Effect of Dexamethasone and Fluticasone on Airway Hyperresponsiveness in Horses With Inflammatory Airway Disease.

BACKGROUND: Airway hyperresponsiveness (AWHR), expressed as hypersensitivity (PC75 RL ) or hyperreactivity (slope of the histamine dose-response curve), is a feature of inflammatory airway disease (IAD) or mild equine asthma in horses. Glucocorticoids are used empirically to treat IAD. OBJECTIVES: To determine whether dexamethasone (DEX) (0.05 mg/kg IM q24h) and inhaled fluticasone (FLUT) (3,000 µg q12h) administered by inhalation are effective in decreasing AWHR, lung inflammation, and clinical signs in horses with IAD. METHODS: A randomized crossover study design was used. Eight horses with IAD were assigned to a treatment group with either DEX or FLUT. Measured outcomes included lung mechanics during bronchoprovocative challenges, bronchoalveolar lavage fluid (BALF) cytology, and scoring of clinical signs during exercise. RESULTS: Dexamethasone and FLUT abolished the increase in RL by 75% at any histamine bronchoprovocative dose in all horses after the first week of treatment. However, after 2 weeks of FLUT treatment, 1 horse redeveloped hypersensitivity. There was a significant decrease in the number of lymphocytes after treatment with both DEX and FLUT (P = .039 for both) but no significant differences in other BALF cell types or total cell counts (P > .05). There was no difference in the scoring of the clinical signs during each treatment and washout period (P > .05). CONCLUSIONS AND CLINICAL IMPORTANCE: Both DEX and FLUT treatments significantly inhibit airway hypersensitivity and hyperreactivity in horses with IAD. There are no significant effects on the clinical signs or the number of inflammatory cells (except lymphocytes) in BALF. The treatments have no residual effect 3 weeks after discontinuation.

Tracheobronchoscopic Assessment of Exercise-Induced Pulmonary Hemorrhage and Airway Inflammation in Barrel Racing Horses.

BACKGROUND: Poor performance is often suspected to be associated with EIPH in barrel racing horses; however, there are no published reports of EIPH for this discipline. The prevalence of EIPH in barrel racing horses is also unknown. OBJECTIVES: This study was performed to determine the prevalence of EIPH and signs of airway inflammation in barrel racing horses under normal racing conditions in Alberta. ANIMALS: About 170 barrel racing horses. METHODS: Observational cross-sectional study. Tracheobronchoscopic examinations were performed at least 30 minutes postrace. Video recordings were scored off-site independently by two observers for EIPH and tracheal mucus accumulation (TMA). Horses with an EIPH score ≥2 were not assessed for TMA. Interobserver agreement was calculated by weighted κ statistics. Run times, environmental variables, and clinical information were also recorded for analysis. RESULTS: 77/170 (45.3%) of horses examined showed evidence of EIPH (grade ≥ 1). Interobserver agreement was 0.94. 140/141 (99.3%) of horses assessed for TMA showed evidence of tracheal mucus accumulation (grade ≥ 1) with 104/141 (73.8%) having a TMA score ≥ 2. Interobserver agreement was 0.73. A weak positive association was found between EIPH scores and average run speed, the presence of cough at rest reported by the riders, increased recovery time, exercise intolerance, and outdoor pattern. CONCLUSIONS AND CLINICAL IMPORTANCE: The high prevalence of EIPH observed in the sampled population indicates that barrel racing induces substantial stress on the lungs. The presence of EIPH did not impact negatively on performance. Factors such as environmental dust and frequent traveling might have contributed to the high prevalence of TMA observed.

Methyl nitrosourea induced unscheduled DNA synthesis in vivo in mice. Effects of background genotype on excision repair during aging.

We studied mice from five genetic strains at two ages (young, 10 +/- 0.49 weeks; old, 74.4 +/- 14 weeks) for the induction of unscheduled DNA synthesis (UDS) in bone marrow cells following in vivo challenge by a known mutagen and carcinogen: methyl nitrosourea (MNU). The data, in the form of mean silver grains/cell and the percentage of cells in UDS, were used to evaluate genotype (strain) dependent differences in response to mutagenic challenge and changes in this response with age in otherwise normal individuals. Increasing doses of MNU significantly increased both mean silver grains/cell and the percentage of cells in UDS. The increase was, however, strain specific. Furthermore, the older animals generally had both lower mean silver grains/cell and a smaller percentage of cells in UDS as compared to their younger counterparts. This reduction in UDS patterns during aging (aging response) was variable among mouse genotypes. We attribute these observations to subtle differences in the genetic backgrounds of the mice which affects both their ability to repair damage induced by MNU and the genotype specific decline in this ability with old age.

DNA sequence analysis of the cytosolic acetaldehyde dehydrogenase gene (Ahd-2) in mouse strains with variable ethanol preferences.

Differences in Ahd-2 at the DNA sequence level were characterized in mouse strains with variable ethanol preferences. The 5' region and the region surrounding the active site of Ahd-2 were compared to detect differences which could affect ethanol sensitivity. Only minor differences were found among the strains in the two regions. These differences cannot explain their variable ethanol preference and the implications of sequence identities among the divergent strains in these regions has yet to be determined.

Genotype-specific reduction in methyl nitrosourea (MNU) induced sister chromatid exchanges (SCE) in vivo during aging.

We studied mice from five strains (BALB/c, C3H/HeSnJ, C57BL/6J, Csb and 129/ReJ) at two ages (young, 10 +/- 1 weeks; and old, 67 +/- 3 weeks) for the induction of sister chromatid exchanges (SCEs) in vivo by methyl nitrosourea (MNU). The SCE frequency is genotype-specific. The F1 phenotype resembles the 'low' responding parent. SCE induction is significantly lower in the older animals of each strain than their younger counterparts, and the reduction of SCE/cell with old age is strain-specific. A general explanation for these results must include strain differences in relative mutagenic sensitivity, genotype-specific pattern of reduction in DNA repair and other such factors affecting SCE formation, with old age.

Studies with cDNA probes on the in vivo effect of ethanol on expression of the genes of alcohol metabolism.

Mice (Mus musculus) from three genetic strains with variable responses to ethanol challenge (BALB/c, C57BL/6J and 129/ReJ) were used to evaluate the effect of ethanol feeding on hepatic mRNA specific to the two primary enzymes of ethanol metabolism; alcohol dehydrogenase (ADH; E.C. 1.1.1.1) and aldehyde dehydrogenase (ALDH; E.C. 1.2.1.3). Adh-1 (ADH) and Ahd-2 (ALDH) specific mRNA were evaluated on the livers of ethanol-fed mice and from their age, sex and genotype matched controls (using an isocaloric liquid diet). C57BL/6J (alcohol resistant) mice show a significant (approx. 200%) increase in ADH-1 mRNA levels after ethanol treatment, compared to their matched controls. BALB/c (alcohol sensitive) mice have approximately a 20% increase with ethanol treatment while 129/ReJ (alcohol sensitive) mice show a slight reduction in the ADH-1 specific mRNA following ethanol feeding. A strain-specific pattern is also apparent in the AHD-2 mRNA as a result of ethanol feeding in the experimental animals. C57BL/6J mice have an increase and BALB/c mice show no apparent change in the AHD-2 mRNA. 129/ReJ mice fed an ethanol diet, on the other hand, appear to have a decrease in the level of AHD-2 hepatic mRNA as compared to their matched controls. The relative mRNA levels of the two genes correlate well with the respective enzyme activity levels, but for mice on the control diet only. Ethanol feeding, which causes an apparent reduction in hepatic ADH enzyme activity in BALB/c and 129/ReJ and an apparent increase in ALDH activity in C57BL/6J (under the experimental protocols used) also alters the mRNA levels specific to the two genes. However, changes in the mRNA levels after ethanol feeding cannot be directly related to the changes seen in enzyme activity. The observed steady state level of AHD-2 mRNA and the increase in ALDH activity after ethanol feeding, which is unique to C57BL/6J mice, is expected to offer a faster clearance (metabolism) of acetaldehyde, the toxic metabolite, and may be responsible for, or contribute to, the relative resistance of this strain to ethanol.

Acetaldehyde dehydrogenase (Ahd-2)-associated DNA polymorphisms in mouse strains with variable ethanol preferences.

The genotype-dependent response of mice to ethanol has been well documented. Cytosolic acetaldehyde dehydrogenase (ALDH-2) increases in some strains while decreasing in others with ethanol treatment. Further work suggests that the mRNA for ALDH-2 (Ahd-2 mRNA) levels are altered following ethanol feeding in a strain-dependent fashion. This report identifies differences in Ahd-2 at the genomic DNA level among different strains of mice. Restriction fragment length polymorphisms (RFLPs) associated with the Ahd-2 locus were found for the restriction enzymes EcoRI, HindIII, Pst I and Rsa I. The mouse strains included in this study could be categorized into two groups based on their overall Ahd-2 associated DNA banding patterns. Strains C57BL/6J, C57BL/6J*, C57BL/10J and BALB/c form group 1 while strains C3H/HeJ, C3H/HeSnJ, 129/ReJ, Csb, SW and DBA/2J form group 2. With the exception of BALB/c, group 1 represents alcohol preferring strains while group 2 are alcohol avoiding strains. Additional work will be required to determine the physiological significance (if any) of these RFLPs and their possible relationship to ethanol preference and avoidance.

Identification of genes differentially expressed in C6 glioma cells transfected with connexin43.

Astrocytes are characterized by extensive gap junctional intercellular communication (GJIC) mediated primarily by channels composed of connexin43. In contrast, C6 glioma cells are deficient in connexin expression and gap junctional communication. Transfection of these glioma cells with connexin cDNAs results in changes in cellular phenotype following increased GJIC. Specifically, connexin expression correlates with reduced cellular proliferation and tumorigenicity. To characterize the role of gap junctions in this growth control, we have screened for changes in gene expression by differential display. We have observed that these changes in GJIC are associated with changes in expression of several genes, including those coding for a number of secreted factors which may play a role in modulating the tumor phenotype of these cells. These include the immediate early gene cyr61, ostoepontin and the KC gene (murine homologue of the human gro gene).

Transfection of C6 glioma cells with connexin32: the effects of expression of a nonendogenous gap junction protein.

C6 glioma cells do not express the gap junction protein connexin32 or its corresponding mRNA. Very low levels of connexin43 protein and mRNA, as well as weak intercellular coupling, have been detected. Studies investigating the role of gap junctions in cell proliferation and tumorigenesis have shown that C6 cells transfected with connexin43 have increased levels of intercellular coupling and reduced cell growth (D. Zhu et al., Proc. Natl. Acad. Sci. USA, 88:1883-1887, 1991). To determine whether this growth inhibition is observed with other connexins, a full-length cDNA for connexin32 was used to transfect C6 cells. A number of transfected clones, expressing various levels of connexin32 mRNA, were obtained. Further analysis of several of these clones has shown that they have a corresponding increase in both the amount of connexin32 immunoreactivity and intercellular coupling. Thus, transfection of the C6 glioma cell line with connexin32, a gene which is normally expressed in the rat brain but not in C6 cells, produces both a functional mRNA and protein. Growth of the transfected clones was reduced in vivo. In vitro, growth of the various clones was not correlated to either levels of connexin32 expression or intercellular coupling. This is in contrast to findings in the previous study, in which cell growth was reduced in response to connexin43 expression both in vivo and in vitro in the transfected cells. These clones provide a unique system to study the role of gap junctions in cell proliferation and other tumor characteristics.

SV40 Tag transformation of the normal invasive trophoblast results in a premalignant phenotype. II. Changes in gap junctional intercellular communication.

Poor gap junctional intercellular communication (GJIC) has been associated with uncontrolled cell growth and neoplasia. We have successfully propagated normal first trimester invasive extravillous trophoblast (EVT) cells, and have produced premalignant EVT lines after SV40 Tag transformation: RSVT-2 is an uncloned line that is long-lived; RSVT2/C is a clonal line that is immortal. Both are hyperproliferative, hyperinvasive and variably refractory to the anti-proliferative and anti-invasive effects of transforming growth factor beta (TGFbeta). Possible changes in gap junctions during the transition of normal invasive EVT cells to the premalignant stage were examined by comparing expression of connexin proteins (by immunolabeling for Cx26, Cx32, Cx40, Cx43), and mRNA (by Northern blot with cDNA probes for Cx26, Cx32, Cx43), and functional GJIC (by dye transfer using the preloading method) in normal parental EVT cells and their SV40 Tag transformants. Results from immunofluorescence and Northern blot analysis revealed that, of the panel of connexins examined, only Cx43 was variably expressed in these cell lines in vitro. Expression of Cx43 protein and mRNA was abundant in normal EVT cell line HTR8, reduced in long-lived RSVT-2 cells and undetectable in immortalized RSVT2/C cells. GJIC, as measured by dye transfer between donor and recipient cells, was also similarly reduced in recipient RSVT-2 cells, and drastically reduced in RSVT2/C cells, irrespective of whether the dye donor was of the same cell type (homocellular coupling) or HTR8 cells (heterocellular coupling). Treatment with TGFbeta reduced Cx43 mRNA expression as well as GJIC in normal EVT cells, but not in the SV40 Tag transformants. Our findings suggest that downregulation of connexins with the resultant impairment in GJIC is an early event in tumor progression, as observed in the premalignant SV40 Tag transformants.

SV40 Tag transformation of the normal invasive trophoblast results in a premalignant phenotype. I. Mechanisms responsible for hyperinvasiveness and resistance to anti-invasive action of TGFbeta.

Invasion of the uterus by first trimester human placental extravillous trophoblast (EVT) cells depends on mechanisms shared by malignant cells. However, unlike tumor invasion, trophoblast invasion of the uterus is stringently controlled in situ by local molecules such as transforming growth factor (TGF)beta. Since EVT cells possess active invasion-associated genes but are nontumorigenic, our objective was to induce premalignant and then malignant phenotype into a normal EVT cell line in order to identify the molecular basis of tumor progression. Simian virus 40 large T antigen (SV40 Tag) was introduced into a normal human first trimester invasive EVT cell line, HTR8, established in our laboratory. Since the HTR8 line has a limited in vitro lifespan of 12-15 passages, SV40 Tag-transformed cells were selected on the basis of extended lifespan. A long-lived line, RSVT-2, was produced and an immortalized subclone, RSVT2/C, was further derived under a forced crisis regimen. We examined transformation-induced alterations in proliferative and invasive abilities, responses to the invasion and proliferation-regulating growth factor TGFbeta and changes in gene expression for invasion-associated enzymes or enzyme inhibitors. RSVT-2 and RSVT2/C cell lines were hyperproliferative and hyperinvasive when compared with the parental HTR8 cell line. They were also variably resistant to the anti-proliferative and anti-invasive signals from TGFbeta. Since both cell lines remained non-tumorigenic in nude mice, these properties indicate that they attained a premalignant phenotype. Both cell lines showed reduced expression of tissue inhibitor of metalloproteases (TIMP)-1, while TIMP-2 and plasminogen activator inhibitor (PAI)-I expression was was also reduced in RSVT2/C cells, thus contributing to their hyperinvasiveness. Their resistance to the anti-invasive action of TGFbeta was explained by the failure of TGFbeta to upregulate TIMPs and PAI-I, in contrast to the TGFbeta-induced upregulation noted in parental HTR8 cells.