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Biomedical subjects

H T Meryman

Publications and source records attributed to H T Meryman.

At least 19 recordsLinked to original sources
PubMed1995

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.

Animals↗
PubMed1991

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.

Adenosine Triphosphate↗
PubMed1991

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.

Blood Preservation↗
PubMed1990

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.

Amides↗
PubMed1990

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.

Antigen-Presenting Cells↗
PubMed1989

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.

Cryopreservation↗
PubMed1989

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.

Antigen-Presenting Cells↗
PubMed1983

Studies of cell separation: a comparison of the osmotic response of human lymphocytes and granulocyte-monocyte progenitor cells.

The feasibility of using hypo- or hypertonic stress to selectively destroy lymphocytes while sparing stem cells was investigated. Lymphocytes were isolated from peripheral blood and exposed to Hanks' balanced salt solutions ranging in concentration from 66 to 2700 mOsm. The Boyle-van't Hoff plot of cell volume versus reciprocal osmolality was linear. Following osmotic stress, viabilities of the lymphocytes and the granulocyte-monocyte progenitor cells (CFUc) were determined. Lymphocyte viability was assessed by tritiated thymidine incorporation following mitogen stimulation. CFUc viability was measured with the soft agar colony assay. Both types of cells were found to possess high osmotic tolerances compared to other blood cells. While progenitor cells in general appeared to survive anisotonic exposure somewhat better than lymphocytes, significant statistical differences were not established for most situations. The highest degree of CFUc enrichment was twofold, but there was a concomitant 50% drop in CFUc survival. These results suggest that osmotic stress is not a useful procedure for the separation of peripheral blood lymphocytes and stem cells.

Bone Marrow↗
PubMed1982

Isolation of large numbers of fully viable human neutrophils: a preparative technique using percoll density gradient centrifugation.

Techniques were developed for the isolation of human neutrophils from large volumes of human blood. This preparative procedure avoids exposure to Ficoll/Hypaque and offers a low cost alternative to centrifugal elutriation. First, leukocyte-enriched suspensions were obtained by treating buffy coats with 2% dextran. The leukocyte suspensions were then fractionated on three steps Percoll density gradients. The gradients were designed to carry large numbers of cells while maintaining resolution of the cell peaks. As a result, at least 320 X 10(6) leukocytes could be processed on a single 12 ml gradient. Extensive testing of the procedure showed that the purity of the neutrophil band was 96.5 +/- 3.16% (n = 50). The band contained 82.4 +/- 14.6% (n = 39) of the neutrophils applied to the gradient. Viability assays (bacterial killing, chemotaxis) demonstrated that neutrophils isolated on Percoll gradients were just as active as those harvested by centrifugal elutriation. Recovery data are presented and compared to the results obtained by others.

Blood Bactericidal Activity↗
PubMed1982

Glycerolization of the human neutrophil for cryopreservation: osmotic response of the cell.

The human neutrophil probably requires a very high intracellular concentration of cryoprotectant to avoid cellular injury during cryopreservation. Accordingly, new techniques were developed so that high concentrations of protectant could be safely used. To accomplish that, it was necessary to overcome the osmotic limitations of the neutrophil as well as problems with cytotoxicity and membrane permeability. To reduce complications from toxicity, glycerol was selected as the protective agent. To minimize osmotic stress, the protectant concentration was changed slowly and gently by a system combining cross-flow filtration and exponential gradient glycerolization. With that system, it was possible to introduce and remove high concentrations of glycerol with little loss of neutrophil viability. For both ascending and descending gradients, calculations were used to follow the changes in intracellular glycerol concentration. Similar calculations were used to determine the relationship between cell volume, gradient slope, and medium osmolality. From those data, guidelines were formulated for the glycerolization and deglycerolization of the human neutrophil.

Cell Membrane Permeability↗
PubMed1981

Freeze preservation of sickle erythrocytes.

The effect of short-term cryopreservation on metabolic, functional, and survival characteristics of erythrocytes from patients with sickle cell disease was examined. Post-thaw hemolysis of glycerolized sickle (SS) erythrocytes was greater (mean 12.9%) than in hemoglobin-AA cells (mean 4.7%). Freeze preservation had no apparent effect on red cell morphology, percent irreversibly sickled cells, and fetal hemoglobin content. There were modest reductions in ATP and 2,3-diphosphoglycerate in thawed, washed sickle erythrocytes (12.7% and 29.7%, respectively). However, the autologous survival of 51Cr-labelled SS red cells was not shortened by cryopreservation. The safety and efficacy of autotransfusion of cryopreserved red cells in alloimmunized sickle cell disease patients with anemic episodes unrelated to sickling need to be determined.

Adenosine Triphosphate↗
PubMed1979

Influence of glygerol on ATP and 2,3-DPG levels of human erythrocytes.

Glycerolization of freshly collected human erythrocytes leads to a reduction in ATP levels which return to their original values following deglycerolization. The reduction in ATP levels is largely prevented by pyruvate but not by inosine and glucose, singly or in combination. This implies that the ATP lesion associated with glycerolization reflects mainly a decrease in the NAD/NADH ratio and accordingly a reduced glyceraldehyde-3-phosphate dehydrogenase activity. 2,3-DPG levels were not influenced by glycerolization and deglycerolization. Erythrocytes depleted of ATP in the presence of glycerol can have their ATP levels repleted by either pyruvate or inosine, but require the presence of both compounds for maintenance during a post-rejuvenation incubation period. This indicates that the prolonged presence of glycerol influences both the NAD/NADH ratio and the carbon-containing intermediates of glycolysis. Glycerol did not influence ATP repletion of erythrocytes stored at 4 degrees C for 20 days by inosine in combination with pyruvate, but substantially decreased the repletion of their 2,3-DPG levels.

Adenosine Triphosphate↗