Cystic fibrosis mouse with intestinal obstruction.
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Animals↗
Biomedical subjects
M J Evans
Publications and source records attributed to M J Evans.
Nuclear respiratory factor 1 activation sites in genes encoding the gamma-subunit of ATP synthase, eukaryotic initiation factor 2 alpha, and tyrosine aminotransferase. Specific interaction of purified NRF-1 with multiple target genes.
Transcription factor nuclear respiratory factor 1 (NRF-1) was originally identified as an activator of the cytochrome c gene and subsequently found to stimulate transcription through specific sites in other nuclear genes whose products function in the mitochondria. These include subunits of the cytochrome oxidase and reductase complexes and a component of the mitochondrial DNA replication machinery. Here we establish that a functional recognition site for NRF-1 is present in the ATP synthase gamma-subunit gene extending the proposed respiratory role of NRF-1 to complex V. In addition, biologically active NRF-1 sites are found in genes encoding the eukaryotic translation initiation factor 2 alpha-subunit and tyrosine aminotransferase, both of which participate in the rate-limiting step of their respective pathways of protein biosynthesis and tyrosine catabolism. The recognition sites from each of these genes form identical complexes with NRF-1 as established by competition binding assays, methylation interference footprinting, and UV-induced DNA cross-linking. Cloned oligomers of each NRF-1 binding site also stimulate the activity of a truncated cytochrome c promoter in transfected cells. The NRF-1 binding activities for the various target sites copurified approximately 33,000-fold and resided in a single protein of 68 kDa. These observations further support a role for NRF-1 in the expression of nuclear respiratory genes and suggest it may help coordinate respiratory metabolism with other biosynthetic and degradative pathways.
Animals↗
Absence of interleukin 1 alpha radioprotection in tumor-bearing animals: elevated plasma levels of prostaglandin E versus a preexisting primed marrow.
Recombinant human interleukin 1 (IL-1) administered as a "priming" agent 24 h prior to hematopoietically lethal doses of total body irradiation (TBI) confers radioprotection to normal C57B1/6 (B6) mice, but not to B6 tumor-bearing animals (TBAs) known to have altered hematopoietic steady states. Using the Lewis lung tumor (LLca) in the B6 mouse, studies were carried out to determine whether the failure of IL-1 to radioprotect the LLca TBA was related to a preexisting "primed" hematopoietic state in the TBA or resulted from inhibition of myelopoietic activity associated with the production of prostaglandin E (PGE) by, or in response to, the tumor. Both normal B6 and LLca B6 TBAs were injected (every 24 h x 1-5) with 100 micrograms of indomethacin (IND) prior to the administration of IL-1. A single treatment with IND was sufficient to reduce the elevated levels of PGE found in the plasma of the TBAs. After five treatments, IND reduced the PGE level to below that of controls. Neither the acute nor the protracted IND treatment, however, affected the expansion of the stem and progenitor cell compartments of the marrow in the LLca TBA. Furthermore, no evidence of restoration of the radioprotective properties of IL-1 was observed in TBAs pretreated with IND. Collectively, these data suggest that the failure of IL-1 to provide radioprotection to the LLca TBA is not a direct result of the elevated plasma PGE levels associated with growth of the LLca tumor. In addition, these studies provide insight into the importance of examining in vivo effects of biological molecules in altered, as well as normal, physiological states.
Animals↗
Cellular sequence of tracheal repair in sheep after smoke inhalation injury.
The cellular repair process of injured tracheal epithelium is described for sheep after exposure to toxic smoke containing high concentrations of acrolein. Fourteen fasted 3-4-year-old ewes had a portion of their cervical trachea exposed to cotton smoke for 20 min and then were sacrificed at various time intervals ranging from 1 to 22 days after exposure. Within 1 day of injury, columnar epithelium sloughed intact from the trachea with a concomitant reduction of nearly 35% in the basal cell population. At 2 days of recovery, the cellularity of the epithelium had increased and mitotic figures were observed in some tracheal epithelial and gland cells. By 8 days, undifferentiated hyperplastic cells increased to 30/100 microns, differentiated nonciliated columnar cells first appeared, and the basal cell population returned to a normal count of 13 cells/100 microns. Thirteen days after exposure, the undifferentiated hyperplastic cell population had declined to 7 cells/100 microns, nonciliated columnar cells were at control values, and some ciliated cells were identified. At 18 and 22 days, epithelium was normal in appearance and the count was 13 cells/100 microns. Data suggest that because the columnar epithelium sloughs intact with the cilia remaining active, toxic smoke may affect their attachment to the basal lamina. Furthermore, the regeneration process involves differentiation of hyperplastic cells in which they elongate down to the basal lamina, thus re-establishing the integrity of tall epithelium in the sheep trachea.
Animals↗
Disruption of the cystic fibrosis transmembrane conductance regulator gene in embryonic stem cells by gene targeting.
We have successfully disrupted the cftr (cystic fibrosis transmembrane conductance regulator) gene at its endogenous locus in embryonic stem cells by gene targeting. We are using a double replacement strategy to introduce subtle mutations into exon 10. We report here the first step of creating a null mutation by insertion of a functional hprt (hypoxanthine phosphoribosyl transferase) mini-gene into exon 10 of the cftr gene. Targeted embryonic stem cell clones were identified by PCR screening and confirmed by Southern blot analysis. One of the cftr targeted clones has been injected into recipient blastocysts and shown to contribute to chimaeras. The targeted clones will now be used as the starting point for a second gene targeting step to remove the hprt gene in exon 10 with the concomitant introduction of the delta F508 mutation or other mutations.
Animals↗
Differential inhibition of human basal keratinocyte growth to silver sulfadiazine and mafenide acetate.
The impact of topical antimicrobial agents on improving the survival of patients with major thermal injuries is significant. However, the effects of these agents on cells responsible for wound healing has only recently received attention. Fresh human basal keratinocytes were grown in serum-free modified MCDB 153 medium under standard tissue culture conditions. Cells were subsequently exposed to concentrations of silver sulfadiazine and mafenide acetate as low as 1/100 of that used clinically over a period of 5-7 days. Cellular responses documented with hemocytometer cells counts, cellular protein assays, phase-contrast microscopy, and transmission electron microscopy show only severe toxicity to mafenide acetate. Such data imply that inhibition of wound epithelialization is greater with the use of mafenide acetate than with the use of silver sulfadiazine.
Cell Count↗
Pulmonary hemodynamics and tissue damage after one lung infusion of Pseudomonas aeruginosa in sheep.
The relative roles of hematogenous mediators and direct bacterial toxicity due to phagocytosis by pulmonary intravascular macrophages were determined by selective bacterial infusion into the left pulmonary artery and comparison of right and left lungs at 24 h. Chronically instrumented sheep received 15-min pulmonary arterial infusions of live Pseudomonas aeruginosa (0.35-2.9 x 10(9), n = 6) or saline (n = 5). The saline group demonstrated stable cardiopulmonary function over time. Left lung blood flow, measured by Doppler flow probe, decreased 15 min into the bacterial infusion, with a concomitant sevenfold increase in left lung pulmonary vascular resistance index. The right lung pulmonary vascular resistance index doubled at 1 h, in association with increased plasma thromboxane B2 levels. An increase in cardiac index and decrease in systemic vascular resistance occurred at 12 h. The wet-to-dry weight ratio of the Pseudomonas-infused left lung was increased compared with that of the sham-infused lung. The tissue count of neutrophils in the lungs was doubled in both sides, but neutrophils on the left were more degranulated. The left lung tissue damage was caused by direct bacterial toxicity, including activation of phagocytic cells. Hematogenous mediators induced pulmonary and systemic hemodynamic changes and right lung neutrophil sequestration, but they did not damage the noninfused lung.
Animals↗
A chimaeric mouse model for autosomal-dominant polycystic kidney disease.
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Animals↗
Differentiation of anchoring junctions in tracheal basal cells in the growing rat.
A function of airway basal cells is to attach ciliated and nonciliated columnar cells to the basal lamina. The significance of the basal cell in attachment is related to the height of the columnar epithelium. In taller epithelia, basal cells are more numerous and differentiated with respect to anchoring junctional adhesion mechanisms (desmosomes, hemidesmosomes, and the cytoskeleton) than in shorter epithelia. In this study, we determined if basal cell anchoring junctional adhesion mechanisms differentiated during growth of the airway. Tracheas from five 3-day-old, five 30-day-old, and five 90-day-old rats were prepared for electron microscopy and morphometrically studied by standard techniques. The circumference of the trachea increased from 2.5 +/- 0.2 to 7.5 +/- 0.4 mm during growth. The height of the columnar cell increased from 13.4 +/- 1.5 to 24.6 +/- 3.9 microns, and the number of basal cells per millimeter increased from 3.2 +/- 0.7 to 9.6 +/- 1.8 during growth. The number of desmosomes per basal cell profile increased significantly from 1.5 +/- 0.1 to 2.1 +/- 0.1, as did keratin filament volume density from 0.046 +/- 0.05 to 0.098 +/- 0.032. The amount of hemidesmosome attachment per basal cell did not increase significantly during growth of the airway. These data demonstrate that as tracheas grow in circumference, the columnar cells increase in height, basal cells increase in number, and anchoring junctional adhesion mechanisms differentiate in the basal cells. These changes are closely related to the height of the epithelium and result in maintaining a constant amount of attachment between the columnar epithelium and the basal lamina as the epithelium increases in height.
Aging↗
Estimating trauma centre workload.
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Emergency Medical Technicians↗
Morphologic changes in basal cells during repair of tracheal epithelium.
Basal cells are differentiated with respect to junctional adhesion mechanisms and play a role in attachment of columnar epithelium to the basal lamina. Although much is known about nonciliated and ciliated cell differentiation during the repair process after injury, little is known about the basal cell. We studied the morphology of basal cells and quantitated junctional adhesion structures during repair of tracheal epithelium exposed to toxic cotton smoke. Ten adult ewes were given a smoke injury to a portion of the upper cervical trachea and were killed at 4, 6, 8, 10, and 18 days after injury for morphometric studies. At 4 days, there was a stratified reparative epithelium over the basal lamina, which was two to four cells in depth. The basal cells were identified by their hemidesmosome (HD) attachment to the basal lamina. Basal cells were about 69% larger than controls and flattened rather than columnar. The amount of HD attachment was 192% greater than controls. In contrast, volume density of cytokeratin filaments had decreased about 47%. Basal cells had returned to normal numbers and size and a columnar shape by day 18. The amount of desmosome (D) and HD attachment and volume density of cytokeratins had also reached control levels by day 18. These data indicate that morphology of basal cells changes during the initial stages of reparative regeneration but returns to normal by 18 days. Morphologic changes appear to reflect changes in size of the cell associated with cell division rather than differentiation of recently divided basal cells.
Animals↗
Synergy between recombinant human IL-1 alpha (rHuIL-1) and M-CSF (rHuM-CSF) during the recovery of murine hematopoietic activity in myelosuppressed animals: abbreviated versus chronic administration of rHuM-CSF.
The ability of highly purified, recombinant human macrophage colony-stimulating factor (M-CSF) and recombinant human interleukin 1 alpha (IL-1) to rescue hematopoietic activity from the myelosuppressive effects of 5-fluorouracil (5-FU) was investigated in the C57Bl/6 mouse. IL-1 (q24 h x 4) stimulated granulopoietic recovery in the 5-FU-treated animals and reduced the period of severe neutropenia associated with this drug by 7 days. Chronic M-CSF administration (q24 h x 14), on the other hand, resulted in a modest retardation of granulocyte recovery, and, when combined with IL-1, the chronic administration of M-CSF significantly dampened the accelerated recovery of granulopoietic activity observed with IL-1 alone. Consistent with their effects on neutrophil recovery, IL-1 alone markedly enhanced the recovery of the granulocyte erythrocyte macrophage megakaryocyte colony-forming units (CFU-GEMM), macrophage colony-forming units (CFU-M), and erythroid burst-forming units (BFUe) in the marrow, whereas M-CSF failed to demonstrate a significant influence on the restoration of these hematopoietic progenitors (with the exception of delaying the recovery of the BFUe). Unexpectedly, the combination of IL-1 plus M-CSF (q24 h, days 1-4) followed by M-CSF (q24 h, days 5-14) resulted in a more than additive stimulation of progenitor recovery in both the marrow and the spleen that was observed as early as day 3 following 5-FU treatment. Furthermore, in the absence of protracted M-CSF administration on days 5-14, the 4-day rescue with a combination of IL-1 plus M-CSF also resulted in a more than additive effect on the recovery from 5-FU-induced neutropenia. Collectively, these observations demonstrated that IL-1 and M-CSF can interact synergistically to stimulate granulopoietic recovery in the 5-FU-treated animal. However, the data also suggest that the continued administration of M-CSF following the 4-day IL-1 plus M-CSF rescue may interfere with the restoration of neutrophils in the myelosuppressed animal.
Animals↗
Detection and purification of antiidiotypic antibody against anti-DNA in intravenous immune globulin.
Pooled normal human IgG for therapeutic use, following depletion of anti-DNA, anti-Fc, and anti-F(ab')2 of normal IgG, expressed antiidiotypic activity against anti-DNA derived from lupus sera. The antiidiotype enriched by elution from anti-DNA affinity columns bound directly to anti-DNA IgG and inhibited the binding of lupus sera to DNA but did not bind to normal IgG or inhibit the binding of anti-tetanus toxoid to tetanus toxoid. Antiidiotypes in pooled normal sera may have a role in the clinical improvement seen in patients with autoimmune diseases receiving intravenous immune globulin.
Antibodies, Anti-Idiotypic↗
Immunocytochemical localization of laminin in hamster tracheal epithelial cell cultures.
EM examination of 28 day cultures of enzymatically dissociated hamster tracheal epithelial (HTE) cells grown on collagen coated millipore filters reveals that fragments of basal lamina may be present at the basal plasmalemma. Since the basal lamina consists of several major components including type IV collagen, heparan sulfate proteoglycans, entactin/nidogen, and laminin, questions naturally arise concerning the presence of such a structure in this cell culture system. When immunocytochemical procedures utilizing anti-laminin antibody and PAP techniques are carried out with paraffin sections of HTE culture at 1,2,3, and 4 weeks in vitro, LM analysis reveals that a thin, dense line of reaction product is present between the basal surface of the HTE cells and the underlying collagen substrate. Immunoblotting evaluation carried out with supernatants of 7d HTE cell homogenates and HTE cell conditioned media also indicate that laminin is being produced by the tracheal cells. Thus, the presence of basal lamina-like fragments, the immunocytochemical localization of laminin, and immunoblot identification of laminin in hamster tracheal epithelial cell cultures, suggest that, although basal lamina components may be produced by HTE cells, at the time points tested, they are not yet being organized into a complete basal lamina.
Animals↗
In situ freezing of embryonic stem cells in multiwell plates.
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Cell Line↗
Interleukin-1 alpha protects against the toxicity associated with combined radiation and drug therapy.
A dose of total body irradiation sufficient to cause 70% mortality within 30 days (9.5 Gy) and maximally tolerated doses of either adriamycin (10 mg/kg) or cis-dichlorodiammine platinum (8 mg/kg) were administered to C57B1/6 mice and animal survival used as an index of toxicity. Whereas the nature of the toxicity resulting from the radiation alone was hematopoietic, the addition of either drug to the total body irradiation resulted in a pattern of animal death more consistent with that of gastrointestinal toxicity (100% dead within 7 days). However, if the radiation was delivered as a regional abdominal exposure, rather than total body, the gastrointestinal death observed following the combination of total body irradiation drug was not observed. The administration of 2.5 x 10(5) U IL-1 v24 hr prior to total body irradiation demonstrated significant protection against this dose of radiation (90% survival vs 30% survival). Similar protection was also observed when the IL-1 was administered 24 hr prior to the combination of total body irradiation with either drug. While these observations suggested that the IL-1 was protecting against gastrointestinal toxicity, subsequent studies demonstrated that IL-1, in addition to accelerating hematopoietic recovery following radiation insult, was equally effective in advancing the repopulation of the stem cell (CFU-GEMM) and progenitor cell (CFU-M and CFU-GM) compartments following drug treatment. Collectively, the data from these studies demonstrate that the lethal effects resulting from combined total body irradiation + drug treatment contain both a gastrointestinal and a hematopoietic component.
Animals↗
Altered radioprotective properties of interleukin I alpha (IL-1) in non-hematologic tumor-bearing animals.
The radioprotective properties of IL-1 were investigated in the respective murine hosts for the Lewis lung (LLca) and EMT-6 tumors. For these studies, doses of total body irradiation were selected for the C57B1/6 (9.5 Gy) and Balb/c (7.5 Gy) mice that resulted in a 60% mortality over a 30-day interval. When a "priming" dose of 2.5 x 10(5) U IL-1 was administered 24 hr prior to the radiation exposure, animal mortality was markedly reduced (60% vs 5-10%). Under identical experimental conditions, however, the presence of either the LLca or the EMT-6 tumors in their respective host strains was found to compromise the level of radioprotection conferred by this priming dose of IL-1. In Balb/c mice bearing the EMT-6 tumor, a priming dose of IL-1 resulted in only a modest level of radioprotection when compared to non-tumor-bearing control animals (median animal survival increased by 11.5 days). In C57B1/6 mice bearing the LLca tumor, IL-1 failed to demonstrate any evidence of radioprotection. Following a sublethal dose of total body irradiation, the appearance of an accelerated repopulation of the stem cell (8d CFUs and CFU-GEMM) and the myeloid progenitor (CFU-M) compartment in the marrow of the IL-1 primed EMT-6, but not the LLca, tumor-bearing animals was consistent with the hypothesis that the mechanism leading to radioprotection in IL-1 primed rodents involves an accelerated recovery of hematopoietic activity. It was also noted that the presence of the EMT-6 tumor was associated with an increase in the "radiosensitivity" of the Balb/c mouse. Collectively, these data suggest that the use of biological modifiers should be examined under altered physiological conditions prior to attempting to translate them into the clinic.
Animals↗
Plasma corticotrophin releasing factor and vasopressin responses to exercise in normal man.
OBJECTIVE: To evaluate the hypothalamo-pituitary-adrenal (HPA) response to both mild and intense exercise. DESIGN: Maximum oxygen consumption (VO2max.) was initially determined. Exercise to 20% (mild) and 90% (intense) VO2max. was undertaken in random order. Subjects were exercised over a 5-minute period to the required level of intensity, which was maintained for a further 10 minutes. SUBJECTS: Six trained male athletes. MEASUREMENTS: Brachial vein blood was sampled before and at 5-minute intervals during and after exercise and assayed for plasma cortisol, ACTH, arginine vasopressin (AVP), corticotrophin releasing factor (CRF), sodium and lactate levels. Haemoglobin and haematocrit were measured before and at the end of exercise. Heart rate was monitored continuously and blood pressure measured whenever blood was sampled before and after exercise. RESULTS: Maximum heart rate was 184 +/- 2 and 82 +/- 5 (mean +/- SEM) beats per minute at 90 and 20% VO2max. respectively. Plasma volume fell by 8.7 +/- 2.4% with intense exercise. Significant increases in plasma concentrations of AVP, ACTH and cortisol, but not CRF, occurred during intense exercise. Plasma sodium concentration, which increased during intense but not mild exercise, peaked at 10 minutes after the onset of exercise and preceded the rise in plasma AVP. The AVP peak clearly preceded the ACTH peak in two subjects and occurred synchronously with it in three subjects. CONCLUSION: These results indicate that although significant HPA activation occurs during intense exercise, CRF does not appear to play a major role in mediating the ACTH response to an acute episode of vigorous exercise in man. AVP may be more important in this regard.
Adrenocorticotropic Hormone↗