In this study, we compared immunoglobulin heavy-chain-gene-based minimal residual disease (MRD) detection by real-time quantitative PCR (RQ-PCR) and next-generation sequencing (NGS) to assess whether ...NGS could overcome some limitations of RQ-PCR and further increase sensitivity, specificity, accuracy and reproducibility. In total, 378 samples from 55 patients with acute lymphoblastic leukemia (ALL), mantle cell lymphoma (MCL) or multiple myeloma (MM) were investigated for clonotype identification, clonotype identity and comparability of MRD results. Forty-five clonotypes were identified by RQ-PCR and 49 by NGS. Clonotypes identified by both tools were identical or >97% homologous in 96% of cases. Both tools were able to routinely reach a sensitivity level of 1 × E-05. A good correlation of MRD results was observed (R=0.791, P<0.001), with excellent concordance in 79.6% of cases. Few discordant cases were observed across all disease subtypes. NGS showed at least the same level of sensitivity as allele-specific oligonucleotides-PCR, without the need for patient-specific reagents. We conclude that NGS is an effective tool for MRD monitoring in ALL, MCL and MM. Prospective comparative analysis of unselected cases is required to validate the clinical impact of NGS-based MRD assessment.
Herpes and herpes-like virus infections have been reported in various marine mollusc species associated with high mortality rates. Following the characterisation and genome sequencing of ostreid ...herpesvirus 1 (OsHV-1), specific diagnostic tools have been developed based on conventional PCR techniques or
in situ hybridisation. We have now developed a real-time PCR assay for rapid, sensitive and quantitative detection of OsHV-1, and compared it with a conventional PCR technique described previously. The new assay utilised SYBR
® Green chemistry with specific primers C
9/C
10 targeting the C region. The melt curve analysis of OsHV-1 DNA or DNA extracted from infected material showed only one melting temperature peak (75.75
±
0.1
°C). The assay had a detection limit of 4
copies/μL of viral genomic DNA and a dynamic range of 5 logs. Using infected oyster samples as template, the assay was about 100-fold more sensitive than single PCR method using C
2/C
6 primers. The assay was applied successfully for rapid diagnosis (100
min) and quantitation of OsHV-1 in different developmental stages of
Crassostrea gigas. Although it already exists a competitive PCR method to quantify OsHV-1 DNA, quantitative data that will emerge in future using the new sensitive and reliable assay will illuminate aspects of pathogenesis, in particular the viral loads in asymptomatic oysters and the kinetics of infection in specific target tissues.
Ocular involvement is common in sarcoidosis. Our study aimed to evaluate the role of screening for uveitis in subjects with sarcoidosis.
Retrospective case series of 88 subjects with a pre-existing ...diagnosis of sarcoidosis, with no previous diagnosis of uveitis, reviewed by Ophthalmology at Auckland District Health Board between January 2016 and May 2022.
Among those undergoing a screening examination, uveitis was observed in 27.8% (15 out of 54 subjects). In those presenting with acute eye symptoms, uveitis was observed in 94.1% (32 out of 34 subjects). Sarcoid uveitis was diagnosed in a total of 50 out of 88 subjects (56.8%). 45 subjects required ocular treatment. Sarcoid uveitis was observed in 6 out of 27 subjects (22.2%) who were entirely asymptomatic at screening. On multivariate analysis, blurring of vision (OR 26.2 p < 0.001), eye pain (OR 7.3 p = 0.014) and respiratory disease (OR 7.1 p = 0.044) were associated with increased risk of sarcoid uveitis. In the 41 subjects with no uveitis at initial examination, 3 subjects (7.3%) subsequently developed uveitis.
Our study highlights the importance of ophthalmic screening of all patients with systemic sarcoidosis, even in asymptomatic patients. With a high correlation of ocular symptoms in diagnosis of sarcoid uveitis, ophthalmologists should educate patients to look out for the development of symptoms of ocular inflammation, and clinicians who continue follow up for systemic sarcoidosis should remind patients to watch carefully for these symptoms to facilitate timely diagnosis and intervention.
•Screening for uveitis in subjects with systemic sarcoidosis is important.•Asymptomatic subjects with good visual acuity were found to have sarcoid uveitis.•Increased risk of uveitis with blurring of vision, eye pain and respiratory disease.•Subjects with no uveitis at initial examination may develop uveitis later on.•Subjects should be monitored for symptoms to facilitate a timely diagnosis.
The liver represents the third most frequent site of metastasis in patients with breast cancer. We performed in vivo selection using 4T1 breast cancer cells to identify genes associated with the ...liver metastatic phenotype. Coincident with the loss of numerous tight-junctional proteins, we observe claudin-2 overexpression, specifically in liver-aggressive breast cancer cells. We further demonstrate that claudin-2 is both necessary and sufficient for the ability of 4T1 breast cancer cells to colonize and grow in the liver. The liver-aggressive breast cancer cells display a claudin-2-mediated increase in their ability to adhere to extracellular matrix (ECM) components, such as fibronectin and type IV collagen. Claudin-2 facilitates these cell/matrix interactions by increasing the cell surface expression of α(2)β(1)- and α(5)β(1)-integrin complexes in breast cancer cells. Indeed, claudin-2-mediated adhesion to fibronectin and type IV collagen can be blocked with neutralizing antibodies that target α(5)β(1) and α(2)β(1) complexes, respectively. Immunohistochemical analyses reveal that claudin-2, although weakly expressed in primary human breast cancers, is readily detected in all liver metastasis samples examined to date. Together, these results uncover novel roles for claudin-2 in promoting breast cancer adhesion to the ECM and define its importance during breast cancer metastasis to the liver.
Herpes- and herpes-like viruses are known to infect a wide range of bivalve mollusc species throughout the world. Abnormal summer mortalities associated to the detection of ostreid herpesvirus 1 ...(OsHV-1) have been currently reported in France among larvae and spat of the Pacific cupped oyster
Crassostrea gigas.
In the present work, we have developed an experimental protocol of horizontal transmission based on the cohabitation between healthy and experimentally infected oysters. Through a cohabitation trial, the kinetics of OsHV-1 detection in different oyster organs and seawater samples were investigated and characterized for the first time using real time quantitative PCR.
In aquaculture management it is important to establish objective criteria to assess health and welfare of the fish. Here we show that European sea bass (
Dicentrarchus labrax) confronted with ...husbandry-associated stress (tank cleaning, i.e. scrubbing, and water temperature variation) during early life stages show poorer survival and disease resistance as juveniles. We evaluated several parameters for stress (plasma cortisol, glucose and lactate, hydromineral status), growth performance, the immune response (plasma IgM levels) and the effects of a nodavirus challenge. Principal component analysis allowed the establishment of a stress panel including plasma cortisol, osmolality, IgM levels and weight. Sea bass juveniles reared during early life in high and constant temperature perform best in terms of stress-related parameters assessed by principle component analysis. Variable water temperature triggers dramatic changes in plasma cortisol, osmolality, IgM levels, body weight and susceptibility to nodavirus that suggest a strong and prolonged activation of the HPI axis. Scrubbing induces some disturbances typical for mild short-term, acute stress, viz. increased plasma osmolality and decreased IgM levels, but does not affect plasma cortisol, growth or susceptibility to nodavirus of sea bass. Our data fit well with the concept of allostasis. We discuss the relevance of our work for sea bass aquaculture.
Reproduction of nodavirus disease was performed by experimental infection of sea bass eggs during fertilization or at larval stage 4 with 2 genetically distinguishable nodavirus strains (Sb1 and Sb2) ...isolated from sea bass collected along the Atlantic and Mediterranean French coast. The pathogenicity of the virus strains was assigned after detection of the virus by ELISA and immunohistochemistry (IHC). The Atlantic (Sb1) strain was more pathogenic than the Mediterranean (Sb2) strain during the fertilization step whilst both strains were pathogenic following experimental exposure of 4 d old larvae. Virus lesions developed in the brain 4 to 6 d following experimental exposure. Experimental ELISA proved very sensitive for detecting the nodavirus in Sb1 or Sb2 experimentally infected larvae, as well as in naturally infected sea bass larvae collected in French hatcheries or in barramundi larvae reared in the Pacific area. The development of an ELISA specific for the 2 nodavirus strains isolated from the sea bass should be useful for the detection of the virus, in addition to other techniques recommended by the Office International des Epizooties (OIE).
Physical microenvironmental parameters conducive to production of flavonoids in vitro from continuous
Vaccinium pahalae suspension cultures were examined first in shake flask culture experiments, and ...results were used to guide adaptations of a bioreactor production environment. Anthocyanin pigments were primarily concentrated in smaller aggregates up to 519 μm diameter. Agitation at 150 rpm and routine use of a mechanical scraper to periodically retrieve cells thrown out of solution was sufficient to keep productive plant cell aggregates in bioreactor suspension. A set up for enhanced irradiance with mercury lamps providing an average of 240 μmol m
−2 s
−1 PPF at the inner surface of the bioreactor vessel was required to sustain anthocyanin productivity through a 10-day production cycle.
Scale-up of a myoblast culture process Boudreault, Pascale; Tremblay, Jacques P.; Pépin, Marie-France ...
Journal of biotechnology,
09/2001, Volume:
91, Issue:
1
Journal Article
Peer reviewed
The effects of different types of cell carriers, strategies for cell transfer on carriers, and of several fusion inhibitors on the growth kinetics of primary human myoblasts culture were studied in ...order to develop a bioprocess suitable for the treatment of Duchenne muscular dystrophy based on the transplantation of unfused cells. Our results indicate that myoblast production is larger on Cytodex 1 and 3 than on polypropylene or polyester fabrics and on a commercial porous macrocarrier. Myoblast growth conditions with Cytodex 1 were further investigated to establish the bioprocess operating conditions. It was found that microcarrier density of 3 g DW l
−1, inoculum density of 2×10
5 cells ml
−1, and continuous agitation speed of 30-rpm result in final myoblast production comparable to static cultures. However, for all the culture conditions used, myoblasts growth kinetics exhibited a lag phase that lasted a minimum of 1 week prior to growth, the end of the lag phase correlating with the appearance of microcarrier aggregates. Based on this observation, we propose that aggregation promotes cell growth by offering a network of very large inter-particular pores that protect cells from mechanical stress. We took advantage of the presence of these aggregates for the scale-up of the culture process. Indeed, using myoblast-loaded microcarrier-aggregates instead of myoblast suspension to inoculate a fresh suspension of microcarriers significantly reduced the duration of the lag phase and allowed the scale-up of the bioprocess at the 500-ml scale. In order to ensure the production of unfused myoblasts, the efficiency of five different fusion inhibitors was investigated. Only calpeptin (9.1 μg ml
−1) significantly inhibited the fusion of the myoblasts, while TGFβ (50 ng ml
−1) and LPA (10 μg ml
−1) increased myoblasts growth but did not affect fusion, sphingosine (30 μg ml
−1) induced a 50% death and NMMA (25 μg ml
−1) had no effect on either growth or fusion. Finally, transplantation trials on severe combined immunodeficient mice showed that microcarrier-cultured human myoblasts grown using the optimized bioprocess resulted in grafts as successful as myoblasts grown in static cultures. The bioprocess, therefore, prove to be suitable for the large-scale production of myoblasts required for muscular dystrophy treatment.
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