Feasibility of allogeneic stem-cell transplantation after azacitidine bridge in higher-risk myelodysplastic syndromes and low blast count acute myeloid leukemia: results of the BMT-AZA prospective study.

BACKGROUND: Allogeneic stem-cell transplantation (HSCT) is the only curative treatment in myelodysplastic syndromes (MDS). Azacitidine (AZA) is increasingly used prior to HSCT, however in Europe it is only approved for patients who are not eligible for HSCT. PATIENTS AND METHODS: We conducted a phase II multicenter study to prospectively evaluate the feasibility of HSCT after treatment with AZA in 70 patients with a myelodysplastic syndrome (MDS), 19 with acute myeloid leukemia (AML), and 8 with chronic myelomonocytic leukemia (CMML). After a median of four cycles (range 1-11): 24% of patients achieved complete remission, 14% partial remission, 8% hematologic improvement, 32% had stable and 22% progressive disease. Ten patients discontinued treatment before the planned four cycles, due to an adverse event in nine cases. RESULTS: A HSC donor was identified in 73 patients, and HSCT was performed in 54 patients (74% of patients with a donor). Main reasons for turning down HSCT were lack of a donor, an adverse event, or progressive disease (9, 12, and 16 patients, respectively). At a median follow-up of 20.5 months from enrolment, response to AZA was the only independent prognostic factor for survival. Compared to baseline assessment, AZA treatment did not affect patients' comorbidities at HSCT: the HCT-CI remained stable in 62% patients, and worsened or improved in 23% and 15% of patients, respectively. CONCLUSIONS: Our study shows that HSCT is feasible in the majority of patients with HR-MDS/AML/CMML-2 after AZA treatment. As matched unrelated donor was the most frequent source of donor cells, the time between diagnosis and HSCT needed for donor search could be 'bridged' using azacitidine. These data show that AZA prior to HSCT could be a better option than intensive chemotherapy in higher-risk MDS. The trial has been registered with the EudraCT number 2010-019673-1.

Identification of prognostic factors predicting outcome in Hodgkin's lymphoma patients relapsing after autologous stem cell transplantation.

BACKGROUND: High-dose chemotherapy followed by autologous stem cell transplantation (ASCT) is the standard of care for patients with relapsed Hodgkin's lymphoma (HL). However, there is currently little information on the predictors of outcome for patients whose disease recurs after ASCT. METHODS: Five hundred and eleven adult patients with relapsed HL after ASCT from EBMT-GITMO databases were reviewed. RESULTS: Treatments administered following ASCT failure included conventional chemotherapy and/or radiotherapy in 294 (64%) patients, second ASCT in 35 (8%), and alloSCT in 133 (29%). After a median follow-up of 49 months, overall survival (OS) was 32% at 5 years. Independent risk factors for OS were early relapse (<6 months) after ASCT, stage IV, bulky disease, poor performance status (PS), and age ≥50 years at relapse. For patients with no risk factors OS at 5 years was 62% compared with 37% and 12% for those having 1 and ≥2 factors, respectively. This score was also predictive for outcome in each group of rescue treatment after ASCT failure. CONCLUSION(S): Early relapse, stage IV, bulky disease, poor PS, and age ≥50 years at ASCT failure are relevant factors for outcome that may help to understand the results of different therapeutic approaches.

Molecular characterization of adenovirus from clinical samples through analysis of the hexon and fiber genes.

Human adenoviruses (HAdVs) are common pathogens associated with a variety of clinical manifestations. Although most infections are self-limiting, HAdVs can cause severe or lethal infections in immunocompromised as well as in healthy individuals. Several HAdVs have recently been characterized as emerging pathogens. In Italy, epidemiological, and especially molecular epidemiological, information on this pathogen is scarce. This study describes the characterization by cell culture, PCR and phylogenetic analysis of HAdV strains originating from a small collection of clinical samples gathered between 2008 and 2009. The distribution of different HAdV species was studied and the possible presence of newly emerging types was ascertained. A broad-range primer pair was used, targeting a portion of the hexon gene, in combination with species-specific primer pairs targeting a portion of the fiber gene. Human and animal reference AdV strains were included in the study. The broad-range assay identified all HAdV strains (study and reference samples), as well as three out of four animal AdV reference strains. Seven different types belonging to three HAdV species (B, C and F) were identified in the study samples. Species C was by far the most frequent. Two co-infections were detected, each with two serotypes within species C (types 1/2 and 2/6). The combined use of these two PCR assays--allowing not only the identification of known types but also, potentially, the discovery of newly emerging ones--can provide valuable epidemiological information on the spread of HAdVs.

Splenic artery transposition for hepatic arterial reconstruction in a locally advanced pancreatic cancer: a case report and literature review.

OBJECTIVE: During pancreatic surgery for malignancies, hepatic revascularization is needed in case of en bloc resection with hepatic artery involvement. In these cases, the use of the splenic artery is described in the literature, including transposition and interposition techniques. PATIENTS AND METHODS: We report the case of pancreatic cancer resection with involvement of the right hepatic artery, anomalous arising from the superior mesenteric artery, and hepatic revascularization with splenic artery reconstruction. A literature review to analyze the use of splenic artery in hepatic revascularization during pancreatic cancer surgery was performed. RESULTS: A 61-year-old man with a 55-mm hypovascular tumor in the pancreatic head, in wide contact with the right hepatic artery, underwent total pancreatectomy and splenectomy. Right hepatic artery was resected, and the distal part of the splenic artery was transposed to the right hepatic artery with a termino-terminal anastomosis. Histopathological examination revealed R0 resection. CONCLUSIONS: Hepatic revascularization with splenic artery should be considered in patients suitable to extend resectability in pancreatic cancer surgery. A multidisciplinary approach and careful pre-operative planning are essential.

Management of mycotic aorto-iliac aneurysms: a 30-year monocentric experience.

OBJECTIVE: This study aims to analyze the early and late outcomes of our 30-year experience with mycotic aneurysms of the abdominal aorta and iliac arteries. PATIENTS AND METHODS: This retrospective cohort study compared the outcomes of all the patients with mycotic aneurysm, by analyzing prospectively collected data between September 1989 and October 2019 from the Unit of Vascular Surgery of Fondazione Policlinico Universitario Gemelli - IRCCS in Rome, Italy. RESULTS: Twenty-three patients with mycotic aneurysm were included. Twenty-two patients underwent surgery; one patient arrived at the emergency room with unstable clinical conditions and died before being treated. Fourteen cases (60.9%) were located at the infrarenal aorta, while three cases (13.0%) were pararenal aortic aneurysms. Six cases (26.1%) had an iliac arteries localization. Seventeen patients (77.3%) underwent open surgical repair aneurysmectomy with in situ reconstruction, while three cases (13.6%) underwent extra-anatomic revascularization. Three patients (13.6%) underwent the placement of an endoprosthesis, of whom two underwent hybrid procedures, and one EVAR. The latter underwent an early conversion to open repair due to a type I endoleak. The mean length of hospital stay was 35 ± 18.7 days. Five patients (22.7%) died in the immediate postoperative period. In the follow-up of 45.5 ± 41.3 months (range 2-156), we documented six deaths (35.3%), of whom two (11.8%) were aortic-related for a 34.8% overall aortic-related mortality. Eleven patients were alive, with an overall survival of 47.8%. CONCLUSIONS: Mycotic aneurysm is an extremely rare and varied pathology. Open surgical repair showed to be a safe approach because of a complete and aggressive debridement of local infected tissues, with an acceptable long-term mortality rate.

The Akt signaling pathway determines the different proliferative capacity of chronic lymphocytic leukemia B-cells from patients with progressive and stable disease.

Chronic lymphocytic leukemia (CLL) B-cells are hyporesponsive to many proliferative signals that induce activation of normal B-lymphocytes. However, a heterogeneous response has recently been observed with immunostimulatory CpG-oligodeoxynucleotides (CpG ODN). We now show that CpG ODN induce proliferation mainly in CLL B-cells from patients with progressive disease and unmutated immunoglobulin V(H) genes, whereas G(1)/S cell cycle arrest and apoptosis are induced in leukemic B-cells from stable/V(H) mutated CLL. Examination of early signaling events demonstrated that all CLL B-cells respond to CpG ODN stimulation by degradation of the NF-kappaB inhibitor IkappaB and activation of the Akt, ERK, JNK and p38 MAPK kinases, but the magnitude and duration of the signaling response was greater in the proliferating cases. Pharmacological inhibition of these pathways showed that simultaneous activation of Akt, ERK and JNK is required for cell cycle progression and proliferation. Conversely, introduction of constitutively active Akt in nonproliferating CLL B-cells resulted in induction of cyclin A following CpG ODN stimulation, indicating that increased Akt activation is sufficient to overcome the hyporesponsiveness of these cells to proliferative signals. Thus, the magnitude of Akt signaling may determine the distinct responses observed in leukemic B-cells belonging to the different prognostic subgroups.

Incidence of the JAK2 V617F mutation among patients with splanchnic or cerebral venous thrombosis and without overt chronic myeloproliferative disorders.

BACKGROUND: Thrombosis of splanchnic or cerebral veins is a typical manifestation of polycythemia vera (PV) or essential thrombocythemia (ET). The recently identified Janus kinase 2 (JAK2) V617F somatic mutation is closely related to chronic myeloproliferative disorders (CMD). OBJECTIVE: To assess the incidence of the JAK2 V617F mutation among patients with splanchnic or cerebral venous thrombosis with or without overt CMD. PATIENTS AND METHODS: We searched for the mutation in 139 adult patients (> 18 years old) with thrombosis of hepatic veins (HVT, n = 15), or extrahepatic portal vein (PVT) and/or mesenteric vein (MVT) (n = 79), or cerebral veins (CVT, n = 45). Only 19 patients fulfilled criteria for diagnosis of PV (n = 8) or ET (n = 11) at the time of thrombosis: four had HVT, 11 PVT and/or MVT, and four CVT. RESULTS: The JAK2 V617F mutation was found in 94.7% [95% CI 75.3-99.0] of the patients with overt CMD at the time of thrombosis, in 21.5% (95% CI 13.8-31.7) of the patients with abdominal venous thrombosis and without overt CMD, and in 4.8% (95% CI 1.3-16.1) of the patients with CVT and without overt CMD. Among the patients without overt CMD or thrombophilia and with unprovoked thrombosis, 29.4% (95% CI 16.8-46.1) with splanchnic venous thrombosis and 42.8% (95% CI 24.4-63.4) with PVT had the JAK2 V617F mutation. CONCLUSIONS: A substantial proportion of patients with splanchnic venous thrombosis and a small, but significant, number of patients with CVT can be recognized as carriers of the JAK2 V617F mutation in the absence of overt signs of CMD. The clinical significance of such findings deserves further investigation.

Kinetics of peg-filgrastim after high-dose chemotherapy and autologous peripheral blood stem cell transplantation.

Peg-filgrastim is a form of G-CSF with a sustained duration of action due to self-limited clearance. We administered 6 mg peg-filgrastim to 18 autograft recipients on day +1 after transplantation for hematologic malignancies. Plasma samples were collected at baseline and during transplantation. Hematopoietic recovery and clinical outcomes were compared to the historical data of 54 patients not receiving G-CSF. Patients receiving peg-filgrastim achieved a serum level of 115 000 pg/ml on day +2, 24 h after drug administration. Drug level maintained a plateau until day +8 and, after day +10, declined concomitantly with myeloid recovery. Patients experienced prompt neutrophil recovery: days +9 and +10 to 500 and 1000 neutrophils per microliter, and 4 days with an absolute neutrophil count <100 cells per microliter. Duration of antibiotic therapy was significantly shortened, but we did not observe significant differences in other end points. In conclusion, peg-filgrastim was well tolerated and efficacious, and hastened myeloid recovery.

Treatment of refractory chronic GVHD with rituximab: a GITMO study.

The anti-CD20 chimaeric monoclonal antibody Rituximab has recently been shown to induce significant clinical response in a proportion of patients with refractory chronic graft-versus-host disease (cGVHD). We now report 38 patients, median age 48 years (22-61), receiving Rituximab for refractory cGVHD, assessed for clinical response and survival. Median duration of cGVHD before Rituximab was 23 months (range 2-116), the median number of failed treatment lines was 3 (range 1 to > or =6) and the median follow-up after Rituximab was 11 months (1-88). Overall response rate was 65%: skin 17/20 (63%), mouth 10/21 (48%), eyes 6/14 (43%), liver 3/12 (25%), lung 3/8 (37.5%), joints 4/5, gut 3/4, thrombocytopaenia 2/3, vagina 0/2, pure red cell aplasia 0/1 and, myasthenia gravis 1/1. During the study period 8/38 died: causes of death were cGVHD progression (n=3), disease relapse (n=1), infection (n=3), sudden death (n=1). The actuarial 2 year survival is currently 76%. We confirm that Rituximab is effective in over 50% of patients with refractory cGVHD and may have a beneficial impact on survival.

Conditioning regimens for allogeneic hematopoietic stem cell transplants in acute myeloid leukemia.

AML is currently the first indication for allogeneic hematopoietic stem cell transplantation (allo-HSCT), as shown by international transplant registries. The conditioning regimens are classified as myeloablative conditioning, non-myeloablative or reduced intensity conditioning. Targeted radioimmunotherapy such as anti-CD45 antibody have also been added to the conditioning regimen in an attempt to improve tumor cell kill. Refinement of standard regimens has led to a reduction of non-relapse mortality, also in the older age group over 60 or 70 years of age. Relapse post allo-HSCT remains an important issue, especially for patients who undergo transplant with residual or refractory disease. In these patients, pre- and post-transplant interventions need to be considered.

Decision analysis of allogeneic hematopoietic stem cell transplantation for patients with myelodysplastic syndrome stratified according to the revised International Prognostic Scoring System.

Allogeneic hematopoietic stem cell transplantation (allo-SCT) represents the only curative treatment for patients with myelodysplastic syndrome (MDS), but involves non-negligible morbidity and mortality. Crucial questions in clinical decision-making include the definition of optimal timing of the procedure and the benefit of cytoreduction before transplant in high-risk patients. We carried out a decision analysis on 1728 MDS who received supportive care, transplantation or hypomethylating agents (HMAs). Risk assessment was based on the revised International Prognostic Scoring System (IPSS-R). We used a continuous-time multistate Markov model to describe the natural history of disease and evaluate the effect of different treatment policies on survival. Life expectancy increased when transplantation was delayed from the initial stages to intermediate IPSS-R risk (gain-of-life expectancy 5.3, 4.7 and 2.8 years for patients aged ⩽55, 60 and 65 years, respectively), and then decreased for higher risks. Modeling decision analysis on IPSS-R versus original IPSS changed transplantation policy in 29% of patients, resulting in a 2-year gain in life expectancy. In advanced stages, HMAs given before transplant is associated with a 2-year gain-of-life expectancy, especially in older patients. These results provide a preliminary evidence to maximize the effectiveness of allo-SCT in MDS.

PO-54 - Clinical and laboratory characterization of platelet dysfunction caused by ibrutinib treatment in patients with chronic lymphocytic leukemia.

INTRODUCTION: Ibrutinib treatment in patients with chronic lymphatic leukemia (LLC) is associated with bleeding-related adverse events. About 50% of treated patients display minor bleedings and about 5% major bleedings. A defect of platelet function has been hypothesized and inhibition of signaling by glycoprotein (GP) VI, which is the receptor for collagen, has been previously described. Ibrutinib-associated bleedings and platelet dysfunction may be relevant in the context of aged patients, who are often under antithrombotic treatments. AIM: Aim of this study is to investigate and characterize the effect of ibrutinib on platelet function in vitro in patients with CLL. MATERIALS AND METHODS: Nine patients recruited in ongoing ibrutinib clinical trials were studied. Assessment of spontaneous bleeding was performed using the WHO bleeding scale. The following tests were performed before and after initiation of treatment with ibrutinib: 1) Light transmission aggregometry (LTA) using platelet-rich-plasma (ADP 2-4µM, PAR1-AP 25 µM, Collagen 10 ug /mL, arachidonic acid 1 mM, ristocetin 0.6-1.2mg/mL) 2) Measurement of vWF antigen and ristocetin cofactor activities by chemiluminescent immunoassay. RESULTS: I) Five patients displayed grade 1 bleeding (cutaneous bleeding) and one patient grade 2 bleeding (rectal bleeding) (66.7%) after initiation of ibrutinib treatment. II) Eight patients displayed abnormalities of the aggregation by 10 ug/ml collagen after initiation of ibrutinib treatment. At high collagen concentration, only significant prolongation of the lag phase was measured (60.4±10.6sec vs basal 38.4±17sec), whereas the maximal aggregation was not impaired (67.9±21.4% vs basal 85.5±5.8%). Compared to previous reports, these results confirmed an impairment of collagen induced aggregation, but at these concentrations only the lag phase was affected. III) Five patients displayed a significant improvement of the aggregation by 2 uM (91.25±5.26% vs basal 39.3±24.62%) and 4 uM (91±2.83% vs basal 65.42±19.43%) ADP after initiation of ibrutinib treatment. IV) vWF antigen and ristocetin cofactor activity were measured in 3 patients. In all patients vWF levels were higher at the onset of the disease (169±38%) and reduced up to normal values under Ibrutinib treatment (111.4±47%). CONCLUSIONS: Ibrutinib treatment in LLC patients causes a mild bleeding phenotype most probably due to platelet dysfunction. In this study, collagen induced aggregation resulted impaired, whereas the aggregation by PAR1-AP, ristocetin and arachidonic acid was not affected. On the contrary, the aggregation by ADP was improved upon ibrutinib treatment. The levels of vonWillebrand factor are significantly higher in LLC patients before treatment and were normalized by ibrutinib.

PML-RARα kinetics and impact of FLT3-ITD mutations in newly diagnosed acute promyelocytic leukaemia treated with ATRA and ATO or ATRA and chemotherapy.

The APL0406 study showed that arsenic trioxide (ATO) and all-trans retinoic acid (ATRA) are not inferior to standard ATRA and chemotherapy (CHT) in newly diagnosed, low-intermediaterisk acute promyelocytic leukaemia (APL). We analysed the kinetics of promyelocytic leukaemia-retinoic acid receptor-α (PML-RARα) transcripts by real-time quantitative PCR (RQ-PCR) in bone marrow samples from 184 patients and assessed the prognostic impact of fms-related tyrosine kinase 3-internal tandem duplication (FLT3-ITD) in 159 patients enrolled in this trial in Italy. After induction therapy, the reduction of PML-RARα transcripts was significantly greater in patients receiving ATRA-CHT as compared with those treated with ATRA-ATO (3.4 vs 2.9 logs; P=0.0182). Conversely, at the end of consolidation, a greater log reduction of PML-RARα transcripts was detected in the ATRA-ATO as compared with the ATRA-CHT group (6.3 vs 5.3 logs; P=0.0024). FLT3-ITD mutations had no significant impact on either event-free survival (EFS) or cumulative incidence of relapse in patients receiving ATRA-ATO, whereas a trend for inferior EFS was observed in FLT3-ITD-positive patients receiving ATRA-CHT. Our study shows at the molecular level that ATRA-ATO exerts at least equal and probably superior antileukaemic efficacy compared with ATRA-CHT in low-intermediaterisk APL. The data also suggest that ATRA-ATO may abrogate the negative prognostic impact of FLT3-ITD.

The risk of thrombosis in patients with acute leukemia: occurrence of thrombosis at diagnosis and during treatment.

BACKGROUND: Thromboembolism can occur during acute leukemia, especially acute lymphoid leukemia (ALL) treated with L-asparaginase. Yet, most reports are anecdotical and scarce data are available on the risk of thrombosis in acute myeloid leukemia (AML). OBJECTIVES: To evaluate the risk of thrombosis in patients with acute leukemia. PATIENTS AND METHODS: Three-hundred and seventy-nine consecutive adult patients with newly diagnosed acute leukemia were recruited in an observational cohort study conducted from January 1994 to December 2003. Diagnosis was ALL in 69 patients, acute promyelocytic leukemia (APL; FAB subtype M3) in 31, and non-M3 AML in 279. All first or recurrent symptomatic thromboembolic events objectively diagnosed were recorded. RESULTS: Twenty-four patients of the overall 379 (6.3%; 95% CI 4.1%-9.2%) had a first thrombosis, venous in 80% of the cases and arterial in 20%. At diagnosis, thrombosis was a presenting manifestation in 13 cases (3.4% of the whole cohort): 1.4% in ALL, 9.6% in APL, and 3.2% in non-M3 AML patients. Follow-up was carried out on 343 patients without thrombosis at diagnosis and further 11 thrombotic events (3.2%) were recorded. At 6 months from diagnosis, the cumulative incidence of thrombosis was 10.6% in ALL, 8.4% in APL, and 1.7% in non-M3 AML patients. The patients who received L-asparaginase had a 4.9-fold increased risk of thrombosis in comparison with those who did not (95% CI 1.5-16.0). The fatality rate due to thrombosis was 0.8%. CONCLUSIONS: In patients with acute leukemia, the risk of thrombosis is not negligible. Thombosis can be a presenting symptom at diagnosis in a significant portion of cases with APL (9.6%) and non-M3 AML (3.2%); a similar rate of thrombosis can occur during the subsequent course of the disease. The incidence of symptomatic thrombosis at diagnosis is relatively low in ALL patients (1.4%), but is significantly increased by further treatment up to 10.6%. Strategies of antithrombotic prophylaxis should be investigated in this setting.

BKV infection and hemorrhagic cystitis after allogeneic bone marrow transplant.

Hemorrhagic cystitis (HC) is a well-known complication after allogeneic bone marrow transplant (BMT) and can be related to adenovirus or human polyomavirus BK (BKV) infections. In this study a group of 20 patients after allogeneic BMT has been examined. BMT urine samples were analysed for the presence of Adenovirus and BKV DNAby means of polymerase chain reaction (PCR). 5/20 BMT patients developed HC after BMT. The presence of BKV DNA in urine samples was evident in 3/15 patients without HC and in 5/5 patients with HC. In 2/5 HC-patients the BKV DNA was not found after therapy with Cidofovir and Ribavirin. The search for adenovirus DNA in all samples was negative. The analysis of BKV non-coding control region (NCCR) isolated from urine samples revealed a structure very similar to the archetype in all samples. The RFLP (Restriction Fragment Length Polymorphism assay) showed the presence of BKV subtypes I and IV, with the prevalence of subtype I (4/5). This study supports the hypothesis that HC is mainly related to BKV rather than to adenovirus infection in BMT patients. Moreover, since BKV subtype I was predominant, it is reasonable to hypothesize that a specific BKV subtype could be associated with the development of HC.

Granulocyte colony-stimulating factor perturbs lymphocyte mitochondrial function and inhibits cell cycle progression.

Sera from healthy subjects receiving recombinant human granulocyte colony-stimulating factor (rHuG-CSF) to mobilize CD34(+) peripheral blood progenitors (PBPC) have been recently shown to induce unresponsiveness of allogeneic lymphocytes to mitogenic challenge. In the present investigation, the effects of rHuG-CSF on the early stages of lymphocyte activation-induced apoptosis and on lymphocyte cell cycle entry were evaluated. Sera were obtained from HLA-identical donors receiving rHuG-CSF to mobilize CD34(+) PBPC for allogeneic transplantation. Normal peripheral blood mononuclear cells (PBMC) were challenged with phytohemagglutinin (PHA) in the presence of serum collected before (preG) or after rHuG-CSF administration (postG). Mitochondrial function, that is, incorporation of 3,3'-dihexyloxacarbocyanine iodide [DiOC(6)(3)] and generation of reactive oxygen species (ROS) as well as expression of c-Myc and Bcl-2 family members (Bcl-2, Bcl-X(L), Bax) were evaluated by multiparameter flow cytometry. The activation-induced fragmentation of genomic DNA was detected by highly sensitive LM-PCR assay.CD4(+)DiOC(6)(3)(low) and CD8(+)DiOC(6)(3)(low) T lymphocytes increased and reached 32% (range 27%-38%) and 20% (range 15%-23%) of circulating T cells, respectively, on day 4 of rHuG-CSF administration. Hypergeneration of ROS could be demonstrated in 65% (range 58%-82%) of CD4(+) T lymphocytes and in 0.4% (range 0.2%-0. 8%) of circulating CD8(+) T cells. rHuG-CSF determined no alteration of mitochondrial function if added to allogeneic PBMC in vitro, thus suggesting indirect effects mediated by soluble factors; on the contrary, when PBMC were challenged with PHA in the presence of postG serum, both perturbation of mitochondrial transmembrane potential (Deltapsi(m)) and hypergeneration of ROS were induced, and lymphocytes were predominantly arrested in a G(0) -like phase of the cell cycle and displayed genomic DNA fragmentation. Interestingly, the preincubation of PBMC with a blocking antibody directed against CD95 abrogated the perturbation of lymphocyte Deltapsi(m), suggesting that the CD95 signaling pathway might play a role in the induction of apoptosis after PHA stimulation in the presence of postG serum. Moreover, Bax protein was overexpressed in postG (median fluorescence intensity = 180, range 168-186) compared with preG cultures (median fluorescence intensity = 75, range 68-80; p < 0.01), while no differences in Bcl-2, Bcl-X(L), and c-Myc staining intensity were observed. Our findings demonstrate a humoral-mediated rHuG-CSF-induced dissipation of lymphocyte mitochondrial Deltapsi(m); these effects might be mediated by Bax overexpression, with imbalance between apoptosis-promoting and apoptosis-inhibiting Bcl-2 family members and with subsequent induction of mitochondrial permeability transition. Whether immune dysfunction will favorably impact on incidence and severity of acute graft vs host disease after allogeneic PBPC transplantation remains to be determined.

Serum of healthy donors receiving granulocyte colony-stimulating factor induces T cell unresponsiveness.

The effects of serum from healthy donors receiving recombinant human granulocyte colony-stimulating factor (rhG-CSF) (G-serum) on blast transformation, expression of activation-related antigens, secretion of interleukin (IL)-2, and proliferation were evaluated in allogeneic lymphocytes stimulated with phytohemagglutinin. Escalating concentrations of G-serum induced 27%, 47%, and 70% suppression of lymphocyte proliferation; interestingly, CD4+ and CD8+ cells underwent blast transformation and up regulated early (CD69) and late (CD25, HLA-DR, and CD71) activation-related antigens. Negligible fractions of apoptotic cells were found after mitogenic challenge, suggesting that the strongly diminished proliferation was not attributable to extensive activation-induced programmed cell death of responding T cells. The levels of IL-2 in cultures containing G-serum were comparable to those in cultures performed without G-serum; however, high concentrations of exogenous IL-2 restored lymphocyte mitogenesis regardless of G-serum concentration. These findings--cell enlargement, upregulation of activation-related antigens, inability to proliferate after mitogenic stimulus, and restoration of cell division by exogenous IL-2--resembled those associated with "partial activation" of lymphocytes, a fundamental control mechanism of tolerance induction in T cell clones. Soluble immunoregulatory mediators infused with allogeneic hematopoietic progenitor products collected after rhG-CSF administration could induce T cell unresponsiveness in vivo, thus preventing clonal expansion and amplification of immune responses, and could account for the unexpectedly reduced incidence and severity of graft vs. host disease compared with allogeneic marrow infusion.