Expression of the Escherichia coli lacZ gene on a plasmid vector in a cyanobacterium.

A biphasic plasmid vector was used to introduce the Escherichia coli K-12 lac operon into the unicellular cyanobacterium Agmenellum quadruplicatum PR-6. The PR-6 transformants expressed beta-galactosidase at nearly as high a level as did Escherichia coli transformants. In order to accomplish this, it was necessary to obtain PR-6 mutants that could be transformed by plasmids with unmodified recognition sites for the endogenous PR-6 restriction endonuclease Aqu I. These mutants were generated by a variation of the ectopic mutagenesis techniques that have been used in other naturally transforming bacteria. The ability to assay the expression of lacZ in PR-6 paves the way for the construction of gene fusions with various PR-6 promoters and quantitation of their expression under specific in vivo conditions.

Cationic lipids reduce time and dose of c-myc antisense oligodeoxynucleotides required to specifically inhibit Burkitt's lymphoma cell growth.

Burkitt's lymphoma is characterized by a translocation of the c-myc gene with one of the immunoglobulin loci which activates overexpression of the c-myc oncogene. Antisense-oligodeoxynucleotides (AS-ODNs) offer the potential to block specific c-myc gene expression within lymphoma cells, but often exhibit a low efficiency of AS-ODN uptake. In this study, a polycationic lipid reagent, Lipofectamine (LFM), was utilized as a vehicle to increase efficiency of delivery, decrease the time needed to observe an inhibitory effect, and decrease the AS-ODN dose. The objective was to develop a more efficient and rapid in vitro AS-ODN strategy to inhibit proliferation of c-myc-dependent lymphoma cells and to test the specificity of Burkitt's lymphoma cell line-directed AS-ODNs for potential use as molecular purging agents in bone marrow transplantation. Proliferation assays were performed to determine the inhibitory effect of the AS-ODNs on two Burkitt's lymphoma cell lines with different chromosomal translocations, Daudi and ST486, in medium containing 8.5 microM LFM. AS-ODNs at a concentration of 0.36 microM induced a significant decrease in proliferation for both cell lines using the specific AS-ODN for each respective translocation. Within 5 h, Daudi responded to its specific AS-ODN/lipid complexes with a 35% decrease in proliferation, compared to cells which received no treatment or Daudi-specific AS-ODN without LFM (P = 0.0001). Daudi showed an insignificant decrease in proliferation when treated with an AS-ODN specific for the ST486 translocation (4%, P = 0.26). ST486 proliferation was decreased by 52% when treated with the specific antisense for ST486 compared to no treatment or ST486-specific AS-ODN without LFM (P < 0.003). Treatment with the AS-ODN specific for Daudi showed an insignificant 4% decrease (P = 0.42). Controls, including sense ODN for structure, reverse AS-ODN for structure and base composition, and AS-ODN without LFM, did not produce a significant change in cells treated with LFM alone or cells receiving no treatment. Clonogenic assays of both Daudi and ST486 treated with their specific AS-ODNs revealed a 50% inhibition of colony formation after the 5 h incubation as compared to no treatment. Confocal laser scanning microscopy verified that cellular uptake of AS-ODN was enhanced by cationic lipids. Immunoblot analysis showed a 63 +/- 5% and a 50 +/- 3% reduction in intracellular c-myc levels for Daudi and ST486, respectively, when their respective AS-ODNs were administered. Normal bone marrow progenitors were unaffected by the ODN/LFM complexes. These results suggest that the specific c-myc AS-ODN/LFM complexes inhibit c-myc-dependent tumor proliferation at an earlier time and at a lower dose compared to no lipid facilitation. This approach may form the basis for utilizing specific AS-ODN/LFM therapy either alone or in a cocktail of other agents as an ex vivo molecular purging approach to autologous stem cell transplantation in Burkitt's lymphoma.

Coordinate regulation of Steel factor, its receptor (Kit), and cytoadhesion molecule (ICAM-1 and ELAM-1) mRNA expression in human vascular endothelial cells of differing origins.

Endothelial cells (EC) are a major component of the bone marrow and peripheral vasculature microenvironments and contribute to the regulation of hematopoiesis. Human EC cultured from umbilical vein (HUVEC) and adult aorta (HAEC) were compared to determine differences in levels of the multipotent cytokine, Steel factor (SLF), its receptor (Kit), intercellular adhesion molecule-1 (ICAM-1), and endothelial leukocyte adhesion molecule-1 (ELAM-1) before and after stimulation with human interleukin-1 beta (hIL-1 beta), human tumor necrosis factor-alpha (hTNF-alpha), recombinant human (rh) SLF, phorbol 12-myristate 13-acetate (PMA), or calcium ionophore (A23187). HUVEC expressed four-fold higher basal levels of Kit and three-fold higher basal levels of SLF transcripts than HAEC. In contrast, the basal level of ICAM-1 mRNA was four-fold lower in HUVEC than in HAEC. These differences in expression persisted following activation. All five agonists downregulated Kit mRNA levels by 50 to 80%, but there remained a three-fold higher level of expression in HUVEC compared to HAEC. While SLF mRNA expression was increased four-fold by IL-1 beta or TNF-alpha and 50-fold by PMA or A23187, there was still a two-fold higher level in HUVEC than in HAEC. Similarly, production of cell-associated SLF was induced two-fold above basal level by PMA in HUVEC and HAEC, with HUVEC producing two-fold more than HAEC before and after stimulation. Production of soluble SLF was also increased six-fold in HUVEC and HAEC by PMA, but the HAEC produced slightly more than the HUVEC. Expression of ICAM-1 mRNA was increased 11-fold in activated HUVEC and HAEC, but the induced levels of both ICAM-1 and ELAM-1 mRNA were three-fold lower in HUVEC. The time course of SLF mRNA upregulation and Kit mRNA downregulation paralleled the upregulation of both cytoadhesion molecules. Differences between HUVEC and HAEC may be related to their vascular sources, but also suggest that disparate regulation of SLF, Kit, ICAM-1, and ELAM-1 expression could indicate a predisposition of neonatal EC toward impaired cytokine signal transmission.

Treatment with UDP-glucose, GDNF, and memantine promotes SVZ and white matter self-repair by endogenous glial progenitor cells in neonatal rats with ischemic PVL.

Periventricular leukomalacia (PVL) is one of the foremost neurological conditions leading to long-term abnormalities in premature infants. Since it is difficult to prevent initiation of this damage in utero, promoting the innate regenerative potential of the brain after birth may provide a more feasible, prospective therapy for PVL. Treatment with UDP-glucose (UDPG), an endogenous agonist of G protein-coupled receptor 17 (GPR17) that may enhance endogenous self-repair potentiality, glial cell line-derived neurotrophic factor (GDNF), a neurotrophic factor associated with the growth and survival of nerve cells, and memantine, a noncompetitive antagonist of N-methyl-d-aspartate (NMDA) receptors that block ischemia-induced glutamate signal transduction, has been reported to achieve functional, neurological improvement in neonatal rats with PVL. The aim of the present study was to further explore whether UDPG, GDNF and/or memantine could promote corresponding self-repair of the subventricular zone (SVZ) and white matter (WM) in neonatal rats with ischemia-induced PVL. SVZ or WM tissue samples and cultured glial progenitor cells derived from a 5 day-old neonatal rat model of PVL were utilized for studying response to UDPG, GDNF and memantine in vivo and in vitro, respectively. Labeling with 5'-bromo-2'-deoxyuridine and immunofluorescent cell lineage markers after hypoxia-ischemia or oxygen-glucose deprivation (OGD) revealed that UDPG, GDNF and memantine each significantly increased glial progenitor cells and preoligodendrocytes (preOLs), as well as more differentiated immature and mature oligodendrocyte (OL), in both the SVZ and WM in vivo or in vitro. SVZ and WM glial cell apoptosis was also significantly reduced by UDPG, GDNF or memantine, both in vivo and in vitro. These results indicated that UDPG, GDNF or memantine may promote endogenous self-repair by stimulating proliferation of glial progenitor cells derived from both the SVZ and WM, activating their differentiation into more mature OLs, and raising the survival rate of these newly generated glial cells in neonatal rats with ischemic PVL.

Genes from the cyanobacterium Agmenellum quadruplicatum isolated by complementation: characterization and production of merodiploids.

The isolation of several biosynthetic genes from a cyanobacterium, Agmenellum quadruplicatum, by complementation of auxotrophic mutations in Escherichia coli, and their partial characterization, is described. Although our search for such genes has not been exhaustive, it appears that complementation of E. coli mutations may be of limited utility for the identification and/or isolation of cyanobacterial genes. Despite some overlap in the complementation abilities of these isolated cyanobacterial DNA fragments, the genes that we have studied in some detail are not located in operons. We have used mutagenized versions of these cyanobacterial DNA fragments to produce mutant phenotypes in the cyanobacterium, but clean auxotrophs were not obtained. Complementation of these mutant phenotypes can be obtained when the appropriate wild-type DNA fragment is introduced into the cyanobacterium on a shuttle vector. Recombination between two copies of a cyanobacterial gene occurs at high frequency in the cyanobacterium.

Decreased endogenous circulating Steel factor (SLF) levels following allogeneic and autologous BMT: lack of an inverse correlation with post-BMT myeloid engraftment.

We have previously demonstrated an inverse relationship between circulating endogenous G-CSF levels and myeloid engraftment post-BMT. A new early-acting hematopoietic growth factor, Steel factor (SLF), has recently been demonstrated to induce the proliferation of early hematopoietic progenitor cells and synergistically stimulate committed progenitor cells in the presence of lineage-specific CSFs. In this pilot study, we determined the temporal relationship between endogenous SLF levels and the circulating absolute neutrophil count (ANC) (myeloid engraftment) in both children and adults undergoing both allogeneic and autologous BMT. Pre-BMT SLF levels were 2600 +/- 100 pg/ml compared to significantly lower levels of G-CSF (30-50 pg/ml). The circulating SLF level was significantly decreased throughout the post-BMT period (ANC < or = 200 x 10(6)/l: 1500 +/- 600 pg/ml; ANC 200-500 x 10(6)/l: 1780 +/- 130 pg/ml; ANC > or = 500 x 10(6)/l: 1690 +/- 110 pg/ml) (p < 0.001). There was a lack of an inverse relationship between the circulating SLF level and the ANC (r = -0.43) (p = NS). For comparison, SLF levels from immune thrombocytopenia (platelet < = or 20 x 10(9)/l) and chemotherapy-induced neutropenia patients (ANC < or = 200 x 10(6)/l) were similar to pre-BMT levels but significantly higher than post-BMT levels (p < or = 0.02 and < or = 0.001, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

A light-regulated DNA-binding activity interacts with a conserved region of a Lemna gibba rbcS promoter.

We have characterized a DNA-binding activity, designated light-regulated nuclear factor (LRF-1), which interacted with a specific sequence located 150 nucleotides upstream from the transcription start site of a phytochrome-regulated Lemna gibba rbcS gene (SSU5B). There was a higher level of LRF-1 activity recovered from nuclei of light-grown plants than from dark-treated plants. In light-grown plants given a 1-day dark treatment, either white light or a single 2-min red illumination caused a rapid twofold to threefold increase in this activity, suggesting that the phytochrome system is probably involved in its regulation. The nuclear extracts also contained an activity that bound specifically to Box II sequences from a pea rbcS gene [Green, P.J., Yong, M.H., Cuozzo, M., Kano-Murakami, Y., Silverstein, P., and Chua, N.-H. (1988). EMBO J. 7, 4035-4044], but this activity was not higher in the light-grown compared with the dark-treated plants. Comparison of about 700 base pairs upstream from the SSU5B transcription start site with the upstream sequences of two other Lemna rbcS genes revealed several conserved regions. One of these regions is found upstream of rbcS genes in other species and is contained in the sequence which was shown to interact with LRF-1.

Plasmid transformation in Agmenellum quadruplicatum PR-6: construction of biphasic plasmids and characterization of their transformation properties.

Biphasic, chimeric plasmids for the transformation of Agmenellum quadruplicatum PR-6 (Synechococcus sp. strain 7002) were constructed by splicing the 3.0-megadalton cryptic plasmid from strain PR-6 into plasmids pBR322 and pBR325 from Escherichia coli. Transformants of either E. coli or strain PR-6 by these plasmids could be detected on the basis of the drug resistance marker(s) carried by the chimeric plasmids. Plasmid DNA isolated from a PR-6 transformant transformed PR-6 much more efficiently than plasmid DNA prepared from E. coli. Plasmids from which the AvaI recognition site was deleted (AvaI is an isoschizomer of the AquI restriction endonuclease of strain PR-6) also transformed strain PR-6 much more efficiently than did plasmids containing the AvaI recognition site. These and other results suggest that AquI strongly effects plasmid transformation when the donor plasmid contains an unmodified AquI recognition site. Multimeric forms of the chimeric plasmids are also much more efficient at transforming strain PR-6 than are the analogous monomeric forms.

Developmental regulation of RNA transcript destabilization by A + U-rich elements is AUF1-dependent.

The developmental immaturity of neonatal phagocytic function is associated with decreased accumulation and half-life (t((1)/(2))) of granulocyte/macrophage colony-stimulating factor (GM-CSF) mRNA in mononuclear cells (MNC) from the neonatal umbilical cord compared with adult peripheral blood. The in vivo t((1)/(2)) of GM-CSF mRNA is 3-fold shorter in neonatal (30 min) than in adult (100 min) MNC. Turnover of mRNA containing a 3'-untranslated region (3'-UTR) A + U-rich element (ARE), which regulates GM-CSF mRNA stability, is accelerated in vitro by protein fractions enriched for AUF1, an ARE-specific binding factor. The data reported here demonstrate that the ARE significantly accelerates in vitro decay of the GM-CSF 3'-UTR in the presence of either neonatal or adult MNC protein. Decay intermediates of the GM-CSF 3'-UTR are generated that are truncated at either end of the ARE. Furthermore, the t((1)/(2)) of the ARE-containing 3'-UTR is 4-fold shorter in the presence of neonatal (19 min) than adult (79 min) MNC protein, reconstituting developmental regulation in a cell-free system. Finally, accelerated ARE-dependent decay of the GM-CSF 3'-UTR in vitro by neonatal MNC protein is significantly attenuated by immunodepletion of AUF1, providing new evidence that this accelerated turnover is ARE- and AUF1-dependent.

Genetic analysis of two new mutations resulting in herbicide resistance in the cyanobacterium Synechcoccus sp. PCC 7002.

Two herbicide-resistant strains of the cyanobacterium Synechococcus sp. PCC 7002 are compared to the wild-type with respect to the DNA changes which result in herbicide resstance. The mutations have previously been mapped to a region of the cyanobacterial genome which encodes oneof three copies of psbA, the gene which encodes the 32 kDa Qb-binding protein also known as D1 (Buzby et al. 1987). The DNA sequence of the wild-type gene was first determined and used as a comparison to that of the mutant alleles. A point mutation at codon 211 in the psbA1 coding locus (TTC) to TCC) results in an amino acid change from phenylalanine to serine in the D1 protein. This mutation confers resistance to atrazine and diuron at seven times and at two times the minimal inhibitory concentration (MIC) for the wild-type, respectively. A mutation at codon 211 resulting in herbicide resistance has not previously been described in the literature. A second point mutation at codon 219 in the psbA1 coding locus (GTA to ATA) results in an amino acid change from valine to isoleucine in the D1 protein. This mutation confers resistance to diuron and atrazine at ten times and at two times the MIC for the wild-type, respectively. An identical codon change conferring similar herbicide resistance patterns has previously been described in Chlamydomonas reinhardtii. The atrazine-resistance phenotype in Synechococcus sp. PCC 7002 was shown to be dominant by plasmid segregation analysis.

Regulation of interleukin-11 protein and mRNA expression in neonatal and adult fibroblasts and endothelial cells.

Interleukin-11 (IL-11), a newly-identified cytokine produced by stromal cells, elevates platelet counts in neonatal rats in vivo and synergizes in vitro with IL-3 in supporting murine megakaryocyte colony formation and stimulating hematopoietic stem cells. Megakaryocytopoiesis is also enhanced by other colony-stimulating factors (CSFs), including IL-3, IL-6, and Steel factor (SLF). Dysregulation of neonatal thrombopoiesis predisposes newborns to develop thrombocytopenia during sepsis, despite increased circulating pools of committed thrombopoietic progenitors in newborn cord blood compared with adult. We previously reported reduced expression of granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte-colony-stimulating factor (G-CSF), and IL-3 from stimulated cord mononuclear cells, but increased expression of SLF in human umbilical vein endothelial cells (HUVEC). Therefore, we hypothesized that IL-3, IL-6, and SLF might modulate megakaryocytopoiesis by inducing IL-11 expression, and newborns might express altered levels of IL-11 mRNA expression during activated conditions, contributing to the difference in circulating colony-forming unit-megakaryocyte (CFU-Meg) cord and adult blood. Phorbol myristate acetate (PMA) induced a twofold greater increase in IL-11 mRNA expression in neonatal fibroblasts (NFb) compared with adult fibroblasts (AFb), and a 3.6-fold greater increase in HUVEC than human adult aorta endothelial cells (HAEC) by Northern blot analysis. PMA also induced a threefold greater increase in IL-11 protein production in NFb than AFb. Physiologic agonists IL-1 alpha, transforming growth factor-beta 1 (TGF-beta 1), and TGF-beta 2 triggered upregulation of IL-11 mRNA expression in both NFb and AFb. However, IL-3, IL-6, PIXY321 (a GM-CSF-IL-3 fusion protein), and SLF failed to upregulate IL-11 mRNA expression from the basal level, while macrophage-colony stimulating factor (M-CSF) mRNA was significantly induced. These data suggest that the hematopoietic effect of IL-6, SLF, and IL-3 on megakaryocytopoiesis is probably not mediated by secondary IL-11 mRNA expression. Similarly, inflammatory agonists IL-1 beta, lipopolysaccharide (LPS), and tumor necrosis factor-alpha (TNF-alpha) alone did not upregulate IL-11 expression from the basal level in endothelial cells, whereas intracellular adhesion molecule-1 (ICAM-1) and endothelial leukocyte adhesion molecule-1 were strongly induced. Minimal basal IL-11 expression was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) in NFb, AFb, HUVEC and HAEC. The quantitative RT-PCR assay also verified that IL-1 beta and TNF-alpha-stimulated HUVEC and HAEC, and IL-3- and IL-6-stimulated NFb and AFb only expressed minimal levels of IL-11 mRNA.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of phosphodiester and phosphorothioate antisense oligodeoxynucleotides on cell lines which overexpress c-myc: implications for the treatment of Burkitt's lymphoma.

The product of the c-myc proto-oncogene is a highly conserved nuclear phosphoprotein whose expression is closely linked to cellular proliferation and differentiation. We have been interested in developing an antisense oligodeoxynucleotide (ODN) strategy to inhibit the proliferation of c-myc-dependent malignancies for use in future specific therapies and bone marrow purging regimens. Our experimental approach was to incubate either antisense or sense ODNs, spanning the 5' cap region of the c-myc gene, with c-myc overexpressing cell lines (HL-60) Raji, MJBL, CA-46) for up to seven days. Proliferation assay to test the inhibitory effect of an unmodified antisense ODN 15-mer (GCACAGCTCGGGGGT) showed that concentrations as low as 50 micrograms/ml significantly decreased proliferation of HL-60 cells by approximately 40% (P < 0.0001; n = 6) compared to controls. Clonogenic assays showed that the same antisense ODN inhibited colony formation by MJBL (40%0 and Raji (52%) cells. Subsequent experiments to study the effect of a more nuclease-stable, phosphorothioate-modified antisense ODN 18-mer (GCAGCACAGCTCGGGGGT) revealed 66% inhibition of HL-60 cell proliferation at 96 and 120 hours at 50 micrograms/ml, whereas sense ODN control had no effect. However, tenfold less of the modified antisense ODN (1 micrograms/ml) was required to inhibit proliferation of HL-60 cells by 50% compared to the unmodified antisense ODN. A decrease in the HL-60 native c-myc protein level was also observed with 100 micrograms/ml of modified antisense ODN, but not with the sense ODN control, by immunoblot analysis. Additionally, concentrations up to 10 micrograms/ml of either modified antisense or sense ODN did not decrease CFU-GM formation (145 +/- 35%, P = 0.27) in human bone marrow, suggesting that these levels of ODN would have a negligible effect on normal hematopoietic cells. These pilot data suggest that modified antisense ODN directed at the cap region of the c-myc gene could specifically inhibit c-myc expression at a single, lower dose than unmodified ODN and may play a future role in inhibiting the growth of c-myc-dependent malignant cells.

Circulating steel factor (SLF) and G-CSF levels in preterm and term newborn and adult peripheral blood.

PURPOSE: An immaturity in humoral, cellular, and phagocytic immunity predisposes the newborn to overwhelming bacterial infection. The maturation and proliferation of early hemapoietic stem cells give rise to all three of these aspects of immunity. Defects in the regulation of early hematopoiesis may account in part for the immaturity of neonatal host defense. A new hematopoietic growth factor, Steel factor (SLF), has recently been demonstrated to induce the proliferation of early hematopoietic progenitor cells. Our purpose was to study SLF and its effect on hematopoiesis. PATIENTS AND METHODS: We measured circulating serum SLF levels in preterm and term newborns and compared them to adults, matched third trimester pregnant mothers, and to circulating granulocyte colony-stimulating factor (G-CSF) levels. RESULTS: There was no significant difference in SLF levels between preterm and term newborns and adults (3000 +/- 200 vs. 2700 +/- 200 vs. 3100 +/- 300 pg/ml) (Pt vs. T vs. A) (p = NS). Also, there was no significant difference between matched third trimester maternal levels and their matched term newborn (2650 +/- 330 pg/ml vs. 3530 +/- 400 pg/ml) (p = NS). However, G-CSF levels were significantly higher in preterm newborns compared to term newborns and adults (p < or = 0.025). The preterm newborn G-CSF levels, however, were significantly lower compared to positive controls obtained from neutropenic patients post bone marrow transplantation (ANC < or = 200/mm3) (174 +/- 86 vs. 669 +/- 82.3 pg/ml) (p < 0.001). Additionally, there were no significant differences in G-CSF levels between matched third trimester maternal samples and matched term newborns. CONCLUSION: These studies suggest that circulating levels of SLF in the preterm and term newborn are similar to adults and do not account for differences in hematopoiesis.

Increased granulocyte-macrophage colony-stimulating factor mRNA instability in cord versus adult mononuclear cells is translation-dependent and associated with increased levels of A + U-rich element binding factor.

The level of granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNA is fourfold lower in phorbol myristate acetate (PMA) + phytohemagglutinin (PHA)-activated mononuclear cells (MNC) from newborns compared with adults. The GM-CSF transcription rate is similar in umbilical cord and adult MNC, but transcript half-life is threefold lower in cord activated MNC. Interaction of RNA binding proteins, such as the cloned adenosine + uridine-rich element, binding factor, AUF1, with eight AUUUA motifs in the human GM-CSF mRNA 3'-untranslated region (GM-3'-UTR) has been implicated in regulating transcript stability. Translational inhibition by cycloheximide (CHX) significantly increased GM-CSF mRNA accumulation and half-life by three-fold in activated cord MNC, but had a minimal effect in activated adult MNC as compared with PMA + PHA alone. Electrophoretic mobility-shift assays with a 32P-labeled, 305-nucleotide RNA comprising the GM-3'-UTR revealed two RNaseT1-resistant, bound complexes that were almost twice as abundant in cord than in adult MNC extracts. Mobility-shift competition assays and RNaseT1 mapping localized the binding site of both complexes to a 52-nucleotide region containing seven of eight AUUUA motifs. Inclusion of AUF1 antiserum produced a supershifted complex at 35-fold higher levels in cord than in adult MNC extracts. Extracts from the carcinoma cell line 5637, with extended GM-CSF mRNA half-life, also had very low levels of anti-AUF1 supershifted complex. Anti-AUF1 immunoblotting showed significantly higher levels of two AUF1 protein isoforms and lower levels of one in cord than in adult MNC or 5637 extracts. These results suggest that destabilization of GM-CSF mRNA in cord MNC is translation-dependent and that increased levels of specific AUF1 isoforms in cord MNC may target transcripts for increased degradation, which could account in part for dysregulation of neonatal phagocytic immunity.

Tissue uptake of circulating thrombopoietin is increased in immune-mediated compared with irradiated thrombocytopenic mice.

We have previously demonstrated a significant inverse correlation between circulating thrombopoietin (TPO) levels and peripheral platelet (PLT) counts in patients with thrombocytopenia secondary to megakaryocytic hypoplasia but not in patients with immune thrombocytopenic purpura (ITP; Chang et al, Blood 88:3354, 1996). To test the hypothesis that the differences in the circulating TPO levels in these two types of thrombocytopenia are caused by differences in the total capacity of Mpl receptor-mediated TPO clearance, thrombocytopenia was induced in female CD-1 mice either by sublethal irradiation (irradiated) or rabbit antimouse PLT serum (RAMPS) for 1 day (1 d RAMPS) and 5 days (5 d RAMPS). A well-characterized murine model of autoimmune thrombocytopenic purpura, male (NZW x BXSB) F1 mice (W/B F1), was also included in this study. All thrombocytopenic mice and their controls received trace amounts of 125I-recombinant murine TPO (125I-rmTPO) intravenously and were killed 3 hours postinjection. Blood cell-associated radioactivity was significantly decreased in all 4 groups of thrombocytopenic mice. Significantly increased plasma and decreased whole spleen-associated radioactivity was observed in the irradiated group compared with controls (P <.05). While a lesser but still significant increase in plasma and decrease in whole spleen-associated radioactivity was observed in the 1 d RAMPS mice (P <.05), there were no significant differences between the 5 d RAMPS nor the W/B F1 male mice compared with controls, although whole spleen-associated radioactivity was higher in the W/B F1 male. A significant inverse correlation of plasma and whole spleen-associated radioactivity was demonstrated in W/B F1 male mice (r = -.91, n = 6, P <.05). There was also a decrease in bone (femur)/blood-associated radioactivity in the irradiated group compared with controls (P <.05), but a significant increase in 1 d and 5 d RAMPS mice (P <.01). Furthermore, the 125I-rmTPO uptake capacity within the spleen and marrow of immune thrombocytopenic mice appeared to be associated with a higher megakaryocytic mass when tissue samples were examined by light microscopy. Internalization of 125I-rmTPO by megakaryocytes and PLTs in the spleens and marrows of ITP mice was also demonstrated directly using electron microscopic autoradiography. Labeled PLTs were also found within splenic macrophages. Additionally, the mean PLT volumes of RAMPS mice were significantly higher than those of the control and irradiated mice (P <.05), as was the bound 125I-rmTPO (cpm) per million PLT (P <.05). Finally, significantly decreased 125I-rmTPO degradation products were only found in the plasma of the irradiated mice compared with control animals (P <.05). These data suggest that the lack of Mpl+ cells in the mice with thrombocytopenia secondary to megakaryocytic hypoplasia (irradiated) results in decreased uptake and degradation of TPO and higher circulating TPO levels. Furthermore, these data also suggest that, after a brief TPO surge in response to immune thrombocytopenia (1 d RAMPS), the lack of an inverse correlation of circulating TPO with PLT counts during steady-state immune thrombocytopenic mice (5 d RAMPS + W/B F1 male) is due, at least in part, to its uptake and degradation by the high PLT turnover and increased mass of megakaryocytes.

Transforming growth factor-beta 1, macrophage inflammatory protein-1 alpha, and interleukin-8 gene expression is lower in stimulated human neonatal compared with adult mononuclear cells.

Hematopoiesis is developmentally immature in the newborn compared with the adult. Diminished gene expression of several positive hematopoietic regulators has been observed in activated cord compared with adult peripheral blood mononuclear cells (MNC; Cairo et al. Pediatr Res, 30:362, 1991 and Cairo et al, Pediatr Res, 31:574, 1992). However, altered expression of negative hematopoietic regulators during states of increased demand may also contribute to the pathogenesis of newborn dyshematopoiesis. To test this hypothesis, we measured protein levels of transforming growth factor-beta 1 (TGF-beta 1) and macrophage inflammatory protein-1 alpha (MIP-1 alpha) in the conditioned media of human umbilical cord and adult MNC using specific enzyme-linked immunosorbent assays. There was significantly less TGF-beta 1 in culture supernatants of cord versus adult MNC after 24, 72, and 120 hours of stimulation (P < .05), and significantly less MIP-1 alpha in cord versus adult supernatants after 72 hours and 120 hours of stimulation (P < .01). We then examined the mRNA expression of the negative regulators TGF-beta 1, MIP-1 alpha, and interleukin-8 (IL-8) in cord and adult MNC using Northern blot hybridization followed by quantitative densitometry. Cord MNC expressed significantly less TGF-beta 1 mRNA than adult MNC 6 hours and 72 hours after stimulation (P < .001). Cord MNC expressed significantly less MIP-1 alpha mRNA than adult MNC 6 hours (P < .01), 24 hours (P < .001), and 72 hours after stimulation (P < .001). Cord MNC also expressed significantly less IL-8 mRNA than adult MNC 6 hours after stimulation (P < .001). Therefore, decreased mRNA accumulation appears to coincide with reduced cytokine expression in the activated cord MNC. There were no significant differences in the transcription rates determined by nuclear run-on assay of either the TGF-beta 1 or MIP-1 alpha gene in cord versus adult MNC after 6 hours of stimulation, suggesting that the reduced TGF-beta 1 and MIP-1 alpha mRNA in activated cord MNC may be secondary to alteration in posttranscriptional regulation. The present results, together with those of our previous studies, suggest that the altered expression of both positive and negative hematopoietic regulators may be involved in the immaturity of host defense in human neonates.

Differential mechanisms in the regulation of endogenous levels of thrombopoietin and interleukin-11 during thrombocytopenia: insight into the regulation of platelet production.

The regulation of megakaryocytopoiesis and thrombopoiesis appears to be under the control of an array of hematopoietic growth factors. To determine the relationship of endogenous thrombopoietic cytokine levels and circulating platelet (PLT) counts, we measured the levels of thrombo-poietin (TPO), interleukin-11 (IL-11), and interleukin-6 (IL-6) in patients with significant thrombocytopenia secondary to both marrow hypoplasia and increased PLT destruction. Increased endogenous levels of TPO and IL-11, but not IL-6, were detected in bone marrow transplant patients with thrombocytopenia following myeloablative therapy (BMT/MAT) (TPO: 1,455.5 +/- 87.3 pg/mL, [PLT 39,600 +/- 7,800/microL], P < .001, n = 12; IL-11: 227.9 +/- 35 pg/mL, [PLT 32,900 +/- 57,000/microL], P < .05, n = 19; IL-6: 25.8 +/- 8.4 pg/mL, [PLT 32,800 +/- 5,057/microL], P > .05, n = 4] v normal donors [TPO < 150 pg/mL, n = 8; IL-11 < 50 pg/mL, n = 9; IL-6 < 10 pg/mL, n = 5 [PLT 203,000 +/- 7,500/microL]. There was a significant inverse correlation between endogenous levels of TPO and IL-11, but not IL-6, and PLT counts in the MAT/BMT patients (TPO: r = -0.57, P < .0001, n = 188; IL-11: r = -0.329, P < .0001, n = 249; IL-6: r = -0.1147, P > .05, n = 62). In patients with immune thrombocytopenia purpura (ITP), with decreased PLT survival, but intact bone marrow megakaryocytopoiesis, endogenous IL-11 levels were significantly increased (328.0 +/- 92.6 pg/mL, [PLT: 20,900 +/- 3,000/microL], P < .05, n = 25). However, endogenous TPO levels remained undetectable (< 150 pg/mL, [PLT 30,500 +/- 5,500/microL], n = 15). These results suggest that there may be differential mechanisms regulating endogenous TPO, IL-11, and IL-6 levels during acute thrombocytopenia and suggest that the absolute number of circulating PLTs may not always be the sole regulator of endogenous TPO levels. Other mpl-expressing cells of the megakaryocyte lineage may contribute to the regulation of circulating TPO levels as well. Our results also suggest IL-11 levels may in part, be regulated by a negative feedback loop based on circulating PLT counts, but also may, in part, be regulated by a variety of inflammatory agonists. Both TPO and IL-11, therefore, appear to be active thrombopoietic cytokines regulating, in part, megakaryocytopoiesis during states of acute thrombocytopenia.