The Role of Lipopeptidophosphoglycan in the Immune Response to Entamoeba histolytica

The sensing of Pathogen Associated Molecular Patterns (PAMPs) by innate immune receptors, such as Toll-like receptors (TLRs), is the first step in the inflammatory response to pathogens. Entamoeba histolytica, the etiological agent of amebiasis, has a surface molecule with the characteristics of a PAMP. This molecule, which was termed lipopeptidophosphoglycan (LPPG), is recognized through TLR2 and TLR4 and leads to the release of cytokines from human monocytes, macrophages, and dendritic cells; LPPG-activated dendritic cells have increased expression of costimulatory molecules. LPPG activates NKT cells in a CD1d-dependent manner, and this interaction limits amebic liver abscess development. LPPG also induces antibody production, and anti-LPPG antibodies prevent disease development in animal models of amebiasis. Because LPPG is recognized by both the innate and the adaptive immune system (it is a “Pamptigen”), it may be a good candidate to develop a vaccine against E. histolytica infection and an effective adjuvant

PD-L1 Expression Induced by the 2009 Pandemic Influenza A(H1N1) Virus Impairs the Human T Cell Response

PD-L1 expression plays a critical role in the impairment of T cell responses during chronic infections; however, the expression of PD-L1 on T cells during acute viral infections, particularly during the pandemic influenza virus (A(H1N1)pdm09), and its effects on the T cell response have not been widely explored. We found that A(H1N1)pdm09 virus induced PD-L1 expression on human dendritic cells (DCs) and T cells, as well as PD-1 expression on T cells. PD-L1 expression impaired the T cell response against A(H1N1)pdm09 by promoting CD8+ T cell death and reducing cytokine production. Furthermore, we found increased PD-L1 expression on DCs and T cells from influenza-infected patients from the first and second 2009 pandemic waves in Mexico City. PD-L1 expression on CD8+ T cells correlated inversely with T cell proportions in patients infected with A(H1N1)pdm09. Therefore, PD-L1 expression on DCs and T cells could be associated with an impaired T cell response during acute infection with A(H1N1)pdm09 virus

Effect of additives in colorimetric end-point RT-LAMP assay performance.

(A) Colorimetric RT-LAMP reactions under optimized conditions using N1 primer set and 40 mM of guanidine hydrochloride (GuHCl) in the reaction buffer. The figure shows the colorimetric determination of each reaction (upper panel) and the electrophoretic profile of the amplification reaction products (lower panel). (B) Colorimetric RT-LAMP reactions under optimized conditions using N1 primer set in absence of GuHCl in the reaction buffer. The figure shows the colorimetric determination of each reaction (upper panel) and the electrophoretic profile of the amplification reaction products (lower panel). (C) Colorimetric RT-LAMP reactions under optimized conditions using N1 primer set and 0.8M of betaine in the reaction buffer. The figure shows the electrophoretic profile of the amplification reaction products. (D) Colorimetric RT-LAMP reactions under optimized conditions using N1 primer set in absence of betaine in the reaction buffer. The figure shows the electrophoretic profile of the amplification reaction products. C+: 1x104 copies of N1 in vitro transcript used as positive control; NTC: non-template control; M: DNA molecular weight marker 1 Kb Plus DNA Ladder (Invitrogen). (TIF)</p

Expression and purification of recombinant <i>Bacillus stearothermophilus</i> DNA polymerase (Bst) and reverse transcriptase (RT) from Moloney Murine Leukemia Virus.

Coomassie blue-stained 8% Tricine-SDS-PAGE electrophoresis gel analysis. (A) Expression of recombinant Bst enzyme and Ni2+-IMAC purification. Lane 1: clarified supernatant loaded; Lane 2: flow-through fraction; Lane 3–6: washing step fractions; Lane 7–9: eluted fractions containing Bst. (B) Heparin column purification of recombinant Bst. Lane 1: desalted sample loaded; Lane 2: flow-through fraction; Lane 3: washing step fraction; Lane 4–6: elution fractions; Lane 7–8: concentrated Bst-containing fractions. (C) Final Bst formulations from three different purification batches (B1, B2, B3). (D) Expression of recombinant RT enzyme and Ni2+-IMAC purification. Lane 1–3: flow-through fractions; Lane 4–5: washing step fractions; Lane 6–10: elution fractions containing RT. (E) Cation exchange column purification of recombinant RT. Lane 1: IMAC elution fraction; Lane 2: desalted sample loaded; Lane 3–5: flow-through fractions; Lane 6–7: washing step fractions; Lane 8–13: elution fractions. (F) Final RT formulations from three different purification batches (B1, B2, B3). M: molecular weight marker; C+: previously purified Bst or RT enzyme, employed as control positive; I: insoluble fraction; S: soluble fraction; C: clarified supernatant. Violet or green arrows indicate the expected size for Bst and RT enzymes, respectively.</p

Chromatographic profiles of the purification steps of Bst and RT by fast protein liquid chromatography (FPLC).

(A) Chromatogram of Bst purification by Ni+2-IMAC. (B) Chromatogram of RT purification by Ni+2-IMAC. (C) Chromatogram of the desalting step of the Bst-containing fractions. (D) Chromatogram of the desalting step of the RT-containing fractions. (E) Chromatogram of the second purification step by heparin affinity chromatography for RT. (F) Chromatogram of the second purification step by cation exchange chromatography for RT. Values expressed in mAU are shown in purple (Bst) or green (RT). The dotted lines correspond to the concentration of the elution buffer used in each case: EB-AI (A), EB-BI (B), DB-A (C), DB-B (D), EB-AII (E), EB-B-II (F). Black arrows indicate the peaks of the chromatograms selected for the following purification steps. (TIF)</p