Accuracy of H. pylori fecal antigen test using fecal immunochemical test (FIT).

BACKGROUND: Gastric and colorectal cancer (CRC) are both one of the most common cancers worldwide. In many countries fecal immunochemical tests (FIT)-based CRC screening has been implemented. We investigated if FIT can also be applied for detection of H. pylori, the main risk factor for gastric cancer. METHODS: This prospective study included participants over 18 years of age referred for urea breath test (UBT). Patients were excluded if they had used antibiotics/bismuth in the past 4 weeks, or a proton pomp inhibitor (PPI) in the past 2 weeks. Participants underwent UBT, ELISA stool antigen test in standard feces tube (SAT), ELISA stool antigen test in FIT tube (Hp-FIT), and blood sampling, and completed a questionnaire on user friendliness. UBT results were used as reference. RESULTS: A total of 182 patients were included (37.4% male, median age 52.4 years (IQR 22.4)). Of these, 60 (33.0%) tested H. pylori positive. SAT and Hp-FIT showed comparable overall accuracy 71.1% (95%CI 63.2-78.3) vs. 77.6% (95%CI 70.4-83.8), respectively (p = 0.97). Sensitivity of SAT was 91.8% (95%CI 80.4-97.7) versus 94.2% (95%CI 84.1-98.9) of Hp-FIT (p = 0.98). Serology scored low with an overall accuracy of 49.7% (95%CI 41.7-57.7). Hp-FIT showed the highest overall user convenience. CONCLUSIONS: FIT can be used with high accuracy and sensitivity for diagnosis of H. pylori and is rated as the most convenient test. Non-invasive Hp-FIT test is highly promising for combined upper and lower gastrointestinal (pre-) cancerous screening. Further research should investigate the clinical implications, benefits and cost-effectiveness of such an approach.

A randomised comparison of two faecal immunochemical tests in population-based colorectal cancer screening.

OBJECTIVE: Colorectal cancer screening programmes are implemented worldwide; many are based on faecal immunochemical testing (FIT). The aim of this study was to evaluate two frequently used FITs on participation, usability, positivity rate and diagnostic yield in population-based FIT screening. DESIGN: Comparison of two FITs was performed in a fourth round population-based FIT-screening cohort. Randomly selected individuals aged 50-74 were invited for FIT screening and were randomly allocated to receive an OC -Sensor (Eiken, Japan) or faecal occult blood (FOB)-Gold (Sentinel, Italy) test (March-December 2014). A cut-off of 10 µg haemoglobin (Hb)/g faeces (ie, 50 ng Hb/mL buffer for OC-Sensor and 59 ng Hb for FOB-Gold) was used for both FITs. RESULTS: In total, 19 291 eligible invitees were included (median age 61, IQR 57-67; 48% males): 9669 invitees received OC-Sensor and 9622 FOB-Gold; both tests were returned by 63% of invitees (p=0.96). Tests were non-analysable in 0.7% of participants using OC-Sensor vs 2.0% using FOB-Gold (p<0.001). Positivity rate was 7.9% for OC-Sensor, and 6.5% for FOB-Gold (p=0.002). There was no significant difference in diagnostic yield of advanced neoplasia (1.4% for OC-Sensor vs 1.2% for FOB-Gold; p=0.15) or positive predictive value (PPV; 31% vs 32%; p=0.80). When comparing both tests at the same positivity rate instead of cut-off, they yielded similar PPV and detection rates. CONCLUSIONS: The OC-Sensor and FOB-Gold were equally acceptable to a screening population. However, FOB-Gold was prone to more non-analysable tests. Comparison between FIT brands is usually done at the same Hb stool concentration. Our findings imply that for a fair comparison on diagnostic yield between FIT's positivity rate rather than Hb concentration should be used. TRIAL REGISTRATION NUMBER: NTR5385; Results.

Precore and core promoter mutants are associated with higher HBeAg seroconversion but low disease remission rates in HBV patients treated with nucleos(t)ide analogues.

HBeAg seroconversion in HBV patients is considered an important event. We determined precore (PC) and base core promoter (BCP) mutations in 137 HBeAg-positive nucleos(t)ide analogues (NA) treated patients by INNO-LiPA HBV PreCore assay (Innogenetics). The majority of patients with nongenotype A had PC/BCP mutants present at baseline (P = 0.02). During 29 months of therapy, 45 patients achieved HBeAg seroconversion. Probability of HBeAg seroconversion was higher in patients with PC and/or BCP mutants (P = 0.01). After HBeAg seroconversion, patients with BCP mutants had more HBeAg relapse (P = 0.07), and PC mutants less often achieved HBV DNA < 2000 IU/mL (P = 0.07).

Sensitive detection of hepatocellular injury in chronic hepatitis C patients with circulating hepatocyte-derived microRNA-122.

As chronic hepatitis C patients with progressive disease can present themselves with normal ALT levels, more sensitive biomarkers are needed. MicroRNAs are newly discovered small noncoding RNAs that are stable and detectable in the circulation. We aimed to investigate the association between hepatocyte-derived microRNAs in serum and liver injury in patients with chronic hepatitis C. The hepatocyte-derived miR-122 and miR-192 were analysed in sera of 102 chronic HCV-infected patients and 24 healthy controls. Serum levels of miR-122 and miR-192 correlated strongly with ALT (R = 0.67 and R = 0.65, respectively, P < 0.001 for both). Median levels of miR-122 and miR-192 in HCV-infected patients were 23 times and 8 times higher as in healthy controls (P < 0.001 for both). Even within the HCV-infected patients with a normal ALT (n = 38), the levels of miR-122 and miR-192 were 12 times and 4 times higher compared with healthy controls (P < 0.001 for both). Multivariate logistic regression analyses showed that only miR-122 was a significant predictor of the presence of chronic HCV infection (P = 0.026). Importantly, miR-122 was also superior in discriminating chronic HCV-infected patients with a normal ALT from healthy controls compared with the ALT level (AUC = 0.97 vs AUC = 0.78, P = 0.007). In conclusion, our study confirmed that liver injury is associated with high levels of hepatocyte-derived microRNAs in circulation and demonstrated that in particular miR-122 is a sensitive marker to distinguish chronic hepatitis C patients from healthy controls. More sensitive blood markers would benefit especially those patients with minor levels of hepatocellular injury, who are not identified by current screening with ALT testing.

Predictors for neoplastic progression in patients with Barrett's Esophagus: a prospective cohort study.

OBJECTIVES: Patients with Barrett's esophagus (BE) have an increased risk of developing esophageal adenocarcinoma (EAC). As the absolute risk remains low, there is a need for predictors of neoplastic progression to tailor more individualized surveillance programs. The aim of this study was to identify such predictors of progression to high-grade dysplasia (HGD) and EAC in patients with BE after 4 years of surveillance and to develop a prediction model based on these factors. METHODS: We included 713 patients with BE (≥ 2 cm) with no dysplasia (ND) or low-grade dysplasia (LGD) in a multicenter, prospective cohort study. Data on age, gender, body mass index (BMI), reflux symptoms, tobacco and alcohol use, medication use, upper gastrointestinal (GI) endoscopy findings, and histology were prospectively collected. As part of this study, patients with ND underwent surveillance every 2 years, whereas those with LGD were followed on a yearly basis. Log linear regression analysis was performed to identify risk factors associated with the development of HGD or EAC during surveillance. RESULTS: After 4 years of follow-up, 26/713 (3.4%) patients developed HGD or EAC, with the remaining 687 patients remaining stable with ND or LGD. Multivariable analysis showed that a known duration of BE of ≥ 10 years (risk ratio (RR) 3.2; 95% confidence interval (CI) 1.3-7.8), length of BE (RR 1.11 per cm increase in length; 95% CI 1.01-1.2), esophagitis (RR 3.5; 95% CI 1.3-9.5), and LGD (RR 9.7; 95% CI 4.4-21.5) were significant predictors of progression to HGD or EAC. In a prediction model, we found that the annual risk of developing HGD or EAC in BE varied between 0.3% and up to 40%. Patients with ND and no other risk factors had the lowest risk of developing HGD or EAC (<1%), whereas those with LGD and at least one other risk factor had the highest risk of neoplastic progression (18-40%). CONCLUSIONS: In patients with BE, the risk of developing HGD or EAC is predominantly determined by the presence of LGD, a known duration of BE of ≥10 years, longer length of BE, and presence of esophagitis. One or combinations of these risk factors are able to identify patients with a low or high risk of neoplastic progression and could therefore be used to individualize surveillance intervals in BE.

Advance notification letters increase adherence in colorectal cancer screening: a population-based randomized trial.

OBJECTIVE: The population benefit of screening depends not only on the effectiveness of the test, but also on adherence, which, for colorectal cancer (CRC) screening remains low. An advance notification letter may increase adherence, however, no population-based randomized trials have been conducted to provide evidence of this. METHOD: In 2008, a representative sample of the Dutch population (aged 50-74 years) was randomized. All 2493 invitees in group A were sent an advance notification letter, followed two weeks later by a standard invitation. The 2507 invitees in group B only received the standard invitation. Non-respondents in both groups were sent a reminder 6 weeks after the invitation. RESULTS: The advance notification letters resulted in a significantly higher adherence (64.4% versus 61.1%, p-value 0.019). Multivariate logistic regression analysis showed no significant interactions between group and age, sex, or socio-economic status. Cost analysis showed that the incremental cost per additional detected advanced neoplasia due to sending an advance notification letter was € 957. CONCLUSION: This population-based randomized trial demonstrates that sending an advance notification letter significantly increases adherence by 3.3%. The incremental cost per additional detected advanced neoplasia is acceptable. We therefore recommend that such letters are incorporated within the standard CRC-screening invitation process.

Screening for colorectal cancer: comparison of perceived test burden of guaiac-based faecal occult blood test, faecal immunochemical test and flexible sigmoidoscopy.

BACKGROUND: Perceived burden of colorectal cancer (CRC) screening is an important determinant of participation in subsequent screening rounds and therefore crucial for the effectiveness of a screening programme. This study determined differences in perceived burden and willingness to return for a second screening round among participants of a randomised population-based trial comparing a guaiac-based faecal occult blood test (gFOBT), a faecal immunochemical test (FIT) and flexible sigmoidoscopy (FS) screening. METHODS: A representative sample of the Dutch population (aged 50-74years) was randomised to be invited for gFOBT, FIT and FS screening. A random sample of participants of each group was asked to complete a questionnaire about test burden and willingness to return for CRC screening. RESULTS: In total 402/481 (84%) gFOBT, 530/659 (80%) FIT and 852/1124 (76%) FS screenees returned the questionnaire. The test was reported as burdensome by 2.5% of gFOBT, 1.4% of FIT and 12.9% of FS screenees (comparing gFOBT versus FIT p=0.05; versus FS p<0.001). In total 94.1% of gFOBT, 94.0% of FIT and 83.8% of FS screenees were willing to attend successive screening rounds (comparing gFOBT versus FIT p=0.84; versus FS p<0.001). Women reported more burden during FS screening than men (18.2% versus 7.7%; p<0.001). CONCLUSIONS: FIT slightly outperforms gFOBT with a lower level of reported discomfort and overall burden. Both FOBTs are better accepted than FS screening. All three tests have a high level of acceptance, which may affect uptake of subsequent screening rounds and should be taken into consideration before implementing a CRC screening programme.

Screening for colorectal cancer: randomised trial comparing guaiac-based and immunochemical faecal occult blood testing and flexible sigmoidoscopy.

BACKGROUND: Screening for colorectal cancer (CRC) is widely accepted, but there is no consensus on the preferred strategy. We conducted a randomised trial comparing participation and detection rates (DR) per screenee of guaiac-based faecal occult blood test (gFOBT), immunochemical FOBT (FIT), and flexible sigmoidoscopy (FS) for CRC screening. METHODS: A representative sample of the Dutch population (n = 15 011), aged 50-74 years, was 1:1:1 randomised prior to invitation to one of the three screening strategies. Colonoscopy was indicated for screenees with a positive gFOBT or FIT, and for those in whom FS revealed a polyp with a diameter > or = 10 mm; adenoma with > or = 25% villous component or high grade dysplasia; serrated adenoma; > or = 3 adenomas; > or = 20 hyperplastic polyps; or CRC. RESULTS: The participation rate was 49.5% (95% confidence interval (CI) 48.1 to 50.9%) for gFOBT, 61.5% (CI, 60.1 to 62.9%) for FIT and 32.4% (CI, 31.1 to 33.7%) for FS screening. gFOBT was positive in 2.8%, FIT in 4.8% and FS in 10.2%. The DR of advanced neoplasia was significantly higher in the FIT (2.4%; OR, 2.0; CI, 1.3 to 3.1) and the FS arm (8.0%; OR, 7.0; CI, 4.6 to 10.7) than the gFOBT arm (1.1%). FS demonstrated a higher diagnostic yield of advanced neoplasia per 100 invitees (2.4; CI, 2.0 to 2.8) than gFOBT (0.6; CI, 0.4 to 0.8) or FIT (1.5; CI, 1.2 to 1.9) screening. CONCLUSION: This randomised population-based CRC-screening trial demonstrated superior participation and detection rates for FIT compared to gFOBT screening. FIT screening should therefore be strongly preferred over gFOBT screening. FS screening demonstrated a higher diagnostic yield per 100 invitees than both FOBTs.

Screening for colorectal cancer: random comparison of guaiac and immunochemical faecal occult blood testing at different cut-off levels.

Immunochemical faecal occult blood testing (FIT) provides quantitative test results, which allows optimisation of the cut-off value for follow-up colonoscopy. We conducted a randomised population-based trial to determine test characteristics of FIT (OC-Sensor micro, Eiken, Japan) screening at different cut-off levels and compare these with guaiac-based faecal occult blood test (gFOBT) screening in an average risk population. A representative sample of the Dutch population (n=10 011), aged 50-74 years, was 1 : 1 randomised before invitation to gFOBT and FIT screening. Colonoscopy was offered to screenees with a positive gFOBT or FIT (cut-off 50 ng haemoglobin/ml). When varying the cut-off level between 50 and 200 ng ml(-1), the positivity rate of FIT ranged between 8.1% (95% CI: 7.2-9.1%) and 3.5% (95% CI: 2.9-4.2%), the detection rate of advanced neoplasia ranged between 3.2% (95% CI: 2.6-3.9%) and 2.1% (95% CI: 1.6-2.6%), and the specificity ranged between 95.5% (95% CI: 94.5-96.3%) and 98.8% (95% CI: 98.4-99.0%). At a cut-off value of 75 ng ml(-1), the detection rate was two times higher than with gFOBT screening (gFOBT: 1.2%; FIT: 2.5%; P<0.001), whereas the number needed to scope (NNscope) to find one screenee with advanced neoplasia was similar (2.2 vs 1.9; P=0.69). Immunochemical faecal occult blood testing is considerably more effective than gFOBT screening within the range of tested cut-off values. From our experience, a cut-off value of 75 ng ml(-1) provided an adequate positivity rate and an acceptable trade-off between detection rate and NNscope.

Pharmacogenetics of thiopurine therapy in paediatric IBD patients.

BACKGROUND: Azathioprine is widely used in the treatment of children with inflammatory bowel disease. The occurrence and type of adverse events to azathioprine may be related to thiopurine S-methyltransferase (TPMT) enzyme activity and to inosine triphophate pyrophosphatase (ITPase) deficiency. AIM: Investigate frequencies of functional TPMT polymorphisms and ITPA polymorphisms and their association with the occurrence of adverse events during azathioprine therapy in a paediatric inflammatory bowel disease population. METHODS: Seventy-two azathioprine treated paediatric inflammatory bowel disease patients, 47% girls, mean age 12.5 years (range 6.5-17.5), were assessed for TPMT and ITPA polymorphisms and for adverse events. The relation between polymorphisms and adverse events is evaluated. RESULTS: Of all azathioprine treated patients, 11 experienced an adverse event for which azathioprine was stopped: pancreatitis (n = 4), leucopenia (n = 2) and 'general malaise' (n = 5). Of the 11 patients who stopped azathioprine because of adverse events, 10 had wild-type alleles for all investigated genotypes. Genotyping of ITPA 94C>A polymorphisms showed that two patients were homozygous, both tolerated azathioprine well. CONCLUSIONS: No association of functional ITPA and TPMT polymorphisms and the occurrence of azathioprine related adverse events could be detected. Pharmacogenetic assessment prior to thiopurine therapy does not seem warranted.

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 villin. CDX2 expression was detected in 23 of 67 (34%) samples with only cardiac-type mucosa. Detection of CDX2 in cardiac-type mucosa increased the likelihood of finding intestinal metaplasia in another biopsy set of columnar-lined oesophagus (odds ratio 3.5, 95% CI = 1.2-10, P = 0.02). MUC2 was positive in 13 of 23 (57%) of CDX2-positive cardiac-type mucosa samples, whereas villin was detected in seven of 23 (30%). CONCLUSIONS: CDX2 expression in cardiac-type mucosa might be able to predict the presence of undetected intestinal metaplasia in columnar-lined oesophagus, and thus may be a putative marker for the presence of intestinal metaplasia in the absence of goblet cells.

Role of Fc receptor gamma chain in inflammation and cartilage damage during experimental antigen-induced arthritis.

OBJECTIVE: To study the role of Fc receptor (FcR) gamma chain in inflammation and cartilage destruction during antigen-induced arthritis (AIA). METHODS: FcR gamma-/- mice and controls were immunized with methylated bovine serum albumin (mBSA) in Freund's complete adjuvant, followed by induction of arthritis by local injection of mBSA into the right knee joint. Joint inflammation was studied by 99mTc uptake and by histology. Breakdown of proteoglycans from the cartilage matrix was determined by loss of red staining in Safranin O-stained knee joint sections, and matrix metalloproteinase (MMP)-mediated aggrecan degradation was determined by immunolocalization using anti-VDIPEN antibodies. Chondrocyte death was measured by determining empty lacunae in hematoxylin-stained sections and with the TUNEL assay in cryostat sections. Erosion was detected as ruffling of the cartilage surface. RESULTS: Joint swelling, as measured by 99mTc uptake on days 1, 3, and 7, was significantly decreased in FcR gamma-/- mice compared with controls. On day 7 after AIA induction, sustained joint inflammation, as seen histologically, was not significantly lower in FcR gamma-/- deficient mice. In various cartilage layers (femur, tibia, patella) of central arthritic knee joints, marked depletion of proteoglycans (40-70%), chondrocyte death (25-50%), and mild surface erosion were found. In FcR gamma-/- knee joints, depletion of proteoglycans was comparable (40-70%). Strikingly, chondrocyte death and matrix erosion were absent. Furthermore, MMP-induced aggrecan neoepitopes, which were abundantly found in controls, were also absent in FcR gamma-/-. Nevertheless, latent MMPs were present in the cartilage matrix as seen in APMA-activated patellae. CONCLUSION: FcR gamma chain is involved in the severity of acute and sustained inflammation and is a crucial factor in cartilage erosion during AIA, probably by regulating activation of latent MMPs present in the cartilage matrix.

Characterization of transgenic mice that secrete functional human protein C inhibitor into the circulation.

Protein C inhibitor (PCI) is a heparin binding serine protease inhibitor in plasma, which exerts procoagulant activity by inhibiting thrombomodulin-bound thrombin or activated protein C (APC). Since the role of PCI in vivo is largely unknown we generated genetically modified mice with expression of human PCI mRNA in hepatocytes only. Three transgenic lines have been characterized. Transgenic mice did not show gross developmental abnormalities. Two lines showed a pericentral and one line showed a periportal expression pattern of human PCI mRNA in the liver. Genetically modified mice secreted a functional transgenic protein into the circulation (3-5 microg/ml plasma in heterozygous mice and 10 microg/ml in homozygous mice), which inhibited human APC activity in the presence of heparin. Interestingly, transgenic mice in which human PCI was expressed periportally in the liver had the highest specific activity. Endogenous mouse PCI mRNA could only be detected in the male and female reproductive system, but not in the liver, indicating that endogenous PCI levels in the circulation are low or even absent in mice. These results demonstrate that the human PCI transgenic mice are a suitable model for studying the in vivo role of PCI in blood coagulation.

Resolution of cutaneous inflammation after local elimination of macrophages.

We constructed an immunotoxin, composed of an antibody directed against the high-affinity IgG receptor CD64 and Ricin-A, with the aim of resolving chronic inflammation through elimination of activated macrophages. In vitro, this immunotoxin proved very efficient in inducing apoptosis in activated macrophages, leaving resting and low CD64-expressing macrophages unaffected. We examined the activity of our immunotoxin in a sodium lauryl sulfate (SLS)-induced cutaneous inflammation model, using transgenic mice expressing human CD64. Upon intradermal injection of the immunotoxin (IT), cutaneous inflammation resolved in 24 h. This was demonstrated histologically by clearance of all CD64-expressing macrophages, followed by clearance of other inflammatory cells. Clinical parameters associated with inflammation, such as local skin temperature and vasodilation, also decreased.

The human Fc receptor for IgA (Fc alpha RI, CD89) on transgenic peritoneal macrophages triggers phagocytosis and tumor cell lysis.

Even though IgA is considered to play an important role in immunity, surprisingly little is known about the presence of IgA Fc receptor (Fc alpha R)-expressing effector cells in tissues. Difficulties in obtaining human tissue macrophages, led us to study peritoneal macrophages in a human Fc alpha RI transgenic (Tg) mouse model. Naive peritoneal macrophages did not express hFc alpha RI. Expression, however, could be induced by overnight culture, and was upregulated by GM-CSF. In addition, the receptor proved functional since macrophage-mediated phagocytosis and tumor cell kill were effectively triggered via hFc alpha RI. To assess necessity of the FcR gamma-chain signaling molecule for hFc alpha RI function in macrophages, Tg mice were crossed with mice deficient in FcR gamma-chain (gamma-/-). Tg, gamma-/- macrophages were unable to kill tumor cells. This, because Tg macrophages failed to express hFc alpha RI in the absence of FcR gamma-chain, and overnight culture did not overcome this lack of expression. Further studies with the transgenic mouse model presented in this study will help to define the precise conditions under which Fc alpha RI is expressed on macrophages. It will, furthermore, represent a useful tool to study Fc alpha RI function in immune defense.

Human immunoglobulin A receptor (FcalphaRI, CD89) function in transgenic mice requires both FcR gamma chain and CR3 (CD11b/CD18).

Even though more immunoglobulin A (IgA) is produced in humans than all other isotypes combined, relatively little is known about receptors that bind the Fc part of IgA. The myeloid IgA receptor, FcalphaRI (CD89), triggers various effector functions in vitro, but its in vivo role remains unclear. Here, a transgenic mouse model is described in which FcalphaRI is expressed under its own regulatory sequences. Receptor expression and regulation by cytokines was comparable to the human situation and hFcalphaRI can trigger phagocytosis and lysis of tumor cells. To analyze the contribution of the FcR gamma chain or the beta2 integrin CR3 (CD11b/CD18) in FcalphaRI biological function, FcalphaRI transgenic mice were crossed with either FcR gamma chain -/- or CR3 -/- mice. In contrast to in vitro data, FcR gamma chain was essential for surface expression of hFcalphaRI in vivo. Functional studies in hFcalphaRI/ gamma-/-mice were, therefore, limited. In vitro studies showed FcR gamma chain to be necessary for phagocytosis. Neither hFcalphaRI expression nor phagocytosis, triggered via hFcalphaRI, were influenced by CR3. Remarkably, the capacity to lyse tumor targets was ablated in hFcalphaRI transgenic/ CR3-/- mice, although binding of neutrophils to tumor cells was intact. This shows a previously unrecognized importance of CR3 for hFcalphaRI-mediated antibody-dependent cellular cytotoxicity (ADCC).

Partial characterization of the DNA repair protein complex, containing the ERCC1, ERCC4, ERCC11 and XPF correcting activities.

The nucleotide excision repair (NER) protein ERCC1 is part of a functional complex, which harbors in addition the repair correcting activities of ERCC4, ERCC11 and human XPF. ERCC1 is not associated with a defect in any of the known human NER disorders: xeroderma pigmentosum, Cockayne's syndrome or trichothiodystrophy. Here we report the partial purification and characterization of the ERCC1 complex. Immunoprecipitation studies tentatively identified a subunit in the complex with an apparent MW of approximately 120 kDa. The complex has affinity for DNA, but no clear preference for ss, ds or UV-damaged DNA substrates. The size of the entire complex determined by non-denaturing gradient gels (approximately 280 kDa) is considerably larger than previously found using size separation on glycerol gradients (approximately 120 kDa). Stable associations of the ERCC1 complex with other known repair factors (XPA, XPC, XPG and TFIIH complex) could not be detected.

Correction of xeroderma pigmentosum repair defect by basal transcription factor BTF2 (TFIIH).

ERCC3 was initially identified as a gene correcting the nucleotide excision repair (NER) defect of xeroderma pigmentosum complementation group B (XP-B). The recent finding that its gene product is identical to the p89 subunit of basal transcription factor BTF2(TFIIH), opened the possibility that it is not directly involved in NER but that it regulates the transcription of one or more NER genes. Using an in vivo microinjection repair assay and an in vitro NER system based on cell-free extracts we demonstrate that ERCC3 in BTF2 is directly implicated in excision repair. Antibody depletion experiments support the idea that the p62 BTF2 subunit and perhaps the entire transcription factor function in NER. Microinjection experiments suggest that exogenous ERCC3 can exchange with ERCC3 subunits in the complex. Expression of a dominant negative K436-->R ERCC3 mutant, expected to have lost all helicase activity, completely abrogates NER and transcription and concomitantly induces a dramatic chromatin collapse. These findings establish the role of ERCC3 and probably the entire BTF2 complex in transcription in vivo which was hitherto only demonstrated in vitro. The results strongly suggest that transcription itself is a critical component for maintenance of chromatin structure. The remarkable dual role of ERCC3 in NER and transcription provides a clue in understanding the complex clinical features of some inherited repair syndromes.