High-level secretion of recombinant monomeric murine and human single-chain Fv antibodies from Drosophila S2 cells

Single-chain variable fragment (scFvs) antibodies are small polypeptides (∼26 kD) containing the heavy (VH) and light (VL) immunoglobulin domains of a parent antibody connected by a flexible linker. In addition to being frequently used in diagnostics and therapy for an increasing number of human diseases, scFvs are important tools for structural biology as crystallization chaperones. Although scFvs can be expressed in many different organisms, the expression level of an scFv strongly depends on its particular amino acid sequence. We report here a system allowing for easy and efficient cloning of (i) scFvs selected by phage display and (ii) individual heavy and light chain sequences from hybridoma cDNA into expression plasmids engineered for secretion of the recombinant fragment produced in Drosophila S2 cells. We validated the method by producing five scFvs derived from human and murine parent antibodies directed against various antigens. The production yields varied between 5 and 12 mg monomeric scFv per liter of supernatant, indicating a relative independence on the individual sequences. The recombinant scFvs bound their cognate antigen with high affinity, comparable with the parent antibodies. The suitability of the produced recombinant fragments for structural studies was demonstrated by crystallization and structure determination of one of the produced scFvs, derived from a broadly neutralizing antibody against the major glycoprotein E2 of the hepatitis C virus. Structural comparison with the Protein Data Bank revealed the typical spatial organization of VH and VL domains, further validating the here-reported expression system

Inhibition of Amebic Cysteine Proteases Blocks Amebic Trogocytosis but Not Phagocytosis

BackgroundEntamoeba histolytica kills human cells by ingesting fragments of live cells until the cell eventually dies, a process termed amebic trogocytosis. In a previous study, we showed that acidified amebic lysosomes are required for both amebic trogocytosis and phagocytosis, as well as cell killing.MethodsAmebic cysteine proteases (CPs) were inhibited using an irreversible inhibitor, E-64d.ResultsInterfering with amebic CPs decreased amebic trogocytosis and amebic cytotoxicity but did not impair phagocytosis.ConclusionsWe show that amebic CPs are required for amebic trogocytosis and cell killing but not phagocytosis. These data suggest that amebic CPs play a distinct role in amebic trogocytosis and cell killing

Studies of the “Chain Reversal Regions” of the Avian Sarcoma/Leukosis Virus (ASLV) and Ebolavirus Fusion Proteins: Analogous Residues Are Important, and a His Residue Unique to EnvA Affects the pH Dependence of ASLV Entry▿

Most class I fusion proteins exist as trimers of dimers composed of a receptor binding and a fusion subunit. In their postfusion forms, the three fusion subunits form trimers of hairpins consisting of a central coiled coil (formed by the N-terminal helices), an intervening sequence, and a region containing the C helix (and flanking strands) that runs antiparallel to and packs in the grooves of the N-terminal coiled coil. For filoviruses and most retroviruses, the intervening sequence includes a “chain reversal region” consisting of a short stretch of hydrophobic residues, a Gly-Gly pair, a CX6CC motif, and a bulky hydrophobic residue. Maerz and coworkers (A. L. Maerz, R. J. Center, B. E. Kemp, B. Kobe, and P. Poumbourios, J. Virol. 74:6614-6621, 2000) proposed a model for this region of human T-cell leukemia virus type 1 (HTLV-1) Env in which expulsion of the final bulky hydrophobic residue is important for early conformational changes and specific residues in the chain reversal region are important for forming the final, stable trimer of hairpins. Here, we used mutagenesis and pseudovirus entry assays to test this model for the avian retrovirus avian sarcoma/leukosis virus (ASLV) and the filovirus ebolavirus Zaire. Our results are generally consistent with the model proposed for HTLV-1 Env. In addition, we show with ASLV EnvA that the bulky hydrophobic residue following the CX6CC motif is required for the step of prehairpin target membrane insertion, whereas other residues are required for the foldback step of fusion. We further found that a His residue that is unique to the chain reversal region of ASLV EnvA controls the pH at which ASLV entry occurs

Inhibition of Amebic Lysosomal Acidification Blocks Amebic Trogocytosis and Cell Killing

Entamoeba histolytica ingests fragments of live host cells in a nibbling-like process termed amebic trogocytosis. Amebic trogocytosis is required for cell killing and contributes to tissue invasion, which is a hallmark of invasive amebic colitis. Work done prior to the discovery of amebic trogocytosis showed that acid vesicles are required for amebic cytotoxicity. In the present study, we show that acidified lysosomes are required for amebic trogocytosis and cell killing. Interference with lysosome acidification using ammonium chloride, a weak base, or concanamycin A, a vacuolar H+ ATPase inhibitor, decreased amebic trogocytosis and amebic cytotoxicity. Our data suggest that the inhibitors do not impair the ingestion of an initial fragment but rather block continued trogocytosis and the ingestion of multiple fragments. The acidification inhibitors also decreased phagocytosis, but not fluid-phase endocytosis. These data suggest that amebic lysosomes play a crucial role in amebic trogocytosis, phagocytosis, and cell killing.IMPORTANCEE. histolytica is a protozoan parasite that is prevalent in low-income countries, where it causes potentially fatal diarrhea, dysentery, and liver abscesses. Tissue destruction is a hallmark of invasive E. histolytica infection. The parasite is highly cytotoxic to a wide range of human cells, and parasite cytotoxic activity is likely to drive tissue destruction. E. histolytica is able to kill human cells through amebic trogocytosis. This process also contributes to tissue invasion. Trogocytosis has been observed in other organisms; however, little is known about the mechanism in any system. We show that interference with lysosomal acidification impairs amebic trogocytosis, phagocytosis, and cell killing, indicating that amebic lysosomes are critically important for these processes

Determination of modifiable risk factors for length-for-age z-scores among resource-poor Indonesian infants.

To reduce the burden of early-life linear growth faltering in low- and middle-income countries, interventions have focused on nutrition strategies, sometimes combined with water quality, sanitation, and hygiene (WASH). However, even when combined, their effects on linear growth have been inconsistent. Here, we investigate potential predictors of length-for-age z-scores (LAZ) in a cohort of resource-poor rural Indonesian infants to inform the optimal strategies to reduce linear growth faltering. Apparently healthy rural breastfed Indonesian infants were randomly selected from birth registries at age 6 months (n = 230) and followed up at 9 (n = 202) and 12 (n = 190) months. Using maximum likelihood estimation, we examined longitudinal relationships among socio-demographic status, maternal height, infant sex, age, water source, sanitation facility, energy, protein, micronutrient intakes and biomarkers (serum ferritin, zinc, retinol binding protein (RBP), selenium-adjusted for inflammation), and α-1-acid glycoprotein (AGP) and C-reactive protein (CRP) (systemic inflammation biomarkers) at age 6 and 9 months on LAZ at age 9 and 12 months. Stunting (LAZ <-2) at 6, 9, and 12 months was 15.7%, 19.3%, and 22.6%, respectively. In the full model, the predictor variable at age 6 months that was most strongly associated with infant LAZ at 9 months was maternal height (0.18 (95% CI 0.03, 0.32) SD). At age 9 months, the strongest predictors of LAZ at 12 months were improved drinking water source (-0.40 (95% CI -0.65, -0.14) vs. not improved), elevated AGP compared to not elevated (0.26 (95%CI -0.06, 0.58), maternal height (0.16 (95% CI 0.02, 0.31) SD), sex (0.22 (95% CI -0.02,0.45) female vs. male), serum RBP (0.12 (95% CI -0.01, 0.25) SD), and protein intake (0.17 (95% CI -0.01, 0.35) SD). Health promotion that includes exclusive breastfeeding up to the first six months and follows microbial water quality guidelines to ensure water intake is always safe should be considered