Genetic analysis of the human tumor necrosis factor alpha/cachectin promoter region in a macrophage cell line.

The 615-bp 5' flanking region of the human TNF-alpha/cachectin gene was isolated and ligated to the luciferase reporter gene. In addition, a series of truncated promoter constructs was generated by exonuclease III digestion. The promoter activity of these constructs was studied in a transient transfection system using the TNF-alpha-producing U937 cell line. Full-length and truncated TNF promoter constructions extending from -615 to -95 bp relative to the transcription start site (TSS) could be induced by phorbol esters. A construct truncated to within 36 bp of the TSS (and within 11 bp of the TATAA box) was inactive. Therefore, the phorbol ester responsive is localized in the TNF/cachectin promoter to a relatively short region proximal to the TATAA box.

Regulation of human eosinophil viability, density, and function by granulocyte/macrophage colony-stimulating factor in the presence of 3T3 fibroblasts.

Normodense human peripheral blood eosinophils were isolated under sterile conditions from the 22/23 and 23/24% interfaces and the cell pellet of metrizamide gradients. After culture for 7 d in RPMI media in the presence of 50 pM biosynthetic (recombinant) human granulocyte/macrophage colony-stimulating factor (rH GM-CSF), 43 +/- 7% (mean +/- SEM, n = 8) of the cells were viable; in the absence of rH GM-CSF, no eosinophils survived. The rH GM-CSF-mediated viability was concentration dependent; increased survival began at a concentration of 1 pM, a 50% maximal response was attained at approximately 3 pM, and a maximal effect was reached at concentrations of greater than or equal to 10 pM rH GM-CSF. In the presence of rH GM-CSF and mouse 3T3 fibroblasts, 67 +/- 6% (mean +/- SEM, n = 8) of the eosinophils survived for 7 d. In a comparative analysis, there was no difference in eosinophil viability after 7 and 14 d (n = 3) in the presence of 50 pM GM-CSF and fibroblasts. Culture with fibroblasts alone did not support eosinophil survival. The addition of fibroblast-conditioned media to rH GM-CSF did not further improve eosinophil viability, indicating a primary role for GM-CSF in supporting these eosinophil cell suspensions ex vivo and a supplementary role for 3T3 fibroblasts. Eosinophils cultured for 7 d localized on density gradient sedimentation at the medium/18, 18/20, and 20/21 interfaces of metrizamide gradients, indicating a change to the hypodense phenotype from their original normodense condition. In addition, the cultured eosinophils generated approximately 2.5-fold more LTC4 than freshly isolated cells when stimulated with the calcium ionophore A23187 and manifested sevenfold greater antibody-dependent killing of S. mansoni larvae than the freshly isolated, normodense cells from the same donor. Thus we demonstrate the rH GM-CSF dependent conversion in vitro of normodense human eosinophils to hypodense cells possessing the augmented biochemical and biological properties characteristic of the hypodense eosinophils associated with a variety of hypereosinophilic syndromes. In addition, these studies provide a culture model of at least 14 d suitable for the further characterization of hypodense eosinophils.

Scrubs contamination, domestic laundry effect and workwear habits of clinical staff at a referral hospital.

OBJECTIVE: To determine contamination rates of scrub suits worn by veterinary surgeons and nurses following a single shift. MATERIALS AND METHODS: Cross-sectional preliminary study at a UK small animal referral centre. Sterilised scrub suits were distributed to veterinary surgeons (n = 9) and nurses (n = 9) at the beginning of their clinical shift and worn for at least 8 hours. They were then analysed for bacterial contamination before and after home laundry at 30°C. A questionnaire was distributed to hospital clinical staff regarding workwear habits. RESULTS: Median bacterial counts were 47 (interquartile range: 14 to 162) and 7 (interquartile range: 0 to 27) colony forming units per cm2 before and after laundering scrub suits. Bacteria identified included Staphylococcus sp., Enterococcus sp., Escherichia coli , Bacillus sp., Pseudomonas aeruginosa , Micrococcus sp., ß-haemolytic Streptococci and a Group G Streptococcus. From 101 staff surveyed, 64.0% reported wearing fresh, clean scrub tops and 58.4% fresh, clean trousers each day, while 64.4% left the workplace wearing the same clothing in which they undertook clinical work. CLINICAL SIGNIFICANCE: Workwear contamination risks spread of pathogens into the community and personnel compliance with workplace guidelines warrants further attention. Home laundry at 30°C significantly decreases, but does not eliminate, the bacterial burden after a single shift.

A new determinant of glucocorticoid sensitivity in lymphoid cell lines.

The SAK cell line, derived from a spontaneous thymic lymphoma in an AKR mouse, is resistant to lysis by glucocorticoids in spite of the presence of functional glucocorticoid receptor. Receptor function was determined by hormone binding analyses, as well as characterization of hormonal effects on cell growth and on the accumulation of murine leukemia virus and metallothionein mRNAs. SAK cells were fused with a receptor-defective (and therefore resistant) variant of a well-characterized murine thymoma line, W7. The resulting hybrids are glucocorticoid sensitive, demonstrating complementation of the receptor defect in W7 cells by the functional glucocorticoid receptor of SAK. This fusion shows that SAK cells are resistant to the hormone due to the absence of another function designated "I" for lysis. SAK cells were also fused with glucocorticoid-sensitive W7 cells (containing wild-type receptor), generating glucocorticoid-sensitive hybrids, which demonstrate that the dexamethasone-resistant phenotype of the SAK cells is recessive. Resistant derivatives of this hybrid were found which still contain the full amount of receptor. Chromosome analysis revealed that, on the average, the resistant derivatives had lost two chromosomes, suggesting segregation of chromosomes carrying genetic material necessary for the "lysis" function. The drug 5-azacytidine (a known inhibitor of DNA methylation) has been shown to cause heritable changes in gene expression. Treatment of SAK cells with 5-azacytidine generated glucocorticoid-sensitive clones at high frequency, suggesting that the gene(s) involved in the "lysis" function are intact and have been inactivated through a process such as differentiation.

Cytokines alter production of HIV-1 from primary mononuclear phagocytes.

Some strains of human immunodeficiency virus type 1 (HIV-1) can infect primary monocytes and monocyte-derived macrophages in vitro. In this report, the effect of cytokines on the production of one of these strains that shows a tropism for mononuclear phagocytes, designated HIV-1JR-FL, was studied. Primary peripheral blood mononuclear phagocytes infected with HIV-1JR-FL were treated with the hematopoietic factors: granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), macrophage colony-stimulating factor (M-CSF), and gamma-interferon (gamma-IFN). The M-CSF, GM-CSF, IL-3, and gamma-IFN were able to alter HIV-1 production under different conditions.

The human gene encoding GM-CSF is at 5q21-q32, the chromosome region deleted in the 5q- anomaly.

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) is a 22,000-dalton glycoprotein that stimulates the growth of myeloid progenitor cells and acts directly on mature neutrophils. A full-length complementary DNA clone encoding human GM-CSF was used as a probe to screen a human genomic library and isolate the gene encoding human GM-CSF. The human GM-CSF gene is approximately 2.5 kilobase pairs in length with at least three intervening sequences. The GM-CSF gene was localized by somatic cell hybrid analysis and in situ hybridization to human chromosome region 5q21-5q32, which is involved in interstitial deletions in the 5q- syndrome and acute myelogenous leukemia. An established, human promyelocytic leukemia cell line, HL60, contains a rearranged, partially deleted GM-CSF allele and a candidate 5q- marker chromosome, indicating that the truncated GM-CSF allele may reside at the rejoining point for the interstitial deletion on the HL60 marker chromosome.

Evidence for the involvement of GM-CSF and FMS in the deletion (5q) in myeloid disorders.

By in situ chromosomal hybridization, the GM-CSF and FMS genes were localized to human chromosome 5 at bands q23 to q31, and at band 5q33, respectively. These genes encode proteins involved in the regulation of hematopoiesis, and are located within a chromosome region frequently deleted in patients with neoplastic myeloid disorders. Both genes were deleted in the 5q-chromosome from bone marrow cells of two patients with refractory anemia and a del(5)(q15q33.3). The GM-CSF gene alone was deleted in a third patient with acute nonlymphocytic leukemia (ANLL) who has a smaller deletion, del(5)(q22q33.1). Leukemia cells from a fourth patient who has ANLL and does not have a del(5q), but who has a rearranged chromosome 5 that is missing bands q31.3 to q33.1 [ins(21;5)(q22;q31.3q33.1)] were used to sublocalize these genes; both genes were present on the rearranged chromosome 5. Thus, the deletion of one or both of these genes may be important in the pathogenesis of myelodysplastic syndromes or of ANLL.

Studies of the putative transforming protein of the type I human T-cell leukemia virus.

The putative transforming protein of the type I human T-cell leukemia virus (HTLV-1) is a 40-kilodalton protein encoded by the X region and is termed p40XI. On the basis of both subcellular fractionation techniques and immunocytochemical analysis, it is now shown that p40XI is a nuclear protein with a relatively short half-life (120 minutes). It is synthesized de novo in considerable quantities in a human T-cell line infected with and transformed by the virus in vitro, and it is not packaged in detectable amounts in the extracellular virus.

Purified human granulocyte-macrophage colony-stimulating factor: direct action on neutrophils.

Neutrophil migration inhibition factor from T lymphocytes (NIF-T) is a lymphokine that acts to localize granulocytes. Medium conditioned by the Mo human T-lymphoblast cell line was used to purify NIF-T, a glycoprotein with a molecular weight of 22,000. The NIF-T was found to potently stimulate the growth of granulocyte and macrophage colonies from human bone marrow and colony formation by the KG-1 myeloid leukemia cell line. Thus a human lymphokine (NIF-T) that modulates the activities of mature neutrophilic granulocytes is also a colony-stimulating factor acting on precursors to induce growth and differentiation of new effector cells.

Impact of Calman system and recent reforms on surgical training in gynaecology.

Specialist training in the UK has been affected by changes in recent years aimed at a reduction in junior doctors' working hours to comply with employment regulations and the introduction of structured training with specified duration. The Calman reforms implemented in 1996 introduced a focussed system with defined competencies and a shorter training period. The previous system was based on experience gained in an apprentice-type setting with no defined duration of training. The European Working Time Directive (EWTD) regulates the number of working hours for junior doctors and aims for a 48-h working week by 2009. In the surgical disciplines a reduction in working hours and shorter duration of training could adversely affect the acquisition of operative skills. The concern among trainees and their trainers was that surgical exposure has been reduced and therefore trainees have limited surgical experience by the time they complete training. We conducted this study in a teaching district hospital to determine the effect of recent changes on gynaecological surgical training. We found that there was a 27% reduction in surgical activity between 1995 and 2005 from 3,789 to 2,781, whereas the number of trainees had increased by 67% from 6 to 10. The proportion of operative procedures performed by trainees decreased from 55% (2,078/3,789) in 1995 to 34% (951/2,781) in 2005 (p < 0.001). The average number of procedures performed by each trainee in 2005 was 95 compared with 346 in 1995, a 73% reduction (p < 0.001). Innovative approaches to surgical training in gynaecology are required to produce a competent surgeon in a shorter time, or the risk of future consultants having limited surgical experience will increase.

Antibiotic prescribing practice and adherence to guidelines in primary care in the Cape Town Metro District, South Africa.

BACKGROUND: Knowledge of antibiotic prescribing practice in primary care in South Africa is limited. As 80% of human antibiotic use is in primary care, this knowledge is important in view of the global problem of antibiotic resistance. OBJECTIVES: To assess antibiotic prescribing in primary care facilities in the Cape Town Metro District and compare it with current national guidelines, and to assess the reasons why prescriptions were not adherent to guidelines. METHODS: A retrospective medical record review was performed in April/May 2016. Records of all patients seen over 2 days in each of eight representative primary care facilities in the Cape Town Metro District were reviewed. The treatment of any patient who raised a new complaint on either of those days was recorded. Prophylactic antibiotic courses, tuberculosis treatment and patients with a non-infection diagnosis were excluded. Treatment was compared with the Standard Treatment Guidelines and Essential Medicines List for South Africa, Primary Healthcare Level, 2014 edition. RESULTS: Of 654 records included, 68.7% indicated that an antibiotic had been prescribed. Overall guideline adherence was 45.1%. Adherence differed significantly between facilities and according to the physiological system being treated, whether the prescription was for an adult or paediatric patient, and the antibiotic prescribed. Healthcare professional type and patient gender had no significant effect on adherence. The main reasons for non-adherence were an undocumented diagnosis (30.5%), antibiotic not required (21.6%), incorrect dose (12.9%), incorrect drug (11.5%), and incorrect duration of therapy (9.5%). CONCLUSIONS: This study demonstrates poor adherence to guidelines. Irrational use of antibiotics is associated with increased antibiotic resistance. There is an urgent need to improve antibiotic prescribing practice in primary care in the Cape Town Metro District.

Granulocyte-macrophage colony-stimulating factor. Sensitive and receptor-mediated regulation by 1,25-dihydroxyvitamin D3 in normal human peripheral blood lymphocytes.

We show that 1,25-dihydroxyvitamin D3 (1,25[OH]2D3), the most hormonally active metabolite of vitamin D3, modulates sensitively and specifically both the protein and messenger RNA accumulation of the multilineage growth factor granulocyte-macrophage colony-stimulating factor (GM-CSF). The regulation of GM-CSF expression is seen in both normal human mitogen-activated T lymphocytes and T lymphocytes from a line (S-LB1) transformed with human T cell lymphotropic virus 1 (HTLV-1). In contrast, cells from a HTLV-1 transformed T lymphocyte line (Ab-VDR) established from a patient with vitamin D-resistant rickets type II with undetectable 1,25(OH)2D3 cellular receptors are resistant to the action of 1,25(OH)2D3. Inhibition of GM-CSF expression by 1,25(OH)2D3 can occur independently of interleukin 2 regulation and is probably mediated through cellular 1,25(OH)2D3 receptors. We conclude that 1,25(OH)2D3 may be important in the physiology of hematopoiesis.

Transcriptional and posttranscriptional modulation of myeloid colony-stimulating factor expression by tumor necrosis factor and other agents.

Granulocyte (G) and granulocyte-macrophage (GM) colony-stimulating factors (CSF) are necessary for proliferation and differentiation of myeloid hematopoietic cells. Fibroblasts stimulated by tumor necrosis factor alpha (TNF alpha) and several other agents are a rich source of these CSF. The GM-CSF synthesized by these cells had the same molecular weight and glycosylation pattern as that produced by activated T lymphocytes, as shown by [35S]methionine labeling studies. Northern (RNA) blot analysis showed that the fibroblasts had trace levels of G- and GM-CSF mRNA. Both G- and GM-CSF mRNA concentrations coordinately increased after exposure of the cells to TNF alpha (greater than or equal to 5 ng/ml), 12-O-tetradecanoylphorbol 13-acetate (TPA) (greater than or equal to 5 x 10(-10) M), or cycloheximide (20 micrograms/ml). Both TNF alpha and TPA increased levels of G- and GM-CSF mRNA in the absence of new protein synthesis. Transcriptional run-on studies demonstrated that fibroblasts constitutively transcribed GM-CSF, and transcription was enhanced 3.0-fold by TNF alpha and 2.5-fold by TPA and was unchanged by cycloheximide. The stability of G- and GM-CSF transcripts was determined after exposure of the cells to actinomycin D; the half-lives of G- and GM-CSF mRNA in unstimulated cells were less than 0.25 h and were increased 2- to 16-fold in cells cultured with TNF, TPA, or cycloheximide. In summary, both transcriptional and posttranscriptional signals acted coordinately to modulate the levels of G- and GM-CSF mRNAs in fibroblasts.

The repeated sequence CATT(A/T) is required for granulocyte-macrophage colony-stimulating factor promoter activity.

The hematopoietic growth factor GM-CSF (granulocyte-macrophage colony-stimulating factor) is expressed by activated but not resting T lymphocytes. Previously, we localized GM-CSF-inducible promoter activity to a 90-bp region of GM-CSF 5'-flanking sequences extending from bp -53 to +37. To more precisely identify the GM-CSF DNA sequences required for inducible promoter activity in T lymphocytes, we have performed mutagenesis within a region of GM-CSF 5'-flanking sequences (bp -57 to -24) that contains the repeated sequence CATT(A/T). Mutations that do not alter the repeated CATT(A/T) sequence do not eliminate inducible promoter activity, whereas mutation or deletion of either of the CATT(A/T) repeats eliminates all inducible promoter activity in T-cell lines and in primary human T lymphocytes. Thus, both copies of the direct repeat CATT(A/T) are required for mitogen-inducible expression of GM-CSF in T cells.

Characterization of the human granulocyte-macrophage colony-stimulating factor promoter region by genetic analysis: correlation with DNase I footprinting.

T-cell activation induces expression of the hematopoietic growth factor granulocyte-macrophage colony-stimulating factor (GM-CSF). To define the molecular events involved in the induction of GM-CSF gene expression more clearly, we prepared and analyzed deletion mutants of GM-CSF promoter recombinant constructs. The results localized inducible expression to a 90-base-pair region (-53 to +37) which is active in uninfected and human T-cell leukemia virus-infected T-cell lines but not in resting or mitogen-stimulated B cells. DNase I footprinting experiments revealed protection of sequences contained within this region, including a repeated nucleotide sequence, CATT(A/T), which could serve as a core recognition sequence for a cellular transcription factor. Upstream of these GM-CSF promoter sequences is a 15-base-pair region (-193 to -179) which has negative regulatory activity in human T-cell leukemia virus-infected T cells. These studies revealed a complex pattern of regulation of GM-CSF expression in T cells; positive and negative regulatory sequences may play critical roles in controlling the expression of this potent granulopoietin in the bone marrow microenvironment and in localized inflammatory responses.

Granulocyte-macrophage colony-stimulating factor and interleukin-3 signaling pathways converge on the CREB-binding site in the human egr-1 promoter.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates myeloid progenitor cell proliferation and enhances the function of terminally differentiated effector cells. Interleukin-3 (IL-3) stimulation results in the proliferation and maturation of early bone marrow progenitor cells. These activities are mediated by non-tyrosine kinase-containing receptors which consist of ligand-specific alpha subunits that complex with a common beta subunit required for signal transduction. Both GM-CSF and IL-3 rapidly and transiently induce expression of early growth response gene 1 (egr-1) in the human factor-dependent cell line TF-1. To define the mechanism of early response gene induction by GM-CSF and IL-3, growth factor- and serum-starved TF-1 cells transfected with recombinant constructs containing sequences of the human egr-1 promoter were stimulated with GM-CSF or IL-3. A 116-nucleotide (nt) region of the egr-1 promoter which contains sequences inducible by GM-CSF and IL-3 was defined. DNase I footprint analysis identified a 20-nt region, including nt -57 to -76, which contains a potential cyclic AMP (cAMP) response element (CRE). Electrophoretic mobility shift assays performed with CREB antibody confirmed the presence of CREB in the DNA-binding complex. Mutational analysis of the cytokine-responsive region of the egr-1 promoter revealed that both the cAMP response and serum response elements are required for induction by GM-CSF and IL-3. Nuclear extracts from GM-CSF- or IL-3-stimulated but not unstimulated TF-1 cells contain factors which specifically bind to the Egr-1-binding site in the nt -600 to -480 region of the promoter. Electrophoretic mobility shift assays were performed with antibodies against the Egr-1 protein to demonstrate the presence of the protein product in the shifted complex. Our studies suggest that the Egr-1 protein may further stimulate transcription of the egr-1 gene in response to GM-CSF as a secondary event.

Characterization of a cell-type-restricted negative regulatory activity of the human granulocyte-macrophage colony-stimulating factor gene.

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates the proliferation and maturation of normal myeloid progenitor cells and can also stimulate the growth of acute myelogenous leukemia (AML) blasts. GM-CSF is not normally produced by resting cells but is expressed by a variety of activated cells including T lymphocytes, macrophages, and certain cytokine-stimulated fibroblasts and endothelial cells. Production of GM-CSF by cultured AML cells has been demonstrated, and GM-CSF expression by normal myeloid progenitors has been postulated to play a role in myelopoiesis. We have investigated the regulation of expression of GM-CSF in AML cell lines, and our results demonstrate the presence of a strong constitutive promoter element contained within 53 bp upstream of the cap site. We have also identified a negative regulatory element located immediately upstream of the positive regulatory element (within 69 bp of the cap site) that is active in AML cell lines but not T cells or K562 CML cells. Competition transfection and mobility shift studies demonstrate that this activity correlates with binding of a 45-kDa protein.

Granulocyte-macrophage colony-stimulating factor and tetradecanoyl phorbol acetate induce a distinct, restricted subset of primary-response TIS genes in both proliferating and terminally differentiated myeloid cells.

Induction of early-response genes (tetradecanoyl phorbol acetate [TPA]-induced sequences, or TIS genes; R.W. Lim, B.C. Varnum, and H.R. Herschman, Oncogene 1:263-270, 1987) by granulocyte-macrophage colony-stimulating factor (GM-CSF) and TPA was examined both in a factor-dependent murine cell line, 32D clone 3, and in mature human neutrophils. When GM-CSF-deprived 32D clone 3 cells were exposed to GM-CSF or to TPA, four TIS mRNAs (TIS7, TIS8, TIS10, and TIS11) were rapidly and transiently induced. However, neither GM-CSF nor TPA could induce accumulation of TIS1 mRNA in 32D clone 3 cells, even under superinducing conditions. Both GM-CSF and TPA also elicited rapid, transient expression of TIS8 and TIS11 mRNA in postmitotic human neutrophils. However, neither agent could induce accumulation of TIS1 mRNA in human neutrophils. TIS1 is a member of the nuclear receptor supergene family that codes for ligand-dependent transcription factors. Cell-type restriction of inducible transcription factors may contribute to developmental specification.

Induction of glucocorticoid-resistant variants in a murine thymoma line by antitumor drugs.

Several antitumor drugs are shown to be mutagenic in murine thymoma lines: mitomycin C, bleomycin, streptonigrin, Colcemid, and BD40, an analog of ellipticine. Using conditions yielding 3 to 40% cell survival, all five drugs tested increase the frequency of glucocorticoid-resistant variants. Mitomycin C is as efficient as the classical alkylating agents N-methyl-N'-nitro-N-nitrosoguanidine and ethyl methanesulfonate. The other drugs, previously untested for mutagenic activity on mammalian cells, are weak mutagens yielding variants at frequencies 1 to 2 orders of magnitude lower than the alkylating agents. All 152 variants obtained result from defects in the glucocorticoid receptor. Variants induced by mitomycin C, streptonigrin, Colcemid, and BD40 have very reduced receptor activity, as measured by dexamethasone binding. In contrast, bleomycin or the combination of mitomycin C and dexamethasone induce a majority of variants having dexamethasone-binding activity comparable to the parental line. However, assays of nuclear transfer capacity and genetic complementation show that these receptors are nonfunctional and may result from point mutations in the gene encoding the glucocorticoid receptor. This study suggests that, in combination therapies, antitumor drugs might induce glucocorticoid-resistant lymphoid cell variants that could be selected by the hormone.