The depletion of T cells from haematopoietic stem cell transplants.

OBJECTIVE: In our laboratory, we have developed an immunorosette technique for the depletion of T cells from bone marrow transplants. Tetrameric complexes of monoclonal antibodies are able to form very stable immunorosettes, which are efficiently depleted with the aid of a blood cell separator. Major improvements over the original sheep red blood cell depletion are the use of human (patient or donor derived) erythrocytes instead of sheep-derived cells, and the possibility of using a closed system for separation in a cell separator. In contrast to bone marrow, mobilized haematopoietic stem cell transplants obtained after leucocytapheresis contain higher numbers of T cells. Therefore, a different approach is necessary. METHOD: We have used two CD34 selection systems (Isolex 300SA and the Clinimacs) to perform T-cell depletions from peripheral blood stem cell (PBSC) transplants. RESULTS: Immunorosette T-cell depletion, with CD2/CD3 tetrameric complexes, of bone marrow transplants resulted in a mean 2.5 log depletion of T cells with a yield of 50% of the CD34+ cell population. Stem cell selection of PBSC transplants using one of the CD34 selection procedures resulted in a 4.5 log depiction of T cells for both systems, but with different results for the recovery of CD34+ cells. An increased yield of CD34+ cells was obtained with the Clinimacs procedure (57.9+/-9.0%) in comparison to the Isolex procedure (40.1+/-12.5%). CONCLUSION: Our own immunorosette depletion technique and the two tested CD34 selection methods for stem cell transplants both resulted in a very efficient T-cell depletion with the recovery of 40-60% of the CD34 haematopoietic stem cells present in the transplant.

In vivo distribution and elimination of hemoglobin modified by intramolecular cross-linking with 2-nor-2-formylpyridoxal 5'-phosphate.

Modified hemoglobin solutions have potential application as plasma expanders with oxygen-transporting capacity. In a previous study it was found that modification of hemoglobin by intramolecular cross-linking with 2-nor-2-formylpyridoxal 5'-phosphate (NFPLP) improves the vascular retention time by a factor of three, and it also improves the oxygen-transporting properties. In the present study we investigated in rats how, after exchange transfusion of a clinically relevant dose, the modified hemoglobin (HbNFPLP) was distributed in the body compared with how the unmodified hemoglobin was distributed. By using a new technetium 99m labeling technique, we found in a scintigraphic study that accumulation of hemoglobin in the kidneys was greatly diminished by the intramolecular cross-linking with NFPLP. These findings were confirmed by light-microscopic observations after diaminobenzidine staining. It was concluded that the impairment of kidney function caused by blockade of the tubuli is not to be expected from HbNFPLP. In the liver and spleen, where the free HbNFPLP is possibly eliminated, some accumulation of 99mTc label was observed, but the major part of the extravascular label was diffusely spread throughout the body. This led to the conclusion that important accumulation of undegraded HbNFPLP does not occur in the liver and spleen. Rapid appearance of both hemoglobin and HbNFPLP in the lymph showed that cross-linking with NFPLP does not prevent the distribution of hemoglobin over the interstitial space in the first hours after administration. However, pharmacokinetic analysis demonstrated that transcapillary transfer contributes only to a limited extent to the disappearance from the circulation. During 24-hour infusions of HbNFPLP, a steady state with a constant plasma concentration was easily reached. The latter experiment indicated that the eliminating system does not become saturated during prolonged administration of large doses of HbNFPLP.

Purification and physical characteristics of a hemoglobin solution modified by coupling to 2-nor-2-formylpyridoxal 5'-phosphate (NFPLP).

Human stroma-free hemoglobin (Hb) was crosslinked with 2-nor-2-formylpyridoxal 5'-phosphate (NFPLP), purified over crosslinked dextran, and eluted with a linear salt gradient. The oxygen dissociation curve of this crosslinked hemoglobin appeared to be shifted to the right with a standard P50 of 49 torr (PO2 for 50% saturation with oxygen at a pH of 7.40, a PCO2 of 40 torr, and a temperature of 37 degrees C) compared with a P50 of 12 to 15 torr for the unmodified Hb. The Hill coefficient n of HbNFPLP was 2.1, versus 2.8 for Hb. The proton Bohr factor of HbNFPLP, calculated from P50 values in the pH range of 7.1 to 7.7, was found to be -0.19, versus -0.29 for unmodified Hb. The oxygen capacity of HbNFPLP was not affected by the crosslinking and was found to be 1.410 ml of O2 per g of HbNFPLP, versus 1.407 ml of O2 per g of Hb for unmodified Hb. Four derivatives of HbNFPLP, i.e., deoxyhemoglobin, oxyhemoglobin, carboxyhemoglobin, and methemoglobin, were prepared, and the light adsorption spectra were recorded in the region of 480 to 680 nm. No differences were detected in comparison with the spectra of unmodified Hb. The alpha and beta chains of the tetramer were separated by reverse-phase chromatography. Comparison of the elution patterns of the chains of Hb and HbNFPLP revealed a retardation of the beta chains due to crosslinking with NFPLP. This indicates that the binding of NFPLP to Hb occurred only between the beta chains.(ABSTRACT TRUNCATED AT 250 WORDS)

Preparation and physical characteristics of a hemoglobin solution modified by coupling to 2-nor-2-formylpyridoxal 5'-phosphate.

Stroma-free hemoglobin (Hb) solutions were prepared on a 20-I scale. In 3-I batches, the beta chains of hemoglobin were crosslinked with 2-nor-2-formylpyridoxal 5'-phosphate (NFPLP) that was synthesized on a gram scale. The coupling efficiency was 60 to 80 percent. The oxygen dissociation curve of these Hb/HbNFPLP mixtures was shifted to the right with P50 (PO2 for 50% saturation with oxygen) values of 26 to 38 torr versus values of 12 to 16 torr for the nonmodified hemoglobin solutions. Both the H+ Bohr factor and the Hill coefficient were lower for the Hb/HbNFPLP mixture than for the original hemoglobin solution. The oxygen-binding coefficient beta was the same for both types of Hb solutions. The viscosity and the colloid osmotic pressure of both solutions were also the same. During storage at 4 degrees C for 18 months, no precipitation or denaturation of hemoglobin was detectable in either solution. There was also no conversion of the modified hemoglobin molecules, HbNFPLP, to the native hemoglobin tetramers, dimers, or monomers. The percentage of methemoglobin remained at 5 percent for about 6 months; it increased to 26 percent over the next 12 months. The results indicate that intramolecular coupling of hemoglobin with NFPLP yields a stable product with physiologic oxygen-carrying properties.

An animal model for the detection of hypotensive side effects of immunoglobulin preparations.

Intravenous administration of certain immunoglobulin preparations may cause severe adverse reactions, especially in immunodeficient patients. These reactions are generally assumed to be related to the anticomplementary activity of the preparations, caused by IgG aggregates. Because the exact mechanism of the adverse reaction is unknown, we investigated the reactions induced in anesthetized rats on rapid intravenous administration of different human immunoglobulin preparations. The most conspicuous observation was a severe, long-lasting hypotension, induced by standard immunoglobulin preparations (for intramuscular use), which appeared to be independent from the concomitant complement and neutrophil activation. The long-lasting hypotension was not related to the presence of prekallikrein activator, which induced a transient hypotensive reaction only after sensitizing the rats to bradykinin. The reactions appeared to be associated with IgG aggregates. It was found that certain aggregates induced mainly complement activation, whereas others had mainly a hypotensive effect or no effect at all. It was concluded that the rat model provides a sensitive and reproducible test system for vasoactive properties of immunoglobulin preparations for intravenous administration that cannot be predicted from in vitro measurements, such as anticomplementary activity, aggregate content or prekallikrein activator activity. It is suggested that the test may also be used for other plasma products for intravenous administration.

Effect of coupling of 2-nor-2-formylpyridoxal 5'-phosphate to stroma-free hemoglobin on oxygen affinity and tissue oxygenation. Studies in the isolated perfused rat liver under conditions of normoxia and stagnant hypoxia.

Hemoglobin in stroma-free solution (7 gm/100 ml) was modified by covalently cross-linking the beta-chains with 2-nor-2-formylpyridoxal 5'-phosphate (NFPLP). The coupling efficiency was approximately 65%. The oxygen dissociation curve of the coupling mixture was shifted to the right, with a P50 of 30 mm Hg vs. 15 mm Hg for the nonmodified solution. The effect of the modification on tissue oxygenation was studied in the isolated perfused rat liver at normoxia and at hypoxia induced by decrease of flow rate at constant Po2 and hemoglobin concentration. The two perfusates used were a nonmodified hemoglobin solution and the coupling mixture. The chemical modification of the hemoglobin molecule did not affect the vascular resistance in the liver tissue. During normoxia the NFPLP-induced decrease in oxygen affinity was reflected in a higher venous Po2. The differences in the other oxygen-sensitive parameters were not significant. The decrease in O2 supply induced by a decrease of perfusion flow rate (stagnant hypoxia) resulted in a decrease in venous Po2, O2 consumption, and bile flow rate, and an increase in the cytoplasmatic redox level (lactate/pyruvate ratio) and the mitochondrial redox level (beta-hydroxybutyrate/acetoacetate ratio). During hypoxia the changed oxygen affinity of the modified hemoglobin solution was reflected in small but significant differences between both perfusates in the venous Po2 and both redox levels. No change in O2 consumption and bile flow rate was observed. When compared with earlier low-flow perfusions of the isolated rat liver with erythrocytes, the oxygen affinity of hemoglobin solutions appears not to be rate limiting for the O2 consumption, probably because of better tissue perfusion with hemoglobin solutions.

Oxygen affinity of hemoglobin solutions modified by coupling with NFPLP and the effects on tissue oxygenation in the isolated perfused rat liver.

From these liver perfusions with Hb and Hb/HbNFPLP solutions the following conclusions can be drawn: In spite of the chemical modification of the hemoglobin molecule, no rheological differences are seen. All parameters measured were sensitive to hypoxia induced by a decrease in perfusion flow rate. The NFPLP-induced decrease in oxygen affinity was reflected in a higher venous PO2. These in-vivo observations are in agreement with the in-vitro measured oxygen dissociation curves. The difference in PO2 did not result in a change in the other oxygen-sensitive parameters in this model under the chosen conditions. Possible causes for these observations are: the level of hypoxia was too low the oxygen supply in the perfusions with the modified hemoglobin solutions was lower than the oxygen supply in the perfusions with normal hemoglobin. Whether or not this observation is due to an intrinsic property of the modified hemoglobin molecule remains to be established.

The influence of the position of the oxygen dissociation curve on oxygen-dependent functions of the isolated perfused rat liver. III. Studies at different levels of anaemic hypoxia.

The influence of a 2,3-diphosphoglycerate (2,3-DPG)-induced displacement of the oxygen dissociation curve (O.D.C.) on the isolated perfused rat liver was studied at different levels of anaemic hypoxia. Rat livers were perfused either with fresh or with 2,3-DPG-depleted human erythrocytes at different haematocrit values (from 30% to 2.5%) at constant Po2 of the inflowing perfusate and at constant blood flow rate. The 2,3-DPG-induced difference in oxygen affinity of the red cells did not cause a significant difference in perfusion pressure during the perfusion experiments. Therefore, there is no evidence that 2,3-DPG did alter the vascular resistance of the liver, since blood flow rate could be adusted at equal values. The decrease in oxygen supply brought about by decrease of haematocrit caused a decrease of O2 consumption, of bile flow rate and of venous Po2 and an increase of lactate/pyruvate (L/P) ratio and of beta-hydroxybutyrate/acetoacetate (betaOH/Acac) ratio. There was no influence of a difference in 2,3-DPG content of the erythrocytes on the above-metioned parameters during severe anaemic hypoxia. At moderate anaemic hypoxia the venous Po2 was higher during perfusion with fresh erythrocytes than during perfusion with 2,3-DPG-depleted erythrocytes. Thus, although 2,3-DPG may play a compensatory role during conditions of mild anaemia, no such effects can be observed during conditions of severe hypoxia.