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.

Allele-specific expression patterns of interleukin-2 and Pax-5 revealed by a sensitive single-cell RT-PCR analysis.

Autosomal genes that are subject to random allelic inactivation (RAI), like imprinted genes [1] and genes subject to X-inactivation [2], require mechanisms that dictate the differential transcriptional regulation of two sequence-identical alleles. RAI genes include olfactory receptor genes [3], and the various genes encoding antigen receptors on lymphocytes (immunoglobulin genes, T cell receptor genes and NK receptor genes [4] [5] [6] [7]). These observations raise the possibility that other genes might be similarly regulated. Moreover, an interesting possibility is that certain genes might be monoallelically expressed in some cells and biallelically expressed in others. Recently, reports of monoallelic expression of interleukin-2 (IL-2) [8] [9] and IL-4 [10] [11] have raised the possibility that the cytokine gene family may be subject to monoallelic expression. Another report suggests that the gene encoding the transcription factor Pax-5, which is involved in B-cell (and cerebellar) development [12] [13], is also subject to monoallelic expression [14]. Using a novel single-cell reverse transcription-polymerase chain reaction (RT-PCR) approach, we have analyzed the IL-2 and Pax-5 genes in mice. We found that IL-2 is monoallelically transcribed in some T cells and biallelically transcribed in others, raising interesting questions regarding cytokine gene regulation. Additionally, our analyses suggest that Pax-5 is consistently biallelically transcribed. Thus, the IL-2 gene and other cytokine genes may be regulated in a stochastic manner that results in 0, 1 or 2 alleles of a given cytokine gene expressed in each T cell. This type of regulation could account for the wide variety of different combinations of cytokine genes expressed in individual T cells and therefore plays a role in the generation of T cells with a range of different effector functions.

CCAAT enhancer-binding protein (C/EBP) and AML1 (CBF alpha2) synergistically activate the macrophage colony-stimulating factor receptor promoter.

Transcription factors play a key role in the development and differentiation of specific lineages from multipotential progenitors. Identification of these regulators and determining the mechanism of how they activate their target genes are important for understanding normal development of monocytes and macrophages and the pathogenesis of a common form of adult acute leukemia, in which the differentiation of monocytic cells is blocked. Our previous work has shown that the monocyte-specific expression of the macrophage colony-stimulating factor (M-CSF) receptor is regulated by three transcription factors interacting with critical regions of the M-CSF receptor promoter, including PU.1 and AML1.PU.1 is essential for myeloid cell development, while the AML1 gene is involved in several common leukemia-related chromosome translocations, although its role in hematopoiesis has not been fully identified. Along with AML1, a third factor, Mono A, interacts with a small region of the promoter which can function as a monocyte-specific enhancer when multimerized and linked to a heterologous basal promoter. Here, we demonstrate by electrophoretic mobility shift assays with monocytic nuclear extracts, COS-7 cell-transfected factors, and specific antibodies that the monocyte-enriched factor Mono A is CCAAT enhancer-binding protein (C/EBP). C/EBP has been shown previously to be an important transcription factor involved in hepatocyte and adipocyte differentiation; in hematopoietic cells, C/EBP is specifically expressed in myeloid cells. In vitro binding analysis reveals a physical interaction between C/EBP and AML1. Further transfection studies show that C/EBP and AML1 in concert with the AML1 heterodimer partner CBF beta synergistically activate M-CSF receptor by more then 60 fold. These results demonstrate that C/EBP and AML1 are important factors for regulating a critical hematopoietic growth factor receptor, the M-CSF receptor, suggesting a mechanism of how the AML1 fusion protein could contribute to acute myeloid leukemia. Furthermore, they demonstrate physical and functional interactions between AML1 and C/EBP transcription factor family members.

Toxicity of subretinal ribozyme to the proliferating cell nuclear antigen and 5-fluorouracil in rat eyes.

PURPOSE: To investigate the subretinal toxicity profile of the ribozyme to the proliferating cell nuclear antigen (PCNA-Rz) and 5-fluorouracil (5-FU), as well as the highest nontoxic subretinal dose of the mixture of the two agents in rat eyes. METHODS: Brown-Norway rats received subretinal injections of 1 microg, 10 microg, and 100 microg/microl PCNA-Rz and 0.06 microg/microl, 0.3 microg/microl, and 1.5 microg/microl 5-FU in the right eyes, and the left eyes were injected with H-BSS as control. Each dose was tested on 5 eyes in a 5 microl volume. In a second study, a combination of 5-FU (1.5 microg/microL) with varying 10-30-50 microg/microl doses of PCNA-Rz was tested in a regimen of four sequential subretinal injections. Toxicity was monitored by biomicroscopy, indirect ophthalmoscopy, electroretinography (ERG), and histology. RESULTS: The highest nontoxic dose for subretinal PCNA-Rz was 10 microg/microl, whereas 100 microg/microl showed disturbance of pigmentation with corresponding histological changes of retinal photoreceptor loss and retinal pigment epithelium proliferation or irregularities. Subretinal injection of all three doses of 5-FU did not show any toxicity. Serial injections of a mixture of 1.5 microg/microl 5-FU with 10 microg/microl of PCNA-Rz was found to be safe in rat eyes. CONCLUSIONS: Subretinal injections of the combination of PCNA-Rz (10 microg/microl) and 5-FU (1.5 microg/microl) demonstrated to be safe in rat eyes during the course of this study, even with a multiple administration of four injections.

Simultaneous use of two retroviral vectors in human gene marking trials: feasibility and potential applications.

Two Moloney murine leukemia virus (Mo-MLV)-based neoR retroviral vectors--LNL6 and G1Na--were used to transduce various human tumor-infiltrating lymphocytes (TIL) populations. These groups included bulk CD(8+)- and CD(4+)-enriched TIL from human renal cell carcinomas and melanomas. Transduction efficiencies averaged 5% for single 4-hr supernatant infections. Integrated provirus could be detected for up to 4 weeks of in vitro culture. LNL6 provirus could be distinguished from G1Na provirus using specific polymerase chain reaction (PCR) primers. A single neomycin phosphotransferase (neoR) gene copy could be detected in 10(5) TIL. Using quantitative PCR, the relative ratio of LNL6 to G1Na copies in the same sample could be determined even at low copy numbers. These preclinical studies demonstrate the feasibility of using two retroviral marking vectors in human gene therapy efforts.

In vivo cancer gene therapy with a recombinant interleukin-2 adenovirus vector.

Recombinant adenovirus (AdV) vectors are highly efficient at in vitro and in vivo gene delivery. In vivo therapy of established murine fibrosarcoma and mammary carcinomas was attempted with intratumoral injections of a recombinant AdV vector in which the human interleukin-2 (IL-2) gene was driven by the cytomegalovirus enhancer/promoter. Delayed growth and rejection of some tumors could be achieved with a cumulative virus dose of 2 to 6 x 10(9) plaque-forming units in two or three divided doses. Lower viral doses were ineffective, and higher doses resulted in animal death due to IL-2 toxicity. Using AdV vectors with the marker genes beta-galactosidase and luciferase, it is clear that even small volume (10 to 20 microL) intratumoral injections result in substantial systemic delivery of a portion of the virus dose. These findings define the potential and limitations of in vivo AdV-based cancer gene therapy and provide support for strategies to develop tumor-specific vectors.

The adenoviral transcription factor, E1A 13S, trans-activates the human tumor necrosis factor-alpha promoter.

The 1311 bp TNF-alpha promoter region fused to a luciferase reporter vector was used in a transient transfection system to study the regulation of TNF-alpha promoter activity by E1A 13S in the U937 macrophage cell line and the MLA 144 T cell line. Co-transfections of the TNF-alpha promoter with an E1A expression vector resulted in a strong trans-activation of the promoter in both cell lines. Sequential truncation of the promoter mapped the E1A responsive region to sequences contained between -120 bp and the transcription start site. Truncation to -95 bp caused a dramatic 87% reduction of E1A activation in MLA 144 cells and further truncation to -36 bp caused a complete loss of E1A activation. In U937 cells, each truncation lowered E1A responsiveness but activity was never completely abolished. Site-directed mutagenesis of putative cis-acting sequences in the TNF-alpha promoter identified the AP-1 site as important for E1A trans-activation in the U937 cell line; the AP-2 and CRE sites also appeared to contribute to a lesser degree. In contrast, only the CRE mutation caused a reduction in E1A induced activity in the MLA 144 cell line. Co-transfection of the E1A expression vector with expression vectors for the cellular transcription factors AP-1, AP-2 and CREB indicated that none of these transcription factors showed any co-operativity with E1A. Thus, cis-acting sequences which contribute to E1A trans-activation of the TNF-alpha promoter have been delineated.

Airsacculitis in turkeys exposed to Mycoplasma synoviae membranes.

In studies to investigate the pathogenesis of mycoplasmal airsacculitis, exudative lesions were produced in turkeys by intra-air-sac inoculation with Mycoplasma synoviae cell membranes and viable organisms. Membrane inocula containing 5 mg of protein produced more severe lesions than inocula containing either 2.5 mg or 1 mg protein. Turkeys exposed to 5 mg of membrane protein developed moderately severe airsacculitis; those exposed to viable organisms developed markedly severe airsacculitis. Microscopic examinations revealed that membrane-induced lesions were generally similar to those resulting from infection but were less severe. At the termination of the study, 8 days after exposure, M. synoviae was isolated from respiratory tract tissues of all turkeys exposed to live organisms, but it was not isolated from any of those exposed to membranes or from unexposed control turkeys. Antibody against M. synoviae was demonstrated with the tube agglutination test in sera from turkeys exposed to membranes and those exposed to live organisms, but it was not demonstrated in sera from unexposed control turkeys.

Comparison of strains of Mycoplasma iowae.

Comparison of biochemical test results and protein electrophoretic patterns of 21 strains of Mycoplasma iowae indicated that all were similar. Comparison of agglutination test results indicated marked within-species antigenic variation. None of 21 antigens prepared from different strains were effective in demonstrating turkey antibody against five reference strains. Examination of sera from turkeys exposed by intra-air-sac inoculation to two pathogenic strains also indicated antigenic variation. Neither the M. iowae type-strain, Iowa 695, nor the other reference strains were effective in demonstrating antibody against both strains used to expose the turkeys. These findings suggest that antigenic variation may be a major problem in effective serodiagnosis of M. iowae infections.

Comparison of Mycoplasma meleagridis antibodies demonstrated by tube agglutination and hemagglutination-inhibition tests.

Groups of turkeys inoculated intravenously with hemagglutinating (HA) and nonhemagglutinating (NHA) strains of Mycoplasma meleagridis developed similar antibody titers as demonstrated by the tube agglutination (TA) test, but use of the hemagglutination-inhibition (HI) test indicated markedly higher titers for turkeys inoculated with the HA strain than for those inoculated with the NHA strain. Adsorption of serum antibody with NHA strain antigen removed most agglutination activity but had little effect on HI activity. These results indicate that the HI and TA tests demonstrated antibody against different antigens, with the HI test detecting antibody directed against the hemagglutinin and the TA test detecting antibody directed against membrane antigens common to both strains.

Turkey sinusitis: synergism between Mycoplasma synoviae and Mycoplasma meleagridis.

Sinusitis was produced experimentally in turkeys after intrasinus inoculation with broth cultures of both Mycoplasma synoviae and Mycoplasma meleagridis. No evidence of sinusitis was observed in other turkeys exposed to each of these organisms separately- indicating pathogenic synergism with these mycoplasmas.

Turkey airsacculitis: effect of mixed mycoplasmal infections.

Turkeys experimentally infected with both Mycoplasma meleagridis and a member of the IJKNQR group of avian mycoplasmas developed more-severe air-sac lesions than did turkeys infected with either of these organisms alone. In addition, exudative air-sac lesions were more widely distributed anatomically in turkeys with mixed infections than in those with single infections. Similar studies with Mycoplasma synoviae and M. meleagridis did not indicate a significant increase in the severity of air-sac lesions. Both M. synoviae alone and M. synoviae and M. meleagridis combined produced severe lesions. Perhaps the severity of lesions produced by M. synoviae alone masked any exacerbating effect caused by the mixed infections.

Pathogenicity of strains of the IJKNQR group of avian mycoplasmas for turkey embryos and poults.

Strains of mycoplasmas isolated from turkey embryos and identified as belonging to the IJKNQR group of avian mycoplasmas were found to produce lethal infections in experimentally exposed turkey embryos. Some strains produced exudative airsacculitis in experimentally exposed poults. Of 4 strains used to inoculate poults, two produced moderate airsacculitis, one produced a slight airsacculitis, and one had little or no effect on turkey poult air sacs. Microscopic examination revealed that the air-sac lesions resulting from infection with these mycoplasmas were similar to those resulting from infections with Mycoplasma gallisepticum, M. meleagridis, and M. synoviae. The results were considered to indicate that some strains of the IJKNQR group of avian mycoplasmas are pathogenic for turkeys.

Selection and comparison of Mycoplasma synoviae hemagglutinating and nonhemagglutinating variants.

Hemagglutinating (HA) and nonhemagglutinating (NHA) variants were selected from each of two strains of Mycoplasma synoviae, WVU-1853 and Neb-3S. The HA titers of antigens prepared by 100-fold concentration of broth cultures were 1:2560 and 1:5120 for the HA variants and less than 1:5 for the NHA variants. Adsorption of erythrocytes to colonies of the variants was directly correlated with HA activity. The HA and NHA characteristics were stable in vitro, and there was no change in HA titers after repeated transfers in broth medium. Comparisons of pathogenicity indicated differences between strains but not between variants of each strain. Air-sac lesions resulting from exposure to variants of strain Neb-3S were marked, whereas those resulting from exposure to variants of WVU-1853 were slight. The HA titers of isolates recovered from turkey air sacs exposed to the Neb-3S variants varied considerably, suggesting in vivo instability of the HA characteristic.

Antibody responses of turkeys experimentally exposed to Mycoplasma synoviae.

The antibody response of turkeys exposed to Mycoplasma synoviae intravenously and by way of air sacs was determined by tube agglutination, plate agglutination, hemagglutination-inhibition (HI), and gel diffusion precipitin tests. The results suggested that continued antigenic stimulation was lacking in most turkeys and that the response was due mostly to IgM-type immunoglobulin. Under those conditions, both types of agglutination tests were effective and were more sensitive indicators of exposure than the HI test. The gel diffusion precipitin test was not considered effective under the conditions of this study. HI activity occurred in serums of intravenously exposed turkeys within 4 days of exposure. The sensitivity of this activity to 2-mercaptoethanol treatment suggested that IgM was responsible.

Capsular groups of Pasteurella multocida isolated from avian hosts.

Avian strains of Pasteurella multocida representing a variety of host types, geographic locations, and somatic serotypes were examined to provide information on the distribution of capsular serogroups. Of the 246 strains studied, 166 were capsular group A, 4 were group B, 4 were group D, and 14 were group F; 58 strains were non-encapsulated and consequently not serogroupable. This is the first report of serogroup B P. multocida from avian hosts in the United States. The 188 serogroupable strains represented 12 variations in somatic serotypes and were isolated from 11 types of avian hosts representing 25 states of the United States, Bangladesh, Singapore, and Canada.

The effects of heat-labile Bordetella avium toxin on turkey poults.

Intraperitoneal exposure of turkey poults to various concentrations of Bordetella avium sonicate containing heat-labile toxin indicated a high degree of toxicity: poults died after exposure to sonicate containing as little as 6.32 micrograms of protein. The toxic effects were dose-related: poults that received sonicate containing 158 micrograms of protein died in 4 to 6 1/2 hours, those that received 31.6 and 6.32 micrograms of protein died in 25 to 30 hours, and those that received 1.2 micrograms survived the 6-day course of the study and were apparently unaffected. Histologic examination of poults that received lethal doses of the toxin revealed degeneration and necrosis of parenchymal cells of the liver and pancreas as well as hyperemia, hemorrhage, and necrosis of the mucosa of the small intestine. No lesions were observed in poults that received the sublethal dose of toxin or in unexposed poults. Repeated intranasal exposure of poults to the sonicate did not produce clinical signs of disease or gross lesions.

Virulence of avian capsular serogroup B Pasteurella multocida for turkey poults.

Two strains of capsular serogroup B Pasteurella multocida isolated from avian hosts (swan and turkey) were evaluated for virulence based on lethality for turkey poults. Groups of poults were exposed intramuscularly to various concentrations of organisms of each strain. Both strains were virulent. The strain isolated from a turkey was highly virulent: all exposed poults died in less than 24 hours, including those exposed to only 79 organisms. This highly virulent strain was neither highly invasive nor highly infective: intrapharyngeal exposure with 7.9 x 10(6) organisms resulted in death of only one of five poults, and attempts to isolate the organism from pharyngeal mucosae and livers of surviving poults were unsuccessful. The high degree of virulence of a B capsular group strain isolated from a turkey indicates a disease-producing potential for members of this uncommon serogroup of P. multocida.

Pasteurella multocida virulence factors: selection of fowl cholera-inducing and non-inducing strains.

Relatively little information is available on Pasteurella multocida virulence factors involved in producing fowl cholera. Because of the complex nature of bacterial pathogenesis, the recommended approach for ascertaining these factors is to compare biological attributes of high- and low-virulence strains. To permit use of this approach for fowl cholera, P. multocida strains of high and low virulence were identified. Turkey poults were exposed intrapharyngeally and intravenously (IV) to two antigenically and biochemically similar strains. Based on mortality, strain P-1059 was highly virulent and strain P-1062 was avirulent. Microbiological examination indicated that only the virulent strain infected the pharyngeal mucosa of intrapharyngeally exposed poults and survived and multiplied in IV-exposed poults. These findings indicate strain differences in those virulence factors concerned with the colonization and multiplication stages of disease development.

Somatic serotypes of Pasteurella multocida strains isolated from avian hosts (1976-1988).

During the period of 1976-88, 733 strains of Pasteurella multocida isolated from avian hosts were submitted to the National Animal Disease Center for somatic serotyping. The serotypes were determined and are presented in a summarized form in this report. The most common serotypes were 3 (29%); 1 (18%); 3,4 (12%); and 3,4,12 (9%). The 733 strains had been isolated from 25 species of avian hosts; 400 (55%) were from turkeys. The most common serotypes of strains from turkeys were 3 (38%); 3,4 (18%); 3,4,12 (11%); and 4 (4%).