New concepts for a more effective anti-cancer therapy are urgently needed. Experimental flaws represent a major counter player of this development and lead to inaccurate and unreproducible data as ...well as unsuccessful translation of research approaches into clinics. In a previous study we have created epithelial cell cultures from head and neck squamous cell carcinoma (HNSCC) tissue.
We characterize primary cell populations isolated from human papillomavirus positive HNSCC tissue for their marker expression by RT-qPCR, flow cytometry, and immunofluorescence staining. Their sensitivity to MDM2-inhibition was measured using cell viability assays.
Primary HNSCC cell cultures showed the delayed formation of spheroids at higher passages. These spheroids mimicked the morphology and growth characteristics of other established HNSCC spheroid models. However, expression of epithelial and mesenchymal markers could not be detected in these cells despite the presence of the HNSCC stem cell marker aldehyde dehydrogenase 1 family member A1. Instead, strong expression of B- and T-lymphocytes markers was observed. Flow cytometry analysis revealed a heterogeneous mixture of CD3 + /CD25 + T-lymphocytes and CD19 + B-lymphocytes at a ratio of 4:1 at passage 5 and transformed lymphocytes at late passages (≥ passage 12) with CD45 + CD19 + CD20 + , of which around 10 to 20% were CD3 + CD25 + CD56 + . Interestingly, the whole population was FOXP3-positive indicative of regulatory B-cells (B
). Expression of transcripts specific for the Epstein-Barr-virus (EBV) was detected to increase in these spheroid cells along late passages, and this population was vulnerable to MDM2 inhibition. HPV + HNSCC cells but not EBV + lymphocytes were detected to engraft into immunodeficient mice.
In this study we present a primary cell culture of EBV-infected tumor-infiltrating B-lymphocytes, which could be used to study the role of these cells in tumor biology in future research projects. Moreover, by describing the detailed characteristics of these cells, we aim to caution other researchers in the HNSCC field to test for EBV-infected lymphocyte contaminations in primary cell cultures ahead of further experiments. Especially researchers who are interested in TIL-based adopted immunotherapy should exclude these cells in their primary tumor models, e.g. by MDM2-inhibitor treatment. BI-12-derived xenograft tumors represent a suitable model for in vivo targeting studies.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Mechanical loading is crucial for bone remodeling and osteoblast differentiation. FosB belongs to the AP-1 family of transcription factors, a group of proteins known to regulate osteoblast ...differentiation and bone formation. In mice, FosB is rapidly induced by mechanical stress at the transcriptional level. The aim of this study was to determine the effect of different mechanical stretch patterns on FosB gene expression and on osteogenic differentiation of human osteoblast precursor cells. Human bone-marrow-derived mesenchymal precursor cells were grown in flexible silicone dishes and stimulated by a daily application of three rounds of 2
h of cyclic stretch of either 2% or 8% elongation at 1
Hz on 3 consecutive days using a special motor-driven apparatus. By real-time PCR, we quantified FosB mRNA and the expression of genes involved in osteoblast differentiation such as Runx2 and collagen 1 to determine the osteogenic effect of mechanical stretch. Stretching induced FosB transcription and the expression of osteoblast markers in partly committed human mesenchymal precursor cells in a stretch- and time-dependent manner. We conclude that cyclic stretch-induced FosB expression and the upregulation of osteoblast genes plays a role in osteogenic differentiation of human mesenchymal precursor cells.
Antigen rapid diagnostic tests (Ag-RDTs) play an important role in the diagnosis of SARS-CoV-2. They are easier, quicker, and less expensive than the ‘reference standard’ RT-PCR and therefore widely ...in use. Reliable clinical data with respect to Ag-RDT performance in SARS-CoV-2 Omicron variants of concern (VOCs) are limited. Consequently, the objective of this study was to determine the impact different VOCs—especially Omicron—have on the clinical performance of an Ag-RDT.
We compared the clinical performance of the Sofia SARS-CoV-2 Ag-RDT to RT-PCR in a real-world, single-centre study in a clinical point-of-care setting in patients admitted to a large hospital via the emergency department from 2 November 2020 to 4 September 2022.
Among 38 434 Ag-RDT/RT-PCR tandems taken, 1528 yielded a SARS-CoV-2 positive RT-PCR test result, with a prevalence of 4.0% (95% CI, 3.8–4.2). Overall sensitivity of the Ag-RDT was 63.7% (95% CI, 61.3–66.1) and overall specificity was 99.6% (95% CI, 99.5–99.6). Ag-RDT sensitivity was dependent on viral load (VL), because the sensitivity increased to 93.2% (95% CI, 91.5–94.6) in samples with a VL > 106 SARS-CoV-2 copies/mL. Furthermore, the Ag-RDT was more sensitive in men, and older patients. Variant-dependent sensitivity assessment showed that the sensitivity was significantly lower in Omicron-VOC (64.1%; 95% CI, 60.5–67.6) compared with SARS-CoV-2 wild-type samples (70.0%; 95% CI, 59,8–78,6) (binomial test; p value < 0.001). Analysing the limits of detection showed a 27 times higher 95% limit of detection for the Omicron-VOC BA.5 compared with the SARS-CoV-2 wild-type.
Ag-RDT sensitivity for detection of patients with lower VLs and with Omicron-VOC is reduced, limiting the effectiveness of Ag-RDTs. However, Ag-RDTs are still an unreplaceable tool for widely available, quick, and inexpensive point-of-care SARS-CoV-2 diagnostics.
Matrix metalloproteinases (MMPs) are central to tissue remodelling; however, little is known about the temporal pattern and differential regulation of hepatic MMP expression in the course of chronic ...human liver disease. Using quantitative reverse transcription-PCR ELISA assays, we studied hepatic mRNA expression of MMP-1, -2, -3, -7, -9, -10, -11, -13 and -14 in patients with chronic hepatitis C and hepatitis C virus-induced end-stage liver cirrhosis and controls. Results were compared with histology, hepatic expression of tissue inhibitor of metalloproteinases (TIMP)-1, -2 and -3, procollagen types I and IV, laminin, and with circulating protein levels of hyaluronate, TIMP-1 and -2 and MMP proenzymes, as measured by ELISA. The impact of the MMP-3(-1171) promoter polymorphism on hepatic MMP-3 expression was analysed. Hepatic mRNA expression data identified differentially regulated groups of MMPs during the course of chronic hepatitis C, showing either steadily increasing mRNA expression with disease progression (MMP-1, -2, -7 and -14) or transiently elevated expression (MMP-9, -11 and -13). The first group closely correlated to the parameters of fibrogenesis. Hepatic MMP-3 expression was unrelated to disease stage, but was determined by the MMP-3(-1171) promoter polymorphism. In conclusion, MMP expression during the course of chronic hepatitis C appears to be a closely regulated process, with different clusters of coordinately regulated MMP genes being identified.
•We evaluated the performance of the Sofia SARS-CoV-2 rapid antigen test (Ag-RDT)•A total of 7877 patients were included in a prospective and monocentric study•Overall sensitivity of Ag-RDT compared ...to RT-PCR was 62.9% and specificity 99.4%•In a subset of symptomatic and RT-PCR positive patients sensitivity was 72.1%•The Ag-RDT proved to be a rapid tool to detect patients with high viral loads (Ct-value ≤25)
The rapid and reliable detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is of high importance for individual patient care and hospital infection prevention. We aimed to evaluate the performance of the Sofia SARS-CoV-2 antigen rapid diagnostic test (Ag-RDT) in comparison to real-time reverse-transcription polymerase chain reaction (RT-PCR). We conducted a prospective, monocentric cross-sectional study in an emergency department of a German university hospital from November 2020 to March 2021. We tested all samples using both Sofia SARS-CoV-2 Ag-RDT and real-time RT-PCR. A total of 7877 patients were included. Overall sensitivity of the Ag-RDT was 62.9% and specificity was 99.4%. Sensitivity varied across study months, whereas specificity remained high. Sensitivity increased to 94.2% in samples with a cycle threshold (Ct)-value ≤25. The Sofia Ag-RDT proved to be a rapid tool to detect samples with high viral loads (Ct-value ≤25) and might thus help to identify infectious patients.
Background: In this study the analytical performance of eight glucose point-of-care testing (POCT) devices was evaluated. For this purpose, POCT measurement of glucose in heparinized blood collected ...from patients was paralleled by determination of the glucose concentration in the respective plasma by an analyzer (Hitachi 917) in the central laboratory, providing traceable results. Methods: Trueness of POCT measurements was studied by comparing the plasma POCT values (mean of five measurements) with the results from the traceable measurement procedure (TMP). Results: The percentage of POCT results within ±6% of the TMP mean value ranged from 24% to 50%, depending on the POCT device. Within the reference interval of plasma glucose (4.4–6.0 mmol/L), up to 67% of the POCT values were lower than 4.4 mmol/L, leading to a false diagnosis of hypoglycemia. In the hypoglycemic range (<4.4 mmol/L) up to 29% of the POCT analyses were false normoglycemic. Conclusions: In conclusion, this study shows an insufficient trueness of glucose measurements by POCT devices in the normo- and hypoglycemic range. To improve quality assessment, sample splitting and simultaneous measurement of blood glucose concentration every 4 weeks by POCT devices and of plasma glucose concentration by a reliable TMP is recommended. Clin Chem Lab Med 2006;44:888–93.
Gilbert's disease leads to intermittent non‐hemolytic hyperbilirubinemia by a reduction of hepatic bilirubin glucuronidation associated with the presence of the UDP‐glucuronosyltransferase (UGT) ...1A1*28 polymorphism. It is considered benign because it does not result in hepatocellular damage. However, pharmacogenetic analyses have linked UGT1A1*28 to drug toxicity and cancer predisposition. The protease inhibitor atazanavir (ATV) is an inhibitor of hepatic UGT activity leading to hyperbilirubinemia in individual patients. Whether this is linked specifically to UGT1A1*28 or to more complex variants influencing glucuronidation is unclear. One hundred and six ATV‐treated patients were characterized and genotyped for UGT1A1*28, the UGT1A3 (‐66C) and UGT1A7 (‐57G) promoter variants, and UGT1A7129K/131K. ATV treatment increased median bilirubin levels from 10 to 41 μmol/L (P = .001) with hyperbilirubinemia exceeding 43 μmol/L in 37%. Hyperbilirubinemia over 43 μmol/L was significantly associated not only with UGT1A1*28 but also with UGT1A3‐66C, UGT1A7‐57G, and UGT1A7129K/131K, although these variants do not naturally occur in linkage dysequilibrium in blood donors. Homozygous combinations of UGT1A1*28 with the other variants increased from 7.4% (normal bilirubin to 42 μmol/L) to 41% to 46.1% (43 to >85 μmol/L), and 100% (>85 μmol/L). All six patients with hyperbilirubinemia greater than 85 μmol/L were homozygous for all four variants identifying a haplotype inherited on a single allele. In conclusion, the genetic variant associated with Gilbert's disease is identified as part of a haplotype of four UGT1A variants spanning three genes at the UGT1A gene locus. This haplotype predisposes to hyperbilirubinemia in ATV treatment and may have an additional role as a pharmacogenomic risk factor for drug therapy. (HEPATOLOGY 2006;44:1324–1332.)
Background/Aims Gilbert’s syndrome is a frequent genetic conjugation abnormality associated with adverse drug effects. Genetic UDP glucuronosyltransferase (UGT)1A gene variants can influence gene ...transcription, inducibility and glucuronidation activity. Protease inhibitors used in human immunodeficiency virus (HIV) infection and chronic viral hepatitis can inhibit UGTs. Indinavir (IDV) can lead to hyperbilirubinemia in Gilbert’s syndrome (UGT1A128), which does not explain interindividual severity differences and may thus involve additional UGT1A variants. Methods One hundred and twenty-five HIV patients receiving IDV and 427 healthy blood donors were genotyped for the presence of UGT1A128, UGT1A3 -66T/C, UGT1A7 -57T/G, UGT1A7N129K/R131K using Taqman 5′ nuclease assays. Results Hyperbilirubinemia was observed in 42%. UGT1A128 frequencies did not differ between HIV patients and controls but were significantly higher in hyperbilirubinemic patients. The frequency of homozygous carriers of the 4 UGT1A marker haplotype increased with hyperbilirubinemia affecting all patients with bilirubin levels >85 μmol/l. Conclusions In IDV treatment the risk of severe hyperbilirubinemia is associated with genetic variants of the UGT1A3 and UGT1A7 genes in addition to Gilbert’s syndrome (UGT1A128). This haplotype is a useful predictor of protease inhibitor-induced side effects.
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