Harold T. Meryman

Vitrification as an approach to cryopreservation

Recent developments have opened the possibilty that the problems of freezing and thawing organs might eventually be overcome by an alternative approach to organ cryopreservation, namely, vitrification. Here we will review some of the principles of vitrification, describe the current state of the art, consider how a practical vitrification scheme might work, and conclude by noting how the principles of vitrification relate to and illuminate the principles and practices of freezing.

Naked DNA and adenoviral immunizations for immunotherapy of prostate cancer : A phase I/II clinical trial

Introduction and Objectives: Animal studies have indicated that the use of syngeneic dendritic cells that have been transfected ex vivo with DNA for tumor–specific antigen results in tumor regression and decreased number of metastases. Additional studies have also suggested the possibility to modulate the dendritic cells in vivo either by ‘naked’ DNA immunization or by injecting replication–deficient viral vectors that carry the tumor–specific DNA. Using the prostate– specific membrane antigen (PSMA) as a target molecule, we have initiated a clinical trial for immunotherapy of prostate cancer. The primary objective of the study was to determine the safety of the PSMA vaccine after repeated intradermal injections.Methods: We have included the extracellular human PSMA DNA as well as the human CD86 DNA into separate expression vectors (PSMA and CD86 plasmids), and into a combined PSMA/CD86 plasmid. In addition, the expression cassette from the PSMA plasmid was inserted into a replication deficient adenoviral expression vector. Twenty–six patients with prostate cancer were entered into a phase I/II toxicity–dose escalation study, which was initiated in spring 1998. Immunizations were performed intradermally at weekly intervals. Doses of DNA between 100 and 800 μg and of recombinant virus at 5×108 PFUs per application were used.Results and Conclusion: No immediate or long–term side effects following immunizations have been recorded. All patients who received initial inoculation with the viral vector followed by PSMA plasmid boosts showed signs of immunization as evidenced by the development of a delayed–type hypersensitivity reaction after the PSMA plasmid injection. In contrast, of the patients who received a PSMA plasmid and CD86 plasmid, only 50% showed signs of successful immunization. Of the patients who received PSMA plasmid and soluble GM–CSF, 67% were immunized. However, all patients who received the PSMA/CD86 plasmid and sGM–CSF became immunized. The patients who did not immunize during the first round were later successfully immunized after a boost with the viral vector. The heterogeneity of the medical status and the presence in many patients of concomitant hormone therapy does not permit unequivocal interpretation of the data with respect to the effectiveness of the therapy. However, several responders, as evidenced by a change in the local disease, distant metastases, and PSA levels, can be identified. A phase II clinical study to evaluate the effectiveness of the therapy is currently underway.

Freezing injury from "solution effects" and its prevention by natural or artificial cryoprotection.

Abstract This paper is concerned with two general areas of basic cryobiology: (I) The mechanism by which the hyperosmolality caused by slow freezing injures the living cell, and (II) the mechanism by which cryoprotectants modify this process.

Blood transfusion and immunomodulation: a possible mechanism.

To induce an immunogenic response in vivo, an antigen‐presenting (stimulator) cell must present both antigen‐specific (class II MHC) and an accessory signal to the CD4 T cell. Failure to express the accessory signal has been shown in vitro to induce a state of specific unresponsiveness (anergy) in the T cell. We have shown that although stimulator cells in blood continue to express class II MHC molecules during refrigerated storage, their ability to present the accessory signal diminishes, reaching zero in all units tested by about 13 days. This implies that blood in excess of 2 weeks old should not alloimmunize but could induce some degree of immunosuppression. UV‐B irradiation and, to a lesser extent, gamma‐irradiation, were also shown to inhibit expression of the accessory signal by stimulator cells in blood.

Prolonged storage of red cells at 4 degrees C.

One of the events associated with red cell storage at 4°C is the development of an increasing proportion of echinocytes. Vesicles also may bud off the spicules, presumably leading to a decreased surface‐to‐volume ratio and decreased deformability. Pursuing the hypothesis that increasing the surface tension of the cells by increasing their volume might reduce the tendency toward echinocytosis and extend refrigerated storage time, packed red cells were resuspended in a solution hypotonic (210 mOsm) with respect to solutes that do not penetrate the cell. Since a reduced ionic concentration results in increased membrane permeability for cations, normal ionic concentration was maintained by the addition of NH4C1, which readily penetrates red cells and therefore contributes no osmotic support. Adenine, glucose, mannitol, citrate, and phosphate also were included. Unexpectedly, the predominant effect of red cell storage in this solution was a remarkable elevation of adenosine triphosphate (ATP). At 4, 8, and 10 weeks, (ATP) levels averaged 165, 135, and 110 percent of initial values, respectively. At 16 weeks, ATP still averaged 50 percent of initial values. Twenty‐four‐hour in vivo survival of red cells measured at 12 to 18 weeks ranged between 70 and 80 percent, and hemolysis ranged from 0.3 to 7.1 percent. Both the hypotonicity and the ammonium salt appear to be necessary for the high ATP.

Refrigerated Storage of Washed Red Cells

Abstract. Red cells washed and stored in a citrate‐phosphate‐glucose‐adenine solution at pH 7.4–7.6 demonstrate excellent maintenance of adenosine triphosphate, elevation of 2,3‐diphosphoglycerate well above normal levels for more than 6 weeks, reduced hemolysis and 24‐hour in vivo survival comparable to that of cells stored in ADSOL. These results can be attributed in part to a chloride shift in which the washout of intracellular chloride is associated with an influx of OH“, which increases intracellular pH and thereby increases the rate of glycolysis. The phosphate functions primarily as a buffer to maintain both extra‐ and intracellular pH. Reducing the effective osmolality of the storage solution reduces hemolysis and improves cell morphology.

Fas and Fas Ligand Expression on Human Peripheral Blood Leukocytes

Objectives: Study of Fas and Fas ligand (Fas-L) expression, as well as sFas-L release, by fresh human peripheral blood leukocytes. Methods: Flow cytometry, cytotoxicity, immunofluorescence staining of fresh smears, Western blotting. Results: Granulocytes and monocytes express a low level of Fas receptor, but no Fas-L. These cells, as well as NK cells, contain presynthesized depots of Fas-L which they express following activation by brief storage (60 min) at room temperature or during separation from whole blood. Such activation also leads to Fas receptor upregulation. NK cells do not express Fas receptor. Once expressed on blood leukocytes, fully functional Fas-L can be released from the membrane and can be detected in plasma-free cell supernatants. Conclusion: Human peripheral blood granulocytes, monocytes and NK cells contain intracellular presynthesized Fas-L which they readily express following blood anticoagulation, blood storage or cell separation. Soluble Fas-L is released from those cells and can be detected in protein-free supernatants by immunoblotting.

MANIPULATING RED CELL INTRA- AND EXTRACELLULAR PH BY WASHING

Abstract. Washing red cells with solutions containing no anions capable of entering the cells is known to result in the loss of intracellular chloride and a counterflow of OH‐ which raises intracellular and lowers extracellular pH. Elevating the intracellular pH improves the quality of the cells during 4°C storage. The extent to which intracellular pH can be raised and extracellular pH reduced depends not only on the inability of extracellular anions to enter the cells but also on the buffering capacity of the wash solution. Both intra‐ and extracellular pH can be manipulated by the judicious selection of anions or combinations of anions used in the wash solution.

Costimulatory signals necessary for induction of T cell proliferation.

Two separate signals are required for induction of T cell proliferation. In an attempt to identify them we used polyclonal T cell activation with Con A, which requires costimulation with autologous accessory cells. The co-stimulatory activity is not constitutively expressed on accessory cells since such cells fixed immediately after separation from whole blood are unable to provide the necessary signal(s), although such activity is readily expressed after activation by incubation and such cells subsequently fixed will support Con A-induced T cell proliferation. Addition of recombinant IL-1 plus IL-6 to T cell cultures in the absence of accessory cells does not result in T cell proliferation but addition of these factors to cultures containing fixed activated accessory cells results in further increase in proliferation. The expression of the costimulatory activity during incubation is inhibited in the presence of cycloheximide or tunicamycin. The costimulatory activity of fixed activated cells is partially inhibited by antibody against ICAM-1. This inhibition is not reversed by the addition of recombinant IL-1 and IL-6. When accessory cells are preactivated in the presence of chloroquine, they are unable to provide costimulation to T cells but addition of recombinant IL-1 and IL-6 restores their ability to support T cell proliferation. Accessory cells preactivated in the presence of colchicine show an increased ability to provide costimulation to T cells in culture.

Frozen red cells.

The popularity and the promise of frozen red cells during the 1970s were largely attributable to logistic problems associated with 21-day storage and to the fringe benefits of white cell and plasma depletion that minimized alloimmunization and febrile transfusions and, it was speculated, reduced the risk of HBV transmission. Filtration, particularly with the new generation of filters now appearing on the market, promises to achieve an equivalent reduction in white cells at a fraction of the cost and inconvenience. Donor testing for HBV and anti-HIV and, as would appear from recent data, the ALT assay as a surrogate test for non-A, non-B hepatitis, have reduced the incidence of transmission of these diseases below the level where either evaluating or utilizing red cell freezing would be practically or economically feasible. The use of frozen red cells following rejuvenation will certainly be replaced by effective resuspension solutions that will permit rejuvenation, washing, and additional weeks of refrigerated storage. Barring some wholly unexpected and improbable development bringing the cost and convenience of frozen red cells close to those of refrigerated cells, there is little reason to believe that frozen red cells will find applications in the civilian market, except for the storage of rare types and, possibly, the prevention of CMV transmission in the foreseeable future. The original goal of red cell freezing, to make long term storage possible, has been fully realized. The rest is history.