Nucleotide excision repair: ERCC1 and TFIIH complexes

DNA is the carrier of genetic information in living organisms. The information stored in the nucleotide sequence of DNA is transmitted to the offspring by generating identical copies of the parental DNA molecules. Damage in DNA can cause loss of genetic information. Nevertheless, the DNA is continuously subject to alterations, and its instability is likely one of the major factors in mutagenesis. The structure of DNA can be modified spontaneously by hydrolysis, oxidation, or by environmental factors such as ultra-violet (UV) light, X-rays, or numerous chemical agents. Replication of unrepaired DNA can cause genetic changes, which may affect proper functioning of proteins encoded by that DNA. As a result cellular malfunction, onset of

The predictive value of Golgi Protein 73 in differentiating benign from malignant liver tumors

Introduction: In the work up of primary solid liver lesions it is essential to differentiate correctly between benign and malignant tumors, such as hepatocellular adenoma (HCA) and hepatocellular carcinoma (HCC) respectively. A promising new marker to detect HCC is Golgi Protein 73 (GP73). Studies comparing patients with HCC and cirrhosis with normal controls suggested that GP73 is specific for patients with HCC; however, patients with other liver tumors were not included. We therefore studied the predictive value of GP73 in differentiating between solid benign and malignant liver tumors. Materials and Methods: This study included 264 patients: 88 patients with HCC, 88 with hepatocellular adenoma (HCA), and 88 with focal nodal hyperplasia (FNH). A blood sample was collected from each patient to measure GP73 levels using a quantitative ELISA assay and differences in outcome between subgroups were compared. The receiver operating characteristic (ROC) curve, sensitivity and specificity of GP73 were calculated and compared to alpha-fetoprotein (AFP) levels. Results: When comparing malignant and benign liver tumors the area under ROC was 0.701 and 0.912 for GP73 and AFP respectively. Test characteristics revealed a sensitivity of 60% for GP73 and 65% for AFP; in addition the specificity was 77% for GP73 and 96% for AFP. Conclusion: Although the literature suggests that GP73 is a valuable serum marker in patients with HCC, the serum concentration may also be increased in patients with solid benign liver tumors. Therefore, a GP73 assay is less suitable for discriminating between primary malignant and benign tumors of the liver

FcgammaR expression on macrophages is related to severity and chronicity of synovial inflammation and cartilage destruction during experimental immune-complex-mediated arthritis (ICA)

INTRODUCTION: Fcγ receptors (FcγRs) present on cells of the haematopoietic lineage communicate with IgG-containing immune complexes that are abundant in the synovial tissue of patients with rheumatoid arthritis (RA). In mice, three classes of FcγR (RI, RII, and RIII) have been described. Binding of these receptors leads to either activation (FcγRI and RIII) or deactivation (FcγRII) of intracellular transduction pathways. Together, the expression of activating and inhibitory receptors is thought to drive immune-complex-mediated diseases. Earlier studies in our laboratory showed that macrophages of the synovial lining are of utmost importance in the onset and propagation of immune-complex-driven arthritic diseases. Selective depletion of macrophages in the joint downregulated both inflammation and cartilage destruction. As all three classes of FcγR are expressed on synovial macrophages, these cells are among the first that come in contact with immune complexes deposited in the joint. Recently, we observed that when immune complexes were injected into the knee joints of mice, strains susceptible to collagen-type-II arthritis (DBA/1, B10.RIII) developed more severe arthritis than nonsusceptible strains did, or even developed chronic arthritis. One reason why these strains are more susceptible might be their higher levels of FcγRs on macrophage membranes. To test this hypothesis, we investigated the role of FcγRs in inflammation and cartilage damage during immune-complex-mediated arthritis (ICA). First, we studied arthritis and subsequent cartilage damage in mice lacking functional FcγRI and RIII (FcR γ-chain(-/-) mice). Next, DBA/1 mice, which are prone to develop collagen-type-II arthritis (`collagen-induced arthritis'; CIA) and are hypersensitive to immune complexes, were compared with control C57BL/6 mice as regards cartilage damage and the expression and function of FcγRs on their macrophages. AIMS: To examine whether FcγR expression on macrophages is related to severity of synovial inflammation and cartilage destruction during immune-complex-mediated joint inflammation. METHODS: ICA was induced in three strains of mice (FcR γ-chain(-/-), C57BL/6, and DBA/1, which have, respectively, no functional FcγRI and RIII, intermediate basal expression of FcγRs, and high basal expression of FcγRs) by passive immunisation using rabbit anti-lysozyme antibodies, followed by poly-L-lysine lysozyme injection into the right knee joint 1 day later. In other experiments, streptococcal-cell-wall (SCW)- or zymosan-induced arthritis was induced by injecting SCW (25 μg) or zymosan (180 μg) directly into the knee joint. At several time points after arthritis induction, knee joints were dissected and studied either histologically (using haematoxylin/eosin or safranin O staining) or immuno-histochemically. The arthritis severity and the cartilage damage were scored separately on an arbitrary scale of 0-3. FcγRs were immunohistochemically detected using the monoclonal antibody 2.4G2, which detects both FcγRII and RIII. Deposition of IgG and C3c in the arthritic joint tissue was also detected immunohistochemically. Expression of FcγRs by murine peritoneal macrophages was measured using a fluorescence-activated cell sorter (FACS). Peritoneal macrophages were stimulated using heat-aggregated gamma globulins (HAGGs), and production of IL-1 was measured using a bioassay. To assess the levels of IL-1 and its receptor antagonist (IL-1Ra) during arthritis, tissue was dissected and washed in RPMI medium. Washouts were tested for levels of IL-1 and IL-1Ra using radioimmunoassay and enzyme-linked immunosorbent assay. mRNA was isolated from the tissue, and levels of macrophage inflammatory protein (MIP)-2, monocyte chemoattractant protein (MCP)-1, IL-1, and IL-1Ra were determined using semiquantitative reverse-transcription polymerase chain reaction (RT-PCR). RESULTS: ICA induced in knee joints of C57BL/6 mice caused a florid inflammation at day 3 after induction. To investigate whether this arthritis was FcγR-mediated, ICA was induced in FcR γ-chain(-/-) mice, which lack functional FcγRI and RIII. At day3, virtually no inflammatory cells were found in their knee joints. Levels of mRNA of IL-1, IL-1Ra, MCP-1, and MIP-2, which are involved in the onset of this arthritis, were significantly lower in FcR γ-chain(-/-) mice than in control C57BL/6 mice. Levels of IL-1 protein were also measured. At 6 h after ICA induction, FcR γ-chain(-/-) mice and control C57BL/6 mice showed similar IL-1 production as measured by protein level. By 24 h after induction, however, IL-1 production in the FcR γ-chain(-/-) mice was below the detection limit, whereas the controls were still producing a significant amount. To investigate whether the difference in reaction to immune complexes between the DBA/1 and C57BL/6 mice might be due to variable expression of FcγRs in the knee joint, expression in situ of FcγRs in naïve knee joints of these mice was determined. The monoclonal antibody 2.4G2, which detects both FcγRII and RIII, stained macrophages from the synovial lining of DBA/1 mice more intensely than those from C57BL/6 mice. This finding suggests a higher constitutive expression of FcγRs by macrophages of the autoimmune-prone DBA/1 mice. To quantify the difference in FcγR expression on macrophages of the two strains, we determined the occurrence of FcγRs on peritoneal macrophages by FACS analysis. The levels of FcγR expressed by macrophages were twice as high in the DBA/1 mice as in the C57BL/6 mice (mean fluorescence, respectively, 440 ± 50 and 240 ± 30 intensity per cell). When peritoneal macrophages of both strains were stimulated with immune complexes (HAGGs), we found that the difference in basal FcγR expression was functional. The stimulated macrophages from DBA/1 mice had significantly higher IL-1α levels (120 and 135 pg/ml at 24 and 48 h, respectively) than cells from C57BL/6 mice (45 and 50 pg/ml, respectively). When arthritis was induced using other arthritogenic triggers than immune complexes (zymosan, SCW), all the mouse strains tested (DBA/1, FcR γ-chain(-/-), and C57BL/6) showed similar inflammation, indicating that the differences described above are found only when immune complexes are used to elicit arthritis. We next compared articular cartilage damage in arthritic joints of the three mouse strains FcR γ-chain(-/-), C57BL/6 (intermediate basal expression of FcγRs), and DBA/1 (high basal expression of FcγRs). Three indicators of cartilage damage were investigated: depletion of PGs, chondrocyte death, and erosion of the cartilage matrix. At day 3 after induction of ICA, there was no PG depletion in FcR γ-chain(-/-) mice, whereas PG depletion in the matrix of the C57BL/6 mice was marked and that in the arthritic DBA/1 mice was even greater. PG depletion was still massive at days 7 and 14 in the DBA/1 mice, whereas by day 14 the PG content was almost completely restored in knee joints of the C57BL/6 mice. Chondrocyte death and erosion of cartilage matrix, two indicators of more severe cartilage destruction, were significantly higher in the DBA/1 than in the C57BL/6 mice, while both indicators were completely absent in the FcR γ-chain(-/-) mice. Again, when arthritis was induced using other triggers (SCW, zymosan), all strains showed similar PG depletion and no chondrocyte death or matrix erosion. These findings underline the important role of immune complexes and FcγRs in irreversible cartilage damage. DISCUSSION: Our findings indicate that inflammation and subsequent cartilage damage caused by immune complexes may be related to the occurrence of FcγRs on macrophages. The absence of functional FcγRI and RIII prevented inflammation and cartilage destruction after induction of ICA, whereas high basal expression of FcγRs on resident joint macrophages of similarly treated mice susceptible to autoimmune arthritis was correlated with markedly more synovial inflammation and cartilage destruction. The difference in joint inflammation between the three strains was not due to different susceptibilities to inflammation per se, since intra-articular injection of zymosan or SCW caused comparable inflammation. Although extensive inflammatory cell mass was found in the synovium of all strains after intra-articular injection of zymosan, no irreversible cartilage damage (chondrocyte death or matrix erosion) was found. ICA induced in C57BL/6 and DBA/1 mice did cause irreversible cartilage damage at later time points, indicating that immune complexes and FcγRs play an important role in inducing irreversible cartilage damage. Macrophages communicate with immune complexes via Fcγ receptors. Absence of functional activating receptors completely abrogates the synovial inflammation, as was shown after ICA induction in FcR γ-chain(-/-) mice. However, the γ-chain is essential not only in FcγRI and RIII but also for FcεRI (found on mast cells) and the T cell receptor (TcR)-CD3 (Tcells) complex of γδT cells. However, T, B, or mast cells do not play a role in this arthritis that is induced by passive immunisation. Furthermore, this effect was not caused by a difference in clearance of IgG or complement deposition in the tissue. In this study, DBA/1 mice, which are susceptible to collagen-induced autoimmune arthritis and in a recent study have been shown to react hypersensitively to immune complexes, are shown to express higher levels of FcγRs on both synovial and peritoneal macrophages. Because antibodies directed against the different subclasses of FcγR are not available, no distinction could be made between FcγRII and RIII. Genetic differences in DBA/1 mice in genes coding for or regulating FcγRs may be responsible for altered FcγR expression. If so, these mouse strains would have a heightened risk for immune-complex-mediated diseases. To provide conclusive evidence for the roles of the various classes of FcγR during ICA, experiments are needed in which FcγRs are blocked with specific antibodies, or in which knockout mice lacking one specific class of FcγR are used. The only available specific antibody to FcγR (2.4G2) has a stimulatory effect on cells once bound to the receptor, and therefore cannot be used in blocking experiments. Experiments using specific knockout mice are now being done in our laboratory. Macrophages are the dominant type of cell present in chronic inflammation during RA and their number has been shown to correlate well with severe cartilage destruction. Apart from that, in humans, these synovial tissue macrophages express activating FcRs, mainly FcγIIIa, which may lead to activation of these macrophages by IgG-containing immune complexes. The expression of FcRs on the surface of these cells may have important implications for joint inflammation and severe cartilage destruction and therefore FCRs may constitute a new target for therapeutic intervention

Colorectal Cancer Stage Distribution at First and Repeat Fecal Immunochemical Test Screening

Background &amp; Aims: For colorectal cancer (CRC) screening to be effective, it is important that screen-detected cancers are found at an early stage. Studies on stage distribution of screen-detected CRC at repeat screening of large population-based fecal immunochemical test (FIT)-based screening programs and the impact of FIT cut-off values on staging currently are lacking. Methods: We obtained data for FIT-positive participants (FIT cut-off, 47 μg hemoglobin/g feces) at their first or second (ie, repeat) screening from the Dutch National Screening Database from 2014 to 2018. Tumor characteristics were acquired through linkage with The Netherlands Cancer Registry. We compared stage at diagnosis (I–II vs III–IV) of CRCs detected at a first or second screening. In addition, we analyzed the hypothetical yield and stage distribution of CRC for different FIT cut-off values up to 250 μg hemoglobin/g feces.Results: At the first and second screenings, respectively, 15,755 and 3304 CRCs were detected. CRCs detected at the first or second screening were equally likely to be stages I to II (66.5% vs 67.7%; relative risk, 1.02; 95% CI, 1.00–1.05). A hypothetical increase of the FIT cut-off value from 47 μg to 250 μg resulted in a reduction of detected CRCs by 88.3% and 79.0% at the first or second screening, respectively. Even then, the majority of detected CRCs (63%–64%) still would be diagnosed at stages I to II. Conclusions: FIT-based screening is effective in downstaging CRC, and also at repeat screening. Increasingly, the FIT cut-off level has a limited impact on the stage distribution of detected CRCs, although it greatly affects CRC detection and thus is important to keep low.</p

Does CDX2 expression predict Barrett's metaplasia in oesophageal columnar epithelium without goblet cells?

Background: Intestinal metaplasia (Barrett's oesophagus), but not cardiac-type mucosa in columnar-lined oesophagus, is regarded as premalignant. As intestinal metaplasia and cardiac-type mucosa are endoscopically indiscernible, it is difficult to take targeted samples from columnar-lined oesophagus with consequently a risk of having undetected intestinal metaplasia. Aim: To investigate whether the intestinal markers CDX2, MUC2 and villin can predict the presence of undetected intestinal metaplasia in columnar-lined oesophagus. Methods: Presence of intestinal metaplasia or cardiac-type mucosa was identified in 122 biopsy sets of columnar-lined oesophagus from 61 patients, collected at two subsequent follow-up upper endoscopies. CDX2, MUC2 and villin expression were determined by immunohistochemistry. Results: All intestinal metaplasia samples (55) were positive for CDX2 and MUC2 and 32 of 55 for vil

Serum hepatitis B virus RNA predicts response to peginterferon treatment in HBeAg-positive chronic hepatitis B

Hepatitis B virus (HBV) RNA in serum is a novel biomarker that reflects cccDNA activity. We investigated whether HBV RNA can predict serological response to peginterferon (PEG-IFN) treatment. Serum HBV RNA levels were retrospectively measured at weeks 0, 12, 24 and 52 of therapy and after treatment discontinuation (week 78) in 266 HBeAg-positive chronic HBV patients who had participated in a global randomized controlled trial (HBV99-01 study). Patients received 52 weeks PEG-IFN monotherapy (n = 136) or PEG-IFN and lamivudine (n = 130). The primary end point was HBeAg loss 24 weeks after PEG-IFN discontinuation. At baseline, the mean serum level of HBV RNA was 6.8 (SD 1.2) log c/mL. HBV RNA levels declined to 4.7 (1.7) log c/mL after one year of PEG-IFN therapy alone and to 3.3 (1.2)log c/mL after combination therapy. From week 12 onward, HBV RNA level was significantly lower in patients who achieved HBeAg loss at the end of follow-up as compared to those who did not, regardless of treatment allocation (week 12:4.4 vs 5.1 log c/mL, P =.01; week 24:3.7 vs 4.9 log c/mL, P <.001). The performance of a multivariable model based on HBV RNA level was comparable at week 12 (AUC 0.68) and 24 (AUC 0.72) of therapy. HBV RNA level above 5.5 log c/mL at week 12 showed negative predictive values of 93/67/90/64% for HBV genotypes A/B/C/D for the prediction of HBeAg loss. In conclusion, HBV RNA in serum declines profoundly during PEG-IFN treatment. Early on-treatment HBV RNA level may be used to predict nonresponse

The use of non-invasive stool tests for verification of Helicobacter pylori eradication and clarithromycin resistance

Background: Clarithromycin resistance of Helicobacter pylori (H. pylori) represents a major challenge in eradication therapy. In this study, we assessed if non-invasive stool tests can be used to verify successful H. pylori eradication and determine clarithromycin resistance. Materials and methods:In this prospective study, patients undergoing urea breath testing (UBT) for confirmation of H. pylori eradication were asked to collect the stool as both a dry fecal sample and fecal immunochemical test (FIT). Stool H. pylori antigen testing (SAT) was performed on these samples and assessed for its accuracy in eradication verification. Type and duration of antibiotic treatment were retrospectively collected from patient records and compared with clarithromycin resistance determined by PCR of stool samples. Results: H. pylori eradication information was available for a total of 145 patients (42.7% male, median age: 51.2). Successful eradication was achieved in 68.1% of patients. SAT on FIT samples had similar accuracy for eradication assessment compared to dry fecal samples, 72.1% [95% CI 61.4–81.2] versus 72.2% [95% CI 60.9–81.7]. Clarithromycin resistance rate was 13.4%. Conclusion: H. pylori antigen testing on FIT stool samples to verify H. pylori eradication is feasible and has similar accuracy as H. pylori antigen testing on dry stool samples. Dry stool, but not FIT, was suitable for non-invasive identification of H. pylori clarithromycin resistance by rt-PCR personalizing antibiotic treatment strategies without the need for invasive diagnostics is desirable, as the cure rate of first-line empirical H. pylori treatment remains low.</p

The second round of the Dutch colorectal cancer screening program: Impact of an increased fecal immunochemical test cut-off level on yield of screening

The Dutch colorectal cancer (CRC) screening program started in 2014, inviting the target population biennially to perform a fecal immunochemical test (FIT). We obtained prospectively collected data from the national screening information-system to present the results of the second round (2016) and evaluate the impact of increasing the FIT cut-off halfway through the first round from 15 to 47 μg Hb/g feces on outcomes in the second round. Second round screening was done with a 47 μg Hb/g feces FIT cut-off. Participants were classified based on first round participation status as either FIT (15,47) or FIT (47,47) participants, and previous nonparticipants. In total, 348,891 (75.9%) out of 459,740 invitees participated in the second round. Participation rates were 93.4% among previous participants and 21.0% among previous non-participants. FIT(47,47) participants had a significantly higher detection rate of AN (15.3 vs. 10.4 per 1,000 participants) compared to FIT(15,47) participants in the second round, while their cumulative detection rate of AN over two rounds was significantly lower (45.6 vs. 52.6 per 1,000 participants). Our results showed that participation in the Dutch CRC screening program was consistently high and that second round detection rates depended on the first round FIT cut-off. The cumulative detection over two rounds was higher among FIT(15,47) participants. These findings suggest that a substantial part of, but not all the missed findings in the first round due to the increased FIT cut-off were detected in the subsequent round