Successful replication of parvovirus B19 in the human megakaryocytic leukemia cell line MB-02

The pathogenic human parvovirus B19 has been shown to undergo productive replication in the erythroid lineage in primary normal human hematopoietic progenitor cells. However, none of the established erythroleukemia cell lines has allowed B19 virus replication in vitro. The remarkable erythroid tissue tropism of B19 virus was evaluated with a human megakaryocytic leukemia cell line, MB-02, which is dependent on the growth factor granulocyte-macrophage colony-stimulating factor but can be induced to undergo erythroid differentiation following treatment with erythropoietin (Epo). Whereas these cells did not support B19 virus DNA replication in the presence of granulocyte-macrophage colony-stimulating factor alone, active viral DNA replication was observed if the cells were exposed to Epo for 5 to 10 days prior to B19 virus infection, as detected by the presence of the characteristic B19 virus DNA replicative intermediates on Southern blots. No replication occurred if the cells were treated with Epo for 3 days or less. In addition, complete expression of the B19 virus genome also occurred in Epo-treated MB-02 cells, as detected by Northern blot analysis. B19 progeny virions were released into culture supernatants that were biologically active in secondary infection of normal human bone marrow cells. The availability of the only homogeneous permanent cell line in which induction of erythroid differentiation leads to a permissive state for B19 virus replication in vitro promises to yield new and useful information on the molecular basis of the erythroid tissue tropism as well as parvovirus B19-induced pathogenesis.     

Inhibition of protein kinase C suppresses megakaryocytic differentiation and stimulates erythroid differentiation in HEL cells

The bisindolylmaleimide, GF109203X (2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide ), a highly selective inhibitor of protein kinase C (PKC), was used to test the role of this enzyme in phorbol ester-induced megakaryocytic differentiation of HEL cells. Treatment of these cells with 10 nmol/L phorbol 12-myristate 13-acetate (PMA) for 3 days caused a complete inhibition of proliferation and a threefold increase in the surface expression of glycoprotein (GP) IIIa, a marker of megakaryocytic differentiation that forms part of the fibrinogen receptor complex, GPIIb/IIIa. A similar effect was observed with phorbol 12,13-dibutyrate, but not with the biologically inactive derivative PMA-4-O-methyl ether. The PMA-induced increase in GPIIIa expression was completely inhibited by GF109203X in a dose-dependent manner (IC50 = 0.5 mumol/L), with a maximal effect at 2.5 to 5.0 mumol/L. GF109203X also blocked the inhibitory effect of PMA on cell growth and inhibited PMA-stimulated phosphorylation of the 47-kD PKC substrate, pleckstrin. Incubation of HEL cells with 25 mumol/L hemin for 3 days caused a fourfold to fivefold increase in expression of the erythroid differentiation marker, glycophorin A. In contrast to the inhibitory effect of GF109203X on GPIIIa expression, hemin induction of glycophorin A was enhanced by this compound. Furthermore, GF109203X alone caused a dose-dependent increase in glycophorin A expression, and induced hemoglobinization. Consistent with these changes, Northern blot analysis revealed that GF109203X treatment reduced the steady-state level of GPIIb mRNA and increased those for glycophorin A and gamma-globin. These results suggest that PKC may act as a developmental switch controlling erythroid/megakaryocytic differentiation.     

Expression and activation of protein kinase C isoforms in a human megakaryocytic cell line

Megakaryocytes undergo a unique differentiation program, becoming polyploid through repeated cycles of DNA synthesis without concomitant cell division. We have shown previously that phorbol 12-myristate 13-acetate (PMA) induces the Dami human megakaryocytic cell line to become polyploid and to express platelet-specific proteins, including von Willebrand factor (vWF) and glycoprotein Ib (GpIb). Phorbol esters are thought to regulate gene expression principally through the activation of protein kinase C (PKC), a family of structurally related kinases with potentially unique activation requirements and substrate specificities. A survey of PKC isoforms in Dami cells revealed that, by both Western and Northern analyses, PKC isoforms alpha, beta, delta, epsilon, eta, theta, and zeta were reproducibly detected. PKC-gamma was not detected. In order to define the role of individual PKC isoforms in megakaryocytic maturation, PMA and 2-deoxyphorbol 13-phenylacetate 20-acetate (dPPA), a putative selective activator of the PKC-beta 1 isotype, were compared for their effects on Dami cell maturation. Treatment with either dPPA or PMA caused Dami cells to cease proliferating, to become polyploid, and to express vWF. We also examined dPPA and PMA for their ability to activate and to downregulate expression of different PKC isoforms. Fifteen-minute treatment with PMA resulted in the translocation of PKC isoforms alpha, epsilon, and theta from the cytosolic to the membrane fraction; twenty-four hour treatment resulted in the downregulation of these isoforms. In contrast, dPPA was found to be a potent activator of PKC-epsilon alone and exhibited weaker effects on alpha and theta. These data suggest that PKC isoforms beta, delta, eta, and zeta, which appear not to be activated by either phorbol ester, are unlikely to be primarily involved in megakaryocytic maturation in response to these agents. The isoforms that are translocated by both phorbol esters-PKC isoforms alpha and theta, and particularly epsilon-are more likely to transduce the signals that stimulate Dami cell differentiation.     

The mitogen-activated protein kinase pathway inhibits ceramide-induced terminal differentiation of a human monoblastic leukemia cell line, U937

This communication describes an extracellular signal-regulated kinase kinase (MEK)-dependent signal transduction pathway that prevents the terminal differentiation of a hemopoietic cell line. Both PMA and the cell-permeable ceramide, C2-ceramide, caused differentiation of U937 cells, but with distinct cell morphology and CD11b/CD14 surface expression. While PMA activated extracellular signal-regulated kinase (ERK), a downstream kinase of Raf-MEK signaling, C2-ceramide activated c-Jun NH2-terminal kinase (JNK), an anchor kinase of stress-induced signaling. Furthermore, only C2-ceramide stimulated an induction of cell cycle arrest that was associated with stable expression of p21CIP1 and retinoblastoma nuclear phosphoprotein dephosphorylation. Expression of p21CIP1 and JNK activation were also observed in sphingosine-treated cells, whereas sphingosine did not induce detectable differentiation. Concomitant stimulation with C2-ceramide and PMA resulted in the PMA phenotype, and cell cycle arrest was absent. ERK activation was enhanced by C2-ceramide plus PMA stimulation, whereas the activation of JNK was aborted. Strikingly, the inhibition of MEK with PD98059 altered the phenotype of C2-ceramide- and PMA-stimulated U937 cells to that of cells treated with C2-ceramide alone. Thus, ERK and JNK pathways deliver distinct signals, and the ERK pathway is dominant to the JNK cascade. Furthermore, differentiation and cell cycle arrest caused by C2-ceramide rely on independent signaling pathways, and JNK is an unlikely signaling element for this differentiation. Importantly, during C2-ceramide and PMA costimulation, the JNK pathway is not simply blocked by ERK activation; rather, cross-talk between these MAP kinase pathways acts to simultaneously augment ERK activity and down-regulate JNK activity.     

4-Oxoretinol, a metabolite of retinol in the human promyelocytic leukemia cell line NB4, induces cell growth arrest and granulocytic differentiation

All-trans-retinoic acid (RA) is used as a differentiation therapy for acute promyelocytic leukemia. Patients can become resistant to RA, and this resistance is thought to be mediated in part by an increase in the rate of RA metabolism. We have characterized the metabolism of all-trans-retinol (ROL; vitamin A) in NB4 cells, which are human promyelocytic leukemia cells. NB4 cells metabolize ROL into a variety of compounds, including all-trans-4-hydroxyretinol, all-trans-4-oxoretinol (4-oxoROL), 14-hydroxy-4,14-retro-retinol, anhydroretinol, and several ROL esters. No metabolism of ROL to RA or to RA derivatives in NB4 cells was detected. The rate of ROL metabolism increased after cell differentiation; in a 24-h period, differentiated cells metabolized 2-fold more ROL than did undifferentiated cells. The major difference in the ROL metabolism pattern between undifferentiated and differentiated cells was an approximately 10-fold increase in the production of all-trans-4-hydroxyretinol and 4-oxoROL in differentiated cells. Furthermore, exogenously added 4-oxoROL was capable of eliciting NB4 cell differentiation, as measured by growth inhibition, nitroblue tetrazolium reduction, nuclear body relocalization of PML, and surface expression of CD11b. In addition, 4-oxoROL synergized with IFN-gamma in the promotion of NB4 cell growth arrest. Following treatment of NB4 cells with 4-oxoROL to induce differentiation, the production of 4-oxoROL from ROL was observed; this indicated that 4-oxoROL induces its own synthesis in NB4 cells. In addition, 48 h after the administration of 1 microM 4-oxoROL, NB4 cells maintained a high intracellular concentration (17 microM) of 4-oxoROL. These unique properties of 4-oxoROL may provide advantages over RA in the treatment of promyelocytic leukemia cells because it may be possible to maintain cytodifferentiating concentrations of 4-oxoROL in the cells for extended periods of time.     

Basophilic differentiation of the human leukemia cell line KU812 upon treatment with interleukin-4

To assess the effect of right ventricular pacing on rate regularity during exercise and daily life activities, 16 patients with sinoatrial disease and chronic atrial fibrillation (AF) were studied. Incremental ventricular pacing was commenced at 40 beats/min until > 95% of ventricular pacing were achieved during supine, sitting, and standing. Thirteen patients also underwent randomized paired submaximal exercise tests in either a fixed rate mode. (VVI) or a ventricular rate stabilization (VRS) mode in which the pacing rate was set manually at 10 beats/min above the average AF rate during the last minute of each exercise stage. The pacing interval for rate regularization was shortest during standing (692 +/- 26 ms) compared with either supine or sitting (757 +/- 30 and 705 +/- 26 ms, respectively, P < 0.05). During exercise VRS pacing significantly increased the maximum rate (119 +/- 5.2 vs 106 +/- 4.2 ms, P < 0.05), percent of ventricular pacing (85% +/- 5% vs 23% +/- 7%, P < 0.05), rate regularity index (5.8% +/- 1.6% vs 13.4% +/- 1.9%, P < 0.05), and maximum level of oxygen consumption (12.4 +/- 0.5 vs 11.3 +/- 0.5 mL/kg, P < 0.05) compared with VVI pacing. There was no change in oxygen pulse or difference in symptom scores in this acute study between the two pacing modes. It is concluded that right ventricular pacing may significantly improve rate regularity and cardiopulmonary performance in patients with chronic AF. This may be incorporated in a pacing device for rate regularization of AF using an algorithm that is rate adaptive to postural and exercise stresses.     

STAT5 activation correlates with erythropoietin receptor-mediated erythroid differentiation of an erythroleukemia cell line

Interaction between erythropoietin (EPO) and its membrane receptor induces the proliferation and differentiation of erythroid progenitors. EPO has been shown to activate the JAK2-STAT5 pathway in various hematopoietic cell lines, although the physiological role of this pathway is unclear. We have previously shown that epidermal growth factor activates a chimeric receptor bearing the extracellular domain of the epidermal growth factor receptor linked to the cytoplasmic domain of the EPO receptor, resulting in proliferation of interleukin-3-dependent hematopoietic cells and erythroid differentiation (globin synthesis) of EPO-responsive erythroleukemia cells. In the present study, we introduced various deletion and tyrosine to phenylalanine substitution in the cytoplasmic domain of the chimeric receptor and expressed these mutant chimeras in an EPO-responsive erythroleukemia cell line, ELM-I-1. Mutant chimeric receptors retaining either Tyr343 or Tyr401 could activate STAT5, judged by tyrosine-phosphorylation of STAT5 and induction of CIS, a target gene of STAT5. These mutants were able to induce erythroid differentiation. However, a chimeric receptor containing both Y343F and Y401F mutations could not activate STAT5 nor induce erythroid differentiation. Thus, Tyr343 or Tyr401 of the EPO receptor are independently necessary for erythroid differentiation as well as STAT5 activation. Moreover, exogenous expression of dominant-negative STAT5 suppressed EPO-dependent erythroid differentiation. These findings suggest that STAT5 plays an important role in erythroid differentiation through the EPO receptor cytoplasmic domain.     

Comparative assessment of the activity of beta-carotene, retinoyl-beta-D-glucuronide and retinoic acid on growth and differentiation of a human promyelocytic leukemia cell line HL-60

We compared the ability of all-transretinoic acid (RA), all-trans-retinoyl-beta-D-glucuronide (RAGL), and all-trans-beta-carotene (BC) to inhibit growth and to induce differentiation of the human promyelocytic leukemia cell line HL-60 into morphologically mature granulocytes. BC was made water-soluble by the solutol-solvent-system. RA (1 microM) could induce differentiation of 85% of the HL-60 cells after a total incubation time of 180 hours, RAGL (5 microM) induced 64% of the cells, whereas 33% of the HL-60 cells were differentiated after incubation with BC (10 microM), which was determined by assessing cell functional capacity to reduce nitroblue tetrazolium dye in response to phorbolesters. The absence of RA in RAG and BC treated cells gives strong evidence that RAG and BC exert intrinsic biological effects.     

K-252a-induced polyploidization and differentiation of a human megakaryocytic cell line, Meg-J: transient elevation and subsequent suppression of cyclin B1 and cdc2 expression in the process of polyploidization

Megakaryocytes are unique haemopoietic cells which undergo DNA replication, giving rise to polyploid cells. However, little is known about the mechanism of megakaryocytic polyploidization. To address this issue, we used the human megakaryocytic cell line Meg-J. In the presence of K-252a (an indolocarbasole derivative), Meg-J cells stopped proliferation and exhibited additional megakaryocytic features, including morphological changes, polyploidization, and increases in the levels of surface expression of platelet glycoprotein (GP) IIb/IIa and GPIb. Thrombopoietin (TPO) promoted the K-2 52a-induced polyploidization and megakaryocytic differentiation. In the process of K-252a-induced polyploidization, levels of expression of both cdc2 and cyclin B1 were elevated transiently and subsequently decreased. This suggested that the polyploidization process in Meg-J cells was at least in part associated with a transient elevation and subsequent decrease in the expression of cdc2/cyclin B1 complex, a critical kinase involved in G2/M cell cycle transition.     

Expression of ion channels during differentiation of a human skeletal muscle cell line

An immortal, cloned cell line (RCMH), obtained from human skeletal muscle was established in our laboratory and shown to express muscle specific proteins. We measured ligand binding to ion channels, ion currents using whole cell patch clamp and intracellular calcium both in cells grown in complete media and in cells grown for 4-40 days in media supplemented with hormones and nutrients (differentiating media). Markers for differentiated muscle, such as the muscle isoform of creatine kinase and the cytoskeletal proteins alpha-actinin, alpha-sarcomeric actin, myosin and titin were present in early stages. Receptors for gamma toxin from Tityus serrulatus scorpion venom, a specific modulator for voltage dependent sodium channels, were present (0.9-1.0 pmol mg-1 protein) during stage 1 (0-6 days in culture with differentiating media) and increased by 50% in stage 3 (more than 10 days in differentiating media). High and low affinity dihydropyridine receptors present in stage 1 convert into a single type of high affinity receptors in stage 3. Both intracellular calcium release and InsP3 receptors were evident in stage 1 but ryanodine receptors were expressed only in stage 3. RCMH cells showed no voltage sensitive currents in stage 1. Between 7 and 10 days in differentiating media (stage 2), an outward potassium current was observed. Small inward currents appeared only in stage 3; we identified both tetrodotoxin sensitive and tetrodotoxin resistant sodium currents as well as calcium currents. This pattern is consistent with the expression of voltage dependent calcium release before appearance of both the action potential and ryanodine receptors.     

Thrombopoietin expands erythroid, granulocyte-macrophage, and megakaryocytic progenitor cells in normal and myelosuppressed mice

Thrombopoietin (Tpo), the ligand for the proto-oncogene receptor c-Mpl, increases megakaryocyte size, ploidy, and surface expression of platelet-specific glycoproteins, is inversely related to platelet mass, and is a potent in vivo stimulus of platelet production. However, several features of c-mpl biology, and that of its viral counterpart v-mpl, suggest that the action of Tpo may not be strictly limited to megakaryocytopoiesis. To investigate the possibility that Tpo might affect a multitude of cell lineages, we studied the effects of in vivo administration of the hormone on multiple types of marrow and splenic clonogenic hematopoietic progenitors. We report that Tpo acts to expand BFU-E, CFU-GM, and CFU-Mk and redistribute CFU-E in normal mice and to hasten the recovery of all of these progenitor cell types in myelosuppressed animals. These findings argue that the hematopoietic progenitor cell compartment responds to Tpo as a whole and that the in vivo effects of Tpo administration may be more wide-ranging than previously anticipated.     

Thrombopoietin does not induce lineage-restricted commitment of Mpl-R expressing pluripotent progenitors but permits their complete erythroid and megakaryocytic differentiation

In this study, we examined the in vitro and in vivo effects of forced expression of Mpl-R (the thrombopoietin receptor) on the progeny of murine hematopoietic stem cells. Bone marrow cells from 5-FU-treated mice were transduced with retroviral vectors containing the human Mpl-R cDNA, or the neomycine gene as a control. After 7 days cocultivation on virus-producer cells, GpE86-Mpl-R or Gp86-Neo, the types of hematopoietic progenitor cells responding to thrombopoietin (TPO) were studied by clonogenic assays. Mpl-R-infected cells gave rise to CFU-GEMM, BFU-E, CFU-MK, but not CFU-GM while Neo-infected cells produced only megakaryocytic colonies. In addition, when nonadherent cells from GpE86-Mpl-R cocultures were grown with TPO as the only stimulus for 7 days, a marked expansion of CFU-GEMM, BFU-E, and CFU-MK was observed, while no change in CFU-GM number was seen. Erythroid and megakaryocytic maturation occurred in the presence of TPO while a block in granulocytic differentiation was observed at the myeloblast stage. The direct effects of TPO on Mpl-R-transduced progenitor cells were demonstrated by single cell cloning experiments. To analyze the effects of the constitutive expression of Mpl-R on the determination of multipotent progenitors (CFU-S) and long-term repopulating stem cells, Mpl-R- or Neo-infected cells were injected into lethally irradiated recipient mice. No difference was seen in (1) the number of committed progenitor cells contained in individual CFU-S12 whether colonies arose from noninfected or Mpl-R-infected CFU-S; (2) the mean numbers of progenitor cells per leg or spleen of mice reconstituted with Mpl-R- or Neo-infected cells, 1 or 7 months after the graft; and (3) the blood parameters of the two groups of animals, with the exception of a 50% reduction in circulating platelet counts after 7 months in mice repopulated with Mpl-R-infected bone marrow cells. These results indicate that retrovirus-mediated expression of Mpl-R in murine stem cells does not modify their ability to reconstitute all myeloid lineages of differentiation and does not result in a preferential commitment toward the megakaryocytic lineage.     

Purification and characterization of a cell growth factor from a human leukemia cell line: immunological identity with ferritin

We have succeeded in long-term cultivation of a human erythroleukemia cell line, K-562-T1 (T. Okabe, M. Fujisawa, and F. Takaku, Proc. Natl. Acad. Sci. USA, 81: 453-455, 1984). The cells grown in a protein-free chemically defined medium have been shown to produce cell growth factors (A. Mihara et al., In Vitro Cell. Dev. Biol., 23: 317-322, 1987). In this study, we have purified a cell growth factor from the conditioned medium that stimulates the proliferation of human leukemia cells, HL-60. In the purified factor, two major protein bands of 24 kDa and 22 kDa were identified on a sodium dodecyl sulfate-polyacrylamide gel. The 22 kDa protein was stained with a monoclonal antibody to the light chain of ferritin. The growth-promoting activity of the purified factor was coprecipitated with a monoclonal antibody to the light chain or heavy chain of human ferritin. These results suggest that K-562-T1 cells produce a cell growth factor that is related to ferritin.     

Regulation of phospholipase D activity in a human oligodendroglioma cell line (HOG)

Oligodendroglial cells express many specific proteins, such as myelin basic protein (MBP), which are physiologically phosphorylated by protein kinase C (PKC). Diacylglycerols are physiological activators of PKC and can be liberated from phospholipids by the direct receptor-mediated activation of phospholipase C (PL-C) or indirectly via the activation of phospholipase D (PL-D). In a well-characterized human oligodendroglioma (HOG) cell line, PL-C (measured by release of [3H]inositol phosphates) and PL-D (formation of [3H]myristoylated or palmitoylated phosphatidylethanol) were activated by both carbachol (blocked by pirenzepine, suggesting an M1 receptor) and histamine (H1 receptor) but not glutamate, bradykinin, or phenylephrine. PL-C stimulation by carbachol or histamine was completely inhibited by short-term treatment (< 30 min) with phorbol ester (TPA), a PKC activator. In contrast, PL-D activation by either carbachol or histamine was stimulated in additive fashion by TPA, suggesting at least two distinct mechanisms for PL-D activation. Down regulation of PKC by prolonged (24 hr) treatment with TPA reversed the inhibitory effects of TPA on PL-C and the stimulatory effects on PL-D. However, the PKC inhibitors H-7 and galactosylsphingosine did not inhibit the TPA-mediated stimulation of PLD while the less-specific PKC inhibitor, staurosporine, was only partially inhibitory. Preexposure of cells to carbachol, greatly reduced both PL-C and PL-D activation by carbachol, suggesting homologous desensitization. Time-course studies indicated that PL-D activation (10 sec or less) was at least as fast as PL-C activation, and the affinity of carbachol and histamine for the receptor coupled to either phospholipase (EC50 = 5-10 microM) was about the same.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of natriuretic peptide (urodilatin) release in a human kidney cell line

1,4,5,6-Tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine) is an excellent osmoprotectant. The biosynthetic pathway of ectoine from aspartic beta-semialdehyde (ASA), in Halomonas elongata, was elucidated by purification and characterization of each enzyme involved. 2,4-Diaminobutyrate (DABA) aminotransferase catalyzed reversively the first step of the pathway, conversion of ASA to DABA by transamination with L-glutamate. This enzyme required pyridoxal 5'-phosphate and potassium ions for its activity and stability. The gel filtration estimated an apparent molecular mass of 260 kDa, whereas molecular mass measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was 44 kDa. This enzyme exhibited an optimum pH of 8.6 and an optimum temperature of 25 degreesC and had Kms of 9.1 mM for L-glutamate and 4.5 mM for DL-ASA. DABA acetyltransferase catalyzed acetylation of DABA to gamma-N-acetyl-alpha,gamma-diaminobutyric acid (ADABA) with acetyl coenzyme A and exhibited an optimum pH of 8.2 and an optimum temperature of 20 degreesC in the presence of 0.4 M NaCl. The molecular mass was 45 kDa by gel filtration. Ectoine synthase catalyzed circularization of ADABA to ectoine and exhibited an optimum pH of 8.5 to 9.0 and an optimum temperature of 15 degreesC in the presence of 0.5 M NaCl. This enzyme had an apparent molecular mass of 19 kDa by SDS-PAGE and a Km of 8.4 mM in the presence of 0. 77 M NaCl. DABA acetyltransferase and ectoine synthase were stabilized in the presence of NaCl (>2 M) and DABA (100 mM) at temperatures below 30 degreesC.     

Down-regulation of human sialyltransferase gene expression during in vitro human keratinocyte cell line differentiation

We studied cytokines and anti-cytokine autoantibodies (Aabs) during T.b.brucei infections in IFN-gamma-/-, IFN-gammaR-/- and wild-type mice. Increased serum levels of IFN-gamma, TNF-gamma and IL-4 with decreased Aabs to these cytokines were recorded early during infections in all mice (except IFN-gamma in IFN-gamma-/- mice). Later, these responses were reversed, and surprisingly Aabs reacting to IFN-gamma in the IFN-gamma -/- mice were detected. To examine the possibility that an IFN-? immunoreactive molecule might be expressed due to infections and upon gene deletion, anti-IFN-gamma antibody was inoculated and resulted in abrogation of such Aabs. The scenario was different for IL-10 and TGF- since IFN-gammaR-/- and wild-type mice showed low cytokines and high Aabs early during infections, but later high cytokines and low Aabs were registered. Interestingly, IFN-gamma-/- mice exhibited reversed levels of both IL-10 and TGF-beta, and also of their Aabs. Fab fragments of purified serum immunoglobulins showed binding and neutralizing effects in biological assays. Pre-absorption of the Fab fragments with a cytokine inhibited the binding and neutralization effects of this cytokine, but not of other cytokines. These results highlight an important role for autoimmunity in cytokine regulation, and that genomic deletion of IFN-gamma modulates cytokines and their Aab responses in experimental African trypanosomiasis.