Bordetella pertussis, the causative agent of whooping cough. PT consists of an active subunit (S1) that ADP-ribosylates the alpha subunit of several mammalian G proteins, and a B oligomer (S2–S5) that binds glycoconjugate receptors on cells. PT appears to enter cells by endocytosis, and retrograde transport through the Golgi apparatus may be important for its cytotoxicity. A previous study demonstrated that proteolytic processing of S1 occurs after PT enters mammalian cells. We sought to determine whether this proteolytic processing of S1 is necessary for PT cytotoxicity.Pertussis toxin (PT) is an exotoxin virulence factor produced by Protease inhibitor studies suggested that S1 processing may involve a metalloprotease, and processing does not involve furin, a mammalian cell protease that cleaves several other bacterial toxins. However, inhibitor studies showed a general lack of correlation of S1 processing with PT cellular activity. A combination of replacement, insertion and deletion mutations in the C-terminal region of S1, as well as mass spectrometry data, suggested that the cleavage site is located around residue 203–204, but that cleavage is not strongly sequence-dependent. Processing of S1 was abolished by each of 3 overlapping 8 residue deletions just downstream of the putative cleavage site, but not by smaller deletions in the same region. Processing of the various mutant forms of PT did not correlate with cellular activity of the toxin, nor with the ability of the bacteria producing them to infect the mouse respiratory tract. In addition, S1 processing was not detected in transfected cells expressing S1, even though S1 was fully active in these cells.S1 processing is not essential for the cellular activity of PT. This distinguishes it from the processing of various other bacterial toxins, which has been shown to be important for their cytotoxicity. S1 processing may be mediated primarily by a metalloprotease, but the cleavage site on S1 is not sequence-dependent and processing appears to depend on the general topology of the protein in that region, indicating that multiple proteases may contribute to this cleavage. Bordetella pertussis, a bacterial pathogen of the human respiratory tract that causes the disease whooping cough. PT holotoxin is a multi-subunit complex with an AB5 structure [i proteins) in mammalian cells [Pertussis toxin (PT) is a complex exotoxin and an important virulence factor produced by tructure ,2: the ean cells ,4, and tan cells . The evean cells -9. Subcean cells , providean cells -9. Traffan cells -13. Aftean cells .Pseudomonas exotoxin A and shiga toxin are all activated after cleavage by the endogenous eukaryotic protease furin [Escherichia coli heat-labile toxin (LT) A subunits are cleaved at a protease-sensitive loop to promote maximal activity [Another observation that may bear on the cell biology and cytotoxicity of PT is that the S1 subunit appears to be proteolytically processed to a lower molecular weight form upon interaction of PT with mammalian cells . This prse furin , a subtise furin , or by cse furin . Choleraactivity ,20, and activity .In this study we extend the analysis of proteolytic processing of cell-associated S1 and conclude that S1 processing is not essential for the cellular activity of PT.125I-labelled PT was used for analysis of S1 processing in mammalian cells [In a previous study, an cells . As an aan cells , which, an cells , so it iWe first ruled out the possibility that furin, a protease that cleaves several other bacterial toxins in mammalian cells, is responsible for S1 cleavage, by finding that S1 processing occurs normally in furin-deficient FD11 cells  data no. The bro1, which inhibits vacuolar proton ATPase and therefore prevents endosome acidification [1 had a significant inhibitory effect on S1 processing, with 51% of cell-associated S1 in the unprocessed form  suggested a cleavage site close to either the N-terminus or the C-terminus of S1, although processing at both termini was also a possibility. Processing at the N-terminus of S1 seems unlikely in view of the fact that the arginine at amino acid 9 (R9) is crucial to enzymatic activity . A proteB. pertussis (data not shown) so that we were unable to determine whether processing was affected. However, other changes at this residue (A203G, A203S, A203V) had no effect on PT secretion, processing or activity (data not shown). In an attempt to locate the processing site, we constructed mutant forms of PT with deletions of various stretches of residues in this region. Each of 2 adjacent deletions of 8 residues  with an insertion of 8 amino acids between residues Q205 and A206 in S1 was processed to the same size S1p as wild type PT  and chymotrypsin W215A)  for 3 h to determine cellular activity. As shown in Fig. B. pertussis virulence [B. pertussis, it is possible that S1 could be processed in vivo by alternative proteases absent from CHO cells.We also tested several strains expressing mutant PT constructs in our mouse infection model of irulence , and nonPreviously we constructed stable CHO cell transfectants expressing S1 either with a signal peptide  or without one , and showed that S1 fully ADP-ribosylated target G proteins in each transfectant . In thatThe data from these transfectants do not rule out the possibility that S1 processing may contribute to the trafficking of PT to the ER. However, the data are consistent with the idea that S1 processing is not essential for its activity in mammalian cells, and therefore that translocation of the full-length S1 across the ER membrane occurs. This would distinguish PT from several other bacterial toxins of similar subunit structure, such as cholera toxin, heat-labile toxin and shiga toxin, for which processing of the A subunit is apparently important for cellular activity . The difIn this study we have further characterized the cellular processing of the S1 subunit after PT interacts with mammalian cells. Our major conclusion is that this processing event is not essential for PT activity in mammalian cells, based on several lines of evidence: (1) protease inhibitor studies showed a general lack of correlation between S1 processing and PT cellular activity; (2) mutant forms of PT in which S1 processing was apparently abolished retained significant (>75% of wild type) cellular activity; and (3) no S1 processing was apparent in transfected cells expressing active S1. Although we have not definitively ruled out a contribution of S1 processing to the cellular activity of PT due to the imperfect nature of our unprocessed mutants, it is possible that the processing event is completely unrelated to PT cytotoxicity and instead is an irrelevant activity occurring, possibly in lysosomes, on the large majority of the intracellular pool of PT molecules that do not enter the putative retrograde transport pathway to the ER and then on to the cytosolic target proteins.B. pertussis strain used in this study was a streptomycin- and nalidixic acid-resistant derivative of W28 (Wellcome). The PT9K/129G (PT*) derivative of this strain was constructed as previously described [B. pertussis was grown on Bordet-Gengou agar (Difco) plates containing 15% defibrinated sheep blood and the following antibiotics at the indicated concentrations where necessary: streptomycin 400 μg ml-1, nalidixic acid 20 μg ml-1, gentamicin 10 μg ml-1; or in Stainer-Scholte liquid medium [Escherichia coli strains used were DH10B [B. pertussis, and these were grown on LB agar plates containing 10 μg ml-1gentamicin where necessary or in LB broth containing 100 μg ml-1ampicillin.The escribed . The othd medium  containire DH10B  for stanre DH10B  for conjEcoRI-BsrGI fragment (containing the PT S1 and S2 genes) into EcoRI/Acc65I-digested allelic exchange vector plasmid pJHC1 [E. coli S17.1 and introduced into the B. pertussis chromosome by conjugation and allelic exchange as described previously [Plasmid pJ-PT was obtained by subcloning a 2.3 kb id pJHC1 . Mutatioeviously . Deletioeviously , and theeviously .Samples were run on 12% SDS-PAGE gels and transferred to nitrocellulose filters. To detect S1, blocked filters were incubated with S1-specific monoclonal antibody X2X5 or 1C7, followed by peroxidase conjugated anti-mouse IgG secondary antibody (Amersham). Blots were developed using ECL Plus (Amersham) and exposed to X-ray film.B. pertussis culture supernatants by the fetuin affinity method of Kimura et al. [E. coli DH10B containing the plasmid pGEX-αC20. For induction of the fusion protein, the strain was grown in LB to A600 1.0 and then IPTG was added to a concentration of 0.5 mM. 2 h after IPTG addition, cells were centrifuged, lysed in BPER lysis reagent (Pierce), cleared by centrifugation, and passed through a GSTrap column (Amersham-Pharmacia). Fusion protein was eluted in reduced glutathione buffer, dialyzed against PBS and analyzed by SDS-PAGE and BCA assay (Pierce) to determine protein concentration.PT and mutant derivatives were prepared from a et al. , resuspe3, and 0.5% Triton X-100) or RIPA buffer (10 mM Tris, pH 7.4, 0.1% SDS, 1% sodium deoxycholate, 1% NP-40, 150 mM NaCl) and incubated 30 min on ice. The lysate was then centrifuged 15 min at 13,000 rpm at 4°C in a microfuge, the supernatant was removed to a fresh tube and the pellet was resuspended in sample buffer. Samples were boiled 5 min and loaded onto an SDS-PAGE gel.CHO cells were grown in 6-well plates to near confluency and then PT was added and incubated for the indicated times at 37°C. Cells were then collected by trypsinization, washed in PBS, resuspended in 50–100 μl of either NET/Triton lysis buffer (150 mM NaCl, 5 mM EDTA, 50 mM Tris, pH 7.4, 0.01% NaN1 (ICN, 0.5 μM). After SDS-PAGE and western blotting of samples, band intensities were measured by densitometry and used to calculate the extent of inhibition of S1 processing.Inhibitors were added to CHO cells 30 min prior to the addition of PT. For protease inhibitors we followed the suggestions of Barrett  to deter2. Trypsin was present at 70 μg/ml. Sample buffer was added and the samples were then boiled 5 min before loading onto an SDS-PAGE gel.PT (100 or 200 ng) was digested with trypsin (Sigma) in a volume of 20 μl at room temperature for 1 h in 50 mM Tris, pH 8, and 2 mM CaClSamples were run on 12% SDS-PAGE gels and transferred to nitrocellulose filters. To detect S1, blocked filters were incubated with S1-specific monoclonal antibody X2X5 (3) (a generous gift from Drusilla Burns) or 3CX4, followed by peroxidase conjugated anti-mouse IgG secondary antibody (Amersham). Blots were developed using ECL Plus (Amersham) and exposed to X-ray film.32P-NAD , 10 ng PT, and an aliquot of the lysate sample. For assessment of in vitro enzymatic activity of PT samples by ADP-ribosylation assay, reactions contained, in 25 μl, 0.1 M Tris, pH 7.5, 20 mM DTT, 0.5 mM ATP, 1 μM 32P-NAD , 10–50 ng PT, and 0.5 μg GST-αC20 protein as substrate. The mixture was incubated 90 min at room temperature, sample buffer was added, and the sample was boiled 5 min and loaded onto 15% SDS-PAGE gels. After electrophoresis, gels were fixed, dried and exposed to X-ray film. Band intensities were measured by densitometry and used to calculate the extent of ADP-ribosylation of target proteins.To determine the cellular activity of PT samples, PT (0.5–2 nM) was added to near confluent CHO cells in 12-well culture plates, and after 3 h at 37°C cells were recovered from plates, washed in PBS and lysed in NET/Triton lysis buffer 30 min on ice. The lysate was then centrifuged 15 min at 13,000 rpm at 4°C and the supernatant was stored at -20°C until the assay. The ADP-ribosylation assay contained, in 25 μl, 0.1 M Tris, pH 7.5, 20 mM dithiothreitol (DTT), 0.5 mM ATP, 1 μM CHO cells were incubated overnight with PT and then lysates were made using Triton X-100 buffer at room temperature (to maximize the proportion of S1 in the soluble fraction). Purified 1C7 monoclonal antibody was allowed to bind to a PS10 chip (Ciphergen Biosystems) for 2 h at room temperature, which was then washed with PBS. The CHO cell lysates (soluble fraction) were diluted 1:1 with PBS and 100 μl of each lysate was added to a spot on the chip and incubated at 5°C for 4 days. The chip was washed 3 times with PBS containing 0.1% Triton X-100, 3 times with PBS, and twice with 5 mM HEPES (pH 7.0). After the chip was air-dried, matrix (Sinapinic acid) was added to each spot and allowed to dry. Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry analysis of these samples was performed in a model PBS-II machine (Ciphergen).Six-week-old female BALB/c mice (Harlan) were used for infection experiments. Bacterial inocula were prepared and intranasal inoculation of mice was performed as previously described . Seven dNHC conceived of the study, performed some experiments, supervised personnel and drafted the manuscript. RMM performed most of the plasmid and strain constructions, protein purification, processing assays and cell culture work. GVA performed the mouse infection experiments, some plasmid and strain constructions, protein purification, processing assays and ADP-ribosylation assays. RDP performed some of the plasmid and strain constructions and processing assays. ZEVW performed the GST fusion constructions and purification, some ADP-ribosylation assays, cell culture work and contributed to supervision of the other personnel.


Colorectal cancer is a common malignancy of the gastrointestinal tract. It is the second cancer cause of death in females and third in males. Production of prolactin has been reported with several tumours. However, elevated prolactin plasma levels in colorectal cancer patients remained unclear.In this cross sectional study serum prolactin and carcinoembryonic antigen (CEA) concentrations were assayed using immunoradiometric assay kits, preoperatively in 47 patients, and the results were compared with 51 age and sex matched controls.Prolactin and CEA concentration in patients were significantly more as compared with controls. Hyperprolactinemia was found in 36 (76.6%) patients, while 28 (59.6%) had high level of CEA.Prolactin may be a better tumour marker than CEA in patients with colorectal malignancy. Colorectal cancer is the third highest cause of cancer mortality . In AustCarcinoembryonic antigen (CEA) has been recognized as a serum marker for colorectal cancer in the past three decades  and has Prolactin (PRL) is a hormone with multiple biological actions, synthesized by the anterior pituitary gland  and is bThe aim of this study was to see whether PRL levels are increased in colorectal cancer and to compare the preoperative serum levels of PRL and CEA concentrations in colorectal cancer patients; also we intended to find the possible correlation between hyperprolactinemia and the stage of colorectal cancer.This cross-sectional study included 47 patients with colorectal cancer  and 51 non-cancer patients , who were candidates of colectomy. All patients were admitted in Shariati general hospital affiliated to Tehran University of Medical Sciences (TUMS) from April 2001 to January 2003. All women in the study were postmenopausal. None of the patients were on any drugs known to increase plasma prolactin levels in the last 6 months prior to the study and were not affected by endocrine, renal, and psychiatric disorders. There was no history of rectal bleeding and family history of colorectal cancer. Peripheral venous blood samples were daily collected from seven days before the operation to measure serum PRL and CEA levels. Blood samples were collected between 07:30 a.m. and 09:30 a.m. to avoid diurnal variation of PRL. Serum prolactin levels were measured with immunoradiometric assay (IRMA) kits produced by Kavoshian co (Tehran-Iran). Serum CEA levels were assayed by IRMA, who used kits from IMMUNOTECH co (Marseille-France). The PRL and CEA assays were carried out within 10 days of sampling. The cut off levels for PRL and CEA were 20 ng/ml and 5 ng/ml serum respectively ,18. StagThe study was approved by the Vice-Chancellor of ResearchEthic Committee of TUMS.The data evaluated by Chi-square test and T test using the Statistical Package of Social Science (SPSS Inc., Chicago, IL) for Windows version 11.5. A P-value of <0.05 was considered statistically significant.The number of male and female participants was 29 and 18 in case and 30 and 21 in control group.We compared serum PRL and CEA level of 47 patients with 51 healthy controls. There was no significant difference between the age and the sex distribution of the patients and control groups.Mean PRL serum level in colorectal cancer group was 21.6 ± 8.1versus 10.5 ± 4.6 in control group, which was significantly different (p < 0.0001). In other words 36 patients showed hyperprolctinemia (76.7%) while there was only one such patient in the control group (1.9%).CEA level in 59.6% of the cancer group was above the normal compared with 1.9% of controls (P < 0.0001).Out of 47 patients in the cancer group 9 were in stage I 19.1%), 19 in stage II (40.4%), 10 in stage III (21.3 %) and 9 in stage IV (19.1%). Hyperprolactinemia and high CEA concentrations were found in all stages  declare that they have no competing interests.Dr Ahmad Reza Soroush and Dr Hosein Mahmood zadeh participated in the design of study, acquisition of data and interpretation of data. Dr Mehrnush Moemeni participated in the design of study, acquisition of data, interpretation of data, and drafting the article. Behnam Shakiba participated in acquisition of data, interpretation of data, drafting the article and final approval of this version. Sara Elmi participated in analysis and interpretation of data, drafting the article and final approval of this version. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Avidity determination of antigen-specific immunoglobulin G (IgG) antibodies is an established serological method to differentiate acute from past infections. In order to compare the avidity of varicella-zoster virus (VZV) IgG in pairs of serum and cerebrospinal fluid (CSF) samples, we developed a new technique of avidity testing, the results of which are not influenced by the concentration of specific IgG.The modifications introduced for the new VZV IgG avidity method included the use of urea hydrogen peroxide as denaturing reagent, the adaptation of the assay parameters in order to increase the sensitivity for the detection of low-level VZV IgG in CSF, and the use of a new calculation method for avidity results. The calculation method is based on the observation that the relationship between the absorbance values of the enzyme immunoassays with and without denaturing washing step is linear. From this relationship, a virtual absorbance ratio can be calculated. To evaluate the new method, a panel of serum samples from patients with acute and past VZV infection was tested as well as pairs of serum and CSF.For the serum panel, avidity determination with the modified assay gave results comparable to standard avidity methods. Based on the coefficient of variation, the new calculation method was superior to established methods of avidity calculation.The new avidity method permits a meaningful comparison of VZV IgG avidity in serum and CSF and should be of general applicability for easy determination of avidity results, which are not affected by the concentration of specific IgG. Toxoplasma gondii. Therefore, avidity testing has been particularly useful for these pathogens.In addition to the determination of immunoglobulin M (IgM) antibodies, the avidity of immunoglobulin G (IgG) antibodies is an important parameter for the diagnosis or exclusion of acute infections. Testing of IgG avidity has been applied for a large variety of pathogens (reviewed in ,2). A coDetermination of antibody avidity is usually based on the separation of low and high avidity antibodies by denaturing agents in enzyme immunoassays (EIA) or immunofluorescence assays. Several agents such as guanidine hydrochloride , diethylDiagnosis or exclusion of acute infection is the most common but not the only application of avidity determination. Determination of antibody avidity in pairs of serum and cerebrospinal fluid (CSF) samples has been suggested as a diagnostic means to detect intrathecal antibody synthesis  and to dThe aim of our study was to establish an avidity assay that can be used for comparison of the avidities of varicella-zoster virus (VZV) IgG in serum and CSF. In order to achieve this aim, several modifications of a commercial VZV IgG assay were introduced. These modifications include the use of a novel denaturing agent and a new method to calculate the avidity results.The serum and CSF samples used in this study had been sent to the virological laboratory at the University of Würzburg for routine VZV testing and were stored in aliquots at -20°C. Two groups of serum samples were analyzed for the evaluation of the standard and modified VZV avidity assay. Group 1 consisted of 28 samples from 19 patients with acute or recent VZV infection and included 5 follow-up samples of patient F19, spanning a period of 11 months. The cases of this group fulfilled the following criteria: the presence of clinical symptoms suggestive of varicella, information on the disease onset, and a positive VZV IgM (Enzygnost Anti-VZV/IgM, Dade Behring, Marburg, Germany). Group 2 consisted of 37 samples from 37 subjects with infection in the distant past. In the subjects of this group, VZV IgG antibodies had been detected in previous samples taken at least 8 months earlier. For initial evaluation of the VZV avidity assay for CSF samples, three sample pairs of serum and CSF were tested, two from patients with VZV encephalitis (V2.1 and V9.2) and one from a patient with multiple sclerosis (M22).VZV IgG avidity determination of serum samples was performed in a semi-automated fashion using the Enzygnost Anti-VZV/IgG test kit (Dade Behring) with some modifications. Each of the sera diluted 1:231 in sample buffer was placed in two antigen coated microtiter plate wells. After 60 min at 37°C, one well was washed according to the instructions with the supplied washing buffer. The other well was washed with a solution of urea, or urea hydrogen peroxide, in the supplied washing buffer (for details, see Results) and once with the washing buffer only. The subsequent steps were carried out according to the instructions in an automated fashion using the Behring Elisa Processor III . Peroxidase-conjugated anti-human IgG was added in a 1:50 dilution and the plate was incubated at 37°C for 60 min. After three further washing steps with the supplied washing buffer, tetramethylene benzidine dihydrochloride was added as substrate and kept at room temperature for 30 min. The reaction was stopped with 0.5 N sulfuric acid. Control samples of acute and past VZV infections were included in each run. Where appropriate, VZV IgG antibodies were quantified by an one-point-quantification method (α-method, Dade Behring) according to the instructions of the manufacturer.To increase the sensitivity of the VZV IgG EIA in order to detect low-titer VZV IgG antibodies in CSF, the standard serum assay was modified as follows. The sample incubation time was increased to 180 min; the anti-human IgG was used in a 1:30 dilution and its incubation time was increased to 90 min. All incubations for the CSF assay were performed at room temperature because variation was found to be lower compared to incubation at 37°C. For the serum samples tested for evaluation purposes, the standard dilution of 1:231 was increased by a factor of 6 to yield a final dilution of 1:1386.Determination of VZV IgG avidity in CSF was always done in parallel with serum samples from the same time-point. Both serum and CSF were tested in at least four dilutions of a two-fold titration series. The starting dilution for each sample was derived from routine determinations of VZV IgG titers. For CSF samples, the starting dilution was at least 1:6.VZV IgG avidity was calculated by various methods. First, it was calculated as the ratio of EIA absorbance values obtained with and without the denaturing washing step. Alternatively, one-point quantification titers (see above) instead of absorbance values were used for avidity calculation.ref) and y the absorbance with the denaturant (absdenat). The m- and c-values of the equation for each sample were calculated with the software Excel (Microsoft) from two or more experimentally derived data points. It is essential, that the absorbance values obtained without denaturant (absref) fall in the linear range of the enzyme immunoassay. For the VZV IgG assay with modified assay conditions, the linear range of the absorbance values without denturant extended from approximately 0.200 to 2.800. Avidity was determined from the linear equation for various virtual x-values as the percent ratio of the absorbance values with and without denaturing agent, i. e. avidity = (absdenat/absref) = (m × x + c)/x. The result was referred to as virtual absorbance ratio.Two additional methods were used for determination of VZV IgG avidity in CSF and the corresponding serum samples. One method involved the use of the software "Avidity 1.2", based on curve-fitting analysis of serial dilutions . SecondlIn order to evaluate the usefulness of the Enzygnost VZV IgG EIA for avidity testing, a panel of serum samples from patients with acute VZV infection of defined onset group 1) and from controls with VZV infection in the distant past (group 2) was tested. In a preliminary experiment with three samples from each group, two different denaturing conditions were employed. The separation of the two sample groups with one 3 min washing step using 4 M urea hydrogen peroxide was superior to three 5 min washing steps with 5 M urea . Because it had been shown previously for the Epstein-Barr virus (EBV) assay that avidity index calculations based on one-point-quantification titers gave results better than calculations using absorbance values , the aviCSF avidity assay.In general, IgG concentrations in CSF are by a factor of 1:200 to 1:1000 lower than in the corresponding serum. Therefore, the detection sensitivity of the VZV IgG assay was increased by prolonging incubations times and increasing the concentration of the anti-human IgG conjugate in order to reliably detect VZV IgG in all CSF samples from patients with positive serum VZV IgG. To study the effect of these assay modifications on avidity determination, all serum samples of group 1 and 17 randomly chosen samples of group 2 were retested with the modified assay. All the serum samples were tested in dilution 1:1386 instead of 1:231 used in the standard serum assay. The results are shown in Figure For meaningful interpretation of avidity values in CSF, it is necessary to test serum samples obtained in parallel with the CSF. Because of the large difference of the IgG concentrations between serum and CSF, comparison of the avidity results necessitates independence of IgG concentration. To study this issue, CSF and serum samples were tested in twofold dilution series with the CSF avidity assay. Avidity was calculated as percent ratio of absorbance values from each dilution. The results for representative CSF and serum samples are shown in Figure Because the two methods for avidity calculation described above were significantly influenced by the chosen working dilutions, we developed a new calculation method of antibody avidity. It is based on our observation that the relationship between absorbance values without and with denaturing agent is nearly linear. This relationship can therefore be described by the linear equation y = m × x + c, where m is the slope, c the intercept, x the absorbance without denaturant and y the absorbance with denaturant. Representative examples from patients with acute and past VZV infection and of serum and CSF pairs are shown in Figure Further dilution series of CSF and corresponding serum were tested with the CSF avidity assay in order to confirm that the influence of the IgG concentration on the avidity indices derived from the virtual absorbance ratio was minimal. Table We established a VZV avidity assay that is suitable for comparative evaluation of antibody avidity in serum and CSF samples. In order to achieve this aim several modifications of a serum VZV avidity assay were necessary. Though there have been a few studies on the use of avidity assays for VZV IgG antibodies -18, no sUrea hydrogen peroxide appeared to be superior to urea in the optimization experiments for the VZV avidity assay and presents a novel option for a denaturing agent in avidity measurements. The denaturing effect of one washing step with a 4 M solution of urea hydrogen peroxide was more pronounced than that of three washing steps with a 5 M urea solution. Thus, the denaturing potential of urea hydrogen peroxide appears to be greater than that of urea. Furthermore, because of better solubility, handling of solutions of urea hydrogen peroxide is easier than handling solutions of urea in high molarity. Nevertheless, the optimal denaturing agent may vary between different antigens necessitating careful evaluation of denaturing conditions for each avidity assay.Under normal conditions, virus specific IgG is present in the CSF in very low concentrations. With an intact blood-CSF-barrier, the concentration gradient between serum and CSF is 200:1 to 1000:1. Thus, the usual serological methods for antibody quantification in serum are not sensitive enough to routinely detect specific IgG in CSF. Therefore, we modified the VZV IgG EIA to increase its sensitivity. This modification made it possible to detect and quantify VZV IgG in CSF samples from virtually all patients with measurable VZV IgG in serum. Corresponding serum samples were always run in parallel with CSF in the same assay. The CSF and serum dilutions were chosen individually for each sample pair in order to achieve similar absorbance values in the modified EIA. This assay formed the basis for avidity determination of CSF and the corresponding serum.For meaningful comparison of avidity values in serum and CSF, it is necessary to apply a calculation method that is independent of the concentration of specific IgG in the samples examined. One-point determination represents the simplest method for avidity index calculation, but such methods did not yield satisfactory results in this respect. The avidity technique based on end-point-titration is not influenced by the IgG concentration and is considered the reference method for avidity determination ,7. Howevdenat) for any virtual absorbance value without denaturation (absref). The avidity index is then calculated as the ratio of the two virtual absorbance values (absdenat/absref). Thus, if a fixed absref is chosen for all samples to be compared, the avidity index becomes independent of the concentration of specific IgG in the samples. These theoretical considerations have been confirmed by the results obtained from dilution series in this study. It will be interesting to test the general applicability of this method for standard avidity assays by comparison with single-point and end-point avidity determinations.Searching further for a simple calculation method of avidity indices independent of IgG concentration and sample dilutions, we observed a linear relationship between the absorbance values of the assays without and with denaturing washing conditions. After performing an avidity assay of two dilutions of a given sample, this relationship can be exploited to calculate a linear equation from the two pairs of absorbance values (with and without denaturation). The only requirement is that all absorbance values should fall in the linear range of the EIA. Once for a given sample the linear equation is experimentally determined, the equation can be used for calculation of the virtual absorbance value with denaturation  using the Avidity 1.2 software in infectious-disease diagnosis, and is a shareholder of an SME (Headman Ltd.) with commercial interest in it.RHK, JS and FT carried out the immunoassays and participated in the design of the study. RHK carried out the avidity index calculations. WZ participated in establishing the standard VZV IgG avidity assay. KH provided the software "Avidity 1.2" and participated in the data analysis. BW conceived of and coordinated the study and drafted the manuscript. All authors contributed to the final editing of the manuscript.The pre-publication history for this paper can be accessed here:


The topic of the paper is to study the stability of gene clusters, defined by various hierarchical clustering algorithms, of microarrays experiments including or not MVs.Microarray technologies produced large amount of data. The hierarchical clustering is commonly used to identify clusters of co-expressed genes. However, microarray datasets often contain missing values (MVs) representing a major drawback for the use of the clustering methods. Usually the MVs are not treated, or replaced by zero or estimated by the k-Nearest Neighbor  are highly sensitive to MVs, and surprisingly, for a very tiny proportion of MVs (e.g. 1%). In most of the case, the MVs must be replaced by expected values. The MVs replacement by the kNN approach constitutes one efficient method for restoring the missing expression gene values, with a low error level. Our study highlights the need of statistical treatments in microarray data to avoid misinterpretation.The presence of MVs (even at a low rate) is a major factor of gene cluster instability. In addition, the impact depends on the hierarchical clustering algorithm used. Some methods should be used carefully. Nevertheless, the  Taking advantage of this knowledge, the microarrays technologies allow the characterization of a whole-genome expression by showing the relative transcript levels of thousand of genes in one experiment . Numerouk-means  and the k-means ,9, or wik-means  and Indek-means .i.e. the gene expression levels for various experimental conditions. Different aggregation methods can be used for the construction of the dendogram generally leading to different tree topologies and a fortiori to various cluster definitions [i.e. genes) of two clusters to create a new cluster. Thus the single-linkage clusters contain numerous members and are branched in high-dimensional space. The resulting clusters are affected by the chaining phenomenon (i.e. the observations are added to the tail of the biggest cluster). In the complete-linkage algorithm, the distance between clusters is defined as the distance between the most distant pair of objects (i.e. genes). This method gives compact clusters. The average-linkage algorithm is based on the mean similarity of the observations to all the members of the cluster. Yeung and co-workers [Among these techniques, the HC approach is a widely held method to group genes sharing similar expression levels under different experimental conditions . The HC initions . For ins-workers  showed t-workers  showed bk-nearest neighbors approach (kNN) computes the estimated value from the k closest expression profiles among the dataset. Troyanskaya and co-workers showed that the weighted kNN approach, with k = 15, is the most accurate method to estimate MVs in microarray data compared to replacement by zero, row average, or Singular Value Decomposition [kNN approach is the most popular approach for estimating the MVs.The microarrays experiments frequently contain some missing values. The missing values are part of the experimental errors due to the spotting conditions (e.g. spotting buffer, temperature, relative humidity...) and hybridization (e.g. dust on the slide...) ,17. The position . A recenposition . In the position  have useSaccharomyces cerevisiae and human. Observing that MVs are widely present in the expression data, we then analyzed the effects of MVs on the results of a hierarchical clustering (HC) according to the chosen clustering algorithm. In the same way, we evaluated the impact of MVs replacement and estimation in the gene cluster definition by using a hierarchical clustering method.To explore the incidence of MVs in gene clustering, we first assessed the proportion of MVs in different sets of public data from 552; 16523) and  respectively. The percentage of MVs varies from 0.8% to 10.6%. No relation had been found between the number of genes and the percentage of MVs or the percentage of genes without MVs. As expected, the percentage of genes with MVs increases in function of the number of experimental conditions. In the Sorlie [Table e Sorlie , Spellmae Sorlie  and Gasce Sorlie  respectiDefinition of a complete datafile: We selected from literature a set of microarray data. This set was filtered to retain only genes without MVs. (ii) Construction of reference gene clusters: A gene clustering was performed by different HC algorithms . Seven types of algorithms were used (see section Methods). For each analysis, we defined a number Kalgo of gene clusters, representing the reference clusters. (iii) Generation of a datafile with MVs: MVs were randomly inserted with a fixed rate τ. This rate correspond to the percentage of genes with 1 missing value. So, we defined data with MVs. (iv) Analysis of the newly generated expression data: We carry out a data processing similar to step (ii), with the construction of dendograms for each algorithm  and the definition of the Kalgo new clusters in the different experiments (no replacement of MVs was carried out). Finally, we assessed the stability of the gene clustering by calculating an index measuring the percentage of conserved genes between the clusters of the reference set and the generated set.Figure kNN approach, and clustering with MVs replaced by zero.Moreover, two other experiments were (v) designed and (vi) evaluated: clustering with MVs estimated by the ca. 6000). The original Ogawa set contained 6013 genes with 230 genes having MVs. The elimination of the genes with MVs (i.e. 3.8% of the genes) leads to a set with 5783 genes. For the GS, the number of MVs is more important and some experimental conditions have more than 50% of MVs. So we have limited the final number of selected experimental conditions from 178 to 42 (see section Methods), it allows to conserve 5843 genes, i.e. only 310 genes are not analyzed, representing 5.0% of all the genes.As we work on the impact of MVs in gene clustering, we need at first biological datasets without MVs. These sets have been extracted from Ogawa set   of the initial OS, GS, GSH2O2 and GSHEAT gene content (see section Methods).To assess the influence of size of the datasets and the number of observations (genes), we have generated smaller sets corresponding to a ratio 1/Figure Kalgo, for each clustering algorithm . The rule consists in determining Kalgo clusters as the 10 most important clusters correspond to 80% of the genes of the dataset (see section Methods). The results for OS are shown in Table To perform the comparison of the gene clustering by HC, the numbers of clusters according to every type of hierarchical clustering algorithms have to be defined. Hence we defined the number of clusters,Conserved Pairs Proportion (CPP) index which corresponds to the percentage of genes found associated in the reference clusters and found again associated in the clusters generated from the data with MVs. Figure CPP decrease, the CPP values are always greater than 95%, (ii) the average-and median-linkage methods are within the range [65%; 80%] and (iii) the mcquitty, Ward and complete-linkage methods show the most striking loss. We observe a drastic loss of the clustering stability since τ = 1% of MVs. For instance, with 5% of genes with MVs, i.e. 40 missing data, the mcquitty, Ward and complete-linkage methods have a CPP of 62%, 57% and 52%, respectively. Beyond a rate of τ equal to 10%, the decrease becomes lower.The clusters defined from the reference data and the data with MVs are compared using H2O2 and GSHEAT and all the generated sets from 1/2 to 1/7. These last results show that the quality of the gene clustering is not disturbed by the reduction of the number of genes.Similar results are observed for all the sets OS, GS, GSCPP values of single-and the centroid-linkage methods remain too stable to observe a clear decrease as seen in Figure CPP values and to compare the different aggregation methods.It must be clearly noted that to limit the effect of the topology of each algorithm, we have fixed that the 10 most populated clusters must represent 80% of the genes. The number of the most populated clusters is fixed at 10 due to the Ward linkage method that gives a very limited number of clusters. Then, the percentage of genes belonging to the 10 most populated clusters have been tested ranging from 70% to 90% (data not shown). For example with a percentage equal to 90%, the kNN method has been described by Troyanskaya and co-workers [kNN approach goal is to compute the expected value of a missing value from the k nearest vectors without a missing value. As no theoretical approach exists to define the optimal k values (kopt), we have assessed every value of k within the range 1 to 100 and, selected the kopt value as the k value which has the minimal error rate. Table kopt values obtained for the four sets used in this study and their corresponding error rates. The kopt values are lower in OS subsets showing a low number of genes. The kopt values of GSH2O2 are within the range 11 to 17. The GS and GSHEAT sets exhibit more important variations within the range 8 to 28. The error rate decreases slightly according to the number of genes, but these variations are not significant. Nevertheless, this poor correlation may be due to the subsets composition. Indeed, they keep an equivalent number of clusters with a smaller number of genes per cluster. In the same way, it may simply be due to the kopt variation.The -workers  for the kNN method gives worse prediction of the extreme values than the values close to zero. The real data distribution follows approximately a normal distribution and the kNN approximation leads to a reduction of the standard deviation of this distribution. So, the prediction of the extreme values increases the global error rate implying a higher kopt to reduce this effect. For instance, in the OS sets, we observe that the values within the range [-1.0; 1.0] are approximated with a mean error rate within the range [0.12; 0.14]. Conversely the values more than 1.5 or less than -1.5 are approximated with a mean error rate superior to 1.8. The misestimating of extreme values has an impact on the clustering. One can notice that the unweighted kNN (mean of the k observations) exhibits worse results compared to the weighted kNN used in this study (data not shown).Figure CPP was computed for the seven agglomeration methods. We have compared the HC results obtained with the reference sets and the generated sets without replacement of MVs, with kNN replacement or with zero-value replacement methods. Table kNN and zero-value replacements both improved the mean CPP whatever the clustering method used, except for the Ward method with the zero value replacement. The kNN approach is the most relevant method to replace MVs. In 55.2% to 66.3% of the simulations, kNN is better and globally gives a mean increase of the CPP within the range [0.7; 2.1]. The centroid-and single-linkage methods have better increase in 74.8% and 99.3% of the simulations respectively due to their particular topologies. The zero value replacement is clearly less efficient.The CPPf to consider the f closest clusters of the selected cluster. This index is similar to the previous one and takes into account that the genes may be relocated in close clusters (see section Methods). It allows the evaluation of the topology conservation. We used f = 5. We observed that the co-associated genes in the reference sets are often displaced to close clusters in the simulated sets. As observed for the CPP, the kNN approach improves the CPPf for all the clustering methods in 51.9% to 60.2% of the simulations within the range [0.2; 1.5]. Due to their high initial CPP values (97.7% and 98.8%), single-and centroid-linkage methods do not have a gain as previously observed for the CPP.Nevertheless, as a slight variation can displace one gene into a close cluster, we have characterized another index named H2O2 have CPP and CPPf close to the one of OS. Conversely, the CPP and CPPf of GSHEAT are better than the ones of OS and GSH2O2 for the complete-linkage and Ward methods, but lower for the others. The GS set has higher CPP and CPPf for single-likage to mcquitty method due to a lower influence of the MVs in a vector with a higher number of experimental conditions. Nevertheless, the complete-linkage and Ward linkage still remain at a very low CPP (close to 50%).Similar results are obtained with the other sets with slight variations. For example, GSCPP values. In addition we have tested the consequences of using k values different of kopt values in the range [kopt-10 ; kopt+10] and we observed a decrease of CPP within the range [1%; 5%] (data not shown).Moreover, we have tested the influence of the number of MVs per gene by introducing more than one missing value per gene. We obtained similar results showing less than 0.2% of variations of the i.e. values superior to 1.5 and lower than -1.5. Table CPP values are superior to the CPP values obtained previously, because only the genes with important variations have MVs and are members of small clusters. We observe that MVs replacement has little effect. Indeed, the kNN and zero – value replacement cannot restore a correct distribution .We have followed the same methodology to analyze the extreme values, MVs are a common trait of microarrays experiments. Few works had been reported about MVs replacements -20 and nkNN replacement method was the most efficient approach to compensate the MVs effects compared to the classical replacement by zero. The kopt depends on the sample size. It is important to keep in mind that the MVs corresponding to extreme values are difficult to estimate with the kNN method. The impact of their approximation upon the clustering is significant. Hence, new approaches like the Bayesian Principal Component Analysis (BPCA) may overcome this problem. In a recent work Liu and co-workers suggest to potentially eliminate the incomplete series of data by using robust Singular Values Decomposition [We showed that the position . In addiposition .algo) a specific number of clusters . This point is one of the main difficulties noted by Yeung and co-workers [Contrary to Yeung and co-workers  and Gibb-workers  to evalu-workers  which sh-workers ,34 or Gr-workers  have comN = 6013 genes and n = 8 experimental conditions about the phosphate accumulation and the polyphosphate metabolism of the yeast Saccharomyces cerevisiae [S. cerevisiae [N = 6153 genes and n = 178 experimental conditions. Due to the diversity of conditions in this set, we focused on two experimental subsets corresponding to heat shock and H2O2 osmotic shock respectively.We used 8 public data sets from the SMD database ; see Tab. Two setrevisiae . The secrevisiae . This seN = 5783 genes and n = 8 experimental conditions. The second set without MVs was taken from Gasch et al. and called GS. The experimental conditions (column) containing more than 80 MVs were removed. The resulting GS matrix contains N = 5843 genes and n = 42 experimental conditions. Two subsets were generated from GS and has been noted GSHEAT and GSH2O2. They correspond to specific stress conditions as described previously. GSHEAT and GSH2O2contain respectively N = 3643 genes with n = 8 experimental conditions and N = 5007 genes with n = 10 experimental conditions.To evaluate the incidence of MVs on hierarchical clustering, we built complete datasets without MVs. All the genes containing at least one missing value were eliminated from the Ogawa set (noted OS). The resulting OS set contains i.e. the number of genes, we built six smaller sets corresponding to 1/2, 1/3, 1/4, 1/5, 1/6 and 1/7 of OS, GS, GSHEAT and GSH2O2. To obtain representative subsets, we can not use a random generation which can bias the results. So, we searched for each subset the series of genes which reflect at best all the genes of the complete set. First, the distance matrices between all the genes were computed. Then, we performed an iterative process by: (i) computing the sum of the distances for each possible t-uplets (t=2 to 7) of the set, (ii) choosing the t genes which have the minimal distance, (iii) selecting 1 representative gene upon the t selected genes, this gene is chosen as the closest to the barycenter of the cluster, (iv) eliminating from the process the t genes. Step (i) to step (iv) are repeated until all the genes are used. All the representative genes constitute the subset (1/t). This procedure allows one to reduce the redundancy of similar genes and to maintain approximately a number of gene clusters constant.To test the influence of the matrix size, τ of genes containing one MV (τ = 1 to 50.0%), these MVs are randomly drawn. Each random simulation is generated at least 100 times per experiment to ensure a correct sampling.From the sets without MVs, we introduced a rate vi, a missing value i for a given expression vector v (i.e. a gene), with the kNN method, the k vectors w corresponding to the k most nearest vectors to v (without taking the ith elements of the w-vectors into account) are searched. The missing value vi is then estimated by a weighted value of the k retained wi values.To fulfill d between the vector v and every vector w. The k minimal distances d(v, w) are selected and the estimated value  is computed as follow [The similar vectors are identified by calculating the Euclidean distance s follow ,19:kNN, s(v, wt) = 1 / d(v, wt), this similarity measure s(v, wt) is deduced from the distance d(v, wt) between v and its neighbors w. In equation (1), more a vector w close to v, more it contributes to the estimation of the missing value. The kNN approach has no theoretical criterion to select the optimal k value (kopt) [kopt using the sets without MVs to ensure a minimal bias in the comparison of the hierarchical clustering results. The determined kopt value is associated with a minimal global error rate as defined by Troyanskaya and co-workers [In the weighted e (kopt) . We have-workers .hclust package in R software [The hierarchical clustering (HC) algorithm allows the construction of a dendogram of nested clusters based on proximity information . The HC software . Seven hsoftware .i.e. genes) is calculated by using an external module written in C language. We used the normalized Euclidean distance d* to take account of the MV:The distance matrix between all the vectors (v and w are two distinct vectors and m is the number of MVs between the two vectors. Thus, (vi - wi) is not computed if vi and/or wi is a missing valueTo assess the influence of missing data rates and different replacement methods into clustering results, we have analysed the co-associated genes of an original dataset (without MVs) compared to these genes location in a set with MVs.reference clusterings (RC). In a second step, we generated MVs in data. The MVs are replaced by using the different replacement methods. Then we performed the hierarchical clustering for each new set. The results obtained by these second analyses are denoted generated clusterings (GC). We compared the resulting clusters defined in RC and GC. We assessed the divergence by using an index named Conserved Pair Proportions (CPP). The CPP is the maximal proportion of genes belonging to two clusters, one from the RC and the other one from the GC :The procedure for computing the index algo), we defined Kalgo, the number of clusters. As every type of hierarchical clustering algorithm gives a particular topology, we cannot use the same number of clusters to compare each aggregative method. So, we defined Kalgo such as its 10 most important cluster must represent 80% of the genes. For this purpose, we defined Kinit, an important initial number of clusters , and counted the number of occurrences associated to the 10 most populated clusters. Then we diminished Kinit by one unit and counted again. We stopped the process when the 10 most important clusters represent 80% of the occurrences . We denote by  the jth cluster for a given clustering algorithm with j = {1, ..., Kalgo}. The  clusters are associated with their corresponding gene list .i) For each reference clustering based on a given clustering algorithm  is used -, the second one after estimating the missing data by the kNN method (Eq.1), and the third one after replacing the missing data by zero. For each resulting tree, Kalgo clusters are defined. The  clusters are associated with their corresponding gene list , with j' = {1, ..., Kalgo }.ii) Three hierarchical clusterings are performed after generating MVs in proportion CPP index, we searched for each  cluster the closest  cluster. For each clustering algorithm , the corresponding  cluster is selected as the maximum number of genes  from the gene list  found in . Then, the Conserved Pairs Proportion (CPP) is computed as follow for one simulation:iii) Finally, to estimate the . The term  is the Kronecker symbol, i.e. it is equal to 1 when the genes i and i' in the two gene lists are identical, otherwise 0. G denotes the total number of genes. This index takes the maximal value 1 when the clusterings RC and GR are identical.where CPP-variation. If the remaining genes of a cluster are in the direct neighbour clusters, the use of CPP can bias the analysis. Thus, we characterized the CPPf ratio to consider the f closest clusters of the  cluster. The computation of CPPf ratio is based on the previous ratio and corresponds to the f clusters which are the closest to the winning cluster. From a selected , the upper node of the dendrogram is examined. If the number of clusters linked to this node is inferior to f, the upper node is selected. This process is performed until the number of clusters is inferior or equals to f. The last node tested  is used to compute the CPPf ratio.In addition, a variation of the tree topology may induce a AdB conceived of the study and carried out the MVs generation and the hierarchical clustering results. SH worked on the statistical analysis. AM has the initial idea of evaluating the MV influence on clustering, and participated in its design and coordination. All authors read and approved the final manuscript.


NDRG1 gene is induced by nickel, a transition metal sharing similar physical properties to cobalt. Nickel may create hypoxia-like conditions in cells and induce hypoxia-responsive genes, as does cobalt. Therefore NDRG1 is likely to be another gene induced by hypoxia. HIF-1 is a transcription factor which has a major role in the regulation of hypoxia-responsive genes, and thus it could be involved in the transcriptional regulation of NDRG1 gene. Hypoxia is such a common feature of solid tumours that it is of interest to investigate the expression of Ndrg1 protein in human cancers.The expression of NDRG1 gene and cause the accumulation of Ndrg1 protein. Protein levels remain high even after cells revert to normoxia. Although HIF-1 is involved in the regulation of NDRG1, long term hypoxia induces the gene to some extent in HIF-1 knock-out cells. In the majority of human tissues studied, Ndrg1 protein is overexpressed in cancers compared to normal tissues and also reflects tumour hypoxia better than HIF-1 protein.Hypoxia and its mimetics induce in vitro expression of NDRG1 gene, and nickel probably causes the induction of the gene by interacting with the oxygen sensory pathway. Hypoxic induction of NDRG1 is mostly dependent on the HIF-1 transcription factor, but HIF-1 independent pathways are also involved in the regulation of the gene during chronic hypoxia. The determination of Ndrg1 protein levels in cancers may aid the diagnosis of the disease.Hypoxia is an inducer of the  Cancer is a leading cause of death in humans, and current studies in the clinical area focus on either the early detection of this disease or on the development of new selective treatment tools. New tumour markers can provide aid in cancer diagnosis and create novel treatment opportunities.NDRG1) in different laboratories, is a stress responsive protein which shuttles between cytoplasm and nucleus upon certain insults [Ndrg1, also named Cap43, Drg 1, RTP and rit42 following the discovery of its gene ( insults . After iNDRG1 gene was nickel exposure, we worked on possible ways by which nickel could change the expression of this gene. Among the mechanisms investigated were epigenetic changes (DNA methylation and histone acetylation), signal transduction pathways , and ROS-mediated activation. However, none of these mechanisms were found to be involved in the induction of NDRG1 gene by nickel compounds [Since our origin for the induction of the ompounds -9.NDRG1 could be another hypoxia-responsive gene, and nickel could induce the expression of this gene creating hypoxia-like state in cells, as does cobalt. In the present study, the effects of hypoxia and hypoxia-mimicking agents on the NDRG1 gene expression have been investigated. Because HIF-1 transcription factor is a major regulator of hypoxia-responsive genes [NDRG1 gene expression and HIF-1 has also been studied. Here, it is reported that hypoxia is an inducer of the NDRG1 gene, and that HIF-1 transcription factor is involved in the regulation of this gene, but HIF-1 independent pathways also exist in the induction of the gene in the case of chronic hypoxia.On the other hand, it was shown that nickel induced the expressions of several hypoxia-responsive genes including vascular endothelial growth factor , erythrove genes ,14, the The process of tumour expansion is characterized by rapid growth of cancer cells as the tumour establishes itself in the host. Accompanying this rapid growth are alterations in the cancer cell microenvironment, typically caused by an inability of local vasculature to supply enough oxygen and nutrients to the rapidly dividing tumour cells. This makes hypoxia one common feature of solid tumours . ExploraNDRG1 gene, A549 cells were exposed to hypoxia (0.5% O2) for different time periods. The RNA transcript of NDRG1 started to appear after 4 hours of incubation and increased up to 18 hours  and deficient (HIF-1α-/-) cells were exploited. Short-term hypoxia (20 hrs) experiment showed that NDRG1 mRNA was induced in HIF-1 proficient but not in deficient cells  is a specific five nucleotide-HIF binding-DNA sequence (5'-RCTCG-3') which is common in all hypoxia-responsive genes ,26. NDRGd region . It is kd region . ConceivHIF-1 modifier enzymes, PHD and FIH-1, both have non-heme iron centres (29), and transition metals nickel and cobalt can interact with these centres, subsequently inhibiting the enzymes (30). By their effects on HIF-1 modifying enzymes, nickel and cobalt have the capacity of creating constitutive hypoxia-like conditions in cells -33.NDRG1 expression may be elaborated as follows: when we expose cells to nickel, internalized nickel inhibits PHD enzyme interacting with its iron centre. This prevents the hydroxylation of proline residue in the ODD domain of HIF-1α and subsequent pVHL binding, rescuing HIF-1α from proteasomal degradation . Rescued and accumulated α subunits of HIF-1 form stable heterodimers with β subunits and translocate to the nucleus, and HIF-1αβ heterodimers bind to hypoxia responsive elements (HRE) of the NDRG1 gene and promote the transcription of the gene. Nickel also inhibits the FIH-1 enzyme and subsequent hydroxylation of C-TAD domain of HIF-1α, and this in turn results in the recruitment of coactivators of HIF-1 to the NDRG1 gene regulatory sequences thereby further stimulating the expression of the gene . However, since the experiments to show the specific interaction between metals and HIF-1 modifying enzymes are yet to be executed, this aspect of the hypothesis remains unproven.Our hypothesis relating nickel, oxygen-sensing, hypoxia, HIF-1, pVHL, and Ndrg1 protein outlasts HIF-1 after hypoxia. Despite being a major regulator of hypoxia response, HIF-1 transcription factor is a very unstable protein which is rapidly degraded under normoxic conditions; the half-life of HIF-1α in post-hypoxic cells is less than 5 minutes. On the other hand our results showed that Ndrg1 protein levels remain high at least 16 hours after returning to normoxic conditions Fig. . SimilarNDRG1 expression indicated that the induction of the gene was primarily dependent on this transcription factor .Our study of relationship between HIF-1 and tor Fig. . NeitherOur work in several human tissues showed that in the majority of these organs Ndrg1 protein was differentially overexpressed in cancers compared to normal tissues Fig. , Table 1Several normal tissue samples showed some Ndrg1 expression albeit at lower levels than in cancer samples of similar tissues. In their comprehensive study, Lachat et al. (34) showed the expression of Ndrg1 protein in normal human tissues, reporting also the intensities and sub-cellular localizations of the stainings. We observed similar staining patterns in several tissues; more emphasized stainings in the glandular, acinar, ductal, and tubular cells of normal breast, prostate, colon, and kidney tissues. We also share the observation that Ndrg1 exist in all three locations of the cells-cytoplasm, nucleus, and membrane. But, with the exception of colon mucosa, when we stained the cancerous tissues of above mentioned organs, the staining was more intense. However, since the differential expression of Ndrg1 between normal and cancer tissues of lung, brain, and skin was much starker . More studies are needed to resolve how Ndrg1 levels are managed in normal cells, during hypoxia, and as well as in cancer cells.Another common finding between our study and Lachat et al's (34) is no expression of Ndrg1 in normal brain and lung epithelium. Lachat et al. (34) showed at the transcriptional level that normal brain and lung express NDRG1 gene by VHL tumour suppressor protein (pVHL). They state that no hypoxia-responsive element exists on the 5' flanking sequence of the gene and thus underplay the role of HIF-1 in the regulation of NDRG1. As mentioned previously, NDRG-1 gene has three HIF-1 binding sites, one in its promoter and two in the 3' untranslated region. Therefore, the down-regulation of NDRG1 gene by pVHL is likely to be mediated through the HIF-1 pathway. The observation of NDRG1 being down-regulated by a major tumour suppressor further supports our observation that it is up-regulated in several cancer tissues and could be used as a marker.Masuda et al.  report tHypoxia-responsive pathway (HRP) allows tumour cells to overcome harsh microenvironment conditions associated with tumour growth. The protein products of induced by this pathway  allow clones of tumour cells to gain growth advantage under unfavourable conditions, and this concept is pivotal in switching to a more malignant phenotype. Although the exact functions of Ndrg1 are still unknown, as another effector of HRP, it is also likely to help tumour cells establish themselves. Therefore, the use of drugs that specifically disrupt the functions of Ndrg1 protein may provide new cancer therapies. It is thus of interest to investigate the effects of the elimination of this protein on the cancer cell survival and proliferation. However, first Ndrg1 expression in normal hypoxic tissue (such as infarct tissues) should be determined to show the cancer specificity of the protein. Second, the potential side effects of its elimination should be assessed since several normal tissues express Ndrg1 ubiquitously.Hypoxia is also an important determinant for the success of chemotherapy and radiotherapy . Masuda NDRG1 gene, and nickel probably causes the induction of the gene by interacting with the oxygen sensory pathway. Hypoxic induction of NDRG1 is mostly dependent on HIF-1 transcription factor. However, regulation of the gene in long term hypoxia involves some other HIF-1 independent pathways.Hypoxia induces the Ndrg1 protein was overexpressed in the majority of cancers studied here. With the exception of colon cancer, staining with Ndrg1 antibody distinguished between normal and tumour cells in most cancerous tissues. The mechanism of this overexpression is related to the hypoxic state of cancer cells. Ndrg1 protein is a better indicator of tumour hypoxia than HIF-1 in immunohistochemical analyses. This is probably due to the stable nature of the Ndrg1 protein compared to the very unstable HIF-1 protein, and also to capacity of Ndrg1 in reflecting HIF-1 independent hypoxia response. Therefore, the determination of Ndrg1 protein in tissue samples may provide a more useful tool for cancer diagnosis. Even though the exact functions of Ndrg1 protein are still unknown, as another effector in hypoxia-response, it can help cancer cells to survive and grow under unfavourable conditions. Therefore, it may also be possible to direct therapy towards Ndrg1 protein using drugs that specifically disrupt the functions of this protein.+/+, and HIF-1α-/- fibroblasts were gifts from Dr. Konstantin Salnikow of NYU. The production of HIF-1α+/+ and HIF-1α-/- fibroblasts was described elsewhere [Human lung cancer cell line A549 (CCL185), human osteosarcoma cell line HOS (CRL 1543), human mammary carcinoma cell line MCF-7 (HTB 22), and human prostate cancer cell line DU-145 (HTB 81) were purchased from American Type Culture Collection (Rockville, MD, USA). Human trachea epithelium (HTE) cells, HIF-1αlsewhere . PW cell2. Cells were passaged by trypsinization when they reached 70–80% confluence. A549 cells were grown in Ham's F-12K nutrient mixture (Kaighn's modification) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin [equivalent to 100 units (U)/ml and 100 μg/ml, respectively]. MCF-7, PW, HIF-1α+/+, and HIF-1α-/- cells were maintained in DMEM (Dulbecco's modified Eagle's medium) with the same supplements. HOS and HTE cells were grown in α-MEM  additionally supplemented with 2 mM L-glutamine. For Northern and Western blot experiments, 5 × 105 cells in 10 ml of media were plated in each of 10-cm dishes (Corning Inc, Corning, NY). Cell numbers were determined using the ZM Coulter Counter (Coulter Electronics, England). To render cells hypoxic, dishes were placed in an incubator chamber flushed with 95% N2 and 5% CO2. This resulted in approximately 0.1–0.5% O2 after several hours. After 20 hours, cells were released from hypoxia and quickly scraped in ice-cold phosphate-buffered saline (PBS), and analyses were performed as described below.The cell lines were maintained at 37°C as monolayers in a humidified atmosphere containing 5% CONDRG1 and actin probes were labelled with [α-32P]dCTP by using a randomly primed-DNA labelling kit (Promega). Cloning of NDRG1 gene was as described previously [NDRG1 probe, and then the membrane was stripped and rehybridized with actin probe to show loading. The bands were visualized by exposing X-ray films (Eastman Kodak Co, NY, USA) to hybridized membranes.Total RNA was extracted from cells immediately after exposures by using TRIzol reagent (Gibco-BRL) according to instructions of the manufacturer. 15 μg of RNA/lane was separated by electrophoresis in 1.0% agarose-formaldehyde gels and then transferred to nitrocellulose membranes . 3VO4), 10 mM sodium fluoride, and 1% NP40] containing protease inhibitors (PMSF 1 mM, aprotinin 1 μg/ml, leupeptin 5 μg/ml, and chymostatin 2 μg/ml). 40 μg or 25 μg of protein was loaded into each lane of a 10% SDS-PAGE gel and separated by electrophoresis. Proteins were then transferred to PVDF membranes (Roche Diagnostics Co, IN, USA), and the membranes were first incubated with rabbit anti-Ndrg1 polyclonal antibody at the dilution of 1:1000 in 5% non-fat dry milk for one hour at room temperature. The antibody production is described elsewhere [4 in 5% milk for one hour at room temperature. At the end the membranes were treated with chemiluminiscent substrate ECL (Amersham Pharmacia Biotech, UK) for 1 min at room temperature, and Biomax MR-1 films (Eastman Kodak Co, NY, USA) were exposed to the membranes. The molecular weight of Ndrg1 was determined using prestained molecular weight markers (Invitrogen Life Technologies, CA, USA).Cells were lysed, and proteins were harvested in 125 μl of TNES buffer [50 mM Tris-HCl (pH: 7.5), 2 mM EDTA, 100 mM NaCl, 1 mM sodium orthovanadate NaVO4, 10 m2O2 for a further 30 min. After incubation with the antibody against Ndrg1 protein overnight, the slides were processed with a second anti-rabbit peroxidase-conjugated antibody (Santa Cruz, CA, USA). Identification of the protein was then achieved using Avidin-Biotin horseradish peroxidase complex and 3,3-diaminobenzidine (DAB) as the chromogen. Negative controls were performed using nonimmune serum instead of primary antibodies.For in vivo detection of Ndrg1 protein in human cancer and normal tissues, immunohistochemical (IHC) staining was exploited, using a rabbit polyclonal antibody against a 30-aminoacid-sequence at the C-terminal end of the Ndrg1 protein [Immunohistochemical detection of HIF-1α protein was achieved by using Catalyzed Signal Amplification System (DAKO Corp., Carpinteria, CA) which is based on streptavidin-biotin-horseradish peroxidase complex formation. Antigen retrieval was done by 10 mM sodium citrate buffer (pH 6.0). The specimens were incubated overnight at +4°C with monoclonal anti-HIF-1α antibody  in a dilution of 1:1000. After the amplification of the signal according to manufacturer's instructions, the slides stained with chromogen DAB, counter-stained with hematoxylin, dehydrated, and then mounted.


Despite the well-documented benefits of using warfarin to prevent stroke, physicians remain reluctant to initiate therapy, and especially so with the elderly owing to the higher risk of hemorrhage. Prior research suggests that patients are more accepting of the risk of bleeding than are physicians, although there have been few qualitative studies. The aim of this study was to employ qualitative methods to investigate the experience and perspective of individuals taking warfarin.We conducted face-to-face interviews with 21 older patients  who had been taking warfarin for a minimum of six months. Participants were patients at a family practice clinic situated in a large, tertiary care teaching hospital. We used a semistructured interview guide with four main thematic areas: decision-making, knowledge/education, impact, and satisfaction. Data were analysed according to the principles of content analysis.Participants tended to have minimal input into the decision to initiate warfarin therapy, instead relying in great part on physicians' expertise. There appeared to be low retention of information received regarding the therapy; half the patients in our sample possessed only a superficial level of understanding of the risks and benefits. This notwithstanding, participants reported a high level of satisfaction with the care provided and a low level of impact on their day-to-day lives.Minimal patient involvement in the initial decision and modest knowledge did not appear to diminish satisfaction with warfarin management. At the same time, care providers exert a tremendous influence on the initiation of warfarin therapy and should strive to incorporate patient preferences and expectations into the decision-making process. Warfarin therapy is an effective anticoagulant indicated for the prophylaxis and/or treatment of venous thrombosis and atrial fibrillation (AF), the most common cardiac arrhythmia in older individuals . Oral anDespite the strong evidence base and the endorsement of warfarin therapy by authoritative guidelines, current prescribing patterns of warfarin remain something of a puzzle. Bungard and associates have described the 'real world' use of warfarin therapy as "sub-optimal"  and haveBungard and his colleagues recently conducted a systematic review of the reasons for the underuse of warfarin, identifying patient, provider, and system factors as well as identifying limitations in research studies and arguing for further research into these factors . StudiesClearly, the decision to initiate warfarin therapy is a complex interaction of many variables, involving patient, provider, and system factors. Most studies examining anticoagulation practices in primary care have used surveys or other forms of quantitative methods; however, given the inherent complexity of this subject matter, it is likely that qualitative research methods could provide significant additional insight. A comprehensive literature search failed to find a qualitative investigation of patient perspectives and experiences taking warfarin. The aim of this study, therefore, was to employ qualitative methods to examine the experience and perspective of individuals on long-term warfarin therapy for atrial fibrillation as a means to assess the extent to which the physician-identified barriers reported by Bungard  are in cThis research was undertaken in a family practice clinic situated in a large, tertiary care teaching hospital. The clinic employs 12 physician full-time equivalents working in three teams; each four-physician team is supported by two registered nurses. The practice, which serves a medium-high socioeconomic status population, has a large proportion of elderly patients and the prevalence of AF is higher than reported elsewhere . ComprehPotential participants were identified by the clinic nursing staff. The inclusion criteria stated that the patient must currently be on warfarin therapy and have been so for a minimum period of six consecutive months. Patients were excluded from the study if a significant co-morbidity prevented their participation, if they were unable to converse in English, or if they were unwilling/unable to provide informed consent. From a pool of approximately 60 eligible candidates, the nurses purposively sampled in order to achieve an even gender split as well as an equivalent number of patients who were both normally in-range and out-of-range on their INR tests. A total of 24 patients were invited to participate in the study. Three patients declined to take part, two of whom expected to be unavailable during the interview phase, whereas the third declined due to lack of interest. All participants signed informed consent forms in advance of their participation in the study, which was approved by the Research Ethics Board of the host institution. A demographic profile of the sample is presented in Table We utilized a semi-structured interview guide that was developed on the basis of salient issues identified in the scientific literature, specifically the various barriers to the prescription of warfarin for atrial fibrillation as reported by Bungard et al . InterviGiven the pre-determined nature of the themes, as described above, we employed a content analysis approach to the analysis of the interview data. Whereas grounded theory is used to develop data-induced themes or hypotheses, content analysis is the better-suited approach in those instances where the codes, categories, or themes of interest to the investigators have been previously discovered and described , as is tPrecise criteria were developed for each of the four pre-determined themes in the codebook, namely decision-making, knowledge/education, impact on daily life, and patient satisfaction. The 21 transcripts were then coded according to these criteria. Each transcript was coded by at least two members of the research team using a standardized coding form. Tests for inter-coder reliability indicated a high level of agreement among the coders; instances of disagreement were resolved through a process of discussion and negotiation that included both the fourth author and the principal investigator (CST & REGU). This process yielded a unit-by-variable matrix that allowed for substantive analysis of the data. In order to strengthen the validity of the findings, the analytic processes of coding and interpretation were reviewed by an independent external reader (DG).The great majority of participants reported that the decision to initiate warfarin therapy had been made by "the doctor" – a term that was used to refer not only to family physicians and general practitioners, but also to specialists and attending physicians in urgent care settings. Typically, there was little or no patient involvement in the decision-making process Table . In mostThe level of knowledge and understanding of the benefits and risks associated with warfarin therapy tended to vary with age. Elderly patients (aged 75+) demonstrated poorer knowledge than their younger counterparts; indeed, the knowledge level among older participants appeared to be quite superficial and scattered Table . WhereasAccording to participants' accounts, educational efforts aimed at informing patients about warfarin were minimal and insufficient Table . Those wWhile there is tremendous range in the perceived impact of warfarin therapy on the lives of these patients, the vast majority reported that they have not experienced complications (e.g., hemorrhage, drug interactions). Typically, the decision to start taking warfarin did not precipitate significant changes in their day-to-day lives; many participants reported experiencing only minor inconveniences Table . For theThe vast majority of interviewees reported a high level of satisfaction with the care they receive from the nurses and physicians in the clinic Table . Most paThe findings of this study provide significant and original insight into the perception and experience of patients taking warfarin. The data indicate that patients tend to have minimal input into the decision to initiate warfarin therapy; many have only a superficial level of understanding of the risks and benefits of warfarin; and the majority retain little from the education they received regarding warfarin therapy. This outcome is balanced, however, by the finding that for these patients there was both a high level of satisfaction with the care provided in the family practice setting and a low level of impact on their day-to-day lives.The principal strength of this study is the insight into the lived experience of warfarin therapy as gleaned from the unique perspective of family practice patients currently taking warfarin. We view this as a significant and novel contribution to the literature as we could find no other such study. It is important to note, however, that our sample was drawn from the patient population of an academic primary care practice that is both well-educated and of medium-high socioeconomic status. The applicability of these findings in other patient populations may therefore be limited.Interviewees revealed clear detachment from participation in the decision-making process around initiating warfarin therapy. In some cases, this detachment appeared to stem from the particular circumstances at the time; for instance, if the patient was involved in a medical emergency or was admitted to hospital and had several medications initiated. For others, there was a general belief or understanding that warfarin is a medication without which the patient faced imminent risk of death – it is therefore not a matter to be discussed or negotiated. This finding may be a function of the age of our participants. As a group, elderly patients tend to prefer a directed rather than shared consultation. Prior research indicates that seniors are more likely to be accepting of medical advice without much questioning, rather than assuming a more active role in the decision-making process . This atPatient knowledge of risks, benefits, and issues related to diet and alcohol intake was low, although younger patients demonstrated greater levels of understanding than did those over age 75. This finding is consistent with previous investigations. Lip and colleagues have detected lower levels of knowledge among elderly patients; moreover, longer duration of anticoagulation does not appear to ameliorate patient understanding significantly ,15. For With regard to the impact of warfarin therapy on daily life, our results indicate that warfarin is for the most part well tolerated and does not pose heavy additional burdens or lifestyle changes. The majority of participants were already taking several medications and visiting more than one doctor on a regular basis. This sample also had a low incidence of complications and previous studies have shown that the potential for complication does not on its own result in a significant impact ,17. ThatThe importance of the association between patient satisfaction and adherence has been established in prior research on anticoagulation. In a case-control study, Arnsten and colleagues  found thIncreasingly, theoretical models of the physician-patient encounter advocate the inclusion of patients in the decision-making process . Of courIn summary, the results of this study suggest that patients tend to have limited input into the decision to initiate warfarin therapy. Moreover, a majority appear to lack a comprehensive understanding of the risks and benefits associated with treatment. These findings, however, were balanced by the minimal impact of warfarin on daily life and the high level of patient satisfaction. Further research is required to assess whether these findings are similar in other patient groups, with different demographic and socioeconomic characteristics, including multi-cultural communities . InvestiNone declared.REGU conceived and initiated the study and will act as guarantor. GCD, JM, and BT conducted the interviews. CST co-ordinated the data analysis process. All authors participated in the analysis of the data and the writing of successive drafts of the manuscript and all have read and approved the final draft.The pre-publication history for this paper can be accessed here: