Rapid detection of Candida albicans in clinical blood samples by using a TaqMan-based PCR assay

We describe a rapid and reproducible PCR assay for quantitation of the Candida albicans ribosomal DNA (rDNA) in clinical blood samples based on the TaqMan principle (Applied Biosystems), in which a signal is generated by cleavage of a template-specific probe during amplification. We used two fluorogenic probes based on universal, fungus-specific primers, one for the detection of C. albicans species DNA and one for the detection of all Candida genus DNA. C. albicans blastoconidia mixed with whole blood in a titration experiment yielded a linear PCR signal over a range of 3 orders of magnitude. The TaqMan-based PCR assay for C. albicans exhibited a low limit of detection (5 CFU/ml of blood) and an excellent reproducibility (96 to 99%). While the C. albicans species-specific probe had 100% specificity for C. albicans, all Candida genus-specific probes cross-reacted with other organisms likely to coinfect patients with C. albicans infections. On the basis of these data, we determined the C. albicans loads with a species-specific probe from 122 blood samples from 61 hematology or oncology patients with clinically proven or suspected systemic Candida infections. Eleven positive samples exhibited a wide range of C. albicans loads, extending from 5 to 100,475 CFU/ml of blood. The sensitivity and specificity of the present assay were 100 and 97%, respectively, compared with the results of blood culture. These data indicate that the TaqMan-based PCR assay for quantitation of C. albicans with a species-specific probe provides an attractive alternative for the identification and quantitation of C. albicans rDNA in pure cultures and blood samples.     

Minor histocompatibility antigen DDX3Y induces HLA-DQ5-restricted T cell responses with limited TCR-Vbeta usage both in vivo and in vitro.

In vitro stimulation of human female T cells with male HLA-identical dendritic cells resulted in the generation of HLA-DQB1*0501/0502-restricted minor histocompatibility H-Y antigen-specific CD4(+) T cell clones. Two clones generated from different HLA-identical pairs were analyzed. Use of HLA-DQ5-expressing female Epstein-Barr virus transformed B lymphoblastoid cell lines transfected with various H-Y genes and loaded with overlapping peptides demonstrated that both T cell clones are specific for a peptide encoded by DDX3Y. Previously, an HLA-DQ5-restricted T cell clone specific for the same peptide was isolated from a patient with graft-versus-host disease. Thus, we compared the T cell receptor (TCR) rearrangements of the 2 in vitro generated T cell clones and the ex vivo isolated T cell clone. All 3 clones shared the same TCRBV5-4* gene segment and 2 of 3 clones also used similar TCR-Valpha segments. Our results suggest that T cells recognizing the HLA-DQ5/DDX3Y T cell epitope might be characterized by a relatively limited TCR-beta repertoire. The differences in the junctional TCR-beta region had no effect on the antigen specificity, but altered the capacity of the TCR to distinguish the HLA-DQ5/DDX3Y complex from its allelic counterpart. The results also demonstrate that in vitro stimulation of T cells with allogeneic HLA-identical dendritic cells may facilitate the characterization of in vivo, potentially relevant HLA class II-restricted minor H epitopes.     

Targeting liposomes with protein drugs to the blood-brain barrier in vitro.

In this study, we aim to target pegylated liposomes loaded with horseradish peroxidase (HRP) and tagged with transferrin (Tf) to the BBB in vitro. Liposomes were prepared with the post-insertion technique: micelles of polyethylene glycol (PEG) and PEG-Tf were inserted into pre-formed liposomes containing HRP. Tf was measured indirectly by measuring iron via atomic absorption spectroscopy. All liposomes were around 100 nm in diameter, contained 5-13 microg HRP per mumol phospholipid and 63-74 Tf molecules per liposome (lipo Tf) or no Tf (lipo C). Brain capillary endothelial cells (BCEC) were incubated with liposomes at 4 degrees C (to determine binding) or at 37 degrees C (to determine association, i.e. binding+endocytosis) and the HRP activity, rather than the HRP amount was determined in cell lysates. Association of lipo Tf was two- to three-fold higher than association of lipo C. Surprisingly, the binding of lipo Tf at 4 degrees C was four-fold higher than the association of at 37 degrees C. Most likely this high binding and low endocytosis is explained by intracellular degradation of endocytosed HRP. In conclusion, we have shown targeting of liposomes loaded with protein or peptide drugs to the BCEC and more specifically to the lysosomes. This is an advantage for the treatment of lysosomal storage disease. However, drug targeting to other intracellular targets also results in intracellular degradation of the drug. Our experiments suggest that liposomes release some of their content within the BBB, making targeting of liposomes to the TfR on BCEC an attractive approach for brain drug delivery.     

Vaccination and Antibody Testing in Cats

Vaccines protect cats from serious diseases by inducing antibodies and cellular immune responses. Primary vaccinations and boosters are given according to vaccination guidelines provided by industry and veterinary organizations, based on minimal duration of immunity (DOI). For certain diseases, particularly feline panleukopenia, antibody titres correlate with protection. For feline calicivirus and feline herpesvirus, a similar correlation is absent, or less clear. In this review, the European Advisory Board on Cat Diseases (ABCD) presents current knowledge and expert opinion on the use of antibody testing in different situations. Antibody testing can be performed either in diagnostic laboratories, or in veterinary practice using point of care (POC) tests, and can be applied for several purposes, such as to provide evidence that a successful immune response was induced following vaccination. In adult cats, antibody test results can inform the appropriate re-vaccination interval. In shelters, antibody testing can support the control of FPV outbreaks by identifying potentially unprotected cats. Antibody testing has also been proposed to support decisions on optimal vaccination schedules for the individual kitten. However, such testing is still expensive and it is considered impractical to monitor the decline of maternally derived antibodies.     

Vasorelaxant effects and mechanisms of action of Heracleum sphondylium L. (Apiaceae) in rat thoracic aorta

Ethnopharmacological relevance

Aerial parts of Heracleum sphondylium L. (HS) are used in traditional medicine to treat hypertension. To provide pharmacological basis for this use, we investigated the vasorelaxant effects of a dichloromethane extract of HS (HSDE) and the mechanisms involved.

Materials and methods

Activity of HSDE was evaluated on rat isolated thoracic aortic rings.

Results

HSDE induced vasorelaxation in phenylephrine (PE, 10⁻⁶ mol/L) and high KCl—(6×10⁻² mol/L) pre-contracted aortic rings that was independent on the presence of endothelium. HSDE markedly decreased extracellular Ca²⁺-induced contraction in high-KCl and PE pre-challenged rings. It also inhibited the intracellular Ca²⁺ release sensitive to PE (10⁻⁶ M). The relaxant effect of HSDE were blunted by 4-amino-pyridine (4-AP, 10⁻³ mol/L), an inhibitor of voltage-dependent K⁺ channels.

Conclusion

Our results provide the first evidence that a dichloromethane extract of Heracleum sphondylium L. exhibits vasorelaxant properties through endothelium-independent mechanisms involving the inhibition of Ca²⁺ mobilization and changes in Kv channel conductances. These data argue for its use as antihypertensive therapy in traditional medicine.     

Highly Divergent SARS-CoV-2 Alpha Variant in Chronically Infected Immunocompromised Person

We detected a highly divergent SARS-CoV-2 Alpha variant in an immunocompromised person several months after the latest detection of the Alpha variant in the Netherlands. The patient was infected for 42 weeks despite several treatment regimens and disappearance of most clinical symptoms. We identified several potential immune escape mutations in the spike protein.     

Rabies Virus Populations in Humans and Mice Show Minor Inter-Host Variability within Various Central Nervous System Regions and Peripheral Tissues

Rabies virus (RABV) has a broad host range and infects multiple cell types throughout the infection cycle. Next-generation sequencing (NGS) and minor variant analysis are powerful tools for studying virus populations within specific hosts and tissues, leading to novel insights into the mechanisms of host-switching and key factors for infecting specific cell types. In this study we investigated RABV populations and minor variants in both original (non-passaged) samples and in vitro-passaged isolates of various CNS regions (hippocampus, medulla oblongata and spinal cord) of a fatal human rabies case, and of multiple CNS and non-CNS tissues of experimentally infected mice. No differences in virus populations were detected between the human CNS regions, and only one non-synonymous single nucleotide polymorphism (SNP) was detected in the fifth in vitro passage of virus isolated from the spinal cord. However, the appearance of this SNP shows the importance of sequencing newly passaged virus stocks before further use. Similarly, we did not detect apparent differences in virus populations isolated from different CNS and non-CNS tissues of experimentally infected mice. Sequencing of viruses obtained from pharyngeal swab and salivary gland proved difficult, and we propose methods for improving sampling.     

Coupling of Metal Containing Homing Devices to Liposomes via a Maleimide Linker: Use of TCEP to Stabilize Thiol-groups without Scavenging Metals

Liposomes for drug delivery are often prepared with maleimide groups on the distal end of PEG to enable coupling of homing devices, such as antibodies, or other proteins. EDTA is used to stabilize the thiol group in the homing device for attachment to the maleimide. However, when using a homing device that contains a metal, EDTA inactivates this by scavenging of the metal. Holo-transferrin (Tf) containing two iron atoms (Fe³⁺), has a much higher affinity for the Tf receptor than apo-Tf (which does not contain any Fe³⁺). To couple Tf to a liposome, the introduction of a thiol group is necessary. During this process, by using N-succinimidyl S-acetylthioacetate (SATA), followed by 2–3 h coupling to the liposomes, Fe³⁺ is scavenged by EDTA. This causes a decreased affinity of Tf for its receptor, resulting in a decreased targeting efficiency of the liposomes.

Tris(2-carboxyethyl)phosphine (TCEP) hydrochloride is a sulfhydryl reductant that is often used in protein biochemistry. We found that TCEP (0.01 mM) does not scavenge Fe³⁺ from Tf and is able to protect thiol groups for the coupling to maleimide. Furthermore, TCEP does not interfere with the maleimide coupling itself.

In this communication, we describe the preparation of liposomes, focussing on the coupling of Tf to the maleimide linker at the distal end of PEG, without loosing Fe³⁺ from Tf. This method can be applied to other metal-containing homing devices as well.     

New insights into the mechanisms of the vasorelaxant effects of apocynin in rat thoracic aorta

Apocynin is a naturally occurring acetophenone widely used as an inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Recent data suggested that apocynin might exert NADPH oxidase‐independent pharmacological properties. Among them, vasorelaxant properties have been described, but the mechanisms still give rise to debates. The present study investigated the mechanisms involved in the vasorelaxant effect of apocynin on the in vitro model of rat isolated thoracic aortic rings. Apocynin (30 μm to 10 mm ) induced a dose‐dependent relaxation in both endothelium‐intact and endothelium‐denuded aortic rings with respective EC₅₀ values of 0.78 ± 0.08 and 1.91 ± 0.21 mm . Endothelium removal or inhibition of nitric oxide (NO) synthase with N^ω‐nitro‐l ‐arginine‐methyl ester (l ‐NAME) significantly decreased but did not abolish the effect of apocynin. By contrast, apocynin‐induced relaxation was unchanged after incubation with indomethacin or charybdotoxin plus apamin. In endothelium‐denuded aortas, the vasorelaxant effect of apocynin was significantly reduced by glibenclamide but not by 4‐aminopyridine nor by iberiotoxin. Apocynin significantly decreased Ca²⁺‐induced contraction and inhibited intracellular Ca²⁺mobilization after contraction with phenylephrine. Finally, the acute intravenous injection of apocynin led to an immediate and transient hypotensive effect in spontaneously hypertensive rats (SHR). In conclusion, our data demonstrated that apocynin induces both endothelium‐independent relaxant effects involving inhibition of Ca²⁺mobilization and activation of K_ATP channels in vascular smooth muscle cells and endothelium‐dependent effects mediated by NO. These results should provide a basis for caution when interpreting results on the vascular effects of apocynin.