The importance of erythroid expansion in determining the extent of apoptosis in erythroid precursors in patients with beta-thalassemia major.

Beta-thalassemia major is characterized by ineffective erythropoiesis leading to severe anemia and extensive erythroid expansion. The ineffective erythropoiesis is in part due to accelerated apoptosis of the thalassemic erythroid precursors; however, the extent of apoptosis is surprisingly variable. To understand this variability as well as the fact that some patients undergoing allogeneic marrow transplantation are resistant to the myeloablative program, we attempted more quantitative analyses. Two groups of patients totaling 44 were studied, along with 25 healthy controls, and 7 patients with hemolysis and/or ineffective erythropoeisis. By 2 flow cytometric methods, thalassemic erythroid precursors underwent apoptosis at a rate that was 3 to 4 times normal. Because thalassemic marrow has between 5- to 6-fold more erythroid precursors than healthy marrow, this translated into an absolute increase in erythroid precursor apoptosis of about 15-fold above our healthy controls. In searching for the causes of the variability in thalassemic erythroid precursor apoptosis, we discovered tight direct correlations between the relative and absolute extent of apoptosis and the extent of erythroid expansion as measured either by the absolute number of marrow erythroid precursors or by serum soluble transferrin receptor levels. These results could mean that the most extreme rates of erythroid proliferation lend themselves to cellular errors that turn on apoptotic programs. Alternatively, extreme rates of erythroid hyperplasia and apoptosis might be characteristic of more severely affected patients. Lastly, extreme erythroid hyperplasia could generate such numbers of apoptotic erythroid precursors that marrow macrophages are overwhelmed, leaving more apoptotic cells in the sample.

Long-term survival of ex-thalassemic patients with persistent mixed chimerism after bone marrow transplantation.

Twenty-six transplanted thalassemic patients out of 295 analyzed, showed the presence of persistent mixed chimerism, over a period of time varying between 2 and 11 years after BMT. Despite the presence of large numbers of residual host cells, these transplanted thalassemic patients no longer require red blood cell transfusions and have a functional graft, producing sufficient levels of hemoglobin A ranging from 8.3-14.7 g/dl. These ex-thalassemic patients with persistent mixed chimerism, although they did not achieve complete donor engraftment are no longer exposed to the risk of graft rejection. The mechanisms underlying this apparent state of tolerance or education in these patients are at the present time unknown. However, these observations may be useful for physicians involved in defining optimal strategies for clinical gene therapy, in utero hematopoietic stem cell transplantation and adoption of less toxic conditioning regimens in mini-transplantation.

Treatment of iron overload in the "ex-thalassemic". Report from the phlebotomy program.

After successful marrow transplantation (BMT) iron overload remains an important cause of morbidity in Thalassemia. After BMT, patients have normal erythropoiesis capable of producing a hyperplastic response to phlebotomy so that this procedure can be contemplated as a method of mobilizing iron from overloaded tissues. Forty-one patients (mean age 16 +/- 2.9 years) with prolonged follow-up (range 2-7 years) after BMT were submitted to a moderate intensity phlebotomy program (6 ml/kg blood withdrawal at 14-day intervals) to reduce iron overload. Values are expressed as mean +/- SD or as median with a range (25th-75th percentile). Serum ferritin decreased from 2,587 (2,129-4,817) to 280 (132-920) micrograms/l (p < 0.0001), total transferrin increased from 2.34 +/- 0.37 to 2.9 +/- 0.66 g/l (p = 0.0001), transferrin saturation decreased from 90% +/- 14% to 39% +/- 34% (p < 0.0001). Liver iron concentration evaluated on liver biopsy specimens decreased from 20.8 (15.5-28.1) to 3 (0.9-14.6) mg/g dry weight (p < 0.0001). Alanine amino-transaminase from 5.2 +/- 3.4 to 1.6 +/- 1.2 (p < 0.0001) times the upper level of normality. The histological grading for chronic hepatitis (Histology Activity Index) decreased from 4.2 +/- 2.4 to 2.3 +/- 1.8 (p < 0.0001). Phlebotomy is a safe, efficient, and widely applicable method to decrease iron overload in "ex-thalassemic."

Kaposi's sarcoma after allogeneic bone marrow transplantation.

A case of Kaposi's sarcoma (KS) in an allogenic BMT recipient is reported. A 26-year-old man underwent allogeneic bone marrow transplantation for microdrepanocytosis. He received prolonged immunosuppressive therapy for mild chronic GVHD. Two years after BMT he developed KS localized to the skin. The KS improved rapidly and outcome was complete remission after cessation of immunosuppression.

Persistence of mixed chimerism in patients transplanted for the treatment of thalassemia.

Molecular genetic techniques permit sensitive assessment of host hematopoiesis after marrow transplantation for thalassemia. Information on this persistence and the cell lines in which it occurs may permit therapeutic intervention in patients at high risk for rejection and/or relapse. The objective of this study, therefore, was to determine the evolution and cell line distribution of persistent mixed chimerism detected in 55 patients treated for beta thalassemia. Our findings indicated that rejection occurred in 20 patients, the host component disappeared in 20, and mixed chimerism without transfusion need persisted for 1 to 7 years in 15. In three patients with stable mixed chimerism for 4, 5, and 7 years, host hematopoiesis fluctuated between 25% and 75%. Despite this, donor pattern beta-globin chain synthesis maintained hemoglobin levels between 10 and 13.5 g/dL without transfusion. In these three patients, the polymerase chain reaction of the VNTR and the fluorescent in situ hybridization analysis revealed the coexistence of donor and host cells in the different peripheral blood cell subpopulations and precursors studied (CD2+, CD4+, CD8+, and CD19+ granulocytes; glycophorin-A+, erythroid burst-forming units, CD33+, granulocyte-macrophage colony-forming units). We found that rejection and disease recurrence occur in approximately one third of patients with early mixed chimerism. High levels of host type hematopoiesis can be present in patients not requiring transfusion.

Marrow transplantation for patients with thalassemia: results in class 3 patients.

Thalassemia patients can be categorized as class 1 (minimal liver damage and iron overload), class 3 (extensive liver damage from iron overload), and class 2 (intermediate). These categories are prognostic for treatment outcome after marrow transplantation. Class 3 patients have more transplant-related mortality than other patients. This study examines transplantation outcome for class 3 patients. Records were reviewed of 215 patients in class 3 who received transplants in Pesaro from HLA-identical related donors between May 1, 1984 and May 1, 1994. The influence of pretransplant, peritransplant, and posttransplant variables on survival, relapse, and transplant-related mortality was examined by product-limit and proportional-hazards multivariate analysis. Age and conditioning regimen were influential on survival, and regimens with less than 200 mg/kg cyclosporine (CY) were associated with 5-year survival probabilities of .74 and .63 patients younger than 17 years and older patients, respectively. Transfusion history and regimen were influential on rejection with 5 year probabilities of .53 and .24 in patients who received less than or greater than 100 red blood cell transfusions before transplantation and regimens containing less than 200 mg/kg CY. Results of transplantation for patients with advanced thalassemia treatment have improved with the introduction of conditioning regimens with less CY. This has been associated with an increase in rejection (particularly in patients who have received < 100 red blood cell transfusions before transplant). Efforts at reducing the rejection rate by modifying the conditioning regimen should be concentrated on younger patients who have received a small number of transfusions. Patients with thalassemia who have HLA-identical family members should be transplanted before they are in class 3.

Allogeneic marrow transplantation in patients with chronic myeloid leukemia in chronic phase following preparation with busulfan and cyclophosphamide.

Thirty-four patients with chronic myelogenous leukemia in chronic phase were treated with busulfan 16 mg/kg and cyclophosphamide 120 or 200 mg/kg before allogeneic bone marrow transplantation from an HLA-identical sibling. Cyclosporine, methotrexate and prednisone were used for graft-versus-host disease (GVHD) prophylaxis. The actuarial probabilities of survival and relapse-free survival at 82 months were 71%. With a maximum follow-up of 2471 days, none of the patient experienced hematologic or clinical relapse. In one patient reappearance of host cells was documented 180 days post-transplant which disappeared 277 days post-transplant and the patient is in complete hematological and cytogenetic remission 5 years after the transplant. The probability of transplant-related mortality was 29% while the probability of moderate to severe acute graft-versus-host disease was 38%. This study indicates that busulfan and cyclophosphamide are a good conditioning regimen for marrow transplantation in patients with chronic myeloid leukemia in chronic phase.

Goat immunoglobulin purification on phosphocellulose and DEAE Affi-Gel blue.

We describe a method for the efficient purification of immunoglobulins G (IgG) to near homogeneity from goat serum. This was achieved by performing first an AS-40 fractionation on goat serum, followed by chromatography on phosphocellulose (P11) equilibrated in citrate buffer at pH 5.7. Peak I, eluted at V0 from P11, contained all IgG and the other serum proteins, except beta-globulins and most of the alpha-2-globulins, which are eluted in a second peak with 0.24 M K-phosphate in citrate buffer at pH 6.0. Peak I, concentrated and dialyzed in 20 mM K-phosphate buffer pH 8.0, was then applied onto a DEAE Affi-Gel Blue column equilibrated in the same buffer. Two peaks were obtained from this column: peak I, eluted at V0 contained a pure IgG fraction, while the other serum proteins were in peak II. We conclude that the P11 step, performed under the conditions we report here, is very useful to retain the alpha-2 and beta-globulins, which contaminate the IgG when only the DEAE Affi-Gel Blue purification step is used.

[Purification from goat antiserum of immunoglobulins against G6PD from rabbits]. / Purificazione da antisiero caprino di immunoglobuline anti-G6PD di coniglio.

DEAE Affi-Gel Blue (Bio-Rad) provides an efficient and rapid fractionation of human serum proteins by a single chromatographic step. When goat serum is applied to the matrix and chromatography is performed following the procedure utilized for the human serum proteins, the elution pattern changes and the Ig purification is not satisfactory. We achieved a better Ig purification from goat serum by the following improved procedure. We performed first an AS-40 fractionation followed by extensive dialysis in 50 mM Na-citrate pH 5.7. The sample was then loaded onto a P11 column equilibrated in the same buffer. The fraction eluted at Vo contained total IgG and the other serum proteins, except beta-globulins which were eluted with 0.24 M phosphate. Peak 1 concentrated and dialyzed in 20 mM phosphate buffer pH 8 was then applied to a DEAE Affi-Gel Blue column, equilibrated in the same buffer. Two protein peaks were eluted from this column and electrophoretically characterized as: peak 1, containing a pure Ig fraction (70% yield), peak 2 with albumin and other contaminating serum proteins. When goat antiserum is obtained against a specific protein, our technique may be suitably employed to purify polyclonal antibodies for immunoprecipitation studies.

Glucose-6-phosphate dehydrogenase activity in dorsal root ganglia of vitamin E-deficient rats.

The effect of dietary vitamin E on the activity of glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) was studied in the dorsal root ganglia of rat. One-month-old male Sprague-Dawley rats were randomly assigned to two dietary treatment groups for 2 months. The first received a standard diet supplemented with vitamin E, the second was fed a basal vitamin E-deficient diet. The activity of G6PD was markedly decreased in ganglia of the deficient animals with respect to the controls. On the other hand, the activity of the 6PGD was not significantly altered in the deficient animals. In the red cells the two enzyme activities presented a similar situation and the level of the reduced glutathione in the red cells was not significantly altered by the status of dietary vitamin E. Kinetic analysis with crude extracts of ganglia or partially purified G6PD demonstrated that there was no direct modulatory effect of the vitamin on the enzyme activity. Moreover, nondenaturing gel electrophoresis performed in this study revealed that none of the three G6PD activity bands which appeared on the acrylamide gel were significantly altered in the deficient animals. At present, the mechanism linking the G6PD activity with the status of dietary vitamin E remains unknown. Our results suggest, however, that a reduced NADPH generation produced by a decay of G6PD activity may limit the glutathione peroxidase, a very active enzyme in detoxifying peroxides, and may predispose the nervous tissue to oxidant injury.

[Hepatic hematopoiesis in phenylhydrazine-induced hemolytic anemia]. / Emopoiesi epatica nella anemia emolitica da fenilidrazina.

Ten adult rabbits were divided into two groups: the control rabbits, which received subcutaneous injections of 0.9% NaCl in three days; the experimental animals which received 3 mg/Kg body weight of phenylhydrazine (PHZ) subcutaneously also in three days. On the 8th day from the initial treatment the control and experimental animals were sacrificed, blood was collected to determine hematological parameters and livers were cut into small pieces. Sections were prepared by pressing the pieces onto slides which were stained with the Giemsa stain. The hematocrit and the reticulocytosis of experimental animals were 25 + 3%, and 70 + 5% respectively. In the liver sections of the PHZ treated animals we found a very rich population of immature erythroblasts. In fact proerythroblasts and basophilic erythroblasts were 19%, polychromatic and orthochromatic erythroblasts were 22% and 13% respectively. On the contrary, these cells were absent in the control livers. The lymphocyte and lymphoblast population, on the other hand, was very rich in control animals with a value of 38.8% compared to 1.62% in the anemic animals. The results clearly indicate the hematopoietic function of the liver in the anemic animals although the low percentage of orthochromatic erythroblasts with respect to their precursors suggests the ineffectiveness of the process.

[Progression of the peripheral blood profile in phenylhydrazine-induced hemolytic anemia]. / Processualità del quadro ematico periferico nella anemia emolitica da fenilidrazina.

Male rabbits were made anemic by subcutaneous injections of 3 mg/Kg body weight of phenylhydrazine (PHZ) for the duration of three days. Blood samples were collected prior to the experiment, on the 2nd and 5th day from the end of the treatment. Hematocrit, reticulocyte count, MCV, osmotic resistance, RBC volume distribution curve and morphological analysis were performed on each sample in order to obtain a picture of the progressive changes of the parameters following the PHZ administration. Hematocrit values changed from 49 to 27%, whereas the reticulocytosis increased from 0.6 to 73%. A significant elevation of MCV was detected and the cell volume distribution curve, which presented the typical bell-shape profile in the normal animals became bimodal on the 5th day. The osmotic resistance of RBC of anemic animals, showed a marked deviation from the normal pattern. In fact initial haemolysis started at 0.75-0.70% NaCl concentration in the 5th day samples, while it was between 0.55-0.50% before the PHZ treatment. Finally, morphological analysis revealed a progressive increase of RBC with Heinz bodies, of macro-megalocytes and of immature erythroblasts thus indicating that the cell population, produced during recovery from PHZ induced anemia, is widely heterogeneous.

[Effect of ATP on glucose-6-phosphate dehydrogenase during erythroblast maturation in the rabbit]. / Effetto dell'ATP sulla glucoso 6 fosfato deidrogenasi durante la maturazione degli eritroblasti di coniglio.

The glucose-6-phosphate dehydrogenase (G6PD) activity of erythroblasts, separated at different advancing stages of development, shows a marked decline of activity. A proteolytic mechanism, strictly controlled, is likely responsible of this decay, since a sufficient level of enzyme activity still remains in the circulating erythrocyte. In this report we suggest a model that could explain what triggers the mechanism of proteolytic degradation. HPLC analysis of the nucleotide content of erythroblasts and reticulocytes, showed a marked decline of adenine and pyridine nucleotides and of their catabolic products during the cell development. From thermostability tests, at fixed temperature, we have seen that ATP and NADP only, significantly protected the enzyme activity. In this light, we incubated 10 min at increasing temperatures, with and without ATP or NADP lysates of erythroblasts, separated at different stage of development and of reticulocytes. In the absence of nucleotides, we determined for all fractions a T degree break at 42 degrees C. In the presence of NADP all fractions were stabilized with no break point in the range 37-50 degrees C. On the contrary, the presence of ATP caused a progressive shift of the T degrees C break from the most immature erythroblasts (T degree break at 46 degrees C) to the reticulocytes (T degree break at 42 degrees C). Since ATP did not show any protective effect on the reticulocyte enzyme, we hypothesize the presence in these cells of a structurally modified G6PD. Furthermore, these data support our belief that the marked decline of ATP during cellular development, may represent the element responsible for the enzyme modification.

Rapid purification of glucose-6-phosphate dehydrogenase from mammal's erythrocytes.

A procedure for rapid purification to homogeneity of glucose-6-phosphate dehydrogenase (G6PD) is herein presented. Our method is not new, but represents a simplification of the method of De Flora et al. (Arch. Biochem. Biophys. 169, 362-3, 1975) which consisted of three steps: DEAE-Sephadex, phosphocellulose (P11) and affinity chromatography on 2'5' ADP-Sepharose. These authors eluted the enzyme from the P11 with phosphate and from 2'5' ADP-Sepharose with KC1 and NADP. By our method, the DEAE-Sephadex step is omitted, the G6PD is eluted from P11 with citrate and NADP, and from 2'5' ADP-Sepharose with KC1, NADP and EDTA. The elution of the enzyme from the phosphocellulose was studied in detail and the temperature effect has been described. We report here an application of this method to a rapid microscale purification starting from 3.5-4 ml of rabbit blood, which can be performed in about 8 hours and a macroscale purification starting from 180-200 ml of human blood, which takes a day and a half.