A novel gene coding for the pre-B-cell colony-enhancing factor (PBEF)

No abstract

Pre-B-cell colony enhancing factor (PBEF) was first isolated from an activated peripheral blood lymphocyte cDNA library and was found to be involved in the maturation of B-cell precursors. It was subsequently identified as one of the genes upregulated by distending the human fetal membranes in vitro. Here we report on the genomic organization of this gene, which is composed of 11 exons and 10 introns, spanning 34·7 kb of genomic DNA. Neither the gene nor the protein has any homology with other cytokines in any currently available database. The use of two promoters (proximal and distal) may result in differential, tissue specific expression of the PBEF transcripts. The 5 -flanking region lacks the classical sequence motif that would place it with the hematopoietic cytokines; however, it has several putative regulatory elements, suggesting that this gene may be chemically and mechanically responsive to inducers of transcription.The three PBEF mRNA transcripts were observed in both normal and infected human fetal membranes but were significantly upregulated (P<0·05) in severe infection. The PBEF protein was immunolocalized, in both normal and infected tissues, to both the normal fetal cells of the amnion and chorion and the maternal decidua of the membranes, and to the invading neutrophils. These stained strongly and were likely to contribute to the increased expression in infection. The amniotic epithelial cell line (WISH cells) has been used as a model to study PBEF gene modulation. Lipopolysaccharide, interleukin (IL)-1 , tumour necrosis factor (TNF) and IL-6 all significantly increased the expression of PBEF in 4 h of treatment. The addition of dexamethasone to IL-1 and TNF significantly reduced the response of PBEF to these cytokines. IL-8 treatment failed to alter PBEF gene expression. Thus PBEF is a cytokine expressed in the normal fetal membranes and upregulated when they are infected. It is likely to have a central role in the mechanism of infection-induced preterm birth.

We investigated the effects of PACAP treatment, and endogenous PACAP deficiency, on infarct volume, neurological function, and the cerebrocortical transcriptional response in a mouse model of stroke, middle cerebral artery occlusion (MCAO). PACAP-38 administered i.v. or i.c. v. 1 h after MCAO significantly reduced infarct volume, and ameliorated functional motor deficits measured 24 h later in wild-type mice. Infarct volumes and neurological deficits (walking faults) were both greater in PACAP-deficient than in wild-type mice, but treatment with PACAP reduced lesion volume and neurological deficits in PACAP-deficient mice to the same level of improvement as in wild-type mice.A 35,546-clone mouse cDNA microarray was used to investigate cortical transcriptional changes associated with cerebral ischemia in wild-type and PACAP-deficient mice, and with PACAP treatment after MCAO in wild-type mice. 229 known (named) transcripts were increased (228) or decreased (1) in abundance at least 50% following cerebral ischemia in wild-type mice. 49 transcripts were significantly up-regulated only at 1 h post-MCAO (acute response transcripts), 142 were up-regulated only at 24 h post-MCAO (delayed response transcripts) and 37 transcripts were up-regulated at both times (sustained response transcripts). More than half of these are transcripts not previously reported to be altered in ischemia.A larger percentage of genes up-regulated at 24 hr than at 1 hr required endogenous PACAP, suggesting a more prominent role for PACAP in later response to injury than in the initial response. This is consistent with a neuroprotective role for PACAP in late response to injury, i.e., even when administered 1 hr or more after MCAO. Putative injury effector transcripts regulated by PACAP include β-actin, midline 2, and metallothionein 1. Potential neuroprotective transcripts include several demonstrated to be PACAP-regulated in other contexts. Prominent among these ☆ Data deposition: The raw data from the expression profiling experiments reported in this paper have been deposited in the Gene Expression Omnibus database (accession no. GSE5902). The GSE5902 will be hyperlinked when it is released. were transcripts encoding the PACAP-regulated gene Ier3, and the neuropeptides enkephalin, substance P (tachykinin 1), and neurotensin.

Lipopolysaccharide (LPS) injected into the trachea of rats was found to induce the secretion of leukemia inhibitory factor (LIF) into bronchoalveolar lavage (BAL) fluid with a maximum expression of LIF after 2-12 h. The acute pulmonary neutrophilic inflammation caused by the intratracheal injection of bacterial endotoxin (LPS) could be inhibited by the intratracheal coinjection of recombinant LIF. Compared with intratracheal injection of LPS alone, intratracheal coinjection of LIF and LPS decreases the number of BAL neutrophils obtained 6 h later by approximately 50% (P < 0.0001). LIF decreased the amount of the proinflammatory cytokine tumor necrosis factor (TNF), but not the amount of the anti-inflammatory cytokine interleukin (IL)-6, in the BAL fluid of LPS-injected rats. Similarly, intravenous LIF was found to decrease TNF expression, but increase IL-6 expression, in the serum of rats receiving intravenous LPS. Intravenous LIF, even in the absence of LPS, was found to cause IL-6 expression. In conclusion, intratracheal LPS initiates the secretion of endogenous LIF into the alveolar space where LIF may contribute to the downregulation of LPS-initiated acute neutrophilic inflammation by downregulating expression of TNF. LIF may down-regulate LPS-initiated TNF expression at least in part indirectly by upregulating expression of IL-6, a cytokine known to downregulate LPS-initiated TNF expression.

No abstract

A novel gene coding for the pre-B-cell colony-enhancing factor (PBEF) has been isolated from a human peripheral blood lymphocyte cDNA library. The expression of this gene is induced by pokeweed mitogen and superinduced by cycloheximide. It is also induced in the T-lymphoblastoid cell line HUT 78 after phorbol ester (phorbol myristate acetate) treatment. The predominant mRNA for PBEF is approximately 2.4 kb long and codes for a 52-kDa secreted protein. The 3' untranslated region of the mRNA has multiple TATT motifs, usually found in cytokine and oncogene messages. The PBEF gene is mainly transcribed in human bone marrow, liver tissue, and muscle. We have expressed PBEF in COS 7 and PA317 cells and have tested the biological activities of the conditioned medium as well as the antibody-purified protein in different in vitro assays. PBEF itself had no activity but synergized the pre-B-cell colony formation activity of stem cell factor and interleukin 7. In the presence of PBEF, the number of pre-B-cell colonies was increased by at least 70% above the amount stimulated by stem cell factor plus interleukin 7. No effect of PBEF was found with cells of myeloid or erythroid lineages. These data define PBEF as a novel cytokine which acts on early B-lineage precursor cells.

Key words: gene therapy * anemia * renal failure * muscle cell transplantation * nude mouse Anemia is an invariable consequence of end-stage renal failure (ESRF) and recombinant erythropoietin has dramatically improved the quality of life of patients with ESRF.As an alternative approach, we developed a myoblast gene transfer system for the systemic delivery of human erythropoietin (EPO). We recently reported that transplantation of 4 x 107 cells of a C2 myoblast cell clone that stably secretes high level of functional human EPO, increased hematocrit from 44.6±3.0 to 71.2±7.9(%) in 2 wk, and the increase was sustained for at least 12 wk in nude mice. A renal failure model was created by a two-step nephrectomy in nude mice, and myoblasts were transplanted 3 wk after the second nephrectomy, when mean blood urea nitrogen level had increased from 26.3±6.1 to 85.4±24.0 (mg/ dl) and the hematocrit had decreased from 45.2±2.7 to 33.9±3.7(%). After transplantation, the hematocrit markedly increased to 68.6±4.2(%) 2 wk, and to 68.5±4.0(%) 7 wk after the transplantation. Serum human EPO concentration determined by ELISA indicated a persistent steady EPO production from the transplanted muscle cells 8 wk after the transplantation. The fate of transplanted myoblasts in uremic mice was monitored by transplanting the EPOsecreting clone which had also been transduced with BAG retrovirus bearing the fi-galactosidase gene. 8 wk later, Xgal positive myofibers were detected in the entire transplanted area. The results demonstrate that myoblasts can be transplanted in uremic mice, and that myoblast gene transfer can achieve sufficient and sustained delivery of functionally active EPO to correct anemia associated with