Correlation between the kinetics of CD3+ chimerism and the incidence of graft-versus-host disease in patients undergoing allogeneic hematopoietic stem cell transplantation.

INTRODUCTION: Graft-versus-host disease (GvHD) remains a significant complication after allogeneic hematopoietic stem cell transplantation (HSCT). Early diagnosis and treatment may improve patient outcomes. A prospective study to investigate the relationship between chimerism kinetics and the development of acute or chronic GvHD was carried out. Split chimerism in association with the onset of GvHD was also analyzed. METHODS: Thirty-three patients with hematologic diseases treated with allogeneic HSCT were analyzed. They were conditioned with myeloablative or reduced intensity regimens and grafted with peripheral blood (PB) or bone marrow stem cells. GvHD prophylaxis consisted of cyclosporine and methotrexate. Chimerism evaluation was performed on PB mononuclear cells and purified cell subsets consisting of separated CD3(+) T cells, monocytes (CD14(+)), and granulocytes (CD15(+)). Chimerism analysis was performed at 30, 60, 120, and a median of 200 days after HSCT. RESULTS: Acute GvHD was diagnosed in 19 patients and chronic GvHD in 16. On day 30, no relation was found between the level of donor chimerism and aGvHD. Upon univariate analysis, decreasing mixed chimerism among CD3(+) and infused CD34(+) cell numbers was significantly correlated with acute GvHD development, while the PB stem cell source, reduced-intensity conditioning regimen, and female donor sex were associated with an increased risk of chronic GvHD. In multivariate analysis, the risk of acute GvHD correlated only with the CD34(+) cell dose, while the risk of extensive chronic GvHD was associated with high CD3(+) donor chimerism on day 30. Patients with versus without split chimerism (T cell vs myeloid lines) did not differ statistically in their incidence of acute GvHD or chronic GvHD. CONCLUSION: Our results supported the belief that chimerism kinetics or longitudinal chimerism evaluation is of greater significance than isolated absolute values of the percentage of chimerism at a single point after HSCT. The observations suggest that longitudinal monitoring of chimerism in CD3(+) T-cell subsets is an acceptable method to predict the development of GvHD among patients undergoing HSCT.

[T lymphocytes from patients with Hodgkin's disease suppress erythroid progenitor growth from autologous marrow]. / Limfocyty T chorych na ziarnice zlosliwa hamuja wzrost klonalny prekursorów erytroidalanych szpiku autologicznego.

The effect of peripheral blood T lymphocytes from 42 patients with advanced Hodgkin's disease (grade III and IV) on the autologous marrow erythroid colony formation was studied in diffusion chamber culture. It was found, that unfractionated T lymphocytes suppress the BFU-E--(burst forming unit erythroid) and CFU-E--(colony forming unit erythroid)--derived colony formation by releasing an inhibitory activity. The suppression of colony formation was noted already at 0.25 x 10(5) cell concentration. In experiments with 0.5 x 10(5) and 1.0 x 10(5) T cells the inhibitory effect was increased. Subsequently it was shown, that this inhibition was generated by radioresistant, CD8+ and HLA-DR- subset of T cells. In control experiments, T lymphocytes from healthy subjects had no influence on the erythroid colony formation.

[Cytokines modify 2-chlorodeoxyadenosine (2-CdA) toxicity in acute myeloid leukemia clonogenic cells (L-CFU)]. / Cytokiny modyfikuja toksycznosc 2-chlorodeoksyadenozyny (2-CdA) wobec komórek klonogennych ostrych bialaczek szpikowych (L-CFU).

We investigated the effect of 2-chlorodeoxyadenosine (2-CdA) in concentrations: 10, 20, 50, 100, 150 and 300 nM/l on the clonal growth of acute myeloid leukemia clonogenic cells (L-CFU), after 24 h preincubation with recombinant granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-1 alpha (IL-1 alpha) or interleukin 3 (IL-3) and in conditioned medium obtained from phytohemagglutin stimulated lymphocytes (PHA-LCM). 2-CdA in concentration 300 nM/l significantly reduced (30%) the number of blast colonies in comparison with control experiments (p < 0.05). After preincubation of blast cells with cytokines (20% PHA-LCM, or GM-CSF--100 ng/ml, or IL-3--5 U/ml) an increase of 2-CdA cytotoxic effect on L-CFU clonal growth was observed. In experiments with 2-CdA (300 nM/l) and GM-CSF or PHA-LCM or IL-3 the number of blast colonies was reduced in 70%, 50% and 50%, respectively, (p < 0.05). Preincubation of blasts with IL-1, did not effect the 2-CdA--induced growth inhibition of CFU.

Activated lymphocytes in the marrow cell suspension decrease the mafosfamide-induced CFU-GM cytotoxicity.

The aim of the study was to assess whether other cells, besides erythrocytes, may influence the cytotoxic effect of mafosfamide (maf) during ex vivo bone marrow purging from residual tumor cells before autologous transplantation. It was shown that the presence of normal granulocytes, blast cells from acute myeloid leukemia-patients (AML) and lymphoma cells from patients with chronic lymphocytic leukemia (CLL) during maf incubation did not change the maf-induced growth inhibition of CFU-GM. Similar observation was made in experiments with resting lymphocytes. However, when phytohaemagglutinin- and pokeweed mitogen-preincubated lymphocytes were present in the marrow cell suspension, significant decline of the maf-related CFU-GM cytotoxicity was observed. These results suggest that besides erythrocytes also the activated lymphocytes in the marrow mononuclear suspension may change the final effect of maf purging.

[Effect of 2-chlorodeoxyadenosine, arabinoside cytosine and doxorubicin on acute myeloid leukemia clonogenic cell proliferation]. / Wplyw 2-chlorodeoksyadenozyny, arabinozydu cytozyny i doksorubicyny na wzrost komórek klonogennych ostrych bialaczek szpikowych.

We examined the influence of 2-chlorodeoxyadenosine (2-CDA) in concentration 300 nM/l alone and in combination with cytosine arabinoside (ara-C; 10(-7) M/l and doxorubicine (drb; 1 microgram/ml) on the proliferation of acute myeloid leukemia (AML) clonogenic cells (L-CFU) in agar/liquid culture system. After preincubation of blast cells with 2-CDA or ara-C as a single agent the number of colonies was reduced, reaching 70% of control (p < 0.05). Exposure of AML blasts to drb resulted in a greater reduction of L-CFU proliferation than in experiments with 2-CDA or ara-C alone (p < 0.05). Coadministration of 2-CDA in conjunction with ara-C or drb, and additionally with ara-C and drb caused the inhibition of L-CFU growth in comparison with control experiments by 52%, 78% and 61%, respectively (p < 0.05). In further experiments we studied the effect of ara-C and drb together with 2-CDA in different concentrations (20, 100 or 300 nM/l). After preincubation with 2-CDA in concentration 20 nM/l no change in blast colony formation was observed in relation to controls which comprised ara-C and drb only (p < 0.05). The increase of 2-CDA concentration to 100 or 300 nM/l in combination with ara-C and drb significantly reduced the growth of AML clonogenic cells (p < 0.05). The greatest, 90%, inhibition of L-CFU proliferation was observed after the exposure of blast cells to ara-C, drb and 2-CDA in concentration 300 nM/l (p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

[Cytochemical evaluation of blast cells in acute myeloblastic leukemia after induction of their maturation in liquid culture--diagnostic usefulness]. / Ocena cytochemiczna komórek blastycznych ostrych bialaczek szpikowych indukowanych do dojrzewania w warunkach hodowli plynnej--przydatnosc diagnostyczna.

We have performed the cytochemical analysis (myeloperoxidase, ASD-chloroacetate and acid alpha-naphthylacetate esterases, Sudan black B) of blast cells from 25 acute leukemia patients, after 3, 5 and 7 days of liquid culture with conditioned medium from phytohemagglutinin stimulated leukocytes (PHA-LCM). In case of acute myeloid leukemia blast cells an increase of percentage of positive cells simultaneously with the enhancement of the cytochemical reactions was observed. This method may be useful for the precise diagnosis of poorly differentiated blasts with weak expression of cytochemical phenotype.

Impaired release of colony stimulating activity by monocytes from Hodgkin's disease in response to phorbol myristate acetate activation.

We assessed the humoral effect of resting and phorbol esters preincubated monocytes from Hodgkin's disease patients (HDMo) and healthy subjects (nMo), on granulocyte progenitors (CFU-dG) growth using a double diffusion chamber technique. The release of colony stimulating activity and indomethacin-dependent inhibitors by resting HDMo and nMo was found to be cell-concentration dependent. However, phorbol myristate acetate preincubated HDMo (PMA-HDMo) in contrast to nMo at low concentrations (2.5 x 10(4] were unable to increase the CFU-dG growth stimulation. On the other hand, at a higher cell number (5 x 10(4], phorbol treated HDMo stimulated the myeloid colony formation, whereas nMo suppressed the CFU-dG proliferation. Further enhancement of HDMo and nMo concentrations induced a pronounced inhibition of CFU-dG-derived colony formation, caused by an increased PGE2 production. After incubation with the cyclooxygenase inhibitor-indomethacin, PMA-HDMo showed considerably more granulocyte colony formation than nMo. Our results suggest that the observed abnormalities in the function of HDMo could be associated with an excessive production of PGE2 and a general dysfunction of these cells in Hodgkin's disease.

[Neutrophils do not affect clonal growth of granulocyte progenitor cells cultured in vivo in diffuse chambers]. / Granulocyty obojetnochlonne nie wplywaja na wzrost klonalny komórek ukierunkowanych szeregu granulocytarnego w hodowli w komorach dyfuzyjnych in vivo.

The effect of polymorphonuclear leukocytes on the growth of granulocytic progenitors in the diffusion chamber culture (CFU-dG) has been studied. It has been shown that peripheral blood neutrophils do not affect the myeloid colony formation in this culture system. The inhibition of CFU-dG growth, observed in the presence of peripheral blood leukocytes was dependent on prostaglandins release from monocytes.

[Formation of mixed colonies by progenitor cells of chronic myeloid leukemia cultured in vivo]. / Wytwarzanie kolonii mieszanych przez komórki macierzyste przewleklej bialaczki szpikowej w hodowli in vivo.

The clonal growth of multipotential progenitor cells of chronic myeloid leukemia (CML) in diffusion chamber implanted into peritoneal cavity of neutropenic and anemic rats was assessed. CML precursors formed in methylcellulose culture enriched with medium conditioned by phytohemagglutinin activated lymphocytes mixed neutrophilic-erythroid colonies, in number significantly higher then normal cells. Mixed colonies produced by CML cells, in contrast to the normal precursors, contained also macrophages, eosinophils and megakaryocytes. We conclude that modified diffusion chamber culture technique may be a suitable tool for multipotential progenitor study in CML.

[Effect of activated monocytes on the growth of cloned granulocytic progenitor cells]. / Wplyw aktywowanych monocytów na wzrost klonalny komórek ukierunkowanych szeregu granulocytarnego.

The humoral effect of resting and phorbol myristate acetate (PMA) preincubated normal monocytes on granulocytic progenitor growth in double diffusion chamber culture (CFU-dG) was assayed. The activation of monocytes by PMA was confirmed in NBT reduction test (increased formazan content) and by enhanced superoxide anions production compared with resting cells. The activatory and inhibitory effect of monocytes on granulocytic colony formation was cell-concentration dependent. The maximal CFU-dG growth promoting effects was seen at 5 X 10(4) of resting monocytes, whereas PMA-treated cells exerted their optimal stimulatory activity at lower cell number (2.5 X 10(4)). Further increase of monocyte concentration resulted in diminished CFU-dG proliferation. It is shown that the decline of colony count at high monocyte number was caused by indomethacin-dependent inhibitors.

Granulocyte progenitors (CFU-D) in neutrophilic leukemoid reaction are hyporesponsive to macrophage-induced inhibition.

The responsiveness of marrow granulocyte progenitors (CFU-D) to macrophage-derived stimulatory and inhibitory factors has been studied using diffusion chamber technique in 12 patients with neutrophilic leukemoid reaction (with granulocyte count in the range between 10-40 G/l) and ten healthy subjects. CFU-D from patients with neutrophilic leukemoid reaction (NLR) revealed a normal reactivity to colony-stimulating activity, whereas they were hyporesponsive to macrophage-derived indomethacin-sensitive inhibition. This altered response was correlated both with the concentration of granulocyte progenitors in the S phase and with blood neutrophilic leukocytosis. In patients with higher granulocyte count and increased concentration of CFU-D during active DNA synthesis a more pronounced hyporesponsiveness of granulocyte progenitors to macrophage-induced inhibition has been found.

Splenic monocyte-macrophage dependent suppression of autologous granulocyte-progenitors growth in Hodgkin's disease.

The studies described compare the effect of spleen cell suspensions from 11 patients with Hodgkin's disease (HD) and 5 healthy subjects on the clonal growth of autologous marrow granulopoietic progenitors in diffusion chamber culture (CFU-G/D). Adherent monocyte/macrophage fraction of splenocytes from HD suppresses the proliferation of autologous CFU-G/D. This inhibition was mediated by an indomethacin-sensitive humoral factor(s). Non-adherent lymphoid cells stimulated myeloid colony formation. Dose response curves demonstrated a markedly increased inhibitory-activity production already by low numbers of splenic monocytes/macrophages from HD whereas a comparable counts of monocytes/macrophages from the spleens of healthy subjects stimulated the CFU-G/D growth. These results may suggest a possible activation of splenic monocytes/macrophages with an enhanced prostaglandin-mediated suppressor activity release for local granulocytopoiesis in the spleens of patients with HD.

Response of granulocyte-committed progenitors from patients with chronic myeloid leukemia to humoral regulators release by macrophages in agar diffusion chamber culture.

We investigated the responsiveness of granulocyte-committed progenitors (CFU-G/D) from patients with chronic myeloid leukemia (CML) and healthy subjects to stimulating and inhibiting activities released by murine macrophages in diffusion chamber culture. CFU-G/D from CML demonstrate a normal response to macrophage-derived stimulation. The responsiveness of CFU-G/D from patients with CML to indomethacin-sensitive inhibition was significantly suppressed. In this regard no difference between CFU-G/D from bone marrow and blood of patients with CML could be observed. Colonies formed both by CFU-G/D from healthy subjects and CML consisted exclusively of cells of granulocyte line: from myeloblasts up to polymorphonuclear granulocytes. Similar cellular composition of colonies could be noted during macrophage-derived stimulation and inhibition of CFU-G/D growth. In conclusion, we have demonstrated that CML CFU-G/D which proliferate and differentiate in diffusion chamber culture show a normal response to macrophage-derived stimulation but are less sensitive to indomethacin-dependent inhibition.