the presence of indicated amounts of LPS. HEL-specific Ig in the supernatant was quantified by ELISA and is presented as REU of a 1: 40 dilution. Error bars show SEM of five replicates. This result is representative for five independent experiments.</p

<i>In vitro</i> stimulation of CD83mu spleen cells.

<p>2×10⁵ spleen cells derived from C57BL/6 (open squares) or CD83mu (closed circles) mice were cultured in the presence or absence of LPS (1 µg/ml) as indicated on the x-axis. 5A: Proliferation was estimated by ³H thymidine incorporation after 48h culture. 5B: Total mouse Ig in the supernatant was measured after seven days by ELISA and results are presented as REU of a 1:200 dilution. 5C: IL-10 in the supernatant was quantified by ELISA after 48 h. 5DE: spleen cells were cultured in the presence or absence of anti-CD3 (1 µg/ml). Proliferation (5D) and IL-2 secretion (5E) were measured after 48h culture. Each dot represents an individual experiment performed in five replicates SD being below 10%, the bars indicate the median of all five experiments performed.</p

Positive correlation of CD83 expression to CD86 and MHC-II expression on CD83Tg and CD83mu B cells.

<p>C57BL/6 and CD83 negative littermates to CD83Tg founder 1 mice (open bars), CD83Tg (black bars) or CD83mu (dark grey bars) derived spleen cells (2×10⁶/ml) were double stained for CD19 and either CD83 (2A), CD86 (2B), MHC-II (2C), CD80 (2D), CD40 (2E), CD69 (2F) or IgM (2G) <i>ex vivo</i> or after 48 h incubation with 10 µg/ml LPS as indicated on the x axis. 2×10⁴ CD19 positive cells were analyzed by FACScan. Please note that each bar represents the combined results of five independent experiments employing two female age matched mice of each group per experiment, error bars show SEM. Asterisks indicate a significant difference of the mean (* p<0.05; ** p<0.005; *** p<0.0005) employing students t test.</p

CD83 is upregulated on activated B cells.

<p>C57BL/6 mice derived spleen cells (2×10⁶/ml) were stimulated with anti-BCR (1 µg/ml) and IL-4 (20 ng/ml) or with LPS (10 µg/ml) as indicated in the headline. Cells were triple stained for CD19, CD83 and CD69 at the indicated time points. 1A: Dot blots show all lymphocytes positive for surface expression of CD83 on the x-axis and CD19 expression on the y-axis. 1BC: Graphs show the percentage of CD83 positive B cells (1B) or the mean fluorescence intensity (MFI) of CD83 on B cells (1C) after stimulation with anti-BCR alone (open circle) anti-BCR and IL-4 (closed circle) or with LPS (closed square) in an independent experiment, error bars show SEM of duplicates. 1D: Dot blot shows 2×10⁴ CD19 positive cells derived from LPS activated spleen cells analyzed for CD83 (x-axis) and CD69 (y-axis) surface expression. 1E: 2×10⁶ purified C57BL/6 spleen derived B cells were stimulated with LPS (10 µg/ml). B cells were lysed at the indicated time points, deglycosylated and separated by SDS-PAGE. CD83 was detected by western blot with a polyclonal rabbit anti-CD83 serum. Results are representative for at least three independent experiments.</p

Purified CD83Tg B cells display reduced Ig and increased IL-10 response to LPS stimulation.

<p>Untouched B cells were isolated from pooled spleens of two 8–10 week old sex matched C57BL/6 (open squares) and CD83Tg (black squares) mice. 2×10⁵ B cells were cultured in 5 replicates in the presence of LPS (10 µg/ml) or no further stimulus, as indicated on the x-axis. Proliferation (4A) or IL-10 secretion (4B) were measured after 48h. 4C: Total mouse Ig in the supernatant (dilution 1∶1024) was measured after seven days by ELISA. Each dot represents an individual experiment, employing B cells purified from two pooled spleens (mean of five replicates the SEM not shown, being below 10%). The bars indicate the median and the asterisks indicate a significant difference of the median (***p<0.0005), employing students t test.</p