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.     

Recycling of arterial grafts during reoperative coronary artery operations

BACKGROUND: To overcome problems of lack of conduit and to maximize the number of arterial anastomoses in coronary reoperations we reused previously placed arterial grafts. METHODS: Sixteen patients were identified from February 1994 to July 1997. Mean age was 62.8 years (range, 44 to 75 years). Fifteen (94%) were in Canadian Cardiovascular Society angina class III or IV. The mean interval from primary to secondary operation was 8.5 years (range, 3 to 12 years). Eleven patients had a patent internal mammary artery graft used as the recipient for a proximal Y anastomosis. In 3 cases an arterial graft was reimplanted distally on the same coronary vessel and in 2 onto different coronary vessels. One patient had a combination of these techniques. Five patients required venous conduit. RESULTS: There were no deaths. Mean length of intensive care stay was 69 hours (range, 24 to 144) and mean hospital stay was 14 days (range, 10 to 28 days). All patients were discharged home. Follow-up averages 13 months (range, 2 to 43 months). Twelve patients (75%) are now in Canadian Cardiovascular Society angina class I and 3 (19%) in class II. CONCLUSIONS: Reusing arterial conduits during coronary reoperations is possible with minimal in-hospital morbidity and satisfactory results in terms of freedom from angina. Using these techniques can help overcome the problems of inadequate conduit and maximize the number of arterial anastomoses that can be made per patient.     

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.     

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.     

Psychoses et cancers : puzzle épidémiologique ou brouillage de l’abord clinique des psychopathologies en oncologie ?

This article is related to the evolution of a specific research, developed during the 20th century. This research is dedicated to assess “the cancer” incidence in the population of “the lunatics”, and more precisely in the “psychotic population”. It describes the paradigm of this research and the progressive definition of its questions. Moreover, it specifies the chosen diagnostic entities that are used in this research development. The highlights that treating the questions as an “epidemiological puzzle” is assuming and reflecting the acquisition of a medical model to think about mental disease. Because of this, in the field of oncology, psychopathology clinical approach has become outdated.     

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.     

Lier l’histoire du sujet à l’histoire de son corps : un principe psychothérapeutique de base en oncologie

Two clinical cases are detailed between three clinical psychologists with a psychoanalytic training. The discussion is about the principles of psychoanalysis with a cancer patient. The tools of psychodynamic psychotherapy are usual: understanding the unconscious origins of the demand, waiting for the narration of the patient, allowing historisation, make subjectivation easier. But when the ego unity has never been done and when the identifications are fragile or ambivalent, when the psychical envelopes have never been containing, the psychotherapist needs to re-create an intersubjective therapeutic space. This intersubjective space will permit the narration held by the transferance. The transferance is like an immersion in the first language (listened by the patient as coming from his progressive unification or coming from the mirroring of the therapist).     

What is important in transdisciplinary pain neuroscience education? A qualitative study

Purpose: The main focus of Pain Neuroscience Education is around changing patients’ pain perceptions and minimizing further medical care. Even though Pain Neuroscience Education has been studied extensively, the experiences of patients regarding the Pain Neuroscience Education process remain to be explored. Therefore, the aim of this study was to explore the experiences in patients with non-specific chronic pain.

Materials and methods: Fifteen patients with non-specific chronic pain from a transdisciplinary treatment centre were in-depth interviewed. Data collection and analysis were performed according to Grounded Theory.

Results: Five interacting topics emerged: (1) “the pre-Pain Neuroscience Education phase”, involving the primary needs to provide Pain Neuroscience Education, with subthemes containing (a) “a broad intake” and (b) “the healthcare professionals”; (2) “a comprehensible Pain Neuroscience Education” containing (a) “understandable explanation” and (b) “interaction between the physiotherapist and psychologist”; (3) “outcomes of Pain Neuroscience Education” including (a) “awareness”, b) “finding peace of mind”, and (c) “fewer symptoms”; 4) “"scepticism” containing (a) “doubt towards the diagnosis and Pain Neuroscience Education”, (b) “disagreement with the diagnosis and Pain Neuroscience Education”, and (c) “Pain Neuroscience Education can be confronting”.

Conclusion: This is the first study providing insight into the constructs contributing to the Pain Neuroscience Education experience of patients with non-specific chronic pain. The results reveal the importance of the therapeutic alliance between the patient and caregiver, taking time, listening, providing a clear explanation, and the possible outcomes when doing so. The findings from this study can be used to facilitate healthcare professionals in providing Pain Neuroscience Education to patients with non-specific chronic pain.

• Implications for Rehabilitation

• An extensive biopsychosocial patient centred intake is crucial prior to providing Pain Neuroscience Education.

• Repetitions of Pain Neuroscience Education, in different forms (verbal and written information, examples, drawings, etc.) help patients to understand the theory of neurophysiology.

• Pain Neuroscience Education induces insight into the patient’s complaints, improved coping with complaints, improved self-control, and induces in some cases peace of mind.

• Healthcare professionals providing Pain Neuroscience Education should be aware of the possible confronting nature of the contributing factors.