Use of hollow fiber membrane filtration for the removal of DMSO from platelet concentrates.

It has been hypothesized that, in addition to freezing injury, some damage to platelets may result from the cell packing that occurs during removal of the cryoprotectant. This study examined DMSO removal by fluid exchange across hollow-fiber (HF) filters as an alternative to centrifugation. The DMSO solution with or without cell suspension was passed once through the filter. The optimum exchange during unloading of DMSO was determined by varying the flow rates in the external and internal compartments of the HF filter. Initially, buffered solutions of a 5% DMSO solution in the absence of platelets were pumped into the fibers and exchanged against PBS. The residual DMSO was determined by osmometry. The exchange of DMSO across the membrane was flow dependent and also influenced by the chemical nature of the HF fibers. No protocol using a reasonable rate flow through the fibers removed more than 95% of the DMSO in a single pass. The optimum protocol was achieved with polysynthane fibers with an internal flow rate of approximately 20 mi/min and an external flow rate of 100 ml/min. Subsequently, frozen/thawed platelet concentrates in DMSO were washed using centrifugation and compared to the HF filtration method. Platelet quality was assayed by flow cytometry, cell count, morphology and osmotic stress test. Both filtration and centrifugal washing techniques resulted in comparable morphological scores and numbers of discoid cells. When agents reducing platelet activation were added, platelet quality was improved after washing by either technique. The lower platelet osmotic response with HF filtration than with centrifugation while using activation inhibitors was attributed to the remaining amount of the inhibitors. All other parameters tested were similar. The expression of CD62P was equivalent with both techniques, and centrifugation did not activate platelets more than filtration contrary to what was originally anticipated. In conclusion, platelet quality was comparable after washing by either technique but hollow fiber filtration does remove cryoprotectant more rapidly than does centrifugation.

In vivo transfection and/or cross-priming of dendritic cells following DNA and adenoviral immunizations for immunotherapy of cancer--changes in peripheral mononuclear subsets and intracellular IL-4 and IFN-gamma lymphokine profile.

In order to provoke an immune response, a tumor vaccine should not only maximize antigen-specific signals, but should also provide the necessary "co-stimulatory" environment. One approach is to genetically manipulate tumor cells to either secrete lymphokines (GM-CSF, IL-12, IL-15) or express membrane bound molecules (CD80, CD86). Furthermore, patient dendritic cells can be loaded with tumor-associated antigens or peptides derived from them and used for immunotherapy. Genetic modification of dendritic cells can also lead to presentation of tumor-associated antigens. Transfection of dendritic cells with DNA encoding for such antigens can be done in vitro, but transfection efficiency has been uniformly low. Alternatively, dendritic cells can also be modulated directly in vivo either by "naked" DNA immunization or by injecting replication-deficient viral vectors that carry the tumor specific DNA. Naked DNA immunization offers several potential advantages over viral mediated transduction. Among these are the inexpensive production and the inherent safety of plasmid vectors, as well as the lack of immune responses against the carrier. The use of viral vectors enhances the immunogenicity of the vaccine due to the adjuvant properties of some of the viral products. Recent studies have suggested that the best strategy for achieving an intense immune response may be priming with naked DNA followed by boosting with a viral vector. We have successfully completed a phase I and phase II clinical trials on immunotherapy of prostate cancer using naked DNA and adenoviral immunizations against the prostate-specific membrane antigen (PSMA) and phase I clinical trial on colorectal cancer using naked DNA immunization against the carcinoembryonic antigen (CEA). The vaccination was tolerated well and no side effects have been observed so far. The therapy has proven to be effective in a number of patients treated solely by immunizations. The success of the treatment clearly depends on the stage of the disease proving to be most efficient in patients with minimal disease or no metastases. A panel of changes in the phenotype of peripheral blood lymphocytes and the expression of intra-T-cell lymphokines seems to correlate with clinical improvement.

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 microg and of recombinant virus at 5x10(8) 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.

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.

Mechanisms of alloimmunization and immunosuppression by blood transfusions in an inbred rodent model.

During refrigerated storage leukocytes in donor blood progressively undergo apoptosis followed by secondary necrosis. Using an inbred rodent transfusion model, recipient animals received viable, necrotic, or apoptotic cells. While transfusion of viable blood MNCs stimulated production of IgM, IgG1 (Th2 type) and IgG2a (Th1-type) antidonor antibodies, leading to a suppression of subsequent DTH to donor antigens, transfusion of apoptotic donor cells led to neither alloimmunization nor immunosuppression. On the other hand transfusion of lysed donor cells resulted in production of IgM and IgG1 (Th2-type) antidonor antibodies and to a strong suppression of subsequent DTH to donor antigens. Intravenously administered spleen cells that had been depleted of professional APCs and enriched for B cells stimulated IgM antidonor antibodies but not IgG antibodies. Transfusion of such cells also led to suppression of subsequent DTH to donor antigens, probably through induction of anergy or apoptosis in alloantigen-reactive recipient cells. Depending on the duration of blood storage any or all of these 4 classes of cells may be present and Th2 and/or Th1 effector mechanisms can be generated following blood transfusion.

Blood transfusion, blood storage and immunomodulation.

Allogeneic blood transfusion is the most frequent allotransplantation procedure performed on a routine basis with no prior HLA-typing. Roughly 50% of the recipients of unprocessed red cells and platelets become alloimmunized. Evidence also exists for some degree of transfusion-induced immunosuppression. Prior transfusion has been shown to enhance kidney transplant survival and evidence of an increase in tumor recurrence and of infectious complications has also been presented. The presence of donor antigen-presenting cells appears to be a prerequisite for alloimmunization and they must be both viable and capable of presenting a costimulatory signal in order to induce IL-2 secretion and proliferation of responding CD4 T cells. APCs presenting antigen but no costimulatory signal can induce non-responsiveness in CD4 T cells, a possible mechanism of transfusion-induced immunosuppression. APCs in refrigerated blood continue to present antigen but progressively lose their ability to provide costimulation. By day 14 costimulatory capacity is absent and transfusion of such blood should not alloimmunize but could induce some degree of immunosuppression. Further refrigerated storage in excess of 2 to 3 weeks leads to induction of apoptosis in contaminating leukocytes. We have found that alloantigens-expressed on such cells do not appear to be recognized by responder T cells and transfusion of blood stored in excess of 3 weeks should neither alloimmunize nor immunosuppress.

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.

Manipulating red cell intra- and extracellular pH by washing.

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 degrees 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.

Refrigerated storage of washed red cells.

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.

Cryoprotectant toxicity and cryoprotectant toxicity reduction: in search of molecular mechanisms.

Cryoprotectant toxicity is a fundamental obstacle to the full potential of artificial cryoprotection, yet it remains in general a poorly understood phenomenon. Unfortunately, most relevant biochemical studies to date have not met the basic criteria required for demonstrating mechanisms of toxicity. A model biochemical study of cryoprotectant toxicity was that of Baxter and Lathe, which demonstrated that alteration of a specific enzyme (fructose diphosphatase, or FDPase) was the cause of impaired glycolysis after treatment with and removal of dimethyl sulfoxide (D). FDPase alteration by D was reported to be preventable by the simultaneous presence of amides. This protection could be due to a "counteracting solute" effect similar to that employed by nature, but we find no meaningful correlation between the general protein stabilizing or destabilizing tendency of the cryoprotectant medium and its toxicity. Baxter and Lathe postulated that the effect of D arises from hydrogen bonding between D and the epsilon amino groups of surface lysine residues on FDPase, and it was found that molecules which resembled this group could block the alteration induced by D, presumably by competing with lysine residues for association with D. However, we find that the interaction between D and lysine in the presence of water is actually thermochemically repulsive, and that the presence of formamide does not affect the interaction between D and lysine, implying no useful complex formation between formamide and D. We were also unable to demonstrate that the blocking compounds consistently reduce toxicity when added to D rather than substituting for D, contrary to predictions based on complex formation between blocking compounds and D. In summary, it seems that present concepts of cryoprotectant toxicity are in need of serious revision.

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 costimulatory 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.

Induction of primary mixed leukocyte reactions with ultraviolet B or chemically modified stimulator cells.

Treatment of stimulator cells with 0.1% paraformaldehyde for 60 sec or ultraviolet-B (UV-B) irradiation (1000 J/m2) eliminates their ability to elicit T cell proliferation in a primary mixed leukocyte reaction. However, a T cell response equal to 20-40% of control value could be elicited by paraformaldehyde fixed or UV-B irradiated cells providing the latter are incubated at 37 degrees C for 18 hr prior to treatment. The incubation also induces a one-log increase in the density of fluorescence when the cells are stained with monoclonal antibodies against class II molecules DR and DP as well as the intercellular adhesion molecule -1 (ICAM-1). We interpret this as an increase in the membrane expression of these structures following incubation. Chloroquine and cerulenin, known to inhibit protein degradation and antigen processing and presentation do not influence the upregulation in membrane expression of these class II and adhesion molecules, but do prevent incubation from overriding the effect of paraformaldehyde treatment. Colchicine, which reduces the traffic through tubular lysosomes, also has no effect on the upregulation but enhances allopresentation. We propose that incubation of stimulator cells in the presence of chloroquine and cerulenin results in the membrane expression of class II molecules without associated peptides. The inability of stimulator cells expressing such "nude" MHC molecules to elicit T cell proliferation after chemical modification could be due to easier crosslinking of the allodeterminants by paraformaldehyde when the binding site is empty but could also mean that nude MHC molecules are not per se immunogenic and become so only after acquisition of a peptide. It is also possible that chloroquine, NH4Cl, and cerulenin block the expression of signals other than the class II and cell adhesion molecules that are essential for induction of T cell proliferation.

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.

Density gradient isolation of peripheral blood mononuclear cells using a blood cell processor.

Large numbers of mononuclear cells (MNC) are needed for hematologic reconstitution using peripheral blood stem cells. The possibility of isolating those cells by discontinuous Ficoll-diatrizoate density gradient centrifugation in two blood cell processors (the Haemonetics V50 [V50] and the Cobe 2991 [2991]) were examined. Buffy coats from peripheral blood containing 6.23 X 10(8) MNC were separated in the V50, resulting in a recovery of 75 percent. The purity of the cells, defined as the percentage of lymphocytes and monocytes among all leukocytes, was 95 percent. With larger cell loads (3 to 7 X 10(9) MNC), the yield was higher in the V50 than in the Cobe 2991 (92 versus 75%). After separation in the V50 or the 2991, the cloning efficiencies of hematopoietic progenitor cells (CFU-GM and BFUe) were not different from those of cells isolated on 5 ml Ficoll-diatrizoate gradients in centrifuge tubes. Both leukapheresis and MNC separation can be carried out with the same bowl and tubing set in the V50. With that approach, an average of 6 X 10(9) MNC were processed in 16 experiments. An average recovery of 82 percent with 95 percent purity was achieved. The authors conclude that, in terms of simplicity of operation, cost effectiveness, and maintenance of sterility, the V50 may be better suited than the 2991 for the purification of MNC from peripheral blood.