Procleix Ultrio transcription-mediated amplification vs. serological blood screening in south-western Greece.

We present here our overall experience after 27 months of performance of the Procleix Ultrio [HIV-1, hepatitis C virus (HCV), hepatitis B virus (HBV)] transcription-mediated amplification (TMA) assay. The aim of this report is to assess the impact of nucleic acid testing (NAT) implementation in blood screening of south-western Greek blood donors. We processed 38,264 units of blood as neat samples with the Procleix Ultrio TMA assay (Chiron/GenProbe, Emeryville/San Diego, CA, USA) between 1 January 2005 and 31 March 2007. NAT results were compared to those obtained from routine serology tests and quantitative polymerase chain reaction (PCR) assays. Overall, 52 units of blood tested positive for HBV (1.4 per thousand), 8 for HCV (0.2 per thousand) and none for HIV or multiple infections. The yield of TMA was 0.183 per thousand for HBV (7/38 264) and 0 for HCV. The TMA HBV-positive donations were tested for HBV DNA by a quantitative PCR assay and were found negative (below the detection limit of the method, 200 copies/mL). Follow-up testing showed that the TMA HBV-positive donations were positive for anti-hepatitis B core antigen immunoglobulin G antibodies. Implementation of the TMA assay in the individual donation configuration increased HBV detection compared to serological screening or a commonly used quantitative PCR assay. Follow-up studies will determine the impact of NAT implementation in HBV transmission in countries with an intermediate HBV incidence.

Apoptosis in patients with myelodysplastic syndromes: differential involvement of marrow cells in 'good' versus 'poor' prognosis patients and correlation with apoptosis-related genes.

Apoptosis has been implicated in the pathogenesis of marrow failure in MDS and the coexistence of marrow hypercellularity along with blood cytopenias was seen as evidence of extreme cell death of mainly mature cells in the marrow (ineffective hematopoiesis). We investigated apoptosis in 53 patients with MDS, by using single-step DNA extraction and gel electrophoresis and then by separating fresh marrow mononuclear cells in CD34+ and CD34- populations and in situ single cell evaluation of the process. We also studied the expression of apoptosis-related genes, in total and separated mononuclear marrow cells and correlated the findings with clinical and laboratory characteristics. Patients with apoptosis had increased marrow cellularity, longer overall survival and a longer period for transformation to AML. In 'good' prognosis MDS patients, total mononuclear marrow cells, as well as isolated populations of CD34+ and CD34- cells showed significant degrees of apoptosis; in 'poor' prognosis cases, however, apoptosis was evident only in a large percentage of CD34+ marrow cells and not in total or CD34- cells. Absence of expression of both c-myc and p53 in total marrow cells was associated with significant degrees of apoptosis and in isolated CD34+ and CD34- marrow cells the phenomenon was inversely correlated with the level of bcl-2 expression. In conclusion, marrow apoptosis is detected in both CD34+ and CD34- cells in early MDS and seems to be restricted to CD34+ cells in advanced MDS cases.

Effect of different types of total parenteral nutrition on T-lymphocyte subpopulations and NK cells.

Long-chain triglycerides (LCTs) contained in total parenteral nutrition (TPN) regimens provide a considerable energy substrate in malnourished patients. Their effect on the immune system is controversial, however. In this randomized, prospective study we investigated the possible differences between the effect of TPN with LCTs or medium-chain triglycerides (MCTs) on T-lymphocyte subpopulations. Our study included 15 normal subjects, 20 patients receiving glucose-based TPN, and 40 patients receiving glucose-fat-based TPN. In the last group 20 patients received TPN that included LCTs and 20 received 50% MCTs and 50% LCTs. T-lymphocyte subpopulations, including total T cells and T-helper, T-suppressor, and NK cells, as well as the ratio of helper to suppressor T cells were determined before and 10 d after initiation of TPN. We found a significant decrease in the ratio of helper to suppressor T cells in the LCT group although no such difference was detected in the MCT-LCT group. No difference whatever was found in total T cells and helper, suppressor, and NK cells.

Cytokine gene expression in the marrow stromal cells of patients with giant cell arteritis.

OBJECTIVE: Anemia of chronic disease is a common feature of giant cell arteritis (GCA). It has been shown that constitutive hematopoiesis is regulated in the bone marrow microenvironment by direct cell-to-cell contact between hematopoietic progenitor cells and marrow stromal cells. Since cytokines are of crucial significance in this process, we decided to examine the role of stem cell factor (SCF), granulocyte macrophage colony stimulating factor (GM-CSF), interleukin 3 (IL-3), granulocyte colony stimulating factor (G-CSF), and interleukin 1 alpha (IL-1 alpha), known for their stimulatory effects on in vitro hematopoiesis; and transforming growth factor beta (TGF beta), interferon gamma (IFN gamma) and tumor necrosis factor alpha (TNF-alpha), known for their inhibitory effect, in the anemia of GCA. METHODS: The expression of these cytokines was examined in the marrow stromal cells from 6 GCA patients with anemia of chronic disease and in 6 healthy individuals by the reverse transcription mediated polymerase chain reaction (RT-PCR). RESULTS: Consistent constitutive gene expression was detected in the marrow stromal cells of all control samples for SCF, GM-CSF, IL-1 alpha, TGF beta and TNF-alpha, while only 2/6 expressed IFN gamma. None of the controls expressed either IL-3 or G-CSF. In all GCA patients SCF, TGF beta and TNF-alpha were expressed, but the level of expression was reduced compared to the control group. IL-1 alpha was expressed in 3/6 patients, and IFN gamma in 2/6. More importantly, GM-CSF expression was not detected in any patient, in marked contrast with the controls. CONCLUSION: The combination of the defective expression of SCF and GM-CSF, cytokines with a known stimulatory effect on in vitro hematopoiesis, may contribute to the pathogenesis of anemia in GCA.

Tumor necrosis factor production by human mononuclear cells during total parenteral nutrition containing long-chain triglycerides.

The effect of fat contained in total parenteral nutrition (TPN) regimens on the immune system is controversial. The purpose of our study was to examine whether the synthesis of tumor necrosis factor (TNF), a macrophage-derived protein with several immunomodulating effects, is influenced by the duration of TPN. We studied 20 patients on glucose- and fat-based TPN with lipid emulsions containing long-chain triglycerides (LCTs). Ten of our patients received TPN for 15 days (group A) and the other 10 for 30-40 days (group B). We measured TNF production by phytohemagglutinin- and endotoxin-stimulated peripheral blood mononuclear cells before and after TPN via enzyme-linked immunosorbent assay. The production of TNF before and after TPN was similar in group A but significantly (p less than 0.01) elevated in group B after long-term TPN. Therefore, long-term TPN containing LCTs may increase TNF production, and this may be the result of an immunomodulating effect of fat.

c-Jun N-terminal kinase activation failure is a new mechanism of anthracycline resistance in acute myeloid leukemia.

Chemotherapy resistance is a major challenge in acute myeloid leukemia (AML). Besides the P-glycoprotein efflux, additional cellular factors may contribute to drug resistance in AML. c-Jun N-terminal kinase (JNK) is activated after exposure of cells to chemotherapeutics. We asked whether chemoresistance in AML is attributed to intrinsic failure of the AML blasts to activate JNK. In vitro treatment of U937 AML cell line with anthracyclines induced a rapid and robust JNK phosphorylation and apoptosis. In contrast, the anthracyline-resistant derivative cell lines U937R and URD40 showed no JNK activation after exposure to anthracyclines, also at doses that resulted in high accumulation of the drug within the cells. RNA interference-based depletion of JNK1 in drug-sensitive U937 cells made them chemoresistant, whereas selective restoration of the inactive JNK pathway in the resistant U937R cells sensitized them to anthracyclines. Short-term in vitro exposure of primary AML cells (n=29) to daunorubicin showed a strong correlation between the in vitro pharmacodymanic changes of phospho-JNK levels and the response of patients to standard induction chemotherapy (P=0.012). We conclude that JNK activation failure confers another mechanism of anthracycline resistance in AML. Elucidating the ultimate mechanisms leading to JNK suppression in chemoresistant AML may be of major therapeutic value.

Gene transfer into human hematopoietic progenitor cells with an episomal vector carrying an S/MAR element.

Episomally maintained self-replicating systems present attractive alternative vehicles for gene therapy applications. Recent insights into the ability of chromosomal scaffold/matrix attachment regions (S/MARs) to mediate episomal maintenance of genetic elements allowed the development of a small circular episomal vector that functions independently of virally encoded proteins. In this study, we investigated the potential of this vector, pEPI-eGFP, to mediate gene transfer in hematopoietic progenitor cell lines and primary human cells. pEPI-eGFP was episomally maintained and conferred sustained eGFP expression even in nonselective conditions in the human cell line, K562, as well as in primary human fibroblast-like cells. In contrast, in the murine erythroleukemia cell line, MEL, transgene expression was silenced through histone deacetylation, despite the vector's episomal persistence. Hematopoietic semisolid cell colonies derived from transfected human cord blood CD34(+) cells expressed eGFP, albeit at low levels. After 4 weeks, the vector is retained in approximately 1% of progeny cells. Our results provide the first evidence that S/MAR-based plasmids can function as stable episomes in primary human cells, supporting long-term transgene expression. However, they do not display universal behavior in all cell types.

Genetic modification of hematopoietic stem cells with nonviral systems: past progress and future prospects.

Serious unwanted complications provoked by retroviral gene transfer into hematopoietic stem cells (HSCs) have recently raised the need for the development and assessment of alternative gene transfer vectors. Within this context, nonviral gene transfer systems are attracting increasing interest. Their main advantages include low cost, ease of handling and large-scale production, large packaging capacity and, most importantly, biosafety. While nonviral gene transfer into HSCs has been restricted in the past by poor transfection efficiency and transient maintenance, in recent years, biotechnological developments are converting nonviral transfer into a realistic approach for genetic modification of cells of hematopoietic origin. Herein we provide an overview of past accomplishments in the field of nonviral gene transfer into hematopoietic progenitor/stem cells and we point at future challenges. We argue that episomally maintained self-replicating vectors combined with physical methods of delivery show the greatest promise among nonviral gene transfer strategies for the treatment of disorders of the hematopoietic system.

A distinct pattern of cytokine production from blood mononuclear cells in multitransfused patients with beta-thalassaemia.

The unstimulated and induced production of granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), IL-3, IL-6, stem cell factor (SCF), IL-1beta, tumour necrosis factor-alpha (TNF-alpha), TNF-beta, interferon-gamma (IFN-gamma) and transforming growth factor-beta (TGF-beta) was determined after culture of blood mononuclear cells from 22 patients with severe beta-thalassaemia in a regular transfusion programme, five non-regularly transfused patients with beta-thalassaemia intermedia and nine normal persons. A distinct pattern of cytokine production in thalassaemic patients was detected, namely a low unstimulated production of all cytokines and a significant increase in the stimulated production of IFN-gamma, TNF-alpha and IL- 1beta; these abnormalities were more pronounced in the more heavily transfused older patients. The increased production of the above cytokines, which usually characterize the acute response to infectious agents and have a negative effect on erythropoiesis, may explain the deterioration of anaemia found in thalassaemic patients during acute infections.

Chronic neutrophilic leukemia with dysplastic features. A new variant of the myelodysplastic syndromes.

We are reporting four patients who presented with a persistent increase of mature neutrophils and band forms, no monocytosis in repeated examinations and with dysplastic features in blood and bone marrow. Two of them developed acute myeloid leukemia (AML; FAB classification M1) and the other two were characterised by a progressively aggravating myelodysplasia. All the patients showed a poor response to treatment and died within a period of time ranging from 14 to 80 months after diagnosis. We are proposing the term chronic neutrophilic leukemia with dysplastic features (CNL-D) for this entity, and we believe that it represents a variant of the myelodysplastic syndromes.

Different haematopoietic growth factors have different capacity in overcoming the in vitro interferon gamma-induced suppression of bone marrow progenitor cells.

Interferon gamma (IFN gamma) inhibits haematopoiesis in vitro and an in vivo role in bone marrow suppression has been implied from clinical studies. We investigated the capacity of three recombinant (r), human (h), haematopoietic growth factors to overcome the in vitro IFN gamma inhibition of bone marrow progenitor cells in a methylcellulose culture system. Granulocyte macrophage-colony stimulating factor (GM-CSF) partially reversed IFN gamma-induced suppression of granulocyte-macrophage colony formation, by increasing colony forming units-granulocyte macrophage (CFU-GM) in a proportion ranging from 54-101%. Interleukin-3 (IL-3) and granulocyte-colony stimulating factor (G-CSF) were much less effective. For erythropoiesis, IL-3 was much more effective and partially reversed IFN gamma-mediated inhibition by increasing burst forming units-erythroid (BFU-E) in a proportion ranging from 52-138%. GM-CSF and G-CSF had no significant effect on IFN gamma-induced suppression of BFU-E. In conclusion, haematopoietic growth factors have different capacity to overcome IFN gamma-induced suppression of marrow progenitor cells in vitro. The findings may have therapeutic implications, as combinations of growth factors may be more effective in treating bone marrow failure syndromes.

The in vitro effect of corticosteroids on T-lymphocyte subpopulations in patients with ulcerative colitis.

T-lymphocyte subpopulations were measured in the blood of 20 patients with ulcerative colitis (11 in clinical remission and 9 in exacerbation) and 9 healthy persons, before and after in vitro incubation with dexamethasone. Although we found no significant differences in the helper/suppressor T cell ratio between patients and controls before in vitro incubation, addition of dexamethasone in culture induced a significant decrease of the ratio only in patients with ulcerative colitis in exacerbation. The percentage of natural killer cells before and after incubation with dexamethasone was not altered.

Immunologic abnormalities in patients receiving multiple blood transfusions.

Concern for the potential risk of the acquired immunodeficiency syndrome among blood cell recipients led us to measure immunologic functions in patients who had received multiple transfusions. Abnormalities of two immunologic tests, natural killer cell function and helper/suppressor (T4/T8) lymphocyte subpopulation ratio, have characterized patients with the acquired immunodeficiency syndrome and are prevalent in populations at risk for this syndrome. Natural killer cell function was severely depressed in multiply transfused patients. However, T4/T8 ratios were normal in this population. The role of chronic antigenic stimulation was studied by measurement of HLA-DR expression on T cells. The expression of HLA-DR antigen is markedly elevated in multiply transfused patients. These results show that chronic exposure to foreign antigens may be associated with abnormalities of immunologic function, but that chronically transfused patients do not have the same immunologic profile as reported in some homosexuals and hemophiliacs.

Interferon is the suppressor of hematopoiesis generated by stimulated lymphocytes in vitro.

Lymphocytes that inhibit hematopoiesis may have a pathogenic role in some forms of bone marrow failure, and lymphocyte-mediated suppression may also be important in the normal regulation of bone marrow function. We have investigated the mechanism of in vitro suppression of hematopoiesis by T cells by using the methylcellulose colony culture system. Total peripheral blood T cells and separated subpopulations of helper (OKT4+) and suppressor (OKT8+) cells that have been stimulated by exposure to lectin suppress autologous colony formation by bone marrow myeloid (CFU-C) and erythroid (BFU-E) progenitor cells. Medium conditioned by these cells is also inhibitory, indicating that the suppressor activity is a soluble factor. A strong correlation existed for the concentration of interferon and the degree of hematopoietic suppressor activity in these supernatants; both activities peaked at days 3 to 5 of incubation and had sharply declined by day 7. Interferon production was enhanced by exposure of lymphocytes to sheep red blood cells during the rosetting procedure. Specific antiserum and a monoclonal antibody directed against gamma-(immune) interferon abrogated the inhibitory activity for hematopoiesis produced by lectin-stimulated T cells; an antiserum to alpha-interferon was generally much less effective in neutralizing activity. We infer from these results that gamma-interferon is the mediator of hematopoietic suppression generated by lectin-treated T-cells.

The effect of dietary omega-3 polyunsaturated fatty acids on T-lymphocyte subsets of patients with solid tumors.

The effect of omega-3 polyunsaturated fatty acids (PUFA) on the immune system seems to be beneficial. There has been a number of studies concerning the effect of dietary omega-3 PUFA on different immune parameters. The aim of our present study was to investigate the effect of dietary omega-3 PUFA on T-cell subsets and natural killer (NK) cells of patients with solid tumors. We studied 20 patients with solid tumors who received 18 g fish oil/day for 40 consecutive days. We detected a significant increase in T-helper/T-suppressor cell ratio 40 days into omega-3 supplementation, due mainly to a decrease in the number of suppressor T cells. We concluded that dietary omega-3 fatty acids may have a beneficial effect on the already compromised immune system of patients suffering from solid tumors.

The effect of parenteral indomethacin on T-lymphocyte subpopulations and cytokine production in patients under major surgical operations.

Major surgical trauma has been considered as a cause of immunosuppression mainly through the production of prostaglandin E2 from activated monocytes/macrophages. In the present study we investigated the effect of parenteral indomethacin--a cyclo-oxygenase inhibitor--on T-lymphocyte subsets and cytokine production in patients under major operations. We studied 20 patients undergoing major surgical procedures, 10 of whom were randomly treated pre- and post-operatively with indomethacin (group 2) and 10 were not (group 2). We measured total T-cells, T-helper, T-suppressor, T-helper/T-suppressor (Th/Ts) cell ratio, NK-cells and interleukin (IL-1) and tumor necrosis factor production by endotoxin- or phytohemagglutinin-stimulated peripheral blood mononuclear cells, before operation and at days 1, 3 and 7 postoperatively. We detected a significant increase in Th/Ts cell ratio and an improvement in delayed type hypersensitivity response in the treated group at day 3. We believe that the above immunomodulating effect of in vivo cyclo-oxygenase inhibition may be beneficial in patients under major surgical procedures with a high susceptibility to postoperative infections.

Lymphocyte subpopulations in megaloblastic anaemia due to vitamin B-12 deficiency.

Lymphocyte subpopulations were measured in the blood of 17 patients with megaloblastic anaemia due to vitamin B-12 deficiency. 14 patients had pernicious anaemia and 3 others were gastrectomized. By using monoclonal antibodies recognizing T cell surface markers and immunofluorescence microscopy, we found a significant decrease in the number of circulating suppressor T cells and an increase in the ratio of helper to suppressor T lymphocytes in pernicious anaemia patients. This finding may be related to other immune abnormalities found in pernicious anaemia, e.g. the presence of multiple autoantibodies.

Dietary omega-3 polyunsaturated fatty acids plus vitamin E restore immunodeficiency and prolong survival for severely ill patients with generalized malignancy: a randomized control trial.

BACKGROUND: The aim of the current prospective, randomized control study was to investigate the effect of dietary omega-3 polyunsaturated fatty acids plus vitamin E on the immune status and survival of well-nourished and malnourished patients with generalized malignancy. METHODS: Sixty patients with generalized solid tumors were randomized to receive dietary supplementation with either fish oil (18 g of omega-3 polyunsaturated fatty acids, PUFA) or placebo daily until death. Each group included 15 well-nourished and 15 malnourished patients. The authors measured total T cells, T-helper cells, T-suppressor cells, natural killer cells, and the synthesis of interleukin-1, interleukin-6, and tumor necrosis factor by peripheral blood mononuclear cells before and on Day 40 of fish oil supplementation. Karnofsky performance status, nutritional state, and survival were also estimated. RESULTS: The ratio of T-helper cells to T-suppressor cells was significantly lower in malnourished patients. Omega-3 PUFA had a considerable immunomodulating effect by increasing this ratio in the subgroup of malnourished patients. There were no significant differences in cytokine production among the various groups, except for a decrease in tumor necrosis factor production in malnourished cancer patients, which was restored by omega-3 fatty acids. The mean survival was significantly higher for the subgroup of well-nourished patients in both groups, whereas omega-3 fatty acids prolonged the survival of all the patients. CONCLUSIONS: Malnutrition appears to be an important predictor of survival for patients with end stage malignant disease. Omega-3 polyunsaturated fatty acids had a significant immunomodulating effect and seemed to prolong the survival of malnourished patients with generalized malignancy.

Interferon is a mediator of hematopoietic suppression in aplastic anemia in vitro and possibly in vivo.

We have investigated interferon as a mediator of hematopoietic suppression in bone marrow failure. Interferon production by stimulated peripheral blood mononuclear cells from patients with aplastic anemia was significantly higher than that observed in controls; spontaneous interferon production by these cells was also high for more than half of aplastic anemia patients. Circulating interferon, not detectable in normal individuals, was detected in 10 of 24 patients. Interferon is a potent inhibitor of hematopoietic cell proliferation and, therefore, may be the mediator of suppression in many in vitro models employing patients' cells and sera. The possible pathogenic importance of interferon in aplastic anemia was suggested by an increase in hematopoietic colony formation in vitro after exposure of bone marrow cells to antiinterferon antisera (277 +/- 71% increase for patients compared to 1.6 +/- 1.6% for normal individuals). Interferon levels in the bone marrow sera of aplastic anemia patients were high (mean = 203 international units (IU)/ml, n = 8), even in comparison to circulating levels in the same patients. Normal bone marrow sera also contained measurable interferon but at lower levels (41 IU/ml, n = 16), indicating that interferon may be a normal bone marrow product. High concentration of bone marrow interferon, possibly due to abnormal immunologic activity or a reaction to virus infection of the bone marrow, may mediate hematopoietic suppression in aplastic anemia patients.

Circulating activated suppressor T lymphocytes in aplastic anemia.

We studied the mechanism of hematopoietic suppression in aplastic anemia by means of two-color flow microfluorometric analysis of lymphocyte subpopulations and correlated the results with the occurrence in vitro of hematopoietic suppression and interferon production. In 12 patients with aplastic anemia a striking increase was observed in a population of "activated" suppressor T lymphocytes, which were defined by binding of both anti-Leu-2 and anti-HLA-DR monoclonal antibodies (patients with aplastic anemia, 6.8 +/- 3.2 per cent [mean +/- S.D.]; normal subjects, 1.7 +/- 1.3; patients given multiple transfusions, 2.5 +/- 1.7). Tac antigen expression, another surface marker of lymphocyte activation, was increased on suppressor lymphocytes in all five patients examined (patients with aplastic anemia, 31 +/- 17 per cent; normal subjects, 0.7 +/- 0.24; patients given multiple transfusions, 2.3 +/- 1.2). When Tac+ and Tac- cells were separated in a cell sorter, only Tac+ cells produced interferon. When lymphocytes of patients with aplastic anemia were cocultured with normal bone marrow, only the Tac+ cell fraction showed hematopoietic suppressor activity. In one patient, in vitro elimination of suppressor lymphocytes by use of OKT8 antibody abolished spontaneous interferon production by bone-marrow cells. These results suggest that activated suppressor lymphocytes producing interferon have a role in the pathogenesis of bone-marrow failure, and indicate the usefulness of defined lymphokine and phenotypic markers in the study of aplastic anemia.