Comparison among differential pulse voltammetry, amperometric biosensor, and HPLC/DAD analysis for polyphenol determination

Polyphenols are widespread in vegetables and fruits. They can play an important role in human diet and health, and they influence the sensorial properties of many foods, and act as natural antioxidants. This study was conducted using HPLC/DAD, tyrosinase biosensor, and differential pulse voltammetry (DPV) analyses to detect polyphenolic compounds in natural complex matrices. The analyses were applied to a series of both standards and natural extracts derived from grape, olives, and green tea. The pure compounds include phenolic acids, flavones, flavonols, catechins, tannins, and oleuropein. HPLC/DAD, DPV, and the biosensor approach were used as independent analytical techniques. Bare graphite screen-printed electrodes were employed in DPV and in the biosensor analysis. The most accurate data were obtained by HPLC/DAD analysis, while the DPV approach using screen-printed electrodes could represent a quick screening method for the determination of polyphenols in natural extracts. Use of the biosensor for the analysis of complex matrices needs further study in order to improve its performance.     

Bronchogenic adenocarcinoma in a cat: an unusual case of metastasis to the skin

A 6-year-old, spayed, female, domestic shorthair cat was presented for decreased activity. A nodular lesion was found in the skin extending into the subcutaneous tissue of the right abdominal flank. On lateral and ventrodorsal radiographs of the thorax, an opacity involving the entire right caudal lung lobe and pleural effusion were noted. Cytologic evaluation of cells in the thoracic fluid and in the mass revealed a population of atypical epipthelial cells with marked anisocytosis and high N:C ratios, organized in acinar-like clusters. Multinucleated cells and several mitotic figures were found. The cytologic interpretation was carcinoma. Because of the progressive severity of clinical signs, the cat was euthanized. Histologic evaluation of tissues obtained at necropsy indicated a bronchogenic adenocarcinoma in the lung, with metastasis to the skin of the right flank, but no involvement of the digits. Based on immunohistochemical stains, the neoplastic cells strongly co-expressed cytokeratin and vimentin, and were negative for S-100 and actin-specific antigen. Bronchogenic adenocarcinoma is an uncommon neoplasm in cats, and the digits are the most common sites of metastasis. This case was unusual in that the skin of the abdominal wall was the primary site of metastasis, with no involvement of the digits.     

Fine-needle biopsy of external ear canal masses in the cat: cytologic results and histologic correlations in 27 cases

BACKGROUND: The cytologic diagnosis of ear canal tumors is difficult or impossible by swab alone because cell exfoliation may be poor and neoplastic cells may be masked by associated inflammation. Fine-needle biopsy (FNB) can be used to obtain a higher yield of cells for diagnosis. OBJECTIVE: The purpose of this study was to assess the efficacy and diagnostic value of FNB and cytologic examination in providing an accurate diagnosis of masses growing in the external ear canal of cats. METHODS: Cytologic specimens from masses in the external ear canal, taken under inhaled, general anesthesia, were classified into 4 groups: 1) ceruminous gland hyperplasia or adenoma, 2) ceruminous gland adenocarcinoma, 3) inflammatory polyps, and 4) other neoplastic and non-neoplastic masses. Cytopathologic diagnoses were compared with the final histopathologic diagnoses, and indices of diagnostic test accuracy (sensitivity, specificity, likelihood ratios, diagnostic-odds ratios) were calculated. RESULTS: Twenty-seven masses (from 25 cats, including 2 cats affected bilaterally) were included in the study. The results showed good correspondence between cytologic and histologic diagnoses with an overall agreement index (kappa) of .74, a diagnostic odds ratio of 22, and 100% (27/27) agreement in the diagnosis of inflammatory polyps versus neoplasia (both benign and malignant). CONCLUSIONS: FNB cytopathology of external ear masses in the cat was sufficiently accurate for distinguishing inflammatory polyps from neoplasia. For differentiation of benign proliferation and malignant neoplasia, however, histopathologic confirmation is recommended.     

Immunohistochemical detection of growth hormone (GH) in canine hepatoid gland tumors

The aim of this study was to detect immunohistochemically means growth hormone (GH) in 24 hepatoid gland adenomas and 5 hepatoid gland carcinomas and to compare the difference of immunoreactivity between types of tumors. The tumors were classified according to the WHO standards. Tissue sections which were prepared from formalin-fixed, paraffin wax-embedded tissues from 25 male and 4 female dogs were carried out immunostaining using polyclonal primary anti-hGH and EnVision method. Of 24 hepatoid gland adenomas (perianal gland adenomas) 23 (95.8%) were positive. All 5 hepatoid gland carcinomas (perianal gland carcinomas) were positive. No statistically significant differences in percentage of labelled cells between malignant and benign tumors were seen. The present demonstration of GH in hepatoid gland tumors adds new data on GH in extra-pituitary tissues and hormon-dependent tumors.     

Procalcitonin gene expression after LPS stimulation in the porcine animal model

Procalcitonin (PCT), recognised as a marker of sepsis, was investigated in a porcine model of endotoxic shock. The results showed that continuous IV infusion (1-4 h) of LPS (40 μg/kg) in pigs was able to induce a generalised increase of PCT expression in lung, heart, kidney and liver. The increase in PCT was significant only in kidney and was accompanied by an increase in IL-6 gene expression. In vitro results demonstrated that peripheral blood mononuclear cells (PBMCs), as well as endothelial cells, were potentially capable of contributing to in vivo extrathyroidal PCT production. These findings support previous data from pigs concerning the occurrence of widespread activation of PCT extrathyroidal gene expression during endotoxic shock in pigs. Nevertheless, the levels of PCT detected were very low, suggesting the need for additional studies to validate the pig as a reliable animal model for investigating the role of PCT in sepsis.     

Evaluation of the antifungal susceptibility of Malassezia pachydermatis to clotrimazole, miconazole and thiabendazole using a modified CLSI M27-A3 microdilution method

Background – In this study, we evaluated the antifungal susceptibility of Malassezia pachydermatis to clotrimazole (CTZ), miconazole (MCZ), and thiabendazole (TBD), azole derivatives employed in aural formulations labeled for treatment of canine otitis. Methods – The procedure for in vitro testing was based on the indications of the Clinical and Laboratory Standards Institute (CLSI) M27‐A3 microdilution method. A lipid‐enriched medium was employed to enhance the yeast growth (Christensen’s urea broth, with 0.1% Tween 80 and 0.5% Tween 40 as the lipid sources), while the inoculums size corresponded to approximately 1–5 × 10⁵ yeast cells/mL. Microplates were incubated at 37°C and read 48 h after inoculation. Azole MICs inhibiting fungal growth were the lowest drug concentrations that showed an optical density of ≤50% of the (drug‐free) growth control, as assessed by spectrophotometer (630 nm filter). Results – All isolates were inhibited by the three azoles, with different minimum inhibitory concentration (MIC) values. Most isolates were inhibited by drug concentrations of 2–8 (CTZ), 1–4 (MCZ), or 16–32 (TBD) μg/mL. These results are partially in agreement with the findings of previous studies, in which substantially higher/lower MICs were occasionally reported. This is likely because of the different methodologies employed. Such discrepancies may not apply to clinical situations, where the compounds are applied topically. Conclusion and clinical importance – The concept that clinical failure is linked to increased MICs is debatable, because significantly higher concentrations (in most cases at least 1,000 × the MIC) of the antifungals that were included in our study are routinely used in formulated products.     

Nutritional and pathogenic fungi associated with the pine engraver beetle trigger comparable defenses in Scots pine

Conifer bark beetles are often associated with fungal complexes whose components have different ecological roles. Some associated species are nutritionally obligate fungi, serving as nourishment to the larvae, whereas others are pathogenic blue-stain fungi known to be involved in the interaction with host defenses. In this study we characterized the local and systemic defense responses of Scots pine (Pinus sylvestris L.) against Ophiostoma brunneo-ciliatum Math. (a blue-stain pathogen) and Hyalorhinocladiella macrospora (Franke-Grosm.) Harr. (a nutritional fungus). These fungi are the principal associates of the pine engraver beetle, Ips acuminatus (Gyll.). Host responses were studied following inoculation with the fungi, singly and as a fungal complex, and by identifying and quantifying terpenoids, phenolic compounds and lignin. Although the length of the necrotic lesions differed between control (wound) and fungal treatments, only two compounds (pinosylvin monomethyl ether and (+)-α-pinene) were significantly affected by the presence of the fungi, indicating that Scots pine has a generic, rather than specific, induced response. The fact that both nutritional and blue-stain fungi triggered comparable induced defense responses suggests that even a non-pathogenic fungus may participate in exhausting host plant defenses, indirectly assisting in the beetle establishment process. Our findings contribute to the further development of current theory on the role of associated fungal complexes in bark beetle ecology.     

Transplacental transmission of field and rescued strains of BTV-2 and BTV-8 in experimentally infected sheep

Transplacental transmission of bluetongue virus has been shown previously for the North European strain of serotype 8 (BTV-8) and for tissue culture or chicken egg-adapted vaccine strains but not for field strains of other serotypes. In this study, pregnant ewes (6 per group) were inoculated with either field or rescued strains of BTV-2 and BTV-8 in order to determine the ability of these viruses to cross the placental barrier. The field BTV-2 and BTV-8 strains was passaged once in Culicoides KC cells and once in mammalian cells. All virus inoculated sheep became infected and seroconverted against the different BTV strains used in this study. BTV RNA was detectable in the blood of all but two ewes for over 28 days but infectious virus could only be detected in the blood for a much shorter period. Interestingly, transplacental transmission of BTV-2 (both field and rescued strains) was demonstrated at high efficiency (6 out of 13 lambs born to BTV-2 infected ewes) while only 1 lamb of 12 born to BTV-8 infected ewes showed evidence of in utero infection. In addition, evidence for horizontal transmission of BTV-2 between ewes was observed. As expected, the parental BTV-2 and BTV-8 viruses and the viruses rescued by reverse genetics showed very similar properties to each other. This study showed, for the first time, that transplacental transmission of BTV-2, which had been minimally passaged in cell culture, can occur; hence such transmission might be more frequent than previously thought.     

The Bio-Functional Properties of Pigmented Cereals may Involve Synergies among Different Bioactive Species

This study was aimed at characterizing the anthocyanins and phenolics profile in different varieties of pigmented corn and wheat and in some of their milling fractions. Acid/ethanol extracts were used to assess total anthocyanins, overall antioxidant activity, the overall polyphenol profile, and for evaluating the inhibition of pancreatic α-amylase and of intestinal α-glucosidase. Both enzymes were inhibited in a dose-dependent manner by all extracts, but individual extracts had specific effects on each enzyme. Anti-inflammatory response was evaluated by using acid-free extracts and Caco-2 cells transiently transfected with a luciferase reporter gene responding to cytokine stimulation. The immune response of interleukin-stimulated cells decreased significantly in a dose-dependent manner in the presence of 20–50 μM/l anthocyanins from all grains extracts, again with a different efficiency. The inhibitory ability and the anti-inflammatory capability of these extracts are in most cases higher than in similar extracts from other sources, suggesting that activities in each extract may imply specific synergies between anthocyanins and other phenolics.