Respiratory applications for the future: one perspective.

Respiratory diagnostic instruments previously available only to referral centers should be converted into miniature, portable, wireless medical devices for use in practice. Internet and wireless connections of these instruments should expedite the process of seeking second opinions and in general information sharing. We are moving into a world of greater objectivity that should free us from speculation in some areas, yet open new avenues for research and innovation in others.     

Alfalfa metabolism of propham

Root-treated alfalfa absorbs, translocates, and metabolizes [phenyl-¹⁴C]isopropyl carbanilate ([¹⁴C]propham). After 7 days of root treatment, the distribution of radiolabel was 73% for shoots and 27% for roots. Shoots and roots were extracted and separated into the polar, nonpolar, and solid residual components using a mixture of chloroform, methanol and water. The insoluble residues accounted for approximately 40% of the ¹⁴C found in shoots and roots. The nonpolar fraction (6.1% of the radiolabel in shoots and roots) was not characterized, but was shown to be some component other than parent propham. Propham was not found in either shoots or roots. The polar metabolites were partly purified on Amberlite XAD-2. Cellulase-liberated aglycones were derivatized and separated by high-performance liquid and gas-liquid chromatography. The infrared, nuclear magnetic resonance, and mass spectral data showed that the polar metabolites of alfalfa shoots and roots were glycoside conjugates of isopropyl 2-hydroxycarbanilate (2-hydroxypropham) and isopropyl 4-hydroxycarbanilate (4-hydroxypropham). Conjugated 4-hydroxypropham accounted for 45.9% of the ¹⁴C in the shoots and 3.4% of the ¹⁴C in the roots. Conjugated 2-hydroxypropham accounted for 3.4% of the ¹⁴C in the shoots and 1.4% of the ¹⁴C in the roots.     

Mercapturic acid formation in the metabolism of propachlor,CDAA, and fluorodifen in the rat

When [¹⁴C]F₃-fluorodifen (2,4′-dinitro-4-trifluoromethyl diphenylether), carbonyl-[¹⁴C]CDAA (N,N-diallyl-2-chloroacetamide), and carbonyl-¹⁴C-propachlor (2-chloro-N-isopropylacetanilide) were fed to rats, 57 to 86% of the ¹⁴C was excreted via the urine within 48 hr. Although very little radioactivity was excreted in the feces of CDAA-treated rats, 15–22% of the ¹⁴C was excreted in the feces of propachlor- of fluorodifentreated rats and an average of 8% of the ¹⁴C remained in these rats 48 hr after treatment. Oxidation of the ¹⁴C label to [¹⁴C]O₂ was not a major process in the metabolism of these herbicides. The only major radioactive metabolite present in the 24-h urine of fluorodifen-treated rats, 2-nitro-4-trifluoromethylphenyl mercapturic acid, accounted for 41% of the administered dose of ¹⁴C. In the metabolism of CDAA, the corresponding mercapturic acid accounted for 76% of the dose; it was the only major metabolite present in the 24-h urine. In contrast, three major metabolites were detected in the 24-h urine of propachlortreated rats, and the mercapturic acid accounted for only 20% of the dose. The mercapturic acid of each herbicide was identified by mass spectrometry.     

Characterization of a wild-type strain of Francisella tularensis isolated from a cat.

Francisella tularensis type A is the primary cause of tularemia in animals and humans in North America. The majority of research on F. tularensis has been done with the attenuated live vaccine strain (LVS), which is a type B, but very few wild-type F. tularensis strains have been characterized. A gram-negative coccobacillus that was isolated in pure culture from the lungs of a cat that died after being lost for 5 days was received for identification at the Virginia-Maryland Regional College of Veterinary Medicine Teaching hospital. The isolate (strain TI0902) was not identified (or was misidentified) by commercial identification systems; however, it was identified as F. tularensis subspecies tularensis (type A) by sequencing a portion of the 16S ribosomal RNA gene. Furthermore, repetitive extragenic palindromic sequences-polymerase chain reaction amplified a 4-kb DNA fragment from TI0902 that was characteristic of F. tularensis type A but not type B. The electrophoretic profile of the lipopolysaccharide of strain TI0902 was identical to that of the LVS by Western blotting with antiserum to LVS. The protein-enriched outer membrane of strain TI0902 contained 6-8 proteins, which were similar in molecular size to those from the LVS. Electron microscopy of negatively stained and alcian blue-stained LVS and TI0902 cells showed that both strains were coccobacillary in shape and may be encapsulated. However, after mouse challenge, the TI0902 strain was clearly more virulent than the LVS strain. Results of this study indicate that the genotype and phenotype of wild-type F. tularensis type A strain TI0902 is similar, but not identical, to that of the LVS strain. Further studies will help determine whether pathogenesis and host-pathogen interactions are also similar between the 2 strains.     

Non-target toxicology of a new mosquito larvicide, trypsin modulating oostatic factor

Trypsin modulating oostatic factor (TMOF), a peptide hormone originally isolated from the ovaries of adult Aedes aegypti, is currently under commercial development as a new pesticide chemistry with a novel mode of action for the control of larval mosquitoes. The objective of the current research is to evaluate potential risks of the use of TMOF as an insecticide on non-target organisms. TMOF (YDPAP₆) was degraded in vitro (as determined by HPLC and LC/MS) to DPAP₆, PAP₆, and then AP₆ by leucine aminopeptidase, a pancreatic enzyme found in the digestive system of vertebrates. The rate of degradation of TMOF and PAP₆ was significantly greater than that of DPAP₆, while no metabolism of AP₆ was found. TMOF technical insecticide was produced on a commercial scale by recombinant yeast (heat-killed before application). The technical TMOF when administered in a single dose by gavage to male and female mice at 2000 mg dry weight/kg body weight produced no negative effects as compared to controls up to 12 days after treatment. When male and female mallard ducks were treated by gavage with 1250 mg dry weight of technical TMOF/kg body weight each day for 5 days, again no toxic effects were noted through 35 days after the last treatment. TMOF technical insecticide was also applied to the shaved skin of male and female rabbits at the rate of 2000 mg/kg for 1-2 days, with no effect. The end point observations in these in vivo experiments were mortality; changes in growth rate, behavior, body structure, and color; and possible lesions observed during necropsy. Finally, Daphnia incubated with technical TMOF in rearing water at the level of 1.0 × 10⁶ yeast cells/ml (10 mg/ml) also demonstrated no negative effects on mortality, growth, molting, time to first brood, and production of viable neonates. It appears from these studies that TMOF can be degraded by vertebrate digestive proteases and technical TMOF is not toxic to the non-target organisms examined.     

Immune Function in Critically Ill Dogs

Background

People with critical illness (CI) commonly develop various forms of immune dysfunction, however, there is limited information concerning immune dysfunction in dogs with CI.

Hypothesis

The immune response in CI dogs differs from that of healthy dogs.

Animals

Immunologic variables were compared between 14 dogs with CI, defined as APPLE_fast score of >20 points, admitted to the University of Missouri Veterinary Health Center Small Animal Clinic Intensive Care Unit and healthy controls (n = 15).

Methods

Cohort study evaluating constitutive and lipopolysaccharide (LPS)‐stimulated TNF‐α, IL‐6, and IL‐10 production, phagocytosis of opsonized E. coli and respiratory burst capacity after opsonized E. coli or phorbol 12‐myristate 13‐acetate (PMA) stimulation, peripheral blood lymphocyte phenotype, and monocyte expressions of HLA‐DR and TLR‐4.

Results

Lipopolysaccharide‐stimulated leukocyte TNF‐α (median, Q1, Q3; CI, 49, 49, 120; control, 655, 446, 1174 pg/mL; P = < 0.001), IL‐6 (median, Q1, Q3; CI, 49, 49, 64; control, 100, 49, 166 pg/mL; P = 0.029), and IL‐10 (CI, 49, 49, 56; control, 96, 49, 203 pg/mL; P = 0.014) production and both E. coli (median, Q1, Q3; CI, 60.5, 43, 88.5; control, 86.6, 81, 89.2%; P = 0.047) and PMA (CI, 40, 11.7, 70; control, 93, 83, 97.6%; P = < 0.001)‐stimulated respiratory burst capacity significantly decreased in CI dogs. Percentage of monocytes expressing TLR‐4 greater in the CI dogs (median, Q1, Q3; CI, 46.9, 24.3, 64.2; control, 16.4, 9.4, 26.2%; P = 0.005).

Conclusion

These findings suggest dogs with CI develop immune system alterations that result in reduced respiratory burst function and cytokine production despite upregulation of TLR‐4.