Micro-computed tomography of pulmonary fibrosis in mice induced by adenoviral gene transfer of biologically active transforming growth factor-β1

Background

Micro-computed tomography (micro-CT) is a novel tool for monitoring acute and chronic disease states in small laboratory animals. Its value for assessing progressive lung fibrosis in mice has not been reported so far. Here we examined the importance of in vivo micro-CT as non-invasive tool to assess progression of pulmonary fibrosis in mice over time.

Methods

Pulmonary fibrosis was induced in mice by intratracheal delivery of an adenoviral gene vector encoding biologically active TGF-ß1 (AdTGF-ß1). Respiratory gated and ungated micro-CT scans were performed at 1, 2, 3, and 4 weeks post pulmonary adenoviral gene or control vector delivery, and were then correlated with respective histopathology-based Ashcroft scoring of pulmonary fibrosis in mice. Visual assessment of image quality and consolidation was performed by 3 observers and a semi-automated quantification algorithm was applied to quantify aerated pulmonary volume as an inverse surrogate marker for pulmonary fibrosis.

Results

We found a significant correlation between classical Ashcroft scoring and micro-CT assessment using both visual assessment and the semi-automated quantification algorithm. Pulmonary fibrosis could be clearly detected in micro-CT, image quality values were higher for respiratory gated exams, although differences were not significant. For assessment of fibrosis no significant difference between respiratory gated and ungated exams was observed.

Conclusions

Together, we show that micro-CT is a powerful tool to assess pulmonary fibrosis in mice, using both visual assessment and semi-automated quantification algorithms. These data may be important in view of pre-clinical pharmacologic interventions for the treatment of lung fibrosis in small laboratory animals.     

The evolution of insulin resistance in muscle of the glucose infused rat

Glucose infusion into rats causes skeletal muscle insulin resistance that initially occurs without changes in insulin signaling. The aim of the current study was to prolong glucose infusion and evaluate other events associated with the transition to muscle insulin resistance. Hyperglycemia was produced in rats by glucose infusion for 3, 5 and 8 h. The rate of infusion required to maintain hyperglycemia was reduced at 5 and 8 h. Glucose uptake into red quadriceps (RQ) and its incorporation into glycogen decreased between 3 and 5 h, further decreasing at 8 h. The earliest observed change in RQ was decreased AMPKα2 activity associated with large increases in muscle glycogen content at 3 h. Activation of the mTOR pathway occurred at 5 h. Akt phosphorylation (Ser⁴⁷³) was decreased at 8 h compared to 3 and 5, although no decrease in phosphorylation of downstream GSK-3β (Ser⁹) and AS160 (Thr⁶⁴²) was observed. White quadriceps showed a similar but delayed pattern, with insulin resistance developing by 8 h and decreased AMPKα2 activity at 5 h. These results indicate that, in the presence of a nutrient overload, alterations in muscle insulin signaling occur, but after insulin resistance develops and appropriate changes in energy/nutrient sensing pathways occur.     

Microbial communities in sugarcane field soils with and without a sugarcane cropping history

Microbial communities in rhizosphere soil from sugarcane (Saccharum inter-specific hybrids) and bulk soil were compared at paired field sites with and without a sugarcane cropping history to determine whether monoculture affects soil microbial community composition. Differences were evaluated for culturable microorganisms and functional diversity indicated by community level physiological profiles (CLPP). Qualitative differences in rhizosphere bacterial communities were detected between sites with no sugarcane cropping history (N_site) and sites with a long-term sugarcane cropping history (L_site). More fluorescent pseudomonads were detected in N_site than L_site rhizosphere soil at two of three sites, and Actinobacteria were more numerous in N_site than L_site rhizosphere soil at one site. Fusarial fungi were more numerous in N_site than L_site rhizosphere soils. Bacteria were more numerous in rhizosphere soil compared to bulk soil. Total bacterial, pseudomonad, and Actinobacteria population densities were greater in bulk soil from an N_site compared to an L_site. CLPP distinguished bulk from rhizosphere soil at one of two sites and N_site and L_site rhizosphere soils at two of four sites. Site affected CLPP similarity more than cropping history. The results demonstrated that sugarcane monoculture can affect the composition of the microbial community in field soil. The findings have possible implications for reduced yields associated with sugarcane monoculture.     

Structural alterations in the Bacillus subtilis Spo0A regulatory protein which suppress mutations at several spo0 loci

Secondary site mutations that restore sporulation to sporulation-defective spo0F or spo0B deletion mutants were found to reside in the spo0A gene. Sequence analysis of 23 such sof mutants showed that the sof mutations fell into six classes of missense codon changes, primarily in the conserved amino-terminal domain of the response regulator Spo0A protein. Changes were observed in codons 12, 14, 60, 92, and 121. The residues affected were predominantly located in the potential turn regions at one end of the amino-terminal conserved domain on the same topological face as the active site aspartate residues. The ability of sof mutations to suppress deficiencies in the transmitter kinases, KinA and KinB, of two-component regulatory systems was tested. All of the sof mutations suppressed the sporulation deficiency of kinA mutants but only two classes among five tested suppressed kinB mutations. sof mutants segregated Spo- colonies at high frequency. Five of these Spo- mutants were found to result from mutations in the spo0A locus that reversed the effect of the sof mutatation. One of these was sequenced and found to have the original sof mutation and a new mutation, sos, at codon 105. The accumulation of sos mutations in sof strains suggested that the sof mutations have a subtle, yet deleterious, effect on the growth of the cell. The results suggested that the sof mutations increase the avidity for or reactivity with transmitter kinases in an allele-specific manner, although in some cases it is possible that the sof mutations obviate the need for phosphorylation to activate the Spo0A protein. An alternative hypothesis is presented in which the sof mutations play the role of bypass mutations for kinases.     

Antigen processing and the human T cell receptor repertoire for insulin

Three human T cell lines specific for the A loop of beef insulin were studied to determine the requirements for Ag processing. The data show that the conformation of the A loop of insulin is required for recognition and that the B chain of insulin per se is not necessary for this response. Processing of native insulin was required for responses of all three T cell lines; however, each displayed a different pattern of sensitivity to inhibition of processing and aldehyde fixation of APC. A peptide comprised of two disulfide-linked A chains was partially stimulatory when presented by fixed APC whereas A chain monomers and disulfide-linked A and B chain peptides were not. The response to native insulin, peptides, and A chain dimers was sensitive to chloroquine suggesting that none of these moieties is the terminal processed peptide recognized by insulin immune T cells. The unique patterns of fine specificity, processing requirements, and recognition of aldehyde-fixed antigen-MHC for each T cell line suggest the hypothesis that Ag processing leads to heterogeneity of the T cell repertoire for a single epitope of insulin.     

Expression of high affinity receptors for murine interleukin 4 (BSF-1) on hemopoietic and nonhemopoietic cells

In this report a method for the affinity purification and radiolabeling of recombinant mouse interleukin (IL)-4 is described. It is shown on the basis of several criteria that IL-4 retains full biologic activity after radioiodination and can therefore be used as a valid model for measuring the binding characteristics of native IL-4. By using Scatchard plot analysis of equilibrium binding data, it is demonstrated that 125I-IL-4 binds to a high affinity cell surface receptor which is expressed by both hemopoietic and nonhemopoietic cells. The dissociation constant for 125I-IL-4 (Kd = 20 to 60 pM) corresponds to the concentration of IL-4 which gives 50% biologic activity (i.e., 10 to 30 pM). Binding of 125I-IL-4 is rapid (t1/2 of 2 min), whereas dissociation occurs at a slow rate (t1/2 approximately 4 hr). The IL-4 receptor shows a high degree of specificity. Whereas unlabeled mouse IL-4 competed with mouse 125I-IL-4 in an equimolar fashion for binding to IL-4 receptors, several other lymphokines, including mouse IL-2, IL-3, interferon-gamma, granulocyte-macrophage colony-stimulating factor, and human IL-1, IL-2, and IL-4 were unable to inhibit, even at molar excesses of 400 to 800-fold. At 37 degrees C, 125I-IL-4 is rapidly internalized (approximately 200 molecules/cell/min) by HT-2 cells, with at least 85% of cell surface receptors being functional in this respect. Receptors for IL-4 were found to be expressed by subclasses of T and B cells, mast cells, macrophages, and by cells of the myeloid and erythroid lineages. This wide distribution of receptor expression closely matches the known spectrum of biologic activities of IL-4, including proliferation and/or differentiation of T and B cells, mast cells and granulocytes, and induction of macrophage antigen-presenting capacity. IL-4 receptors were also found on a variety of nonhemopoietic cells such as cloned stromal cell lines from the bone marrow, spleen, thymus, and brain, and on muscle, brain, melanoma, fibroblast, and liver cells. Indeed, only 5 of more than 90 cell types tested have undetectable numbers of IL-4 receptors. The biologic effects of IL-4 on nonhemopoietic cells have not yet been reported and await elucidation.     

Effect of some food sources associated with cassava in Africa on the development, fecundity and longevity of Euseius fustis (Pritchard and Baker) (Acari: Phytoseiidae)

Various foods associated with cassava were tested for their effect on the development, fecundity and longevity of Euseius fustis, the most common phytoseiid species found on cassava in Africa. Euseius fustis developed successfully to adulthood on the spider mite prey species Mononychellus tanajoa (Bondar) and Oligonychus gossypii (Zacher) and on pollen from maize, castor bean and cassava. Euseius fustis also completed development on water-diluted phloem exudate from cassava, diluted honeydew from the cassava mealybug and on various pollen and prey combinations. When reared on Tetranychus urticae Koch prey or free water only, E. fustis did not develop past the deutonymphal stage. All larvae held on clean leaf discs on water-soaked cotton died without moulting, suggesting that E. fustis must feed in order to moult to the nymphal stages. Diets of maize plus castor bean pollen and maize pollen plus M. tanajoa resulted in the highest rate of development, the highest fecundity and the greatest longevity. Castor bean pollen alone and maize pollen alone produced a higher fecundity and greater longevity than M. tanajoa tested alone. A colony of E. fustis reared continuously for seven generations on castor bean pollen produced nine times more adult females than a colony of E. fustis reared continuously on M. tanajoa. No negative effects on the development and fecundity of E. fustis were observed after seven generations were reared on pollen.     

Laboratory Evaluation of a Laboratory-selected Organophosphate-resistant Strain of Amblyseius finlandicus (Acari: Phytoseiidae) for Possible Use in Finnish Apple Orchards

The survival of adults and immatures, and reproduction at rates of 150, 300 and 900 ppm a.i. of azinphosmethyl were compared among four types of Amblyseius finlandicus females and their offspring; a laboratory-selected colony (SEL16) resistant to azinphosmethyl, an unselected base colony (Tuorla), reciprocal F1 females from crosses between SEL16 and a very susceptible colony (Hiekkaharju). The SEL16 and reciprocal F1 females had a higher survivorship than the unselected Tuorla base colony after 2 and 4 days on leaves treated with the three concentrations of insecticide. Azinphosmethyl decreased oviposition by the surviving females of all types, but the decrease was smaller in the SEL16 strain than in the base colony. The survivorship of immatures of SEL16 on fresh azinphosmethyl residues after 6 days was low at all three concentrations, but no survivors were found in the base colony at any of the concentrations. Females deposited significantly more eggs on the untreated areas of the leaves. The importance of this behavior for biological control is discussed. There were no significant differences in developmental rate, ovipositional rate, sex ratio and ovipositional period between the SEL16 and unselected base colony on untreated leaf substrates, suggesting that resistance was not associated with decreased fitness. A mode of inheritance study indicated that azinphosmethyl resistance was incompletely dominant. This was also suggested by the results of bioassays, where the reciprocal F1 females performed nearly as well as the SEL16 strain on leaves treated with field rates of azinphosmethyl.     

Monitoring Laboratory and Field Biotypes of the Walnut Aphid Parasite, Trioxys pallidus, in Population Cages Using RAPD-PCR

A discriminate analysis, based on the frequencies of occurrence of six random amplified polymorphic DNA (RAPD) fragments, distinguished individuals of a pesticide-resistant laboratory biotype (SEL) of the walnut aphid parasite, Trioxys pallidus Haliday (Hymenoptera: Aphidiidae), from individuals of a wild orchard biotype (RB). In mixed populations in the laboratory, the SEL biotype was heavily favored, irrespective of pesticide treatment, suggesting a high degree of laboratory adaptation. The resistance levels in untreated and pesticide-treated mixed (SEL + RB) populations were not different after eight generations. Analyses of RAPDpolymerase chain reaction (PCR) data suggested that the selective advantage of the SEL biotype may have been aided by a partial mating incompatibility between the biotypes. The data can be explained by either of two types of reproductive incompatibility: (1) RB females mating with SEL males produced only male offspring; or (2) the RB biotype could not successfully find mates under laboratory conditions. Successful discrimination of T. pallidus biotypes and their hybrid progeny in population cages suggests that RAPD-PCR offers a method to determine the fate of the pesticide-resistant SEL biotype after release into orchards populated by the RB biotype.