Performance of a recombinant fusion protein linking coagulation factor IX with recombinant albumin in one-stage clotting assays.

Essentials Performance of the one-stage clotting (OSC) assay varies with the clotting activator used. Recombinant FIX-albumin fusion protein (rIX-FP) was reliably monitored with most OSC reagents. rIX-FP shows comparable reagent-dependent variability to other rFIX products in the OSC assay. Actin® FS and kaolin-based reagents underestimated rIX-FP activity by around 50% in the OSC assay. SUMMARY: Background Measuring factor IX activity (FIX:C) with one-stage clotting (OSC) assays, based on the activated partial thromboplastin time (APTT), is the current mainstay of diagnostic techniques for hemophilia B. Assessing the performance of new recombinant FIX (rFIX) products in OSC assays is essential, as APTT reagents from different manufacturers yield different potency estimates for rFIX. Objectives To evaluate the extent to which choice of reagent composition influences rFIX potency measurements of recombinant FIX-albumin fusion protein (rIX-FP, IDELVION) activity in OSC assays. Methods rIX-FP was added to FIX-deficient plasma, and FIX:C was assessed centrally and locally in a multicenter international field study with a variety of commercial OSC APTT reagents. Paired sample analysis of clinical samples was performed to compare values of FIX:C from local and central laboratories. In-house bioanalytical investigations with spiked samples were conducted to compare the APTT-reagent dependent variability of rIX-FP with unmodified rFIX and rFIX Fc fusion protein (rFIXFc). Results Central and local assessments of FIX:C from 10 countries and 21 participating centers showed comparable results to those from the central laboratory across the majority of 18 different APTT reagents from both clinical and spiked samples. There was a consistent underestimation of rIX-FP activity of ≈ 50% with OSC assays using Actin FS or kaolin-based APTT reagents. In the bioanalytical study, rIX-FP showed comparable variability in OSC assays to unmodified rFIX and rFIXFc. Conclusions rIX-FP activity can be accurately measured by the use of OSC assays with the majority of commercial reagents. Actin FS or kaolin-based reagents will probably lead to a 50% underestimation of activity.

International comparative field study evaluating the assay performance of AFSTYLA in plasma samples at clinical hemostasis laboratories.

Essentials AFSTYLA exhibits ≈50% underestimation in activity when the one-stage (OS) assay is utilized. A field study compared the performance of AFSTYLA with Advate in factor VIII activity assays. AFSTYLA activity can be monitored with both the chromogenic substrate and the OS assay. The consistent OS underestimation allows for a conversion factor to be applied to OS results. SUMMARY: Introduction AFSTYLA (antihemophilic factor [recombinant] single chain) is a novel B-domain truncated recombinant factor VIII (rFVIII). For AFSTYLA, an approximate 50% discrepancy was observed between results of the one-stage (OS) and chromogenic substrate (ChS) FVIII activity assays. An investigation was undertaken to test whether there is a linear relationship between ChS and OS assay results that would allow reliable clinical interpretation of results independent of the assay method used. Aims To provide confidence in future clinical monitoring, this field study investigated the performance of AFSTYLA and a full-length rFVIII (Advate® ) in FVIII activity assays routinely performed in clinical laboratories. Methods The comparison of AFSTYLA and Advate was performed in an international, multicenter and blinded field study of simulated post-infusion samples. The study documented the extent of variability between methods and laboratories and characterized the relationship between the ChS and OS assays. Results Results from 23 laboratories demonstrate that intra and interlaboratory variability in OS assays were similar for both products. When comparing within the OS assay format, there was a similar and reagent-correlated variability in response to different activators for both AFSTYLA and Advate. The OS underestimation was highly predictable and consistent across the complete range of FVIII plasma concentrations. Conclusion Post-infusion plasma AFSTYLA levels can be monitored in patients by the OS and ChS assays. The consistent and predictable difference between the two assay formats provides clinicians with adequate guidance on how to interpret the results of the OS assay using a single conversion factor.

Pharmacokinetics and safety of fibrinogen concentrate.

BACKGROUND: Although fibrinogen concentrate has been available for the treatment of congenital fibrinogen deficiency for years, knowledge of its pharmacokinetics comes from only two small studies. OBJECTIVES: To assess the pharmacokinetic (PK) profile, clot integrity and safety of fibrinogen concentrate (human) (FCH) in patients with afibrinogenemia. PATIENTS AND METHODS: A multinational, prospective, open-label, uncontrolled study of patients with afibrinogenemia > or = 6 years of age was conducted in the USA and Italy. Plasma was collected before and after infusion for PK analyses and evaluation by rotational thromboelastometry of maximum clot firmness (MCF) to assess clot integrity. Safety was assessed on the basis of adverse events and laboratory parameters. RESULTS: After a single dose of 70 mg kg(-1) body weight (b.w.) FCH in 14 patients, median incremental in vivo recovery was a 1.7 mg dL(-1) increase per mg kg(-1) b.w., and median levels were 1.3 g L(-1) for fibrinogen activity and antigen 1 h after infusion. Median half-life (t(1/2)) was 77.1 h for fibrinogen activity and 88.0 h for antigen. Plasma recovery in children < 16 years old was similar to that in adults aged 16 to < 65 years, but the t(1/2) and area under the curve were decreased, with an increased steady-state volume and clearance. MCF increased by a mean of 8.9 mm from baseline to 1 h after infusion of FCH (P < 0.0001). All four adverse events reported were mild, and none was serious or related to study drug. CONCLUSIONS: These PK findings confirm a rapid increase in plasma fibrinogen levels after infusion with FCH. Together with the clot integrity and safety data and published data on efficacy, the results support the idea that FCH substitution can restore hemostasis with a good safety profile.

Biochemical comparison of seven commercially available prothrombin complex concentrates.

AIMS: A number of prothrombin complex concentrates (PCCs) are commercially available but they differ in terms of composition. We performed a series of studies to compare the biochemical properties of seven PCCs. METHODS: The following products were investigated: Beriplex P/N, Octaplex, S-TIM 4, PPSB Solvent Detergent, Uman Complex DI, Kaskadil and Cofact. Assays were performed to investigate levels of coagulation factors and their inhibitors, activated coagulation factors and heparin. The thrombin inhibitory capacity of each PCC was determined. Protein content was assessed using the Lowry method and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. RESULTS: The data indicated little difference between most of the products in their levels of factors II, VII, IX and X, with the exception of Uman Complex which had no detectable factor VII. In all cases, the measured levels of coagulation factors were broadly similar to those labelled. Beriplex P/N showed the greatest capacity for thrombin inhibition, a reflection of the observed high levels of the coagulation inhibitors protein C, protein S, protein Z, and small amounts of antithrombin III and heparin in this product. All of the PCCs tested were negative for activated coagulation factors. Purity (i.e. therapeutic protein as a percentage of total protein) was highest in Beriplex P/N, and the second purest product was Uman Complex. CONCLUSION: This in vitro study showed considerable differences between PCCs in terms of coagulation inhibitory capacity and purity.

Prothrombin complex concentrate (Beriplex P/N) for emergency anticoagulation reversal: a prospective multinational clinical trial.

BACKGROUND: Prothrombin complex concentrate (PCC) can substantially shorten the time needed to reverse antivitamin K oral anticoagulant therapy (OAT). OBJECTIVES. To determine the effectiveness and safety of emergency OAT reversal by a balanced pasteurized nanofiltered PCC (Beriplex P/N) containing coagulation factors II, VII, IX, and X, and anticoagulant proteins C and S. PATIENTS AND METHODS: Patients receiving OAT were eligible for this prospective multinational study if their International Normalized Ratio (INR) exceeded 2 and they required either an emergency surgical or urgent invasive diagnostic intervention or INR normalization due to acute bleeding. Stratified 25, 35, or 50 IU kg(-1) PCC doses were infused based on initial INR. Study endpoints included INR normalization (

Prednisolone induces interleukin-18 expression in mononuclear blood and myeloid progenitor cells.

OBJECTIVE: Interleukin (IL)-18 is involved in host defense mechanisms and inflammatory diseases, among them rheumatoid arthritis (RA). High levels of IL-18 expression in RA joints are contrasted by reduced IL-18 expression in RA peripheral blood mononuclear cells (PBMC). Here, we investigated a putative IL-18 regulating role of corticosteroids. METHODS: IL-18 transcript and protein levels in PBMC from untreated and prednisolone treated RA patients, and from healthy donors were assessed by semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) and immunoblotting. IL-18 regulation was determined in PBMC and U937 cells upon exposure to prednisolone in vitro by RT-PCR and Northern Blot analysis, by ELISA in cell culture supernatants, and in transiently transfected THP-1 cells by IL-18 promoter activity luciferase assays. RESULTS: In RA PBMC, IL-18 transcript levels were dose dependently restored, in parallel with administered prednisolone treatment, to subnormal levels. The corresponding intracellular IL-18 deposits in contrast were depleted. In cultured PBMC and promonocytic cell lines, prednisolone up-regulated IL-18 transcription in parallel with increasing the IL- 18 protein release into cell culture supernatants. CONCLUSION: Prednisolone increases IL-18 expression and release in PBMC and monocytic cell lines.

TGF beta-induced SMAD2 phosphorylation predicts inhibition of thymidine incorporation in CD34+ cells from healthy donors, but not from patients with AML after MDS.

Cells from patients with MDS-derived AML display heterogeneous proliferative responses to transforming growth factor beta (TGF beta). We analyzed growth inhibition and SMAD2 phosphorylation by TGF beta in CD34+ cells from nine patients, as compared to normal controls. While TGF beta consistently inhibited thymidine incorporation of normal cells (41% of control, P < 0.05), cells from patients with AML were growth-inhibited in only four of seven cases (40%), whereas TGF beta stimulated thymidine incorporation in the three other samples (166%). Remarkably, TPO reverted the stimulatory effect of TGF beta to profound growth inhibition. Upon exposure to TGF beta, SMAD2 protein was phosphorylated in normal CD34+ cells (n = 3), CD34+ leukemic blasts from all examined patients with AML (n = 4), and in the myeloid leukemic cell lines M-07e and HEL. TGF beta inhibited TPO-mediated thymidine incorporation, cell proliferation and survival in all samples analyzed. In M-07e cells and CD34+ cells from healthy donors, this inhibition was enhanced by an antagonist of JAK2 (AG490), but not a MEK-1 antagonist (PD098059). Conversely, in CD34+ cells from a patient with AML, both AG490 and PD098059 significantly enhanced TGF beta-mediated suppression of TPO-induced thymidine incorporation. Thus, in MDS-derived AML, altered responses to TGF beta may be due to defects downstream of SMAD2 and may involve MAPK activation.

Transforming growth factor-beta1 interferes with thrombopoietin-induced signal transduction in megakaryoblastic and erythroleukemic cells.

OBJECTIVE: Thrombopoietin (TPO) and transforming growth factor-beta(1) (TGF-beta(1)) have been shown to exert opposite effects on proliferation and megakaryocytic differentiation of hematopoietic cells. To determine whether TGF-beta(1) interferes directly with TPO-induced signal transduction in hematopoietic cells, we compared the regulatory effects in the TPO-responsive cell lines Mo-7e and HEL. MATERIALS AND METHODS: The cells were stimulated by 100 ng/mL TPO and/or 100 ng/mL TGF-beta1 and analyzed for proliferation (3H thymidine incorporation), viability (trypan blue exclusion), and protein expression and phosphorylation (Western blot). RESULTS: TPO enhanced the proliferation of Mo-7e cells as determined by 3H-thymidine incorporation, whereas TGF-beta1 suppressed baseline cell growth and antagonized the proliferative effect of TPO. TPO-induced proliferation also was reduced by a specific inhibitor of the mitogen-activated protein kinase (MAPK) pathway (PD098059), which inhibits activation of the MAPK extracellular signal-regulated kinases (ERK) ERK1 and ERK2, and AG490, an inhibitor of Janus kinase-2, which completely blocked TPO-induced proliferation. As demonstrated by Western blotting, TGF-beta1 reduced the TPO-stimulated ERK1/ERK2 and STAT5 phosphorylation in Mo-7e and HEL cells. This effect was completely reversed by preincubation with a tyrosine phosphatase inhibitor (Na3VO4), which suggests that TGF-beta1 activated a phosphatase. Although STAT3 also was activated by TPO, STAT3 activation remained unaltered by TGF-beta1. CONCLUSION: Taken together, these data suggest that TGF-beta1 modulates TPO-mediated effects on megakaryocytic proliferation by interfering with TPO-induced signal transduction, particularly by reducing the activities of MAPK ERK1/ERK2 and STAT5.

Murine M2-10B4 and SL/SL cell lines differentially affect the balance between CD34+ cell expansion and maturation.

The ability of bone marrow stroma to modulate hematopoietic progenitor cell expansion is of considerable interest for gene transfer strategies and transplantation of limited stem cell numbers. We compared the capacity of 2 murine stromal cell lines to affect the balance between maturation and proliferation of human CD34+ cells in short-term expansion cultures. In 7-day serum-free cultures, cytokine-induced amplification of granulocyte-macrophage colony-forming cells (CFC-GM), erythroid burst-forming units (BFU-E), and total cells was significantly increased by the presence of genetically engineered Sl/Sl and M2-10B4 stromal cells in a 1:1 ratio (Sl/M2 cells) compared with stroma-free cultures (P < .05). Sl/M2 cultures generated 21-fold more mature CD15+ cells than stroma-free cultures, without further amplifying the number of CD34+ cells. The addition of serum led to a further increase of CFC-GM, total cells, and CD15+ cells, whereas BFU-E were no longer maintained. Pure Sl/Sl stromal layers were likewise superior to stroma-free cultures in expansion of CD34+ cells and total cells when serum was present. However, the differentiation of CD34+ cells was less pronounced in Sl/Sl cultures compared with Sl/M2 layers, as demonstrated by a lower content of CD15+ cells. Neutralization experiments revealed differential contributions of Flt3 ligand and thrombopoietin to the support of total cell and CFC expansion by Sl/M2 and Sl/Sl stromal feeders.

Expression of interleukin-18 and its monokine-directed function in rheumatoid arthritis.

OBJECTIVES: To investigate the expression of and monokine induction by interleukin 18 (IL-18; also called interferon-gamma inducing factor, IGIF), in peripheral blood mononuclear cells (PBMC) and cultured synoviocytes from rheumatoid arthritis (RA) patients. METHODS: We carried out IL-18 Western blotting and semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) of cytokines in PBMC [IL-18, IL-1beta and tumour necrosis factor alpha (TNF-alpha)] and long-term cultured fibroblast-like synoviocytes (FLS) [IL-18, IL-1beta, TNF-alpha, IL-6, interferon gamma (INF-gamma) and [granulocyte-macrophage colony stimulating factor (GM-CSF)] from RA patients and controls. FLS were isolated from RA synovial membranes (FLS(SM)) and RA synovial fluids (FLS(SF)), osteoarthritis (OA) FLS(SM) and FLS(SF) from spondyloarthropathy patients. FLS were characterized by fluorescence-activated cell sorting of the FLS. PBMC and FLS from RA patients and control subjects were stimulated with recombinant human IL-18 and IL-1beta (rHuIL-18/rHuIL-1beta), and TNF-alpha, IL-1beta and MMP-1 were measured by ELISA in supernatants. RESULTS: Constitutive expression of IL-18 mRNA was significantly reduced whereas that of TNF-alpha was enhanced in RA PBMC. Persistent low expression of IL-18, TNF-alpha, GM-CSF and IL-1beta was observed in RA and OA FLS(SM) as well as spondyloarthropathy FLS(SF). In contrast, high constitutive expression of IL-18 in FLS (CD90/Thy-1- and CD54-positive, CD14- and CD86-negative), accompanied by persistent high levels of TNF-alpha, GM-CSF and IL-1beta expression, was restricted to synovial fluid-derived FLS obtained from RA patients. IFN-gamma was not detectable in any culture, but IL-6 mRNA was equally expressed in all FLS cultures. rHuIL-18 was effective in stimulating TNF-alpha and IL-1beta secretion in PBMC from healthy controls, but failed to stimulate TNF-alpha and IL-1beta secretion from PBMC in 11 of 12 RA patients, and all FLS cultures. rHu-IL-1beta, but not rHu-IL-18, induced interstitial collagenase (MMP-1) in FLS. CONCLUSIONS: Persistent high production of proinflammatory cytokines in RA-FLS(SF) may be relevant for chronic progression in RA synovitis. Levels of TNF-alpha and IL-1beta expression are increased in RA-FLS(SF), but are independent of IL-18. The pathological function of enhanced IL-18 expression in RA-FLS(SF) remains to be further elucidated.

Regulation of interleukin-18 (IL-18) expression in keratinocytes (HaCaT): implications for early wound healing.

Keratinocytes display a high basal level expression of IL-18. Tumor necrosis factor-alpha (TNF-alpha) mediated a large decrease in IL-18 mRNA levels in the human keratinocyte cell line HaCaT, which was accompanied by a subsequent accumulation of IL-18 protein in the cell culture supernatants, which was shown to be biologically active. By contrast, epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha), respectively, strongly decreased IL-18 mRNA expression in HaCaT keratinocytes in the absence of IL-18 protein release from the cells. Notably, a pre-treatment of the cells with EGF, or TGF-alpha clearly attenuated TNF-alpha-induced IL-18 protein, release and bioactivity. For the in vivo situation of cutaneous wound repair, we observed an increase in IL-18 protein, 10 hours post-wounding, that closely correlated to infiltration of neutrophils which are known as producers of TNF-alpha. Our data suggest that bioactive IL-18 might be tightly counter-regulated by platelet- and neutrophil-derived factors at the onset of repair.

Genomic organization and regulation of the human interleukin-18 gene.

The human interleukin(IL)-18 is a key regulator of interferon(IFN)-gamma production and T-cell differentiation. Here we report the complete genomic structure and characterization of the 5'untranslated promoter region of the human IL-18 gene. The gene is composed of six exons and five introns, spanning approximately 19. 5kb. Promoter activity of the 5'-flanking region was investigated with a luciferase reporter gene assay. Transient transfection studies demonstrate a constitutive expression of the IL-18 gene in monocytic U937 and THP-1 cells. For this constitutive expression at least 92 base pairs of the promoter region are essential as shown by consecutive 5' promoter deletions in both cell types. DNA protein binding experiments revealed specific binding of activated signal transducer and activator of transcription factor-5 (STAT5) but not of STAT3 to three consensus sequences upstream in the promoter region. Cotransfection of STAT5 resulted in increased induction of the IL-18 promoter in the U937 and THP-1 cells.

Alteration of c-mpl-mediated signal transduction in CD34(+) cells from patients with myelodysplastic syndromes.

OBJECTIVE: Megakaryocytic differentiation is frequently defective in patients with myelodysplastic syndromes (MDS). As underlying mechanisms, deregulated thrombopoietin receptor (c-mpl)-mediated signaling pathways have been suggested. This study therefore examined whether the impaired signaling in MDS and AML cells includes alterations of c-mpl itself or postsignaling events. METHODS: Bone marrow-derived CD34(+) cells from healthy donors, patients with MDS (RA, RAEB-T), and patients with AML after MDS were isolated by MACS. Expression of c-mpl cDNA was studied by RT-PCR. Thrombopoietin dependent activation of STAT proteins and MAP Kinase p42(erk-2)/44(erk-1) was analyzed by Western blot. RESULTS: Both splicing isoforms of c-mpl (c-mpl-p and c-mpl-k) were expressed in all of the CD34(+) cells examined. Analysis of the c-mpl cDNA revealed no sequence abnormality. We show c-mpl dependent activation of the transcription factors STAT3 and STAT5 as well as MAP Kinase p42(erk-2)/44(erk-1) in CD34(+) cells from healthy individuals. Cells derived from RA patients revealed low basal levels of phosphorylated STAT3 and STAT5 molecules. This phosphorylation was enhanced by stimulation with recombinant thrombopoietin (PEG-rHuMGDF). STAT1 failed to be activated by PEG-rHuMGDF in CD34(+) cells from healthy donors as well as from patients with MDS. In RAEB-T and AML M7 the constitutive expression levels of STAT1, 3, 5, and MAPK were markedly upregulated, resulting in a strong activation of STAT3 and 5 by PEG-rHuMGDF. Despite its high expression, the level of MAPK phosphorylation was not increased in RA or RAEB-T compared to the normal control, and was completely undetectable in AML M7. CONCLUSION: These results suggest that the defective megakaryopoiesis in MDS is not caused by a lack of c-mpl and that STAT3 and STAT5 may contribute to the malignant phenotype of the leukemic cells.

IL-18 activates STAT3 in the natural killer cell line 92, augments cytotoxic activity, and mediates IFN-gamma production by the stress kinase p38 and by the extracellular regulated kinases p44erk-1 and p42erk-21.

IL-18 is a regulator of NK cell function which utilizes the serine-threonine IL-1R-associated kinase signal transduction pathway and may activate additional not yet characterized signaling pathways. Here we evaluated IL-18-mediated signal transduction using the human NK cell line NK92 as a model. NK92 cells were shown by RT-PCR to express all three IL-18 receptor chains (IL-18R, accessory protein-like chain, IL-18-binding protein). Stimulation by IL-18 strongly enhanced tyrosine phosphorylation of STAT3 and of the mitogen-activated protein kinases (MAPK) p44erk-1and p42erk-2. In contrast, STAT5 was not activated. The cytolytic activity of NK92 against K562 target cells, which was augmented in a dose-dependent manner by IL-18 in the presence of trace amounts of IL-2, was suppressed by the specific inhibitors of MAPK pathways (PD098059 and SB203580). Similarly, the stimulatory effect of IL-18 on IFN-gamma protein production, given in conjunction with IL-2, was counteracted by inhibition of MAPK. IL-18 alone failed to stimulate IFN-gamma protein production despite inducing expression of IFN-gamma mRNA. IL-2 alone stimulated neither IFN-gamma mRNA expression nor IFN-gamma protein production. IL-18 did not stimulate proliferation of NK92 cells, either alone or in combination with IL-2 or IL-12. Inhibition of the MAPK pathway did not significantly alter the IL-2- and IL-12-induced proliferation of NK92 cells, whereas the Janus kinase/STAT pathway inhibitor AG490 strongly suppressed proliferation. MAPK activation appears to play a prominent role in IL-18 signaling, being involved in transcription and translation of IL-18-induced IFN-gamma mRNA and IL-18-induced cytolytic effects. In contrast, proliferation of NK92 cells is not affected by MAPK p44erk-1 and p42erk-2.

Effect of treatment with amifostine used as a single agent in patients with refractory anemia on clinical outcome and serum tumor necrosis factor alpha levels.

Amifostine increases in vitro burst-forming unit-erythroid and colony-forming unit-granulocyte/granulcoyte-macrophage cultured from bone-marrow cells from patients with myelodysplastic syndrome (MDS). Several small clinical studies give divergent informations about the potential of amifostine as single agent to improve hematopoiesis in MDS patients. In these studies, patients with refractory anemia (RA), RA with excess of blasts (RAEB), and RAEB in transformation (RAEB-T) were analyzed together, resulting in response rates varying from 8% to 30%. The present multi-center study evaluated whether treatment with amifostine is of clinical benefit in patients with RA who are transfusion dependent. The effect on transfusion frequency as well as on platelets and absolute neutrophil count (ANC) was examined in 14 patients with RA [median age 67 years (55-72 years), male:female 9:5]. Four treatment cycles were planned, each consisting of intravenous amifostine at 200 mg/m2/day three times per week followed by a 2-week interval. Since tumor necrosis factor (TNF) alpha is a main suppressive cytokine for hematopoiesis in RA patients, serum samples for analyzing endogenous levels of TNF alpha were collected prior to the study and after four treatment cycles. In three patients (21%), reduced transfusion requirement with prolongation of the transfusion interval from 4 weeks to 8 weeks (two patients) and 4 weeks to 6 weeks was seen. An increase in ANC from 400/microliter to 2600/microliter and 200/microliter to 3400/microliter was observed in two patients. Platelets increased from 129,000/microliter to 277,000/microliter in an additional patient. In one patient, disease progression from RA to RAEB was observed. Serum TNF alpha levels were increased in MDS patients compared with normal controls (18.8 pg/ml vs 9.1 pg/ml), and there was no change during the treatment with amifostine (17.5 pg/ml). In conclusion, treatment with amifostine as a single agent was of limited benefit in patients with RA. The serum TNF alpha levels were unchanged during treatment with amifostine in RA patients.

Defective megakaryocytic development in myelodysplastic syndromes.

Megakaryocytic proliferation and differentiation is typically abnormal in patients with myelodysplastic syndromes (MDS). The underlying mechanisms for this finding are not known, but may involve defects at the level of the thrombopoietin-receptor (c-mpl) or post-receptor signaling pathways in megakaryocyte progenitor cells. Premature apoptosis of the bone marrow cells and inhibitory effects of cytokines such as tumor necrosis factor alpha have been implicated as contributing to altered megakaryopoiesis in MDS, but their significance remains unclear. The availability of thrombopoietin (TPO) has facilitated more detailed analysis of megakaryocytic biology using several experimental in-vitro systems. However numerous studies have shown that the developmental abnormalities of MDS megakaryocytes could not be corrected by TPO. Increasing investigations are being extended to the evaluation of signal transduction pathways of c-mpl both in cell lines and human hematopoietic cells in order to identify the molecular mechanisms responsible for the defective megakaryocytic development in MDS.

Interleukin-18 stimulates HIV-1 replication in a T-cell line.

Interleukin-18 (IL-18) is a recently identified proinflammatory cytokine. Its ability to induce interferon-g suggests a potential virustatic effect. On the other hand, it stimulates NFkB - an activator of HIV replication. Recently, stimulation of HIV-1 in monocytic cells has been demonstrated. In the present study, the influence of IL-18 on HIV-1 replication in lymphatic cells was investigated. Hut78 cells were infected with HIV-1 in the presence of recombinant human IL-18 expressed either in E. coli or eucaryotically by baculovirus in Sf9 cells. HIV-1 replication was monitored by p24 ELISA and endpoint titration of culture supernatants on C8166 cells. The addition of IL-18 led to a 3- to 15-fold enhancement of HIV replication in Hut78 cells. By addition of neutralising monoclonal anti-IL-18 antibodies, this effect of IL-18 was reduced by 75%. Exposure of Hut78 to IL-18 prior to HIV infection could exclude the possibility that IL-18 promotes infection of cells. Taken together, these data provide direct evidence for an IL-18-mediated enhancement of HIV-1 replication in lymphatic cells.

Counterregulation of interleukin-18 mRNA and protein expression during cutaneous wound repair in mice.

Recent work has suggested interleukin-18 to represent a proinflammatory cytokine that contributes to systemic and local inflammation. As the process of cutaneous wound healing crucially involves an inflammatory phase of repair, we investigated the regulation of interleukin-18 during the repair process. In non-wounded skin we observed high levels of interleukin-18 mRNA, whereas corresponding interleukin-18 protein was expressed only at low basal levels. Upon injury, we found a rapid and large induction of interleukin-18 protein expression, which is directly correlated with decreasing mRNA levels within the wound. Immunohistochemical analysis revealed different sites of expression in the wounded area, with keratinocytes as one major source of interleukin-18 production. The counterregulation of interleukin-18 mRNA and protein expression during wound repair in vivo might represent a general mechanism for interleukin-18 expressional regulation, as cytokine-stimulated keratinocytes exhibit a similar downregulation of interleukin-18 mRNA that is directly associated with increasing interleukin-18 protein levels in vitro. The rapid induction of interleukin-18 during wound healing suggests a role for interleukin-18 within the early phase of repair rather than a role in costimulation of interferon-gamma release from T cells, which are present in high numbers within the wounded area only during the late inflammatory phase of repair.

Megakaryocytic growth in patients with refractory anemia is suppressed by treatment with interferon alpha.

IFN alpha alone or in combination with retinoids or haematopoietic growth factors has been used to treat patients with early MDS because of its properties as a differentiation inducing agent. We investigated whether treatment of patients with refractory anemia (RA) with IFN alpha (1.5x10(6) IU twice a week) and intermittent all-trans retinoic acid (ATRA, 25 mg/m2/d) influences in-vitro megakaryocytic (MK) proliferation and differentiation stimulated by PEG-rHuMGDF. Low-density non-adherent bone marrow (BM) cells from 8 patients with RA were assayed prior to any treatment other than supportive and after a period of 6 months of treatment. MK development was assayed in suspension cultures in the presence of PEG-rHuMGDF and SCF for 7 d using morphological criteria and flow cytometric analysis of CD42b (GP1b) positive cells. BM-cells from 10 healthy individuals served as control. Following stimulation with PEG-rHuMGDF 23+/-7% and 16+/-4% of control cells were CD42b positive after 5 and 7 d of cultures, respectively. In cultures of cells from MDS patients prior to treatment 8+/-2% and 7+/-3% of cells were CD42b+ on days 5 and 7. In the course of IFN alpha treatment cultures of all BM samples from these MDS patients revealed a significant reduction of MK precursor cells (3+/-2%, CD42b+, p=0.03 and 0.04). In conclusion, treatment with TFN alpha and ATRA did not result in improved megakaryocytopoiesis as assessed by in-vitro cultures. On the contrary, low-dose IFN alpha appears to suppress cell proliferation as well as MK development.

Quantification of human interleukin 18 mRNA expression by competitive reverse transcriptase polymerase chain reaction.

Interleukin 18 (IL-18) is a recently identified cytokine, originally called interferon gamma inducing factor, due to its capacity to induce interferon gamma production in Th1 type cells. IL-18 is expressed by a wide variety of cell types including mononuclear phagocytes, osteoblasts, keratinocytes and adrenal cortex cells. To quantify human IL-18 mRNA expression in small-scale cell samples the authors developed a competitive reverse transcriptase polymerase chain reaction using a competitive template as an internal standard. This assay was demonstrated as a valid, sensitive and precise tool to quantify human IL-18 mRNA expression. IL-18 mRNA expression of primary peripheral blood monocytes, CD4(+)T cells, CD8(+)T cells, B cells and NK cells was assessed by competitive RT-PCR. Basal IL-18 expression could be detected in all types of peripheral blood mononuclear cells (PBMC). The kinetics of IL-18 mRNA expression in PBMC from healthy donors was defined in vitro after monocyte-specific (lipopolysaccharide LPS), T-cell-specific (anti-CD3) and polyclonal-unspecific stimulation (phytohaemagglutinin PHA). Only LPS led to a strong increase of IL-18 mRNA expression peaking after 2 h. These results indicate that IL-18 is expressed constitutionally by all major PBMC subtypes. However, only monocyte specific stimulation resulted in a significant induction of IL-18 mRNA expression suggesting activated monocytes e.g. in inflammation as the main source of IL-18 expression.