Comparison of two prepill cortisol concentrations in dogs with hypercortisolism treated with trilostane

Background

The ideal method for monitoring trilostane therapy in dogs with hypercortisolism is still open to debate. Recently, determination of the pre-trilostane (prepill) cortisol concentration has been proposed to be more repeatable than either post-trilostane or post-ACTH cortisol. The aim of this study was to compare two prepill cortisol concentrations in dogs with hypercortisolism during trilostane therapy. Sixteen client-owned dogs with naturally occurring hypercortisolism were prospectively included and cortisol concentrations were measured twice, 1?h apart, before the morning trilostane dose (prepill 1 and 2 cortisol).

Results

A total of 47 prepill cortisol measurement pairs were included. Compared to prepill 1, prepill 2 cortisol was higher in 15, equal in 8 and lower in 24 pairs. Group agreement between prepill 1 and 2 cortisol was 70% (moderate agreement - weighted kappa 0.55). In 30% of the pairs, group assignment was discrepant, implying a different therapeutic decision. In some dogs certain circumstances (e.g. excessive barking, difficulties during blood collection, excitement at arrival) were identified as potential factors explaining the discrepancy between prepill 1 and 2 cortisol measurements.

Conclusions

In a substantial number of dogs treated with trilostane, the two prepill cortisol concentrations differed. Part of this difference might be ascribable to stressful events during test performance. When using prepill cortisol measurements to monitor trilostane therapy, recording of any incident during handling that might affect cortisol release might be helpful to make a reliable decision about a trilostane dose adaptation.

     

Emergence of Resistance to Fluconazole in <Emphasis Type="Italic">Candida albicans</Emphasis> Isolated From Vaginal Discharge

Purpose of Review

To provide information about the emergence of fluconazole resistance in Candida albicans isolated from vaginal discharge, in a global context, and to update the in vitro susceptibility profile of this species from Argentina.

Recent Findings

Vulvovaginal candidiasis is the second most common vaginal infection after vaginal bacteriosis. C. albicans remains the prevalent etiological yeast species, and despite antifungal treatment, the rate of recurrence remains high, which may be associated to antifungal resistance.

Summary

Data here presented were obtained from the study of C. albicans strains isolated from patients with clinical signs of vulvovaginal candidiasis from 1996 to 2017. Data obtained could represent the susceptibility profile of C. albicans strains circulating in Argentina and could be of potential usefulness to monitor and guide therapy, and also suggests the need for greater surveillance programs to detect fluconazole resistance over time.

     

Correlative Microscopy of Vitreous Sections Provides Insights into BAR-Domain Organization In Situ

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Preliminary studies on the effect of Anaplasma marginale antibodies ingested by Dermacentor andersoni ticks (Acari:Ixodidae) with their blood meal on infections in salivary glands

The effect of Anaplasma marginale antibodies ingested with the tick blood meal was tested on infected male ticks that were allowed to feed on cattle immunized with the erythrocytic stage of A. marginale. The experiments were done in two trials. Trial 1 was done using splenectomized calves (two calves per treated and control groups) while ticks in trial 2 were fed on intact yearling cattle (four cattle per treated and control groups). The cattle were immunized with purified outer membrane proteins of erythrocyte-derived A. marginale using saponin (trial 1) or monophosphoryl lipid-A-trehalose dicorynomycolate adjuvant (trial 2). The corresponding control cattle received adjuvant only. All cattle were challenged using Dermacentor andersoni males infected as adults that were allowed to feed for 7 days. In trial 1, the ticks were allowed to feed a second time on susceptible calves to test whether exposure of ticks to immunized cattle affected their ability to transmit anaplasmosis. Infections in fed ticks were monitored by determining the infection rates in salivary glands with an A. marginale-specific RNA probe and light microscopy. Vaccine-derived antibodies ingested with the tick blood meal did not appear to affect the development of A. marginale in previously infected ticks. The infection rates in the salivary glands were not significantly different among ticks fed on immunized versus adjuvant control cattle. When the vaccine-exposed ticks in trial 1 were allowed to feed a second time on susceptible calves, the resulting clinical symptoms of anaplasmosis were similar to those of the controls. There was no statistically significant effect of tick exposure to the anti-erythrocytic stage antibody on the development of salivary gland infection or transmission of A. marginale by ticks.     

Purification, characterization, and specificity of dextranase inhibitor (Dei) expressed from Streptococcus sobrinus UAB108 gene cloned in Escherichia coli.

The dextranase inhibitor gene (dei) from Streptococcus sobrinus UAB108 was previously cloned, expressed, and sequenced. Its gene product (Dei) has now been purified as a single band with apparent molecular mass of 43 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The specific activity of Dei increased 121-fold upon purification. Most Dei activity (91.2%) was located in the periplasmic fraction from recombinant Escherichia coli cells. Dei competitively inhibits dextranase (Dex). This competitive inhibition mechanism has been further shown by detection and recovery of the intermediate enzyme-inhibitor (Dex-Dei) complex by gel filtration technology using fast protein liquid chromatography. Calibration of their molecular masses indicated that native Dei exists as a tetramer, Dex exists as dimer, and the Dex-Dei complex consists of two Dex molecules with two Dei molecules. Deletion analysis indicates that the intact Dei molecule is essential for Dei activity but not for glucan binding and immune cross-reaction. Dei is a special kind of glucan-binding protein with ability to inhibit Dex with high specificity. It can inhibit endogenous Dex, which can make more branches in glucan with the cooperation of the glucosyltransferase GTF-I. This inhibition cause the accumulation of water-soluble glucan. The latter reaction product can inhibit plaque formation and adherence of the mutans group of streptococcal cells. Dei derived from S. sobrinus UAB108 can inhibit only Dex from S. sobrinus (serotypes d and g), S. downei (previously S. sobrinus, serotype h), and S. macacae (serotype h). This finding suggests that Dei is another important protein existing in some serotypes of the mutans group of streptococci which participates in sucrose metabolism through its interaction with Dex.     

Production of parthenolide in organ cultures of feverfew

In vitro culture ofTanacetum parthenium (L.) Sch.Bip. was initiated from aseptically germinated seedlings. culture was derived from nodal explants of the seedlings on MS medium containing 4.44 μM (1.0 mg 1⁻¹ ) 6-benzylaminopurine (BA) and 0.54 μM (0.1 mg 1⁻¹) of α-naphthaleneacetic acid (NAA). Transformed roots were obtained by infection of the stems of aseptically grown seedlings withAgrobacterium rhizogenes LBA 9402. The parthenolide content in the cultivated plant organs was investigated by RP-HPLC. The production of the compound was strongly influenced by the genotype of the parent plant and ranged from 0.13% to 0.75% dry weight in the shoots of the rooted plantlets grownin vitro. The yield of the compound in multiple shoot cultures ofT.parthenium reached 60% of that found in the shoots of rooted plantlets. In contrast to shoots, only trace amounts of parthenolide could be detected in some clones of transformed roots and the roots of plantlets.     

Higher‐order assemblies of oligomeric cargo receptor complexes form the membrane scaffold of the Cvt vesicle

Selective autophagy is the mechanism by which large cargos are specifically sequestered for degradation. The structural details of cargo and receptor assembly giving rise to autophagic vesicles remain to be elucidated. We utilize the yeast cytoplasm‐to‐vacuole targeting (Cvt) pathway, a prototype of selective autophagy, together with a multi‐scale analysis approach to study the molecular structure of Cvt vesicles. We report the oligomeric nature of the major Cvt cargo Ape1 with a combined 2.8 Å X‐ray and negative stain EM structure, as well as the secondary cargo Ams1 with a 6.3 Å cryo‐EM structure. We show that the major dodecameric cargo prApe1 exhibits a tendency to form higher‐order chain structures that are broken upon interaction with the receptor Atg19 in vitro. The stoichiometry of these cargo–receptor complexes is key to maintaining the size of the Cvt aggregate in vivo. Using correlative light and electron microscopy, we further visualize key stages of Cvt vesicle biogenesis. Our findings suggest that Atg19 interaction limits Ape1 aggregate size while serving as a vehicle for vacuolar delivery of tetrameric Ams1.     

HIV-1 Nef promotes migration and chemokine synthesis of human basophils and mast cells through the interaction with CXCR4

Background

The Nef protein can be detected in plasma of HIV-1-infected patients and plays a role in the pathogenesis of HIV-1. Nef produced during the early stages of infection is fundamental in creating the ideal environment for viral replication, e.g. by reducing the ability of infected cells to induce an immune response.

Aim

Based on previous experience showing that both Tat and gp41 of HIV-1 are potent chemotactic factors for basophils and mast cells, and gp120 is a powerful stimulus for the release of histamine and cytokines (IL-4 and IL-13) from basophils, in this study we aimed to verify if the HIV Nef protein can exert some effects on basophils and mast cells purified from healthy volunteers through the interaction with the CXCL12 receptor, CXCR4.

Methods

Basophils purified from peripheral blood cells of 30 healthy volunteers and mast cells obtained from lung tissue of ten healthy volunteers were tested by flow cytometric analysis, chemotaxis and chemokine production by ELISA assays.

Results

Nef is a potent chemoattractant for basophils and lung mast cells obtained from healthy, HIV-1 and HIV-2 seronegative individuals. Incubation of basophils and mast cells with Nef induces the release of chemokines (CXCL8/IL-8 and CCL3/MIP-1α). The chemotactic activity of Nef on basophils and mast cells is mediated by the interaction with CXCR4 receptors, being blocked by preincubation of FcεRI⁺ cells with an anti-CXCR4 Ab. Stimulation with Nef or CXCL12/SDF-1α, a CXCR4 ligand, desensitizes basophils to a subsequent challenge with an autologous or heterologous stimulus.

Conclusions

These results indicate that Nef, a HIV-1-encoded α-chemokine homolog protein, plays a direct role in basophils and mast cell recruitment and activation at sites of HIV-1 replication, by promoting directional migration of human FcεRI⁺ cells and the release of chemokines from these cells. Together with our previous results, these data suggest that FcεRI⁺ cells contribute to the dysregulation of the immune system in HIV-1 infection.

     

Colorimetric Assay for Lysine Decarboxylase in Escherichia coli

A new assay is described for lysine decarboxylase. It is rapid and reproducible in assaying large numbers of samples, a situation in which earlier methods were less convenient. The new method is valuable in the study of peptide fractions and amino acid mixtures which stimulate induction of lysine decarboxylase. It may be useful for work on enzyme structure and modification, genetics, and kinetics.     

Pediococci Residing on Plants

The pediococci residing on plants resemble the lactobacilli, but they differ from the streptococci in their limited distribution and low population level on plants. They are a subgroup within the genus Pediococcus which grow freely in neutral media and require neither NaCl nor CO(2). They are most readily recognized by the ability to initiate growth in liquid media, acidified to pH 5.0, which contain 1.5% sodium acetate. In stained preparations the cells occur singly and in pairs, short chains, and clusters. The occurrence of two-dimensional tetrads may be rare; this varies with the individual culture and with the culture medium. The terminal pH in 2% glucose broth varies from 3.6 to 4.3. Ability to initiate growth at 45 C, production of ammonia from arginine, dissimilation of malate, and fermentation of arabinose are confirmatory characteristics. The subgroup contains only two quite similar, but differentiable, species. P. acidilactici initiates growth at 50 C and produces catalase on heated blood medium but does not produce acid-sensitive catalase; a majority of the strains fail to initiate growth at 10 C and many fail to ferment maltose and lactose. P. pentosaceus initiates growth at 10 C but not at 50 C and produces acid-sensitive catalase; catalase production on heated blood medium is transient; a majority of the cultures ferment maltose, salicin, and trehalose. No carbohydrate serves reliably to differentiate between the species. The guanine plus cytosine ratio of P. pentosaceus deoxyribonucleic acid (DNA) was determined to be 35.1 +/- 1.2 and that of P. acidilactici DNA is 38.5 +/- 0.8.