Detection of enhanced cytotoxic T lymphocyte response with spleenic dendritic cells enriched by a simple sequential adherence method.

Dendritic cells (DC), which are present in many tissues, play a critical role in the initiation of the immune response by presenting antigens to T and B lymphocytes. DC are present in tissues as a minority cell type as compared to other APCs. Although clearly distinguished by morphology and surface markers, dendritic cells are difficult to isolate and multiple step procedures including Percoll/Hypaque or BSA gradient centrifugation have been used. Here we describe a simple method for isolating an enriched population of dendritic cells from mouse spleen by sequential adherence to petri dishes. The purity of dendritic cells enriched thereby was found to be above 70%. The identity of these cells was confirmed to be DC by morphology, presence of surface markers, and their functions, e.g., strong allogeneic MLR reaction and induction of antigen-specific cytotoxic T lymphocyte response.

Influenza A virus-specific H-2d restricted cross-reactive cytotoxic T lymphocyte epitope(s) detected in the hemagglutinin HA2 subunit of A/Udorn/72.

BALB/c mice immunized with a vaccinia virus recombinant expressing the influenza A virus (A/Udorn/72; subtype H3) hemagglutinin HA2 (H3HA2) induced a strong CD8+ cytotoxic T lymphocyte (CTL) response which was H-2d-restricted. Two peptides, derived from the primary sequence of the H3HA2 and consistent with the predictive motif for Kd-restricted epitopes, were tested for their ability to be presented to the CTLs by P815 cells. Peptides corresponding to the amino acids 93SYNAELLVAL102 and 181GYKDWILWI189 of the HA2 primary sequence sensitized target cells for lysis by the HA2-specific CTLs. Secondary in vitro stimulation with dendritic cells as a source of antigen presenting cells treated with peptide 93-102, or infected with recombinant vaccinia virus (HA2-VACC), induced type A cross-reactive CTLs from A/Udorn/72 immune spleen cells. This CD8+ CTL epitope overlaps with an H3HA2 I-Ad-restricted T helper epitope 96-104 recently reported by others. The H3HA2 epitope described here is the first CTL epitope in the influenza hemagglutinin which is found in all three subtypes of influenza A virus.

Effector-assisted refolding of recombinant tissue-plasminogen activator produced in Escherichia coli.

Recombinant tissue-plasminogen activator (r-tPA), expressed in Escherichia coli cells in an aggregated form, was solubilized with a strong chaotrope in the absence of any reducing agent. The solubilized molecule was reactivated by a procedure that was developed to mimic the physiological conditions optimal for the functional folding and activity of the native protein. The use of partially purified fibrinogen, as a source of fibrin (the effector), is shown to facilitate the reactivation process and increase its yield by at least a factor of two. The yield of the process is also shown to be particularly dependent on the recombinant protein concentration. At a concentration level of 3-3.7 mg r-tPA/L in the reactivation mixture, up to a 90% yield of activity was obtained. Purification of the activated form of r-tPA was achieved with a two-step column-chromatography scheme. This included a gel filtration step on a Sephadex G-50 column followed by an affinity chromatography step on a lysine-sepharose column. The product was composed of roughly equal amounts of one-chain and two-chain t-PA. The feasibility of using a two water-soluble polymeric phase system, with a centrifugal partition chromatography (CPC), in scaling up the reactivation process or the purification step was also evaluated.

Affinity purification of tissue plasminogen activator using transition-state analogues.

The search for a simple affinity ligand to purify tissue plasminogen activator (tPA) was facilitated by a solid-phase synthesis approach. A large variety of tripeptide ligands containing argininal were synthesized on agarose gels containing a spacer with carboxy terminal. The immobilized ligands were easy to test with urokinase, and tPA. While a number of sequence combinations showed initial binding by tPA, only a few resulted in tight binding corresponding to a hemiacetal linkage with the active site serine. Hydrophobic residues, especially aromatics, flanking the N-side of argininal gave rise to ligands which were bound strongly by tPA. A gel containing D-Phe-D-Phe-Argal (an aldehyde derivative of arginine) was very effective in purifying tPA derived from cell culture media at small scale (milligrams) and at large (multi-grams).

Large-scale isolation of equine liver alcohol dehydrogenase on a blue agarose gel.

Equine liver alcohol dehydrogenase (EC 1.1.1.1) has been purified by a new scheme using a blue agarose gel (Blue Sepharose) as an affinity sorbent. Starting amounts of 0.6 to 10 kg liver have been processed to enzyme possessing 1.5 U/mg average specific activity, in about three to four days. Some parameters concernining adsorption of enzyme to the blue gel as well as recovery therefrom have been explored.

Affinity purification methods. V1. A novel and rapid isolation procedure for lysozymes.

Lysozymes from animal and plant sources have been purified on agarose derivatives containing a phenylacetyl ligand. While this adsorbent can also bind chymotrypsin, the use of imidazole permits separation of lysozymes from the protease. The phenylacetyl-agarose is a general affinity sorbent bearing a ligand of non-biochemical origin in contrast to those, for example, which use nucleotide cofactors. By careful selection of desorption conditions, different enzymes can be purified with the same sorbent. Results also suggest that binding of lysozymes to an affinity ligand does not require a leash structure between ligand and support matrix.