The carcinogenesis of colorectal cancer has been accepted by a model for a cascade of genetic alterations, named the adenoma-carcinoma sequence. In order to elucidate the carcinogenesis of the colorectal cancer more clearly, the genetic abnormalies of the non-neoplastic mucosal epithelium of the colon and rectum should be investigated. It has been speculated that colonic Paneth cell metaplasia (PaM) is one of the pre-neoplastic mucosa of colonic cancer. Therefore, we studied the propria mucosa of the right colon with PaM from the standpoints of the frequency of the K-ras codon 12 mutations (K-ras), which is initial genetic abnormality in colorectal cancer, and the loss of heterozygosity of microsatellite markers (LOH-MS), which has a relationship to development of colorectal cancer.Fifty-two regions with PaM histopathologically from 12 surgically resected right colon specimens were studied. DNA extraction of the colonic mucosa with PaM was obtained using a microdissection method, and the frequency of the K-ras of PaM was investigated by enriched polymerase chain reaction-enzyme linked mini-sequence assay, and the frequency of the LOH-MS  of PaM was examined by high resolution fluorescenced labeled PCR primers.K-ras mutation was detected in fifteen regions among 52 PaM (28.9%). All mutations were a single mutation and GGT changed to AGT in eleven and GAT in four. LOH-MS were detected in twenty-one regions among 52 PaM (40.4%) . No K-ras mutations and LOH-MS were detected in the controls .Colonic mucosa with Paneth cell metaplasia may be one of the pre-neoplastic mucosa in the development of the colonic epithelial neoplasia. Vogelstein et al.  have repHowever, the genetic abnormalities of the non-neoplastic mucosal epithelium of the colon and rectum has not been investigated, except the aberrant crypt foci  and hypeWe have speculated previously that the colorectal Paneth cell metaplasia (PaM) is one of pre-neoplastic mucosa on the development of the colorectal epithelial neoplasias , becauseThe main purpose of the present study was to investigate the frequencies of the K-ras codon 12 mutations (K-ras) and the loss of heterozygosity of dinucleotide microsatellite markers (LOH-MS) in the propria mucosa with PaM of the right colon.The materials were 12 surgically resected specimens of right colon, which had the colonic carcinomas were present, and histological diagnosis was assessed at the Department of Pathology, Juntendo Izunagaoka Hospital. Although we have intended to investigate the PaM in the normal colon of the individuals without cancer, using the biopsies specimens, it was difficult to detect the PaM in these specimens. No inflammatory bowel diseases were included in the materials. Informed consent was obtained from all the patients to investigate the genetic alterations in the current study.The specimens were fixed in 10 % buffered formalin solution and prepared by cutting the non-neoplasitic area into 3 – 5 mm sections. Each section was embedded in paraffin and stained with hematoxylin and eosin (HE), followed by immunohistochemical staining for anti-lysozyme . Immunohistochemical stainings were performed by the avidin-biotin-peroxidase-complex method, at dilution of 1 : 100.Fifty-two colonic mucosal regions with PaM, distant from neoplastic lesion, aberrant crypt foci and hyperplastic polyp, were detected by HE staining and anti-lysozyme antibody staining, and these mucosa were used as the target regions in the current study.All paraffin blocks of the target regions and 12 paraffin blocks with no PaM as controls of each materials were used for the DNA extraction.Paraffin blocks with the target foci mentioned above were prepared for DNA extraction. The target foci were microdissected using a 20-gauge needle, comparing the slide with HE staining in the same position. The extracted DNA was diluted with 5 ml of TaKaRa DEXPAT .2, 1 μM each primer, 0.625 U Taq DNA polymerase and 1 × PCR buffer  in a thermal cycler. Then, 10 μL of the denatured second PCR product was hybridized with probes to detect the K-ras codon 12 wild-type (GGT) and six mutant  DNAs were immobilized, at 55 degrees of temperature for 30 minutes, and 100 μL of biotinylated A and 0.01 U of TdqDNA polypmerase were added and incubation was continued at 55 degrees of temperature for 30 minutes.Mutation of K-ras was analyzed and compared by enriched polymerase chain reaction-enzyme linked mini-sequence assay (PCR-ELMA). In PCR-ELMA ,9, upstrFor development, 100 μL of avidin-horseradish peroxidase conjugate was added and the reaction was performed at room temperature for 30 minutes. Then, 100 μL of tetramethyl-benzidine (TMB) substrate was added and the plates were left to develop in the dark at room temperature for 20 minutes. Finally, 100 μL of stop solution was contained and the light absorbance of each sample was measured by spectrophotometry  with a 450 nm filter wavelength  were selected for LOH among the microsatellite markers, recommended by National Cancer Institute, because commonly it has been thought to be difficult to study the LOH using mononucleotide microsatellite markers.2, 1 μM each primer marked with fluorescent dye of three colors as blue, green and yellow, 0.625 U Taq DNA polymerase and 1 × PCR buffer  in a thermal cycler. 2. The electrophoresis was conducted 2 hours by means of an ABI-377 DNA auto-sequencer . 3. A comparison was made of peaks of same marker arising from normal tissue and the propria mucosa with PaM, using the Gene Scan TM waveform analyzed softwave, and LOH were assessed. That is to say, LOH (+) was assessed when the ratio of the peak area of  was less than 70 % or was more than 143 %.These LOH-MS were investigated using high resolution fluorescenced labeled PCR primers in proportion to the method of Tsuchida et al. . The outThe data were analyzed statistically with Student's t-test (t-test) and chi-square test ; a p-value of less than 0.05 was considered to be significant.K-ras mutation was detected in fifteen regions among 52 PaM (28.9 %). All mutations were a single mutation. For K-ras mutation patterns, 11 showed GGT to AGT, and four showed to GAT.LOH-MS was detected in twenty-one regions among 52 PaM (40.4 %) . No K-ras mutations and LOH-MS were detected in the controls .Thus, the frequency of both K-ras mutation and LOH-MS in the colonic mucosa with PaM were significantly higher than those of the controls .K-ras mutations have been detected in several human neoplasias -14, and In order to conclude the carcinogenesis of the colorectal cancer more clearly, the genetic abnormality of the non-neoplastic mucosal epithelium of the colon and rectum should be investigated, however, because the colorectal cancers are derived from the colorectal mucosa. It is also important for the preventive medicine of the colorectal cancer to know the carcinogenesis of it.However, until now, the genetic abnormalities of the colorectal non-neoplastic mucosa is unclear, except the aberrant crypt foci  and hypeWe have already reported that the colorectal Paneth cell metaplasia (PaM) is one of pre-neoplastic mucosa on the development of the colorectal epithelial neoplasias , becauseTherefore, K-ras mutation and the loss of heterozygosity of microsatellite markers of PaM were investigated in this study, and the current study is thought to be the first report focusing on this.Our results showed that K-ras mutation was detected in fifteen regions among 52 PaM (28.9%), and LOH-MS was detected in twenty-one regions among 52 PaM (40.4%).Namely, K-ras mutation and LOH-MS of PaM were not rare and the frequency of those of PaM were higher than those of the normal colonic mucosa, and it came to light that some PaM had the genetic abnormalities which had a relationship to the development of colorectal cancer.Paneth cells, which are usually situated at the base of the glands of the small intestine, were first identified by Scwalbe  in 1872,Now, these cells have been one of the most famous cells in the gastro-intestinal tract, however, the detail function of these is not clear. Paneth cells are sometimes seen in the colorectal tubules, for example in the proximal colonic mucosa of elderly subjects ,17, in pDescribed above, many riddles about Paneth cell are still remained. And the gene abnormalities of colonic mucosa with PaM in the current study may be not equal to those of single Paneth cell in the colonic mucosa, because it is difficult to obtain only single Paneth cell in the colonic mucosa, even if microdissection method is used.However, we have been able to investigate the colonic mucosa with PaM, and we think it very interesting that some PaM have K-ras mutation and the loss of heterozygosity of microsatellite markers, and these PaM may be thought to be the pre-neoplastic mucosa in development of the colonic epithelial neoplasia. Further molecular studies concerning Paneth cell metaplasia in the large bowel should be warranted.Colonic mucosa with Paneth cell metaplasia may be one of the pre-neoplastic mucosa in the development of the colonic epithelial neoplasia.PaM, colonic Paneth cell metaplasia; K-ras, K-ras codon 12 mutations; LOH-MS, loss of heterozygosity of microsatellite markers; HE, hematoxylin and eosin; PCR, polymerase chain reaction; ELMA, enzyme linked mini-sequence assay


Since their discovery a decade ago, microRNAs (miRNAs) have emerged as major regulators of gene expression in eukaryotes of all kinds. Only 20 to 40 nucleotides long, a miRNA binds to a specific target sequence within a much longer messenger RNA (mRNA), inhibiting its translation and thus controlling expression of the corresponding gene even after the DNA itself has been read. Within the human genome, there are about 250 genes that code for miRNAs. Each miRNA has the potential to bind to many different transcripts. Variations in miRNA sequence dictate the gene transcripts to which each will bind most strongly.Caenorhabditis elegans, growth control and apoptosis in the fruitfly Drosophila melanogaster, hematopoietic differentiation in mammals, and leaf development, flower development, and embryogenesis in the plant Arabidopsis thaliana. Despite their significance, the full range of genes miRNAs target is unknown, as is the best method for discovering them. In a new study, Debora Marks, Chris Sander, and colleagues describe an algorithm for determining the targets of miRNAs, and show they include more than 10% of all human genes.It has become clear that miRNAs play a critical role in controlling gene expression, for example, in larval developmental transitions and neuronal development in the worm The algorithm uses three factors to evaluate whether a potential target site is likely to actually be regulated by miRNA. First, the target site must have some degree of sequence complementarity to one or more of the known miRNAs. Second, the strength with which the predicted target and its miRNA bind together, which can be calculated from the sequence and other structural factors, must be higher than some threshold. Finally, evolutionary conservation—the presence of the target–miRNA pair in different organisms—is factored in, because the likelihood that the target and miRNA actually pair in vivo is greater if the pair is found in multiple types of organisms.Using these principles, and the specific weighting they assigned to each factor, Marks and colleagues identified 2,273 genes in humans, rats, and mice that are likely targets for miRNA regulation. This is probably an underestimate of the total, since the researchers required each candidate gene to have at least two miRNA target sites. The authors identified another 2,128 genes with only one target site, but note that the false-positive rate here is likely to be high. Whatever the final number, the implication is that several thousand of our approximately 30,000 genes are under the control of miRNAs. Of special interest is that these putative targets include many genes known to be associated with the fragile X mental retardation protein, a crucial but still poorly understood player in mRNA regulation, whose absence leads to a type of mental retardation called fragile X syndrome.Drosophila, 28 have close relations in mammals. Second, an individual miRNA may regulate multiple genes—Marks and colleagues found that the average miRNA interacts with seven distinct mRNAs, with a range from 0 to 268. Third, the genes regulated by a single miRNA may be functionally related, such as components of the protein degradation system or specific signal transduction pathways. Fourth, single genes may be regulated by multiple miRNAs—the gene that encodes amyloid precursor protein, for example, has at least eight miRNA sites—suggesting that expression may be combinatorially controlled by numerous cellular influences.The researchers' findings also reinforce several emerging principles of miRNA-based regulation. First, it is widespread among multicellular eukaryotes, and sequences are surprisingly conserved. Of the 78 known miRNAs in These results provide resources for a host of experiments to elucidate the mechanism of miRNA action, which is not well understood. Several of the identified mammalian miRNA–target pairs have near-perfect matching sequences. In both plants (where miRNAs were first discovered) and animals, such matches are associated with degradation of the mRNA.www.microrna.org), who can modify its parameters as experimental results and new models dictate.The authors fully recognize that their algorithm, called miRanda, is not the last word in miRNA target identification. In order to improve both the search for targets and the algorithm itself, they are making the algorithm and full sets of results in vertebrates available free to other researchers (


PLoS Medicine addressed the issue of ensuring equity in distributing AIDS medications. Reis and Capron discuss the study's implicationsA study by Wilson and Blower in the February issue of  Of the roughly 40 million people living with HIV , an estiGiven this gap between what can be done and what needs to be done, the people who set policies and administer programs to provide ART in high-burden countries are faced with difficult questions of distributive justice. Decisions regarding the pricing of ARTs and other care for patients with HIV/AIDS, the distribution of treatment centers, and potential measures to overcome barriers for vulnerable populations will determine who will get access to treatment and who will die. In order to deal with these crucial issues, decision-makers need guidance on how to design policies on equitable access to ART that respect human rights norms and ethical standards.PLoS Medicine [A new study by Wilson and Blower, published in the February issue of Medicine , addressMedicine . Wilson While the authors suggest that their method could be adapted to take other objectives into account, here they have taken an exclusively egalitarian approach to equity. Although this notion of equity is broadly accepted, other important approaches could have been taken into consideration. Simple equality in access can actually produce inequities (because a fair approach would differentiate among groups in the population according to their different needs); further, under some theories, those who are least advantaged generally should receive a disproportionate share of newly distributed benefits (the maximin principle) . In geogIn determining equitable spatial accessibility for the application of their model to KwaZulu–Natal, the authors used a rather rough estimation of HIV prevalence . As prevalence greatly varies between specific communities, future studies would certainly benefit from using more disaggregated data where available . SimilaPlace matters, but spatial accessibility is only one factor to be overcome in ensuring equitable access to health services. Studies show that even when services are available at a near distance, factors such as temporal accessibility, disease perception, stigmatization, and outright discrimination heavily influence “effective demand” . MoreoveWilson and Blower have developed a mathematical model to determine the fair geographical distribution of ART treatment sites and have applied it to the specific setting of KwaZulu–Natal. Despite some methodological and data limitations, such studies can inform policy-makers' decisions regarding the location of HIV services. Since distance to a treatment center is strongly determinant of patients' ability to access care, WHO is developing a service availability mapping tool to monitor relative equity between districts and identify major gaps in service availability, for example, availability of ART and prevention of mother-to-child transmission programs.Not only is further research needed to refine the spatial accessibility model presented by the authors but careful attention must be paid to other factors that affect access to HIV services and to the underlying assumptions as to what would constitute fair distribution. In a recent guidance document, WHO and UNAIDS recommended that ART programs include special measures to ensure access of vulnerable and marginalized populations and women to ART . The dec


Leukemia is a clonal disorder characterized by uncontrolled proliferation of haematopoietic cells, and represents the most common form of cancer in children. Advances in therapy for childhood leukemia have relied increasingly on the use of high-dose chemotherapy often combined with stem-cell transplantation. Despite a high success rate and intensification of therapy, children still suffer from relapse and progressive disease resistant to further therapy. Thus, novel forms of therapy are required.This study focuses on dendritic cell (DC) vaccination of childhood leukemia and evaluates the in vitro efficacy of different strategies for antigen loading of professional antigen-presenting cells. We have compared DCs either loaded with apoptotic leukemia cells or transfected with mRNA from the same leukemia cell line, Jurkat E6, for their capacity to induce specific CD4+ and CD8+ T-cell responses. Monocyte-derived DCs from healthy donors were loaded with tumor antigen, matured and co-cultured with autologous T cells. After one week, T-cell responses against antigen-loaded DCs were measured by enzyme-linked immunosorbent spot (ELISPOT) assay.DCs loaded with apoptotic Jurkat E6 cells or transfected with Jurkat E6-cell mRNA were both able to elicit specific T-cell responses in vitro. IFNγ-secreting T cells were observed in both the CD4+ and CD8+ subsets.The results indicate that loading of DCs with apoptotic leukemia cells or transfection with tumour mRNA represent promising strategies for development of cancer vaccines for treatment of childhood leukemia. Leukemia represents the most common form of cancer in children. There are two main types of childhood leukemia, acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). The success rate in treatment of childhood leukemia has improved continuously over the past decades , and todImmunotherapy based on vaccination with dendritic cells (DCs) has emerged as an attractive new form of therapy for cancer in general, and DC-based vaccines have already shown promise in follicular non-Hodgkin's lymphoma, and in other hematological malignancies -7. DCs aVaccination with tumor antigen-loaded DCs has been shown to induce both Th and CTL responses, and tumor regression in some patients ,17. An i2), IL-7, IL-2 and IL-12 from R&D Systems . Staurosporin was obtained from .GM-CSF was purchased from Novartis , IL-4, TNFα, IL1β and IL-6 from CellGenix , and Prostaglandin E2 . Non-adherent cells were collected and frozen for later use as responder cells. Adherent cells were cultured in 3 ml CellGro DC medium, supplemented with 800 U/ml GM-CSF and 10 ng/ml IL-4 every second day, until day 5 when maturation of DCs was induced by addition of maturation cocktail  for 24 h. Characterization of DC phenotype was done by staining 0,5 × 106 cells with fluorochrome-labelled antibodies against the Lin1 panel , HLA-DR, CD1a, CD80, CD83, and CD86 , and analyzing by FACSCalibur flow cytometry (Becton Dickinson). The mAb isotypes used were IgG1 FITC, IgG2a PE, IgG1 APC .PBMC from healthy donors  were obtained by density gradient centrifugation . Monocyte-derived DCs were generated under serum-free conditions from the adherent fraction of PBMCs cultured in six-well plates at a density of 4 × 10Jurkat E6 cells obtained from American Type Culture Collection (ATCC) were exposed to 1 μM staurosporin for 3 h to induce apoptosis. Apoptotic cell death was assessed using Annexin V-FLUOS as described by the manufacturer . For assessments of phagocytosis, Jurkat E6 cells were stained with the green fluorescent dye PKH-67 (Sigma Aldrich) as described in the kit manual, exposed to 1 μM staurosporin for 3 h and incubated with immature DCs at a ratio of 3:1. After 6 h, immature DCs were labelled with a red fluorescent antibody (mAb CD1a-PE). Phagocytosis of apoptotic cells was measured quantitatively by flow cytometry. Similarly, phagocytosis was visualized by confocal laser microscopy  using apoptotic Jurkat E6 cells pre-stained with PKH-26 red fluorescent dye (Sigma Aldrich) and DCs stained with the green fluorescent dye PKH-67.6 cells using Trizol Reagent as described by the manufacturer . Poly (A)+ mRNA was isolated from total RNA using the GenoPrep Direct mRNA kit . Purified mRNA was used fresh or stored at -80°C until use. Transfection of DCs with mRNA was performed as described previously [6 cells) were mixed with mRNA, transferred to a 4-mm-gap cuvette and pulsed with a BTX ECM-830 square-wave electroporator  using instrument settings 500 V and 1 ms. Transfected cells were incubated on ice for 30 s followed by addition of 2.0 ml cold CellGro DC medium supplemented with 10 ng/ml IL-4, 800 U/ml GM-CSF and maturation cocktail (see above), and transferred to standard culturing conditions. Transfection with EGFP-pCIpA102 mRNA (10 μg/400 μl) encoding the green fluorescence protein [Jurkat E6 cells were used as a source of tumor material. Total RNA was isolated from 20–25 × 10eviously , with mi protein  was usedThe Negative Isolation Kit  was used for isolation of CD4 and CD8 T cells according to the manufacturer's protocol. Isolation was performed on day 7 after in vitro priming, before setting up the ELISPOT assay.6) expressing Jurkat E6-cell mRNA or loaded with apoptotic Jurkat E6 cells, were co-cultured with 3 × 106 autologous non-adherent PBMC for 7 days in 1.0 ml CellGro DC medium without serum, before setting up the ELISPOT assay. The cultures were tested for INF- production in an ELISPOT assay [5, 1.0 × 105, 2.0 × 105 and 4.0 × 105 responding cells and 0.5 × 104 DCs per well. Mock transfected DCs were used as control. The assay was done in duplicate. Spots were counted manually, and the frequency of reactive T cells was calculated according to the formula: (spots with transfected DC - spots with non transfected DC)/number of T cells added.Mature DCs  and early apoptotic cells were accordingly used in all experiments. To verify apoptosis after 3 h exposure to staurosporin, cells were stained with annexin V-FLUOS and examined by flow cytometry. The assessments showed that more than 90% of the cells were annexin-V positive Fig. .To study uptake of apoptotic leukemia cells by immature DCs, Jurkat E6 cells were stained with the red fluorescent dye PKH-26 before induction of apoptosis. Immature DCs were stained with the green fluorescent dye PKH-67. Apoptotic Jurkat E6 cells were then co-incubated with immature DCs for various periods of time to determine the optimal conditions for internalization of apoptotic Jurkat E6 cells. We observed that immature DCs ingested apoptotic Jurkat E6 cells within 6 h of co-incubation. Phagocytosed apoptotic Jurkat E6 cells (red stained) were observed inside or in the process of being phagocytozed by immature DCs (green stained) by confocal microscopy Fig. .Flow cytometry was used to further determine the efficiency of DC loading with apoptotic leukemic cells. In these experiments, apoptotic Jurkat E6 cells had been pre-stained with the green fluorescent dye PKH-67 and DCs were identified by staining with PE-conjugated anti-CD1a. Highly efficient uptake of apoptotic Jurkat E6 cells was confirmed, since virtually all CD1a positive cells showed green PKH-67 staining Fig. .Following antigen loading, DCs were matured in the presence of pro-inflammatory cytokines for 24 h. Assessments by flow cytometry confirmed that this treatment led to up-regulation of CD83, and the co-stimulatory molecules CD80 and CD86, in compliance with a mature DC phenotype Fig. .Transfection of cells with tumor-derived mRNA is an alternative method for loading of DCs with tumor antigens. mRNA from the Jurkat E6 cells was isolated and electroporated into DCs according to previously optimized methods . FollowiELISPOT assay of INF-γ producing cells is the method of choice for assessments of T-cell responses against cancer vaccines representing a heterogeneous mixture of antigens. This assay measures in a quantitative way the number of reactive T cells in pre and post-vaccination samples and thus directly relates the effect of vaccination to in-vivo expansion of reactive T cells. Autologous T cells were stimulated with tumour-mRNA transfected DCs and with DCs loaded with apoptotic Jurkat E6 cells. Fig.  secreting T cells. However, it appeared that DCs loaded with tumour-mRNA in general were most potent in inducing T-cell responses.Immunotherapy for childhood leukemia has the potential to contribute to long-term control or cure of the disease. Until now immunotherapeutic approaches for leukemia have been limited to trials of cytokine therapy . FurtherWe observed that the frequencies of induced INF-γ producing T cells depended on the individual donor, the method of antigen-loading and the subset of T cells studied. Such variations are not surprising, since the model system used employs allogeneic cells and no effort was done to HLA match the blood donors with the Jurkat cell line in these experiments. Since the experimental system is based on the use of an allogeneic cell line, we expect multiple antigens, encoded by a broad array of polymorphisms, including other HLA alleles to be involved. We have therefore taken advantage of the genetic differences between responding cells and the leukaemia cell line by using the combined repertoires of membrane expressed and cross presented allo-antigens as a sensitive readout for immunological response in our experiments.We expected that the two different procedures would provide some differences in loading of HLA molecules with tumour-derived antigens and subsequently in the responding T-cell subsets. According to the current dogma, processed peptides from phagocytosed apoptotic cells would be directed to HLA class I molecules by a process known as cross-presentation and to HLA class II molecules by the classical pathway. Cross-priming of CTL with antitumor activity has been demonstrated with DCs loaded with apoptotic tumour cells ,21,35. SThe aim of the present study was to determine if loading of DCs with antigens derived from a tumour cell line, either as apoptotic cells or as mRNA would provide a basis for an efficient vaccine, using ELISPOT as a read-out of immune responses. It has been shown that DCs transfected with antigens encoded in tumor mRNA is capable of inducing potent T-cell responses against tumour-specific epitopes . While p secreting T cells. However, it appeared that DCs loaded with tumour mRNA in general were most potent in inducing T-cell responses.In our study we demonstrate that both DCs loaded with apoptotic Jurkat E6 cells or transfected with mRNA isolated from Jurkat E6 cells, can induce T-helper and CTL responses against antigens derived from allogeneic leukemic T-cells. We also show that the two different methods of antigen-loading did not result in any apparent differences in the phenotype of the mature DCs. In terms of immune responses both methods of antigen loading produced DCs capable of inducing INF-The author(s) declare that they have no competing interests.SJJ performed preparation of DCs and T cells, assessments of apoptosis and phagocytosis of apoptotic cells, preparation of Jurkat E6-cell mRNA and transfection of DCs, isolation of T-cell subsets CD4 and CD8 and induction of primary T cell responses. SSL did the transfection of DCs with EGFP mRNA and fluorescence microscopy of DCs. RP, FW and GG planned the project.The pre-publication history for this paper can be accessed here:


Late referral to specialist nephrological care is associated with increased morbidity, mortality, and cost. Consequently, nephrologists' associations recommend early referral. The recommendations' effectiveness remains questionable: 22–51% of referrals need renal replacement therapy (RRT) within 3–4 months. This may be due to these recommendations addressing the specialist, rather than the primary care providers (PCP).The potential of specialist intervention aiming at slowing progression of chronic renal failure was introduced individually to some 250 local PCPs, and referral strategies were discussed. To overcome the PCPs' most often expressed fears, every referred patient was asked to report back to his PCP immediately after the initial specialist examination, and new medications were prescribed directly, and thus allotted to the nephrologist's budget.In retrospective analysis, the stage of renal disease in patients referred within three months before the introductory round , was compared to referrals two years later .Relative number of patients remained stable (28%) for mild/ moderate chronic kidney disease (MMCKD), while there was a noticeable shift from patients referred severe chronic kidney disease (SCKD)  to patients referred in moderate chronic kidney disease (MCKD) .Individually addressing PCPs' ignorance and concerns noticeably decreased late referral. This stresses the importance of enhancing the PCPs' problem awareness and knowledge of available resources in order to ensure timely specialist referral. Late referral to specialist care for renal failure is associated with increased morbidity, mortality, and cost . ConsChronic renal disease may be asymptomatic for a very long time. This stresses the importance of primary care providers in ensuring timely referral to a renal care center. Levin reviewedThe problem of late referral seems to be ubiquitous, if discussions with colleagues world- wide may be believed, and has led to numerous local, regional and national initiatives aiming at ensuring timely referral into specialist care. Some initiatives,8 succesThis article summarizes findings from a local initiative started by one of the authors (K.H.) in 1997 in the city of Dortmund, Germany.In Germany, about 90% of the population are covered by the mandatory health insurance system ("Gesetzliche Krankenversicherung"). All physicians who wish to treat these patients are organized in associations, which negotiate a budget with the insurance companies. Depending on the specialty, a typical per capita budget is then assigned per patient for a period of three months. This budget covers consultation fees, and the cost of medication prescribed. If a physicians exceeds the total budget thus calculated for his practice, the insurance companies can demand restoration of funds. Typically, a GP's per patient budget would be much lower than a nephrologist's. During the time period described here, the nephrologist's budget per patient was about ten times as much than a general practitioner's. While this budget strategy is intended as a safeguard against excess prescriptions, it has been criticized by many physicians for inducing sub- optimal treatment as practice owners comply rather with budget demands than with best practice guidelines.Dortmund, a town of 589000, has a physician to patient ratio of 141/ 100000 (German average: 156/ 100000). There are five nephrological centers, including two hospitals. Currently, 172 general practioners ("Allgemeinärzte und Praktische Ärzte"), 123 internists ("Ärzte für Innere Medizin") and 21 urologists are listed in Dortmund. One of the internist practices supervises a dialysis center, but does not provide specialized nephrological consultancy. GPs, internists and urologists were considered primary care providers, as all referrals for treatment of chronic renal failures came from one of these specialties. There were no major fluctuations of physician numbers between 1997 and 1999, the period for which data was analyzed here.Joint initiatives by all five nephrology centres in Dortmund to give PCPs a series of lectures on treatment of chronic renal disease met with little response, as the presenters regularly outnumbered the audience. In the spirit of the Declaration of Helsinki, part II.1.: , K.H. deIn order to do so, education was brought to the primary care providers, as opposed to bringing the PCPs to education. Over a period of 18 months, K.H. introduced himself and the potential of timely nephrological care in delaying or even halting the progression of chronic renal disease to PCPs- General Practitioners, and specialists in Internal Medicine and Urology. This introductory round included participation in local PCPs' round tables and PCP organized continuous medical education events, but also individually meeting some 250 PCPs for discussion. During these sessions, the topic of renal disease was subtly introduced during discussions on the more common diseases and risk factors, such as cardiovascular disease, hypertension and diabetes mellitus.During these teaching sessions and discussions, three arguments were repeatedly brought forward by the PCPs:1. Mild- moderate renal failure should be treated by the primary care providers, while the nephrologists' task was seen in providing renal replacement therapy.2. Referral to specialist care meant risking the loss of the patient to the specialist's practice .3. If patients did return from specialist consultation, they were usually carrying recommendations for costly permanent medication, such as ACE inhibitors, that put a heavy burden on the PCP's budget.The first of these arguments was addressed by the "teaching module" of the visits. PCPs were given an executive summary of the complexities of diagnostic and therapeutic procedures used in state- of- the- art nephrology, and the potential of intensified treatment by specialists aiming at slowing the progression of renal failure was explained in detail.Secondly, all patients referred were asked to report back to the referring PCP within one week of the consultation, by which time a summary of findings and appropriate counsel had been sent ahead.recommendations, drug prescriptions were handed out directly and so allotted to the much higher nephrologist's budget, thus easing the financial pressure on the primary care providers. More importantly, also the follow- up prescriptions were done in the renal care center, so that at no time the PCPs budget became endangered.Thirdly, instead of drug The present study investigates the question whether improving the PCP's knowledge about the potential of timely treatment of chronic renal failure, in combination with addressing their economic concerns, succeeded in encouraging timely referral to specialist care.2, moderate chronic kidney disease (MCKD), 60 ml/min/1.73 m2 > ECC > 20 ml/min/1.73 m2, and severe chronic renal disease (SCKD), ECC < 20 ml/min/1.73 m2. Due to small proband numbers in the subgroups, the null hypothesis ("no inter- group differences") was tested by the non- parametric, two sided Chi- square test. Survival was estimated by Kaplan- Meier analysis. Statistical analysis was carried out using the SPSS v11.5 package.Retrospective analysis of patients' records. Analysis of patient records was carried out by a final year medical student (TDV). All patients had agreed to have their data used for quality control measures at the time of referral. Specifically informed consent was obtained from all patients still alive at the time of data collection. Two groups were selected by date of first contact with the nephrologist: Group A, third quarter 1997, immediately before the start of the initiative, and Group B, third quarter 1999, six months after the last visit to a primary care provider had taken place. All new patients whose records indicated referral for nephrological specialist treatment were included in the study. Criteria were subnormal creatinine clearance (ECC) or elevated serum creatinine, elevated blood pressure, proteinuria, or erythrocyturia. The patients in each of the two groups were divided into three subgroups according to their renal function: mild/moderate chronic kidney disease (MMCKD), ECC > 60 ml/min/1.73 mTable The present study is a retrospective analysis of patient data from a single nephrological referral centre, and statistics are carried out on a data set of limited size (n = 18 only in the pre- intervention group A). As such, the statistical significance of the findings is doubtful, and the results might be interpreted as akin to a case report, or a medical anecdote.As such, however, it may have a value quite different from, but equal to that of a randomized, controlled study.Aronson recently discussed the value of medical anecdotes; of the More often than not, observational studies give similar results to controlled randomized trials,13, and The positive finding of the present study is that active recruiting strategies may improve the referral pattern. Following the discussions with individual PCPs, more patients were referred to specialist care at a stage where appropriate treatment may slow or even halt the progression of chronic renal failure. This should benefit first the patient, and then in the long run society as a whole. Recent studies,15 have Late referral to specialist care in chronic renal disease remains a problem world- wide. As early as 1984, focusing on the question under which circumstances renal replacement therapy should be initiated or not, questionnaires showed significant differences in the attitudes of nephrologists and non- nephrologists towards referral. SimilarIt has been speculated that this may be because these recommendations address the specialist, rather than the primary care providers (PCP),2.At present, a medline search using the keywords "referral" and "chronic renal failure" listed a total of 81 references since 1974, with only 48 in specialist, not necessarily nephrological journals, while adding "guideline" reduced the reference list to a disappointing three references from the years 1998 and 1999, which included, however, a fully accessible online guideline in the Canadian Medical Association's journal. Neither the American, nor the European Renal Associations' guidelines,4 were fBetter results were obtained using the same search terms on a general search engine (Google), leading at first hit to the DOQI webpage, which includes the free access to full text guidelines. An extensive search using the same terms in German, however, showed no relevant results but an abundance of lay articles of dubious quality. This scarcity of qualified information might exclude those physicians not fluent in English from gathering relevant and up- to- date information. National renal care associations should therefore consider extending their educational efforts beyond their specialist members and selectively target PCPs and their respective professional associations.In the present study, two arguments brought forward by PCPs to explain their reluctance to refer early focused on the economic burden this presented to their own practice, rather than society as a whole. These two arguments concerned budget penalties if too much money was spent on the (costly) pre- dialytic patients with chronic kidney disease, and loss of patients after referral. This is an unusually frank statement.macro- economic level. Micro- economic considerations in the distribution of health care are only hinted at[The majority of 22 medline hits using the search terms "economy" and "renal disease" address the question of best cost- efficiency of treatment strategies on a Due to the retrospective design of this study, one cannot analyze to what extent the PCPs' economic considerations rather than their educational state were responsible for their referral pattern. The fact that financial concerns were the tenor of the individual discussions, however, indicates that health care planners may profit from taking this into account.The authors hope that this single- centre study will entice multi- centre RRT providers to conduct a prospective, large scale study to further investigate the relative contribution of the factors discussed here.The initiative presented here shows that timely specialist referral in renal care can be achieved. National and regional differences in the organization of health care provision may lead to variant strategies to implement optimal health care, but close collaboration with the primary care providers is essential.ECC: endogenous creatinine clearanceMCKD: moderate chronic renal diseaseMMCKD: mild/moderate chronic renal diseasePCP: primary care providerRRT: renal replacement therapySCKD: severe chronic renal diseaseThe authors declare that they have no competing interests.K.H. started the initiative, carried out all the teaching modules and gave access to the data.T.D.V. retrieved the data from archived filesZ.A.L. did statistical analysisM.L. did statistical analysisA.M. did statistical analysis and wrote the manuscript.The pre-publication history for this paper can be accessed here:


Caenorhabditis elegans. We show tight conservation of the HIF-1/VHL-1/EGL-9 hydroxylase pathway. However, persisting differential gene expression in hif-1 versus hif-1; vhl-1 double mutant worms clearly distinguished HIF-1–independent effects of VHL-1 inactivation. Genomic clustering, predicted functional similarities, and a common pattern of dysregulation in both vhl-1 worms and a set of mutants , with different defects in extracellular matrix formation, suggest that dysregulation of these genes reflects a discrete HIF-1–independent function of VHL-1 that is connected with extracellular matrix function.The von Hippel-Lindau (VHL) tumor suppressor functions as a ubiquitin ligase that mediates proteolytic inactivation of hydroxylated α subunits of hypoxia-inducible factor (HIF). Although studies of VHL-defective renal carcinoma cells suggest the existence of other VHL tumor suppressor pathways, dysregulation of the HIF transcriptional cascade has extensive effects that make it difficult to distinguish whether, and to what extent, observed abnormalities in these cells represent effects on pathways that are distinct from HIF. Here, we report on a genetic analysis of HIF-dependent and -independent effects of VHL inactivation by studying gene expression patterns in  Besides its known function of inactivating hypoxia-inducible factor (HIF), genetically engineered worms clearly demonstrate that there exist HIF-independent effects of the von Hippel- Lindau tumor suppressor Oxygen-Caenorhabditis elegans. Whereas mammalian cells possess three HIF-α isoforms that are targeted by VHL, C. elegans has a single HIF-α homolog (HIF-1) and a single VHL homolog (VHL-1), simplifying the genetic approach  bearing defects in genes involved in extracellular matrix function, supporting the existence of a conserved non-HIF pathway connecting VHL with an as yet unknown extracellular matrix function.To address this we have used a genetic approach in approach . Since hC. elegans, a whole-genome microarray was probed to compare transcript patterns in vhl-1 versus wild-type worms (n = 1). From this array a set of genes  was assayed quantitatively by ribonuclease (RNase) protection  were strikingly downregulated by VHL-1  and the egg-laying defective phenotype of egl-9, none of the worm strains showed obvious phenotypic abnormalities. Interestingly, hif-1 corrected the phenotype of egl-9.To determine the extent to which disruption of the conserved EGL-9/HIF-1 pathway mediates these effects we studied wild-type, egl-9 worms  of the six genes contained a potential HIF-1 binding core motif (RCGTG) within an arbitrarily defined region  that was conserved in Caenorhabditis briggsae  expression in normoxia was entirely dependent on HIF-1, whereas other genes retained substantial normoxic expression in hif-1 worms  showed modest upregulation, and one gene (phy-2) showed modest downregulation, by hypoxia that was independent of HIF-1, VHL-1, and EGL-9 , suggesting that the VHL-1–independent repressive effects on nhr-57 may be nonenzymatic.Though the six genes all conformed to the above patterns to demonstrate regulation by the EGL-9/HIF-1 pathway , there w-1 worms A. Second pathway C. Interehif-1; vhl-1 and hif-1 worms (n = 3). Fewer genes showed differential expression than in the vhl-1 versus wild-type array; however, persisting differential expression did suggest the existence of VHL-1 pathways that are independent of HIF-1  contained a potential HIF-1 binding site (HBS) within an arbitrarily defined region that was conserved in C. briggsae  have shown that the entire 110-kb region containing the coregulated gene cluster is arranged in tandem with a second nearly identical segmental duplication of the locus . At this level of identity, our microarray and RNase protection analyses cannot discriminate between the two copies of each gene, so for all of our analyses we have only used the names of the distal copy and genes from the proximal copy were excluded from computational analyses.Interestingly, all six genes validated by RNase protection assay to be negatively regulated by VHL-1 in a HIF-1–independent manner localize within 45 kb on Chromosome V . We applied single-linkage clustering (nearest-neighbor method)  to identp ≪ 10−5 B. Recentegl-9; hif-1 versus hif-1 worms  . Further experiments on dpy-18; hif-1 double mutant worms clearly indicated that the effects of DPY-18 on this group of genes were (like the effects of VHL-1) HIF-1 independent   and the e (DMOG)  were tesgon-1 (heterozygote), mig-17, and unc-6 mutant worms but not in the cuticle-associated dpy-11, bli-4, or sqt-3 mutant worms  that are distinct from protein hydroxylation. These experiments indicated that the six genes were, to varying extents, upregulated in the basement membrane-associated nt worms . Conversnt worms  and SQT-vhl-1 inactivation in different genetic backgrounds, these data clearly distinguish HIF-1–dependent and –independent effects of VHL-1 on gene expression. Somewhat surprisingly, all of the VHL-regulated genes analyzed fell into one of two patterns: independent of HIF-1 and EGL-9 and dependent on DPY-18, LET-268, GON-1, MIG-17, and UNC-6, or the reverse, suggesting that they reflect perturbation of two discrete aspects of VHL-1 function.By comparing the effects of vhl-1 versus wild-type array underlines the importance of the HIF-1 pathway in VHL-1 function. Computational analysis revealed that five of these genes  have at least one HIF-1 binding core motif (RCGTG) that is conserved in C. briggsae within an arbitrarily defined  promoter region, suggesting that they are direct HIF-1 transcriptional targets. Several genes  have mammalian homologs that are HIF targets  regulation by VHL-1 were located within 45 kb on Chromosome V. Analysis of the microarray data revealed that there was indeed a single, highly significant (p < 10−5) chromosomal cluster of genes negatively regulated by VHL-1 in a HIF-1–independent manner and that in total this cluster extended over 110 kb to include F56A4.9, Y45G12C.9, Y45G12C.12, and Y45G12C.2 in addition to the six genes validated by RNase protection assay. The chromosomal localization of genes in C. elegans is not random, with functionally related genes located close to one another     , gon-1 aoteases) , and unc netrin) —suggest  netrin) . These aronectin . Most turonectin . Howeverronectin . Though Escherichia coli strain HT115(DE3) expressing double-stranded (ds) RNA on Nematode Growth Medium containing 1 mM isopropyl-β-D-thiogalactopyranoside (ITPG) and 50 μg/ml ampicillin for 72 h. Plasmids for ds RNA production were derivatives of the L4440 vector and were obtained from J. Ahringer ; ds RNA sequences are available on WormBase (http://www.wormbase.org). Wild-type worms were Bristol strain (N2); mutant strains were obtained from the Caenorhabditis Genetics Center (gon-1 worms (maintained at 18 °C). The double mutants hif-1; vhl-1, egl-9; hif-1, and dpy-18; hif-1 were constructed using either fog-2 or unc-51 to mark hif-1(+); the double mutant egl-9; vhl-1 was constructed using unc-42 to mark egl-9(+); PCR was used to confirm homozygosity.Worms were studied as mixed-stage populations or as synchronized populations following brief exposure to sodium hypochlorite. Exposure to hypoxia (2% or 0.1% oxygen), DIP (200 μM), and DMOG (1 mM) was for 18 h . RNA ints Center . Strainsvhl-1 worms and hif-1 versus hif-1; vhl-1 worms were performed on independent samples of RNA , using near full-genome C. elegans DNA microarrays ; primary array data are available on the SMD (http://genome-www.stanford.edu/microarray) and are also shown in hif-1 versus hif-1; vhl-1 microarray comparisons (n = 3) the log2 fold change was calculated as the mean of the three log2 transformed fold changes. To test for significant upregulation, the mean log2 fold change was compared with zero using a Student's t test. The genes were ranked by amplitude of fold upregulation and a subset of genes was selected for potential validation by RNase protection assays ; (b) mean Cy3-dUTP and Cy5-dUTP background-corrected signal intensities exceeding 300 and 100 U, respectively (lower intensities than these were difficult to detect by RNase protection assay); and (c) high spot quality as judged by manual inspection. For the hif-1 versus hif-1; vhl-1 microarray comparisons (n = 3), genes (which had been filtered as described above) were considered to be differentially expressed if the mean fold change was greater than 2.0  as reciprocal best matches by BLASTN, initiated with the C. elegans gene coding sequence (a single ortholog of F56A4.2 could not be defined). Translation initiation sites  were inferred in both C. briggsae and C. elegans through alignment with the C. elegans coding sequence . Sequences encompassing the 1,000 nucleotides upstream to 250 nucleotides downstream of the translation start sites for orthologous genes were aligned using DNA Block Aligner (C. elegans and C. briggsae.(1) Identification of potential HBSs see . Ortholo Aligner  with the Aligner , identifhttp://genome.ucsc.edu/). The software used for simulations and clustering was implemented in Perl and is available on request.(2) Single-linkage analysis to determine spatial clusters of VHL-1–dependent, HIF-1–independent genes see . A maximhttp://genome-www.stanford.edu/microarray.Primary microarray data can be viewed at Table S1vhl-1  versus wild-type  comparison.Primary microarray data for the (6.5 MB XLS).Click here for additional data file.Table S2hif-1; vhl-1  versus hif-1  microarray comparisons. Continued in Tables Primary microarray data for the three independent (6.6 MB XLS).Click here for additional data file.Table S3Continuation of (6.6 MB XLS).Click here for additional data file.Table S4Continuation of Tables (6.6 MB XLS).Click here for additional data file.http://www.ebi.ac.uk/arrayexpress/) under accession number E-SMDB-23.Primary array data have been deposited in ArrayExpress (H. sapiens VHL gene discussed in this paper can be found in Online Mendelian Inheritance in Man (OMIM) under accession number 608537 (http://www.ncbi.nlm.nih.gov:80/entrez/dispomim.cgi?id=608537).The C. elegans genes discussed in this paper  can be found in the WormBase database by including the name of the gene at the end of the URL .The


When pathogens enter your body, most wind up engulfed (by phagocytes), poisoned (by stomach acids), or flushed out of your system. These defenses kick in when a bacterium's toxic secretions bind to cellular receptors that trigger a chain of events ending in an innate response. A wide variety of cells and chemicals, including phagocytes and cytokines, initiate the inflammatory reaction that redirects innate defenses in the bloodstream to the infected site.If all goes well, the innate system successfully contains and eliminates a pathogen at the site of infection. But if infection persists, so will the inflammatory response, and these first responders turn traitor. It's thought that the immune response rather than the bacteria, for example, causes diarrhea in food poisoning. And when infection leads to death—as happens in septic shock—the immune response may be just as culpable as the infectious microbe.Drosophila and the food-borne pathogen Salmonella. Working with mutant strains of both fly and bacteria, the authors identified genes important to the development of infection and disease and showed that the host's reaction can indeed be lethal.To better understand the interplay between pathogen and host in the onset of infection and disease, David Schneider and colleagues turned to the genetically compliant fruitfly Drosophila phagocytes find, engulf, and kill invading microbes and then alert the rest of the immune system—and how Salmonella circumvents these defenses to initiate disease.Pathogens possess various means to infect their host, unleashing toxins and secreting molecules that enhance virulence by breaching cell membranes and altering the intracellular environment. Fruitflies combat these intrusions with various innate responses, including phagocytosis. In this study, the authors investigated how Salmonella strain (S. tyhphimurium) that produces two pathogenicity complexes, called type III secretion apparatuses, which shuttle virulence molecules through the host's cell membrane and into its cytoplasm. One complex, SPI1, facilitates cell entry while the other, SPI2, retools the intracellular environment to suit bacterial growth. The authors created a series of less virulent Salmonella strains and examined their effects on wild-type (nonmutant) flies. In addition, they looked at infections of wild-type Salmonella in flies carrying mutations in two critical immune response pathways .Schneider and colleagues used a Salmonella died much faster than the Toll mutants and had far more bacteria in their blood. When Schneider and colleagues correlated Salmonella population numbers with fly fate, they discovered something surprising. In other bacteria models, flies die when bacterial pathogen numbers reach a critical mass. Here, Salmonella populations hit a ceiling and the flies died with comparatively few bacteria. In flies infected with Salmonella strains lacking either one or the other virulence complex, the flies survived despite increased bacterial growth.Imd mutants infected with nonmutant eiger gene—a homolog of the human TNF gene—live longer with Salmonella infections. This is the same phenomenon seen in TNF-induced septic shock, when patients die as much from their immune system's response to infection as from the bacteria itself. Since many of the proteins involved in the Drosophila imd pathway have counterparts in the mammalian antibacterial immune response, the model described here can help identify the genetic agents of metabolic collapse associated with bacterial infections in humans.The authors explain this surprising finding with a model in which the fly's immune response produces substances that ultimately engineer its own destruction. Their model is supported by experiments showing that flies carrying a mutation in the


Knowledge of how synapses alter their efficiency of communication is central to the understanding of learning and memory. The most extensively studied forms of synaptic plasticity are long-term potentiation (LTP) and its counterpart long-term depression (LTD) of AMPA receptor-mediated synaptic transmission. In the CA1 region of the hippocampus, it has been shown that LTP often involves a rapid increase in the unitary conductance of AMPA receptor channels. However, LTP can also occur in the absence of any alteration in AMPA receptor unitary conductance. In the present study we have used whole-cell dendritic recording, failures analysis and non-stationary fluctuation analysis to investigate the mechanism of depotentiation of LTP.We find that when LTP involves an increase in unitary conductance, subsequent depotentiation invariably involves the return of unitary conductance to pre-LTP values. In contrast, when LTP does not involve a change in unitary conductance then depotentiation also occurs in the absence of any change in unitary conductance, indicating a reduction in the number of activated receptors as the most likely mechanism.These data show that unitary conductance can be bi-directionally modified by synaptic activity. Furthermore, there are at least two distinct mechanisms to restore synaptic strength from a potentiated state, which depend upon the mechanism of the previous potentiation. These2+/calmodulin-kinase II (CaM-KII)-mediated phosphorylation of GluR1 which occurs during LTP [Recent studies have provided information on the possible mechanisms underlying postsynaptic alterations in synaptic strength. There is evidence that AMPA receptors are inserted into the postsynaptic membrane during LTP -15 and rring LTP , and whiring LTP . Therefode novo LTD .We have recently used peak-scaled non-stationary fluctuation analysis  of synaathways; ,23). In athways; . Indeed,N) [N never involves a change in γ (DPN). These data show, firstly, that there are two distinct molecular mechanisms for the reduction of synaptic strength that are dependent on the nature of the preceding LTP and, secondly, that γ can indeed be bi-directionally modified in response to synaptic activity.In the present study we have investigated a second form of LTD known as depotentiation (DP), which is a reversal of pre-established LTP -27. LTP N) . We wereUsing whole-cell recordings from the proximal apical dendrites of hippocampal CA1 pyramidal cells, minimal stimulation of nearby afferents evoked EPSCs that could be reliably resolved from failures  with a holding potential of 0 mV. This resulted in stable LTP .N). As noted previously [In agreement with a previous study , cells feviously , there wFigure rise: baseline = 1.6 ± 0.2 ms, LTPγ = 1.7 ± 0.3 ms, DPγ = 1.7 ± 0.2 ms, n = 11; τdecay: baseline = 8.3 ± 0.6 ms, LTPγ = 8.2 ± 0.6 ms, DPγ = 8.9 ± 0.7, n = 11; Figure For this group of cells, DP, induced by pairing stimulation (baseline frequency) with a holding potential of -40 mV, always resulted in a reversal of the γ increase, as indicated by the current-variance plot Figure . SimilarN), as indicated by the current-variance plot , a small depression of less than 50 %  was associated with no change in success rate  as well as a decrease in potency (potency ratio = 0.54 ± 0.04). This suggests that in these cells there was an additional decrease in Pr or n. Therefore, for DPγ, the change in γ did not fully account for the amplitude change in every cell . This il Figure  but did l Figure .N). As reported previously for different data sets from this age of rats [N). In the present study we saw a similar proportion of LTP expressed by changes in γ versus N. Strikingly, we observed a precise relationship between the mechanism of DP and the form of preceding LTP; LTPγ was always reversed by DPγ, and LTPN was always reversed by DPN.In this study we have shown that there are two mechanisms for NMDA receptor-dependent DP, a reduction in γ (DPγ) and a decrease in the number of activated AMPA receptors ? It is unlikely that this type of depression is due to a change in channel kinetics because there was no change in EPSC kinetics . The. TheN)? ics see ,35. Post. PostN? ee [open or in theee [open .N are indistinguishable from those that we and others have reported recently for de novo LTD [de novo LTD were mutually occlusive, indicating a convergence of mechanisms. These data argue strongly for a postsynaptic mechanism of expression. Furthermore, since pep2m causes the removal of AMPA receptors from the membrane surface, as determined immunocytochemically [de novo LTD is due to the physical elimination of synaptic AMPA receptors. Other evidence for a postsynaptic mechanism for de novo LTD includes a reduction in the postsynaptic sensitivity to glutamate [N  a change from a single low conductance state to a single high conductance state, 2) changes in open times within a burst and, 3) changes in the proportion of time spent in different conductance states. Since AMPA receptors are known to have multiple conductance states ,33 we hade novo LTD was induced there was never a change in γ [de novo LTD and DP. Moreover, we have also investigated these possibilities using our standard compartmental model [decay which was never observed experimentally, and 2) pronounced changes in asynchrony necessary to cause an artefactual change in γ cause large deviations from the parabolic relationship in the current-variance plot (unpublished observations) and alterations in τrise [rise and τdecay [Other theoretical possibilities exist to explain DPγ. For example, the silencing of synapses close to the patch electrode leaving more distant synapses contributing a lower net γ due to electrotonic filtering, or a decrease in trial-to-trial asynchrony of transmitter release. We believe that these possibilities are unlikely because in every cell in which nge in γ . If suchal model . This shin τrise , also nein τrise . Such chin τrise , howeverd τdecay , which wIn summary, we have shown that there are two distinct molecular mechanisms for the reduction of synaptic strength. Although previous studies have provided evidence that LTD is associated with dephosphorylation of serine 845 on GluR1 ,17 it is4, 26 NaHCO3, 2 CaCl2, 1 MgSO4, 15 glucose, 2 ascorbic acid, 0.05 picrotoxin, saturated with 95% O2 / 5% CO2, at room temperature (23–25°C). Individual dendrites were visualised using infrared illumination and DIC optics and approached under visual control. Whole-cell dendritic recordings of synaptic currents were obtained at a holding potential of -70 mV using patch electrodes (6–10 MΩ) filled with a solution containing (in mM): 135 CsMeSO4, 8 NaCl, 10 HEPES, 0.5 EGTA, 4 Mg-ATP, 0.3 Na-GTP, 5 QX-314, pH 7.25, 285 mOsm. Schaffer collateral-commissural fibers were stimulated at 0.5 Hz using a platinum monopolar or concentric bipolar electrode, which was positioned 20–40 μm from the dendrite parallel to the input pathway. The stimulus intensity was set to evoke some failures to enable a failures analysis to be performed and to ensure that the majority of EPSCs were, for any given trial, evoked by release from a single site. However, to elicit sufficient EPSCs to obtain a baseline estimate of γ before "washout" of LTP it was usually necessary to set the success rate fairly high . As a result it is likely that multiple release sites contributed to the recordings. LTP was induced by pairing 40–60 stimuli (baseline frequency) with a holding potential of 0 mV. DP was induced following the induction of stable LTP by 100–200 stimuli (baseline frequency) at -40 mV holding potential. All recordings were made using an Axopatch 1B amplifier, signals were filtered at 5 kHz (8 pole Bessel filter), digitised at 10 kHz and stored on computer. EPSC amplitude and input resistance were analysed and displayed on-line using the 'LTP' program [Hippocampal slices (400 μm) were obtained from 12–15 day old rats and perfused with an extracellular solution containing (in mM): 124 NaCl, 3 KCl, 1.25 NaHPO program ). Seriesvs. the mean current amplitude and the single channel current was estimated by fitting the data to: σ2 = iI - I2/N + bl, where σ2 is the variance, I is the mean current, N is the number of channels activated at the peak, i is the single channel current and bl is the background variance. The single channel conductance (γ) is then γ = i/V, where V is the driving force . Response amplitude was measured in two ways. For failures, amplitude was estimated by measuring the difference between the average current over two time windows of equal length, one immediately before the stimulus artefact and the other centred on the peak of the mean EPSC. For estimation of the amplitude of successes the peak amplitude over a set time window was determined. Failures were identified visually, and potency was calculated as the mean EPSC amplitude excluding failures. For analysis of EPSC kinetics, rise time was estimated by the time-constant of a single exponential fit of the rising phase of the mean EPSC waveform. For decay, the time-constant of the single exponential fit to the decay phase was used. For display of individual EPSC traces in the figures, the stimulus artefact was digitally subtracted using an average of identified failures. All histograms are represented as smoothed line plots (SigmaPlot ver 5.0). Data are expressed as % of baseline . Statistical significance was assessed using the Student's t-test . All values are expressed as mean ± s.e.m.Non-SFA was performed as described previously . BrieflyAL performed most of the electrophysiology. MJP and MAW also contributed some recordings. PM provided some information that guided the work. TB performed the modelling that provided the theoretical frame-work. JI helped with the design and analysis of experiments. GLC coordinated the work and contributed to its design. All authors read and approved the manuscript.


The notion of a neurally encoded “reward system” that reinforces pleasure-seeking behaviors first emerged fifty years ago. Psychologists James Olds and Peter Milner discovered this phenomenon when their “lack of aim” landed an electrode outside their target while studying the behavioral responses of rats given electrical stimulation to a particular brain region. It was known that stimulation of certain brain regions would induce an animal to avoid the behavior that produced the stimulus. But in the rat with the “misplaced” electrode, stimulation of this new region caused the rat to repeat the behavior that caused the stimulus. Stimulation of certain brain regions provides a very strong incentive to restimulate, creating a feedback loop that reinforces both the behavior and the neural response to it. When gentle shocks were delivered to the rat hypothalamus, for example, the animals would “self-stimulate” 2,000 times per hour by pushing a lever. The neurotransmitter dopamine, it was later discovered, plays an important role in the brain's reward system—and in laying the biochemical foundation of drug addiction.Essential for normal central nervous system function, dopamine signaling mediates physiological functions as diverse as movement and lactation. The dopamine transporter (DAT) is involved in terminating dopamine signaling by removing the dopamine chemical messenger molecules from nerve synapses and returning them into the releasing neurons . DAT can also bind amphetamine, cocaine, and other psychostimulants, which inhibit dopamine reuptake, and, in the case of amphetamine, also stimulate the release of dopamine through DAT. It's thought that abnormal concentrations of dopamine in synapses initiate a series of events that cause the behavioral effects of these drugs. The biochemical steps underlying amphetamine-induced dopamine release, however, are not well characterized. Now, a team led by Jonathan Javitch and Aurelio Galli has identified a chemical modification of DAT that is essential for DAT-mediated dopamine release in the presence of amphetamine. Since this modification does not inhibit the ability of DAT to accumulate dopamine, it may suggest a molecular target for treating drug addiction.Embedded in the membrane of nerve cells, the dopamine transporter has a “tail,” called the N-terminal domain, that protrudes into the cell interior and consists of a stretch of about 60 amino acids. Many of these amino acids are potential sites of phosphorylation, a chemical reaction in which a phosphate group is added through the action of enzymes called kinases. Amphetamine has been shown to increase kinase activity and Margaret Gnegy, a coauthor of the current research article, showed previously that inhibiting protein kinase C activity blocks amphetamine's ability to release dopamine. Therefore, Javitch, Galli, and Gnegy hypothesized that N-terminal phosphorylation of DAT might play a critical role in the dopamine overload caused by amphetamine.The researchers found that amphetamine-induced dopamine release was reduced by 80% in cells expressing a mutant dopamine transporter in which the first 22 amino acids of the N-terminal domain had been removed (del-22). Surprisingly, this truncated transporter displayed normal dopamine uptake. In a full-length DAT, mutation of the five N-terminal serine amino acids to alanine amino acids, which prevented phosphorylation, produced an effect similar to removing the 22 amino acids. In contrast, replacing these five serine residues with aspartate residues to mimic phosphorylation led to normal dopamine release as well as normal dopamine uptake.These findings suggest that phosphorylation of one or more of these serine residues is necessary for amphetamine to flood the synapses with dopamine. While phosphorylation is a normal mechanism for regulating protein activity in a cell—and DAT is “significantly phosphorylated” under normal conditions—amphetamine could increase the level of DAT phosphorylation. Elucidating the mechanisms through which phosphorylation of DAT's N-terminus facilitates dopamine overload could lead to the development of drugs that block the “rewarding” effects of amphetamines and other addictive psychostimulants without interfering with normal dopamine clearance.


Combination therapy of irinotecan, folinic acid (FA) and 5-fluorouracil (5-FU) has been proven to be highly effective for the treatment of metastatic colorectal cancer. However, in light of safety and efficacy concerns, the best combination regimen for first-line therapy still needs to be defined. The current study reports on the bimonthly FOLFIRI protocol consisting of irinotecan with continuous FA/5-FU in five German outpatient clinics, with emphasis on the safety and efficiency, quality of life, management of delayed diarrhea, and secondary resection of regressive liver metastases.2), L-FA (200 mg/m2) and 5-FU bolus (400 mg/m2) on day 1, followed by a 46-h continuous infusion 5-FU (2400 mg/m2). One cycle contained three fortnightly administrations. Staging was performed after 2 cycles. Dosage was reduced at any time if toxicity NCI CTC grade III/IV was observed. Chemotherapy was administered only to diarrhea-free patients.A total of 35 patients were treated for metastatic colorectal cancer. All patients received first-line treatment according to the FOLFIRI regimen, consisting of irinotecan . Dose reduction was necessary in ten patients. Grade III/IV toxicity was rare, with diarrhea (14%), nausea/vomiting (12%), leucopenia (3%), neutropenia (9%) and mucositis (3%). The overall response rate was 31% (4 CR and 7 PR), with disease control in 74%. After primary chemotherapy, resection of liver metastases was achieved in three patients. In one patient, the CR was confirmed pathologically. Median progression-free and overall survival were seven and 17 months, respectively.The FOLFIRI regimen proved to be safe and efficient. Outpatient treatment was well tolerated. Since downstaging was possible, combinations of irinotecan and continuous FA/5-FU should further be investigated in neoadjuvant protocols. ColoRectal Cancer (CRC) have been demonstrated to benefit from chemotherapeutic treatment in terms of both quality and duration of life [Patients with advanced  of life . Of thes of life ,3. 5-Flu of life  -, was a of life -9. TheseIrinotecan (CPT-11), a potent inhibitor of the enzyme topoisomerase I, has demonstrated anti-tumorogenic activity in metastatic colorectal cancer, when used alone or in combinations with FA/5-FU, as adjuvant or palliative treatment (for review see 10 or 11). In randomized phase III clinical trials, second-line therapy with irinotecan significantly improved survival compared to supportive care  or to inHowever, the best regimen of irinotecan and FA/5-FU has yet to be defined. Altogether, irinotecan combined with continuous FA/5-FU infusions seemed to be superiorly active and less toxic than combination with FA/5-FU bolus regimens ,7. RecenFurthermore, irinotecan schedules of weekly and of once every two or three weeks demonstrated similar efficacy and quality of life, as well as significantly lower incidences of severe diarrhea in patients with 5-FU-refractory, metastatic colorectal cancer -17. In cTo date, little data is available regarding irinotecan combined with the simplified bi-monthly LV5-FU2 regimen as a first-line treatment in patients with metastatic colorectal cancer . TherefoAfter approval by the local ethical committee, patients were consecutively recruited from five German outpatient clinics . Eligibility requirements included (1) histologically documented adenocarcinoma of the colon or rectum and progressive measurable metastatic disease, (2) minimum life expectancy of three months, (3) Karnofsky performance status ≥ 60, (4) adequate hematologic, hepatic, and renal function, and (4) no prior chemotherapy for metastatic disease. Participants needed to be between 18 and 75 years of age. This study required that previous adjuvant 5-FU-based therapy with or without radiation therapy be completed at least 6 months prior to entry. Patients with CNS metastases, bowel obstruction, or ileus were excluded from the study. The study was approved by the ethics commission board responsible for all participating institutions. Prior to treatment, all patients gave written informed consent.2 d1, 5-FU bolus 400 mg/m2 d1, followed by 5-FU 46 h continuous infusion 2400 mg/m2 (simplified LV5-FU2 schedule). To prevent expected toxicities, patients were carefully informed of the potential risk of delayed diarrhea and neutropenia and the need for early intervention with loperamide [As previously described -16, treaperamide  and metoperamide . Atropinperamide . AntiemePrimary measures of the study were the overall objective response rate , overall survival, and quality of life. Secondary measures included the disease control rate (ORR + stable disease), time to progression, and frequency and severity of toxicities. Quality of life was assessed after inclusion into the study and as often as possible during the course of treatment, using the EORTC QLQ-C30 (version 2) questionnaire .Safety assessments and complete blood counts were performed weekly. Toxicity was graded according to National Cancer Institute Common Toxicity Criteria (NCI CTC). Toxicities not defined by NCI CTC criteria were classified as grade 0 (none), grade I (minor), grade II (moderate), grade III (severe), and grade IV (life-threatening). In case of any toxicity grade II, with the exception of hand-foot syndrome or alopecia, the next scheduled doses of irinotecan, folinic acid and 5-FU were delayed for a maximum of 1 week (or resolution of diarrhea for at least five days). In case of toxicity grade III/IV or if improvement from grade II to I (or resolution of diarrhea) was not achieved by two weeks, the following chemotherapy doses were reduced by 20 percent. If grade III/IV toxicity did not improve by 2 weeks, treatment was discontinued. Dose reductions were mandatory from the first cycle of chemotherapy in case of toxicity higher than grade II, and chemotherapy was resumed only after complete recovery from diarrhea.Tumor response was assessed according to World Health Organization (WHO) criteria. Tumor reassessment by the same imaging method used to establish baseline tumor measurement was generally performed after every two courses of therapy until progression. A complete response (CR) was defined as complete disappearance of evidence of cancer. A partial response (PR) was defined as a reduction in the sum of the products of the bi-perpendicular diameters of all measurable lesions by at least 50%. Progressive disease (PD) was defined as an increase in the sum of the products of the greatest bi-perpendicular diameters of all measurable lesions by at least 25% or the appearance of new lesions. Stable disease was defined as any reduction or increase in measurable lesions which did not meet the criteria for PR or PD. Confirmed objective responses were those for which a follow-up scan obtained at least four weeks later demonstrated the persistence of the response. The assessment of response and progression was based on investigator-reported measurements.Statistical analysis including survival analysis according to Kaplan-Meier was performed with the SPSS software package. The deadline for data evaluation was March 15, 2004. Survival was measured from the time of diagnosis to the date of death or last follow-up. Progression-free survival was calculated from treatment onset to the time of progression, study withdrawal or death of any cause. Patients who received at least one dose of the treatment regimen were evaluated for toxicity, and patients who completed at least two chemotherapy cycles were evaluated for response.Between 10/2001 and 5/2003, 35 consecutive patients  with metastatic colorectal cancer were enrolled into the study. The median age of these patients was 62 years, ranging from 38 to 73. Baseline characteristics of all patients are summarized in table The 35 patients received a total of 151 chemotherapy cycles , consisting of 451 administrations. Overall, 51  of all administrations had to be delayed for one week. During the complete study period, 19 patients had a delay of therapy for a median of nine days and in 10 patients (29%) a dose reduction was necessary at some point during the treatment period. The most common cause for discontinuation of study treatment was disease progression . In case of discontinuation, 16 (46%) patients received a second line treatment with either oxaliplatin plus a FA/5-FU consisting regimen or an epidermal growth factor receptor antagonist.Hematologic toxicity was mild to moderate in the majority of patients table . Only onWith regard to response, four complete (CR) and seven partial responses were seen, and thus an overall response rate of 31% was observed table . In addiQuality of life data were obtained before and at least once during treatment from 13 patients . The 13 In the current phase IV study we evaluated toxicity and efficacy of the FOLFIRI combination of irinotecan with FA/5-FU as first-line chemotherapy for metastatic colorectal cancer. This combination was established within a phase I clinical trial with a recommended irinotecan dose of 180 mg/qm ,16. IrinIn the majority of our patients the FOLFIRI regimen was well tolerated. Gastrointestinal toxicity or thrombembolic events were never fatal. Most hospitalizations were for prevention rather than treatment of life-threatening conditions. Delayed diarrhea, a well known side effect of irinotecan , was genOverall, we observed relatively low toxicity in our study, with NCI CTC grade III leucopenia amounting to 3%, diarrhea to 14% and nausea/vomiting to 11%. The toxicity observed in our study was lower than that reported by Douillard et al. in the pivotal European first-line trial in the patient group receiving weekly irinotecan (80 mg/qm), 24-h HD-5-FU (2300 mg/qm) preceded by 2-h FA 500 mg/qm . In thisThe response rate achieved in our study (31%) was quite comperable tp previously published data ,7,14,26.Three reasons may account for these differences in survival between the studies. The most important reason may be that a higher percentage of patients  in our study had two or more metastatic sites, indicating a larger tumor burden and consequently a worse prognosis regarding survival . Second,It appears particularly noteworthy that after chemotherapy three of our patients achieved surgical resectability of their metastases. To our knowledge these results are the first ever reported to suggest a potential role for the FOLFIRI regimen in the neoadjuvant setting. Thus far, studies of regional chemotherapy for initially unresectable colorectal liver metastases could demonstrate some success with secondary curative surgery. In two recently published retrospective studies chronomodulated chemotherapy with oxaliplatin and FA/5-FU was used as neoadjuvant treatment for patients with unresectable colorectal liver metastases ,28. TherThe FOLFIRI regimen, consisting of irinotecan with continuous FA/5-FU over 48 h, given on an outpatient basis was safe and well tolerated in our study. The rate of severe side effects was comparably low with this bi-monthly regimen. As tumor control was achieved in about 75% and downstaging of metastatic disease was possible in some cases, combinations of irinotecan plus continuous FA/5-FU should be further investigated in neoadjuvant protocols for initially unresectable liver metastases.CRC Colorectal CancerFOLFIRI chemotherapeutic regimen consisting of irinotecan combined with continuous FA/5-FU infusionsFA Folinic Acid5-FU 5-FluorouracilHD-5-FU high dose 5-FUNCI CTC National Cancer Institute Common Toxicity CriteriaCR Complete remissionPR Partial responseSD Stable diseasePD Progressive diseaseTTP Time to progressionOS Overall survivalMM, SS and AT drafted the manuscript. MM and MH initiated the study. JS, CZ, HH, BA, MS, OK, T, PG and MH were involved in the patient's treatment as well as the documentation of response and side effects.The pre-publication history for this paper can be accessed here:


Keratins are members of the intermediate filaments (IFs) proteins, which constitute one of the three major cytoskeletal protein families. In hepatocytes, keratin 8 and 18 (K8/18) are believed to play a protective role against mechanical and toxic stress. Post-translational modifications such as phosphorylation and glycosylation are thought to modulate K8/18 functions. Treatment of mouse with a diet containing griseofulvin (GF) induces, in hepatocytes, modifications in organization, expression and phosphorylation of K8/18 IFs and leads, on the long term, to the formation of K8/18 containing aggregates morphologically and biochemically identical to Mallory bodies present in a number of human liver diseases. The aim of the present study was to investigate the relationship between the level and localization of the stress inducible heat shock protein 70 kDa (HSP70i) and the level and localization of K8/18 phosphorylation in the liver of GF-intoxicated mice. The role of these processes in Mallory body formation was studied, too. The experiment was carried out parallely on two different mouse strains, C3H and FVB/n.GF-treatment induced an increase in HSP70i expression and K8 phosphorylation on serines 79 (K8 S79), 436 (K8 S436), and K18 phosphorylation on serine 33 (K18 S33) as determined by Western blotting. Using immunofluorescence staining, we showed that after treatment, HSP70i was present in all hepatocytes. However, phosphorylated K8 S79 (K8 pS79) and K8 S436 (K8 pS436) were observed only in groups of hepatocytes or in isolated hepatocytes. K18 pS33 was increased in all hepatocytes. HSP70i colocalized with MBs containing phosphorylated K8/18. Phophorylation of K8 S79 was observed in C3H mice MBs but was not present in FVB/n MBs.in vivo, there is no direct relationship between GF-induced stress and K8/18 phosphorylation on the studied sites. The K8/18 phosphorylation pattern indicates that different cell signaling pathways are activated in subpopulations of hepatocytes. Moreover, our results demonstrate that, in distinct genetic backgrounds, the induction of K8/18 phosphorylation can be different.Our results indicate that GF intoxication represents a stress condition affecting all hepatocytes, whereas induction of K8/18 phosphorylation is not occurring in every hepatocyte. We conclude that,  Intermediate filaments (IFs) with microtubules and actin microfilaments are the major cytoskeletal components of most vertebrate cells -4. IF prIt is now generally accepted that, in multilayered epithelia, one of the function for keratins IFs is to protect the tissue from mechanical stress -9. The fAs for epidermal keratins, the production of transgenic mice targeting K8 or K18 has been necessary to unravel the role of IFs in simple epithelium such as in the liver. In hepatocytes, K8/18 is the only keratin pair and thus both keratins are necessary to form an IF network. Transgenic mice expressing K8 or K18 carrying mutations that affect filament formation, develop mild hepatitis and display greater liver sensitivity to mechanical and toxic stress than wild type animals ,13.Recent studies from Ku et al. -16 have Long-term treatment of mice with a diet containing griseofulvin (GF) induces the development of an hepatitis associated with the formation of MBs, which are biochemically and morphologically similar to those found in humans ,28. ThisIn the present study, we investigated the effect of chronic GF intoxication on hepatocytes from C3H and FVB/n mouse strains. We monitored the expression of the stress inducible form of the heat shock protein 70 kDa (HSP70i) and the induction of K8/18 phosphorylation at specific sites: K8 on serine 79 (K8 S79), K8 on serine 436 (K8 S436), K18 on serine 52 (K18 S52) and K18 on serine 33 (K18 S33) . We The modifications in the amount of HSP70i, K8/18, and phosphorylated keratins  were analyzed by Western Blotting of total proteins from control and GF-treated C3H and FVB/n mouse livers . GF intoxication induced an increase in keratin levels in livers from both mouse strains Fig. . HSP70i,Total proteins from C3H and FVB/n livers were probed with antibodies against K8 pS79, K8 pS436 and K18 pS33 Fig. . SignifiWe analyzed at the cellular level, by double immunofluorescence staining, the distribution of HSP70i and IFs on liver sections of control and GF-treated C3H and FVB/n mouse livers.In control hepatocytes, IFs formed a complex cytoplasmic network that was denser at the cell membrane and particularly around the bile canaliculi Fig. . Our bioCryosections of control and GF-treated C3H and FVB/n mouse livers were fixed with 4% paraformaldehyde and processed for double immunofluorescence staining. As mentioned above, control mice showed hepatocytes with a cytoplasmic IF network, which was denser at the cell periphery Fig. . K8 pS79After 2 weeks of GF-treatment, hepatocytes were enlarged and an increase in cytoplasmic IF network was observed Fig. ,4,5C. ThIn the case of K18 S33, its phosphorylation was increased in most  hepatocytes. Most of the staining was observed at the periphery of the cells, delimitating clearly the bile canaliculi. A few hepatocytes showed high levels of cytoplasmic K18 pS33 Fig. .After 6 weeks of GF-treatment, the distribution of hepatocytes containing K8 pS79 and K8 pS436 was different from the one observed after 2 weeks of treatment. Clusters of labeled cells were smaller, whereas labeled isolated cells became more prominent Fig. ,4F. SingAfter 5 months of GF-treatment, MBs were present in some hepatocytes in both mouse strains Fig. ,4G,5G. MDouble immunostaining with anti-HSP70i and anti-phosphorylated keratins (K8 pS79 or K8 pS436) was performed for studying the localization of HSP70i in relation to keratin phosphorylation. The results showed that HSP70i and phosphorylated K8 species colocalized in some cells  or clusters of cells. Labeled singlet or doublet cells were more numerous after staining with the anti-K8 pS79 than after staining with the anti-K8 pS436. These cells could correspond to cells that are undergoing mitosis. This is supported by previous studies which showed that the phosphorylation of K8 on S79 and S436 occurs during mitosis ,48. ThisThe presence of K8 pS79 and K8 pS436 was also detected in islets of cells. Interestingly, both antigens were present in the same clusters of cells surrounding unstained cells that were most likely undergoing apoptosis. These unstained cells are evocative of detached cells during anoikis, an apoptotic process that can be induced by loss of cell-cell anchorage. Stress and apoptosis has been shown to modulate K8 S79 and K8 S436 phosphorylation ,48. The via K8. Our study show that colocalization of HSP70i and IFs occurs only in a few hepatocytes. Since the hepatocytes, in which colocalization was observed, contained K8 pS79 or K8 pS436, HSP70i binding to IFs in these cells may be related to the presence of keratin phosphorylation and participates to cellular pathways involving phosphorylated K8/18 on specific sites. Ku et al. [Liao et al.  have shou et al.  have shou et al. , HSP70i u et al. ,53. HoweChronic intoxication of mice with GF induces the formation of MBs. Numerous studies have demonstrated the presence of different phosphorylated K8/18 species within MBs, suggesting that K8/18 phosphorylation could participate in the MB formation processes ,54. In oThe treatment with GF that represents a toxic stress, most likely, involves the activation of stress activated protein kinases (SAPKs) in some hepatocytes. SAPKs p38 and JNK are physiologic kinases for K8 S79 and K8 S436 ,55. We pOur results show that increases in HSP70i, K8/18 expression and K8/18 phosphorylation constitute early events in the response of hepatocytes to the presence of GF. These observations support a role for keratins in preserving cellular integrity during stress conditions induced by the presence of a chemical agent ,35. HSP7ad libitum. GF-treated mice were fed a diet containing 2.5% (w/w) GF  for different periods of time: 2 weeks, 6 weeks and 5 months according to the method of Denk et al. [Experiments were performed with adult C3H mice  and FVB/n mice  weighing 25 to 30 g. Two mouse strains were used to minimize the potential effect of different genetic background on the response of hepatocytes and to facilitate the interpretation of the data. All animals were housed with a 12-hour light-dark cycle and allowed the consumption of water and of a standard mouse semi-synthetic diet , both k et al. . Control® Laboratories Canada, Burlington, ON) were used to perform immunolabelling with mAbs LJ4 and 5B3. The secondary antibodies used for Western blotting were as follows: biotinylated goat anti-rat IgG, biotinylated donkey anti-mouse IgG and peroxydase donkey anti-rabbit IgG . Other reagents used were: Horseradish Streptavidin Peroxydase-conjugated (SPC) , Bovine Serum Albumin (BSA) , Leupeptin , Pepstatin , Aprotinin , Normal Horse Serum (NHS) , Luminol .The antibodies used were as following: Troma 1, a rat monoclonal antibody (rAb) that recognizes K8 ; LJ4, a ®) in PBS , incubated with the primary antibodies for 45 min, at room temperature, washed in PBS containing 0.2% (v/v) Tween 20 and incubated for 45 min with the appropriate secondary antibody: biotinylated goat anti-rat IgG , biotinylated donkey anti-mouse IgG  and horseradish peroxydase donkey anti-rabbit IgG . When biotinylated secondary antibodies were used, membranes were washed with PBS-Tween 20 and incubated with streptavidin conjugated with horseradish peroxydase  for 30 min and washed with PBS-Tween 20. The chemiluminescent horseradish peroxydase substrate Luminol  was added to the membranes according to recommendations of the company, and membranes were exposed to Blue X-Omat X-ray film sheets  to localize antibody binding.Livers were homogenized in 62.5 mM Tris-HCl, pH 6.8 containing 2.3 % (w/v) SDS, 50 mM sodium fluoride, 10 mM EDTA, 1 mM sodium pyrophosphate, 1 mM DTT, 1 mM PMSF, 1 μM leupeptin , 1 μM pepstatin , 2.5 μg/ml aprotinin . Proteins were separated by electrophoresis on 10% SDS-polyacrylamide gels . Protein® Laboratories Canada, Burlington, ON) and an Avidin/Biotin blocking kit  according to recommendations of the company. Normal horse serum  was added to solution during incubation step with secondary antibody. For heat shock proteins staining, liver sections were incubated with anti-HSP 70i diluted in TBS containing 10% BSA for 45 min at 37°C, washed in TBS and incubated for 45 min at 37°C with a FITC conjugated donkey anti-rabbit IgG  diluted in TBS containing 10% BSA. For detection of HSP70i, K8 pS79 and K8 pS436, the sections were treated with 1% (v/v) Nonidet P-40  following fixing step with 4% paraformaldehyde.Cryosections (4 μm) of fresh liver were fixed with 4% (w/v) paraformaldehyde in PBS pH 7.2 for 20 min, at room temperature, and rinsed in PBS or TBS  upon staining protocols. Since fixation can affect antibody-binding capacity, cryosections were also fixed with cold acetone (-20°C) for 10 min. For the detection of K8, sections were incubated with rAb Troma 1 at room temperature, washed in PBS and incubated with a FITC or a TRITC conjugated goat anti-rat IgG  for 45 min, at room temperature. For immunostaining of K18 pS33, sections were incubated for 1 hour, at room temperature with anti-K18 pS33 (8250) diluted in PBS containing 10% (w/v) BSA, washed in PBS and incubated for 45 min with a FITC conjugated donkey anti-rabbit IgG in PBS containing 10% BSA . Immunostaining with anti-K8 pS79 (LJ4) and anti-K8 pS436 (5B3) mAbs, was done using the M.O.M. (mouse on mouse) detection kit (Vector® BX60 photomicroscope.The tissues were mounted in P-phenylene diamine diluted in 50% (v/v) glycerol. The slides were kept at -20°C and photomicrographs were collected using an OlympusHSP70i – inducible form of 70 kDa Heat shock protein. GF – griseofulvin. IFs – intermediate filaments. K8 – keratin 8. K8/18 – keratin 8 and keratin 18. K8 S79 – serine 79 on keratin 8. K8 pS79 – phosphorylated serine 79 on keratin 8. MBs – Mallory bodies.MF carried out all western blotting analyses, performed the immunofluorescence studies and participated in drafting the manuscript. LV participated in the design of the study. MC participated in the design of study, its coordination and drafting the manuscript. All authors read and approved the final manuscript.