Sc. In a cell-based assay, quinacrine inhibits the conversion of normal host prion protein (PrPC) to PrPSc at a half-maximal concentration of 300 nM. While these data suggest that quinacrine may be beneficial in the treatment of prion disease, its penetration into brain tissue has not been extensively studied. If quinacrine penetrates brain tissue in concentrations exceeding that demonstrated for in vitro inhibition of PrPSc, it may be useful in the treatment of prion disease.Prion diseases are caused by the accumulation of an aberrantly folded isoform of the prion protein, designated PrPOral quinacrine at doses of 37.5 mg/kg/D and 75 mg/kg/D was administered to mice for 4 consecutive weeks. Plasma and tissue (brain, liver, spleen) samples were taken over 8 weeks: 4 weeks with treatment, and 4 weeks after treatment ended.Sc in cell culture. Particularly extensive distribution was observed in spleen (maximum of 100 μg/g) and liver (maximum of 400 μg/g) tissue.Quinacrine was demonstrated to penetrate rapidly into brain tissue, achieving concentrations up to 1500 ng/g, which is several-fold greater than that demonstrated to inhibit formation of PrPC) to PrPSc will be critical toward its potential benefit.The documented extensive brain tissue penetration is encouraging suggesting quinacrine might be useful in the treatment of prion disease. However, further clarification of the distribution of both intracellular and extracellular unbound quinacrine is needed. The relative importance of free quinacrine in these compartments upon the conversion of normal host prion protein (PrP While the precise site of antiprion action is unknown (intracellular versus extracellular) adequate quinacrine penetration into brain tissue might result in an effective agent for the treatment of prion diseases.Prion diseases, while rare, invariably result in fatal neurodegeneration. At present, no therapy has been proven to be useful in the treatment of prion disease. However, acridine and phenothiazine derivatives have been evaluated ,2 using 0 μg/ml) . HoweverThe objective of this study was to characterize the achievable plasma concentrations and associated tissue (brain, liver, spleen) penetration associated with quinacrine.ad lib as a chocolate flavored liquid diet. From weeks 5–8 animals were given regular feed without quinacrine. At weekly intervals, 3 animals of each strain were euthanized and tissues were collected for analysis.The protocol was approved by the institutional animal care and use committee. The Animal Facility of the Institute for Neurodegenerative Diseases provided animal samples from 2 different strains of mice, FVB and CD1. Twenty four animals of each strain  were fed 37.5 mg/kg/D and 24 animals of each strain were fed 75 mg/kg/D of oral quinacrine over a 4-week period (weeks 1–4), given Quinacrine dihydrochloride (purity: 98.6%) was purchased from Fluka, while sulfadimethoxine sodium salt (purity: 98%) which served as the internal standard (IS) was obtained from Sigma. Quinacrine stock solution was prepared as 1 mg/ml in methanol. Sulfadimethoxine stock solution was prepared as 1 mg/ml in 50% methanol. The working solutions were prepared by diluting the respective standard and control stock solutions with 50% methanol and 0.1% formic acid to 2 μg/ml and 100 ng/ml, respectively. All solutions were stored in a 4°C refrigerator in silinized brown glass containers.3OH/H2O/TFA (45:55:0.05) with 1 mM ammonium formate. The flow rate was 0.8 ml/min with 1/4 split into the mass spectrometer. The injection volume was 5–10 μl with a run time of 3.5 min.The HPLC system employed a Shimadzu LC-10 AD pump and a Waters intelligent Sample Processor 717 Plus autosampler/injector. A BDS Hypersil C18 column, 50 × 4.60 mm, 5 μm particle size was directly coupled to a Micromass Quattro LC Ultima triple quadrupole tandem mass spectrometer using electrospray ionization in positive ion mode. The sample cone voltage and collision energy were 25 V and 20 eV respectively for both quinacrine and the internal standard and the source block and desolvation temperature were 100°C and 400°C, respectively. The mass scanning mode employed multiple reaction monitoring (MRM) with the singly charged quinacrine ion selected at m/z 400.5 giving a fragment ion at m/z/142.0, and the internal standard at m/z 311.0→156.0. The mobile phase consisted of CHAll samples were stored at -70°C until analyzed. Each tissue sample was subjected to a specific method, as described below, for drug extraction and for the determination of concentration. All samples were analyzed by LC/MS/MS.Accuracy and precision was demonstrated throughout the working range with interday and intraday coefficient of variation and relative error <10%.Each plasma sample was thawed at room temperature for 10–15 min; then 20 μl of plasma was aliquotted to a new test tube. To each tube 200 μl of 70% acetonitrile solution, containing 0.1% formic acid and 50 ng/mL of internal standard, was added. The test tubes were vortexed at high speed for 1 min and centrifuged at 10,000 rpm for 10 min. The supernatant was transferred into the autosampler for LC/MS/MS analysis. A set of standard curves with a duplicate set of quality control (QC) samples was generated for sample analysis.Prior to the in vivo study, the stability of quinacrine in mouse brain tissue was determined. Using 3 different doses of quinacrine (4 mg/kg/d, 80 mg/kg/d, 160 mg/kg/d), mouse brain tissue was soaked in 100% methanol for 7 days. Samples were taken on days 0,1,2,3, and 7 to analyze quinacrine concentrations using LC/MS/MS. Complete equilibration was observed by Day 1 and no degradation in quinacrine was noted through day 7.Brain samples were thawed (in plastic tubes) at room temperature for 15–20 min and weighed. To each sample 0.5 ml of 0.9% NaCl was added followed by incubation at room temperature for 1 h. For the standard curve and quality control samples, a brain tissue sample from an untreated mouse was spiked with different amounts of quinacrine and incubated at room temperature for 1 h. After the addition of 100 μl of 1 μg/ml internal standard solution in 50% methanol and 0.1% formic acid, 5 ml of 100% methanol was added into each sample and soaked at 4°C for two weeks. On the last day, 200 μl was aliquotted from each brain sample and placed into the autosampler for analysis via LC/MS/MS.Liver samples were thawed at room temperature for 15–30 min and weighed. Considering the increased size of the liver samples and that the increased connective tissue in liver samples could potentially prevent the complete distribution of quinacrine into methanol, homogenization was used for these samples. To each sample 100% methanol was added (10 ml/g of tissue) and the tissue was homogenized in ice water for 1 min at speed 3 (Tissue Tearor, model 985-370, Biospec Products, Inc). The internal standard (100 μl of 10 μg/ml in 50% methanol, 0.1% formic acid) was added to 0.2 ml of each homogenized liver sample in a glass test tube. Samples were vortexed for 1 min, centrifuged at 3000 rpm for 10 min and 20 μl of each supernatant was transferred into a new test tube. Each sample was further diluted with 4 ml of 50% methanol, vortexed, and 200 μl was transferred to the autosampler for LC/MS/MS analysis. A set of standard curve with a duplicate set of QC samples was generated for sample analysis.Spleen sample preparation was similar to the preparation of brain samples using a methanol soak. Prior to the in vivo study, the stability of quinacrine in mouse spleen tissue was determined. Using 3 different doses of quinacrine (4 mg/kg/d, 80 mg/kg/d, 160 mg/kg/d), mouse spleen tissue was soaked in 100% methanol for 7 days. Samples were taken on days 0, 1, 2, 3, and 7 to analyze quinacrine concentrations using LC/MS/MS. Complete equilibration took place by Day 1 and no degradation in quinacrine was observed through day 7.Samples from the in vivo analysis were soaked for 14 days at 4°C. On day 14, 50 μl was aliquotted from each spleen sample and diluted with 1 ml of 50% methanol. Each sample was vortexed for 1 min and 200 μl was transferred to the autosampler for LC/MS/MS analysis. A set of standard curves with a duplicate set of QC samples was generated for sample analysis.The mass spectrum of quinacrine Q1) and its tandem spectrum Q3), showing the fragment ion selected for MRM, are shown in Figure  and its , showingIn plasma, quinacrine concentrations with the 37.5 mg/kg/D dose ranged from 75 to 175 ng/ml for both FVB and CD1 strains mice and remained at this level during the 4-week dosing interval. Once discontinued, quinacrine was completely eliminated from plasma, with no drug detectable within one week. At 75 mg/kg/D quinacrine, plasma concentrations reached >2 × those observed with the lower dose, averaging 300 to 400 ng/ml.Quinacrine levels in brain tissue for both mice strains were determined to be substantially greater than those achieved in plasma. Similar to observations with plasma, steady-state levels in the brain with the 37.5 mg/kg/D dose were achieved by the end of the first week, averaging 400 to 600 ng/g brain tissue. Figure th week. Similarly, spleen tissue levels in both mice strains were very elevated, averaging 5 to 10 μg/g with the 37.5 mg/kg/D dose and 40 to 100 μg/g with the 75 mg/kg/D dose. Figure The analysis of liver samples showed particularly high quinacrine levels, at many times those observed in plasma and brain. At the end of the first week of treatment with 37.5 mg/kg/D, steady-state levels of 70 to 90 μg/g were achieved in liver tissue in both mice strains, which remained throughout the course of treatment. Figure Of note, in contrast with plasma and brain tissue, in which dose dependency was somewhat linear, increasing quinacrine doses in spleen and liver were associated with substantially greater tissue levels. In addition to the isolation of quinacrine, numerous metabolites were identified in all tested tissue samples (data not shown).The current study is the first comprehensive evaluation of quinacrine distribution in plasma and brain tissue. Shannon et al. evaluated the pharmacokinetics of quinacrine in the treatment of malaria . Using ain vitro study, the EC50 for quinacrine was 300 nM, which approximates 120 ng/ml. We report here that quinacrine levels in brain tissue averaged 400 to 600 ng/g with the 37.5 mg/kg/D dose and 1500 ng/g with the 75 mg/kg/D dose, concentrations that exceed the in vitro EC50 by 3- to 10-fold.The concentrations we report in brain in terms of concentration/gm of tissue are several-fold greater than that shown for effective antiprion activity in ScN2a cells [in vitro, including clearance of PrPSc (5) and/or inhibition of PrPSc formation [in vitro benefit from quinacrine, found the drug to be ineffective in the animal model under the conditions employed [Others have reported the antiprion function of quinacrine ormation . Barret employed . In contemployed ,8.50, it important to note critical limitations in their interpretation. Quinacrine has been determined to be highly protein-bound [While the current study has confirmed brain tissue concentrations in excess of the reported ECin-bound , measurein-bound . The brain-bound  evaluatein vitro antiprion activity similar to or greater than that of the parent compound. These tests warrant further evaluation.In addition to steady-state levels of quinacrine, we observed a number of quinacrine metabolites in all tissue samples studied, which raises the possibility that these metabolites may have in vitro activity, quinacrine may be a useful agent in the treatment of prion disease. However, more detailed tissue distribution analyses linked with the critical sites of prion action are warranted.Based upon its oral bioavailability, favorable tissue distribution characteristics, and The author(s) declare that they have no competing interests.LY, YH, ETL developed the assay, performed sample analyses, contributed to the writing of the paper. PL, GL, MB, CR developed the protocol and performed the animal model experiments. SBP conceived of the study and participated in its coordination. BJG drafted the manuscript, participated in its coordination and mentored LY. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


WT) and Syt VII knockout (KO) mouse embryonic fibroblasts (MEFs) using total internal reflection fluorescence microscopy. In WT MEFs, most lysosomes only partially released their contents, their membrane proteins did not diffuse into the plasma membrane, and inner diameters of their fusion pores were smaller than 30 nm. In Syt VII KO MEFs, not only was lysosomal exocytosis triggered by calcium, but all of these restrictions on fusion were also removed. These observations indicate that Syt VII does not function as the calcium-dependent trigger for lysosomal exocytosis. Instead, it restricts the kinetics and extent of calcium-dependent lysosomal fusion.Synaptotagmin is considered a calcium-dependent trigger for regulated exocytosis. We examined the role of synaptotagmin VII (Syt VII) in the calcium-dependent exocytosis of individual lysosomes in wild-type ; but in MEFs in which synaptotagmin VII has been deleted, lysosomes fuse completely A varieocytosis . Howeverocytosis . It is eocytosis . Syt VIIocytosis . Furtherocytosis . To undet1/2 for FITC in our setup is 28.5 s). Absence of any subsequent decrease in its fluorescence, even over longer periods, indicated that cessation of release was the result of closure of the fusion pore prior to complete release of the lumenal cargo. Quantitation of the lumenal contents retained in all exocytosed lysosomes analyzed in the WT MEFs revealed that only 21% completely released their lumenal content  (To monitor the fate of exocytic lysosomes in MEFs, we labeled their lumen using fluorescent dextran (FITC–dextran). Treating MEFs with calcium ionophore A23187 or the IP3 agonist bombesin or thrombin caused lysosomal exocytosis A and 1B.7 cells) . A compa7 cells) .2+-triggered lysosomal exocytosis, we simultaneously imaged the lysosomal lumenal (TRITC–dextran) and membrane (CD63–GFP) cargoes in WT MEFs. We observed that the membrane proteins were delivered to the plasma membrane during complete release of lumenal content   fraction of lysosomes completely released their lumenal contents when the cellular calcium was raised using ionophore (p = 0.001) (p = 0.001), or bombesin (p = 0.003)  . AbsenceKO MEF) . Upon ex= 0.001) , thrombi003) see . Second,rane see . These pWT MEFs, all the lysosomes that released the TRITC–dextran of 10 or 70 kDa also released similarly sized FITC–dextran . Lysosomes were loaded with dextrans of two different sizes, each tagged with a different fluorophore (FITC or TRITC). In –dextran A and 3B.–dextran . This in–dextran A–1C. ThuWT MEFs, each lysosome that partially released the 10 or 70 kDa TRITC–dextran at the same time also partially released the 145 kDa (data not shown) and 250 kDa FITC–dextran , this occurred in less than 0.75 s  in both WT and KO MEFs, there was a 2-fold decrease (p = 0.02) when bombesin was used to raise cellular calcium in KO MEFs.While all the behaviors of individual lysosomes described above were independent of the agent used to trigger calcium increase, the bulk cellular behavior, such as the total number of lysosomal fusion events that occur at the basal surface of the cell, was dependent on the agent used to increase the cellular calcium level D. While WT MEFs, partial fusion is the predominant mode of lysosomal exocytosis. Unlike partial fusion of secretory granules in PC12 cells, which results in incomplete release of the large lumenal cargo  or truncated Syt VII (KO MEF), as described elsewhere (MEFs were prepared from day 13.5 embryos of lsewhere . Cells wlsewhere  was carrlsewhere .2). The camera and mechanical shutters (Uniblitz, Vincent Associates, Rochester, New York, United States) were controlled using MetaMorph . Images were acquired at 5–10 frames/s. Images containing a region of interest of the cell were streamed to memory on a PC during acquisition and then saved to hard disk. The depth of the evanescent filed for the Apo 60× N.A. 1.45 lens (Olympus Scientific, Melville, New York, United States) was typically approximately 70–120 nm (The illumination and image acquisition using TIR-FM was done as previously described . For sim0–120 nm .WT and fewer than 2 min in Syt VII KO MEFs).For dual-color video sequences, the images acquired through the emission splitter were separated, subtracted for background fluorescence, aligned within accuracy of a single pixel, and analyzed using MetaMorph. For measuring fluorescence intensity, a region was drawn around the vesicle and the peak and average intensity was measured in this region. The minimum and maximum average intensities were normalized on a scale of 0 to 1. For measurement of total number of exocytic lysosomes in a cell, fusion events were counted starting from the addition of ionophore until the cells start to lift off the coverslip  elapsed since the start of the video.Lysosomes of (6.8 MB AVI).Click here for additional data file.Video S2KO MEFs were loaded with 70 kDa FITC–dextran and the cells were treated with 10 μM calcium ionophore. The video shows a single cell immediately following ionophore addition. The images were acquired at five frames per second, and alternate frames are displayed in the video at five frames per second. The time stamp indicates the time (mm:ss) elapsed since the start of the video.Lysosomes in Syt VII (4.9 MB AVI).Click here for additional data file.


Immunosuppression is documented in several malignant diseases, including breast cancer. Subsequently, future therapeutic concepts might include immunological approaches. However, detailed knowledge about tumor immunogenicity and host immunoreactivity, and how to assess these adequately, is still limited. We studied CD28 and CD3-ζ expression in sentinel node biopsies (SNB) from breast cancer patients to analyze tumor-related changes in T cell activity.25 women underwent surgery for primary breast cancer, including SNB. Frozen sections from 21 sentinel nodes could be analyzed with a double-staining technique. CD28 expression was studied in CD4+ and CD8+ T-lymphocyte subsets and compared with CD3-ζ expression in three specified nodal regions.The degree of CD28 expression varied between the different lymph node areas. The lowest degree of CD28 expression was observed in CD4+ T-lymphocytes in the paracortex and germinal centers. Here, a good agreement with CD3-ζ expression was found. A higher CD28 expression was noted in CD4+ T-cells in the primary follicles, where concordance with CD3-ζ expression was weaker. The CD8+ T-lymphocyte subset displayed generally a higher degree of CD28 expression than the CD4+ subset.Sentinel lymph nodes from breast cancer patients displayed local immunosuppression of varying extent. In the areas with the lowest degree of CD28 expression an accordingly low CD3-ζ expression was found. The SNB might prove an important diagnostic tool for the evaluation of interactions between tumor and the host immune system, helping to select patients who might benefit from adjuvant immunotherapy. Numerous studies  portray The CD28 surface receptor is normally expressed on 95% of CD4+ T-cells and approximately 50% of CD8+ T-cells in human peripheral blood . Its natDecreased CD28 expression is described in dysfunctional peripheral T-lymphocytes from patients with hairy cell leukemia  and chroThe expression of the zeta chain of the T-cell receptor (CD3-ζ) is decreased in sentinel node biopsies from breast cancer patients . This doThe study comprised 25 patients who underwent surgery for primary breast cancer, using the sentinel node biopsy technique. Inclusion criteria for enrolment in the study protocol were informed patient consent and a newly diagnosed palpable invasive breast cancer. Exclusion criteria were palpable axillary metastases, multifocality of the cancer, ongoing pregnancy or preoperative cytotoxic treatment.In two cases, the sentinel node could not be immunologically analyzed due to lack of technical quality. In two other cases, nodal tumor growth was too abundant and remaining lymphoid tissue too little to analyze the sections. Thus, the remaining study population comprised 21 patients. Patient and tumor characteristics are presented in Table The study protocol was approved by the ethics committees at the University of Uppsala and the University Hospital of Linköping.Sentinel nodes were identified by injection of a radioactive tracer (Tc-99-nanocolloid) close to the tumor site, preoperative lymphoscintigrams, and injection of Patent blue dye (Guerbet, Paris, France). After biopsy, the sentinel nodes were sent fresh to the pathology department. Specimens were snap-frozen and tissue sections (6–7 μm thick) were obtained for immediate diagnostic analysis regarding the occurrence of metastasis in the sentinel node. Additional frozen sections were wrapped in parafilm and stored at -70°C until processed further at University Hospital of Linköping. Routine diagnostic studies of both tumor tissue and lymph nodes were performed at the pathology department of Central Hospital, Västerås and comprised all parameters listed in Table CD4 (Clone SK3, Becton-Dickinson, Stockholm, Sweden), CD8 (Clone SK1, Becton-Dickinson, Stockholm, Sweden), CD28 (Clone L293, Becton-Dickinson, Stockholm, Sweden), CD3 (Clone UCHT1, DAKO Stockholm, Sweden), TCR-zeta (Clone 2H2D9 (TIA-2), Immunotech, Stockholm, Sweden).The monoclonal antibodies used were as follows: Tissue sections obtained as described above were fixed with 4% paraformaldehyde (pH 7.4) (Riedel-de Haen AG, Seelze, Germany), supplemented with 5.4 g/L of glucose for 5 min and soon afterward washed three times in Hanks' balanced salt solution  supplemented with 0.01 M HEPES solution. To avoid unspecific binding, sections were blocked with normal rabbit serum before the first staining and subsequently incubated with the primary antibodies CD3 (1/40), CD4 (1/25) and CD8 (1/50) for 30 minutes. After the slides had been washed in BSS/saponin, biotinylated rabbit anti-mouse immunoglobulin was added at a 1/100 dilution in BSS/saponin. Mouse IgG (Sigma, Stockholm, Sweden) was used as a negative control. The slides were then incubated with peroxidase-labeled streptavidine  at a 1/100 dilution in BSS/saponin for 30 minutes. DAB (3,3'-diaminobenzidine, D-5637, Sigma, Stockholm, Sweden) was used as a substrate, which resulted in a brown color. The expression of the zeta chain or CD28 was identified using mouse monoclonal antibodies to these substances. The sections were first blocked by incubation with normal goat serum and subsequently incubated with the primary antibodies. Sections stained for the zeta chain (1/2.5) were incubated for 30 minutes while sections stained for CD28 (1/25) were incubated over night. The slides were again washed in BSS/saponin and incubated with goat anti-mouse immunoglobulin for 30 minutes and then with the alkaline phosphatase-anti-alkaline phosphatase (APAAP) mouse monoclonal antibody (Dakopatts D 651) at a dilution of 1/25 in BSS/saponin. After washes in BSS/saponin and Tris-buffered saline (TBS) and incubation with the alkaline phosphatase substrate (Naphthol AS-MX), 2 mg phosphate (Sigma N4875), 0.2 ml dimethylformamide, 9.8 ml 0.1 M Tris buffer pH 8.2, 50 μl 1 M levamisole (Sigma L-9756) and 10 mg Fast-Red TR salt (Sigma F 1500) for 20 minutes, the sections were again washed in TBS. Thereafter, the sections were counterstained in Mayer's haematoxylin for 15 minutes and mounted in Glycergel (Dakopatts, Sweden). All antibody solutions contained 2% normal blood donor AB serum and all incubations were performed in a moist chamber. The APAAP technique resulted in bright red staining for CD3-ζ and CD28. Double-stained cells appeared as red-brown; those with down-regulated CD3-ζ or CD28 were dominated by a brown color.Two independent investigators (A.H., B.G.) analyzed whole sections of the sentinel nodes. The degree of CD28 expression was evaluated in both CD4+ and CD8+ T-cell subsets within three lymph node areas: primary follicles, secondary follicles  and paracortex. The number of CD4+ and CD8+ T-lymphocytes expressing CD28 was semiquantitatively scored as "high" (>75% CD28+), "moderate" (50–75% CD28+) and "low" (<50% CD28+). The regional scores were then put into correlation with our earlier data on expression of CD3-ζ in parallel sections of the same patients where the same scoring system was applied . There wFor testing correlations between the patient data depicted in Table The degree of CD28 expression varied considerably, both between individual patients and between different regions of the sentinel lymph nodes. The lowest degree of CD28 expression was seen in CD4+ T-lymphocytes located in the paracortex and the secondary follicles. CD8+ T-lymphocytes displayed a significantly higher degree of CD28 expression than CD4+ T-cells in both paracortex (p < 0.001) and primary follicles (p < 0.001). There were statistically significant differences in CD28 expression in CD4+ T-cells comparing the three nodal regions (p < 0.01), with CD28 expression being higher in primary follicles than in secondary follicles and paracortex. No such differences were found analyzing CD28 expression in CD8+ T-lymphocytes. No significant correlation could be found between the degree of CD28 expression and tumor size, histological type, hormone receptor status, DNA ploidy status, Elston histological grade, S-phase, patient age, nodal status or tumor stage following the TNM classification of malignant tumors, neither were there any significant correlation detected regarding clinical data and the degree of CD3-ζ expression in our previously published material .Primary follicles are cortical lymph node areas housing predominantly B-lymphocytes. The analysis could be performed in 20 cases. In the CD4+ subset Table , 5 of 20In the CD8+ subset of T-lymphocytes Table , a high These B-cell areas, which develop as secondary follicles upon antigenic stimulation, could be analyzed in 16 patients. In the remaining five cases, no or very few germinal centers were found. In the CD4+ subset Table , 4 of 16A markedly low expression of CD28 was observed in the paracortex, the principal T-cell activation area. Sections of 20 sentinel nodes could be analyzed. In the CD4+ subset Table , and lowAs described in a previous paper of our group , a low eThe expression of CD28 and CD3-ζ was always compared in the same area of the same lymph node. Degrees of expression were only considered corresponding if a patient displayed exactly the same degree of expression (i.e. low, moderate or high) of both markers in that area.In primary follicles, corresponding degrees of expression were found in 12 of 20 patients analyzing the CD4+ subset Table , resultiIn secondary follicles, 11 of 15 patients had the same degrees of CD28 and CD3-ζ in the CD4+ subset  subunit CD3-ζ are more effective to activate an anti-tumor response in vivo than T-cells with TCR-CD3-ζ only . WithoutSeveral studies point towards a role for immunological treatment approaches in breast cancer -39. The The axillary lymph node status is of utmost importance for breast cancer prognosis and the choice of adequate adjuvant treatment. Today, the sentinel node biopsy technique is widely used as a diagnostic tool to gain that valuable information while limiting the operative procedure to a necessary minimum. However, it is also in the sentinel node that the immunoreactivity against the tumor is initiated.In this study, a varying extent of immunosuppression was observed, represented by a decreased expression of CD28 and CD3-ζ in certain areas of the lymph node. The concordance between both markers was highest in the areas of most pronounced down-regulation, namely the paracortical region and the germinal centers. As immunological reactivity was not distributed evenly over the different areas of the lymph node, this might illustrate the dynamic nature of the immunological response to malignant disease within the complex functional anatomy of the lymph node. Thus, the immunohistochemical analysis of the sentinel node biopsy with respect to its preserved architecture might prove an important instrument for the evaluation of interactions between the tumor and the host immune system, helping to select patients who might benefit from adjuvant immunotherapy.All authors participated in the study design and contributed with the collection of material and data. The microscopic analysis was carried out by AH and BG. All authors read and approved the manuscript.


UBE4B, KIF1B, PGD, APITD1, DFFA and PEX14 in the 1p36.22 region in order to find an explanation for a possible down-regulation of this region.A common feature of neuroblastoma tumours are partial deletions of the short arm of chromosome 1 (1p-deletions). This is indicative of a neuroblastoma tumour suppressor gene being located in the region. Several groups including our have been studying candidate neuroblastoma genes in the region, but no gene/genes have yet been found that fulfil the criteria for being a neuroblastoma tumour suppressor. Since frequent mutations have not been detected, we have now analyzed the expression and promoter CpG island methylation status of the genes APITD1. Methylation of this gene is also seen in blood from control individuals and is therefore not believed to participate in tumour development.The current study shows that gene transcripts in high stage neuroblastoma tumours are significantly down-regulated compared to those in low stage tumours in the 1p36.22 region. CpG island methylation does not seem to be the mechanism of down-regulation for most of the genes tested, since no methylation was detected in the fragments analyzed. One exception is the CpG island of UBE4B, KIF1B, PGD, APITD1, DFFA and PEX14 are down-regulated in high stage NB tumours, a feature that can not be explained by CpG island methylation.The genes  Neuroblastoma (NB) is the most common paediatric solid tumour, responsible for 15% of cancer deaths of childhood. It is a tumour of the postganglionic sympathetic nervous system that develops from immature or dedifferentiated neural crest-derived cells -12 and wIn search of tumour suppressor genes, the focus has in the last years moved towards epigenetics and methylation of promoter regions. Methylation of cytosines in CpG-dinucleotides is a common modification in mammalian genomes. Methylated cytosines are more susceptible to deamination, which have lead to an erosion of the number of CpG-dinucleotides. The vast majority of CpGs resides within repetitive elements and is methylated. There are also stretches of DNA rich in CpG that are gene associated, i.e. CpG islands, which are normally unmethylated . MethylaThe most common way to analyze methylation status is based on bisulphite modification of DNA . In thisCASP8, on 2q33, was one of the first genes found to be methylated in neuroblastoma, with a frequency of about 40 % in primary NB tumours [RASSF1A and BLU have been shown to be frequently methylated [CASP8, as already known, was one of these genes, also FLIP at 2q33 was methylated as well as four genes in the chromosome region 8p21; DR4, DR5, DCR1 and DCR2. Genes on 1p36 were also included in this study, none of which were found to be methylated. No genes in the NB tumour suppressor candidate region on 1p36.22 were included. Alaminos and co-workers have in a study screened 45 genes in NB cell lines [UBE4B, located in 1p36.22, was included in this study and found to be unmethylated.Some genes have been analyzed in NB tumours focusing on methylation status. For example,  tumours . On 3p21thylated -29. In athylated . A totalll lines . 34 geneCORT, associated with a CpG island in their respective promoter regions. The fact that this region is deleted in a subset of NB tumours and that frequent mutations are not found, makes it a possible candidate region for hypermethylation of genes. In the current study, we wanted to analyze the expression pattern of these genes in the 1p36.22 region using TaqMan technology and the methylation status of promoter regions associated with CpG islands, in a panel of NB cell lines and primary NB tumours.The genes in the 1p36 consensus deleted region are all, except for GUSB was selected as an endogenous control for real-time PCR quantification, and further used as an internal reference for normalization. Real time-PCR studies of all the genes in the region on cDNA-samples showed a significant reduction of mRNA levels in high stage NB tumours compared to low stage tumour mRNA levels, ranging from 49% for PEX14 to 79% for APITD1  to 25 cM in our tumour material ,7. By inCpG methylation is not the only possible explanation for the down-regulation in gene expression shown in high stage tumours. Analysis of chromatin modifications could be the next step in a further analysis of this region, in search of a mechanism that could lead to down-regulation of the genes. Deficiencies in transcription factors and upstream elements could also account for the decrease in gene transcripts. Another possibility is that the variations are due to differences in the treatment of the tumours before and after surgery. Radiation and chemotherapy could probably have a vast affect on the expression patterns of a substantial amount of genes in the tumour cell. It is however unlikely that all the genes in the analyzed region would be affected by the treatment alone, rather this might explain the differences in the level of down-regulation between different genes.in vivo. In primary tumours, there are always some non-NB cells in the RNA preparations that might affect the gene expression results. On the other hand, genetic events happen to the cells when they get immortalized in cell culture, some genes tend to be down-regulated and others up-regulated. We can also speculate that some of these genes, for example APITD1, might even be so crucial that only cells with expression of this gene are capable of surviving in culture.The difference in expression between NB cell lines and primary NB could be explained by the difference in cell environment between cells in culture and UBE4B, KIF1B, PGD, APITD1, DFFA and PEX14 are down-regulated in high stage NB tumours, a feature that can not be explained by methylation, rather by a mechanism still remaining to be discovered.In conclusion, in our aim to find a mechanism that could inactivate the genes in the candidate gene region we analyzed the expression status and the methylation profile of six genes. The techniques used were TaqMan real-time-RT-PCR technology and bisulphite DNA sequencing. Of the genes analyzed all were down-regulated in high staged NB tumours as compared to low stage tumours. Promoter methylation was not detected in the genes analyzed, except for the CpG island of APITD1. This methylation does not seem to be tumour specific, since methylation was also detected in healthy blood controls. Hence, the six genes A panel of 10 tumours from primary NBs  and 7 NB cell lines (IMR-32, SK-N-AS, SK-N-BE (2), SK-N-SH, SK-N-DZ, SK-N-F1 and SH-SY-5Y) were analyzed  NB tumour tissue using RNeasy RNA extraction kit (Qiagen, Hilden, Germany). 2.4 μg total-RNA of each sample was reversed transcribed to cDNA using Superscript II (Amersham, Buckinghamshire, UK) and random hexamer primers, all according to supplier's protocol. All cDNAs were quality tested by amplification of the housekeeping genes GUSB (β-glucuronidase) and B2M (β2-microglobulin) showed least variations in ΔCT levels, and were expressed at constant levels in all samples regardless of NB-stage. GUSB was selected, and further used as an internal reference for normalization in the real-time PCR quantification analysis .To select the most appropriate endogenous control for the real-time PCR quantification analysis, we tested eight different primary NB samples of different stages for their expression levels of ten commonly used housekeeping genes with TaqMan Human Endogenous Control Plate, (Applied Biosystems, Foster City, CA). Analysis was performed according to supplier's protocol. ® Assays-on-Demand™ Gene Expression Products" (URL:). Real-time PCR was performed in 384-well plates using ABI PRISM® 7900HT Sequence Detection System (Applied Biosystems). Amplification reactions (10 μl) were carried out in duplicate with 0.1 μl template cDNA according to manufacturers protocol (Applied Biosystems). In each assay, a standard curve with six cDNA dilutions was recorded and two non-template controls were included.TaqMan primers and probes were derived from the commercially available "TaqManT-value for duplicates were calculated, and the gene concentration (or gene copy numbers) of test samples was interpolated based on standard curves. All samples were normalized by dividing the concentration of the test gene with the concentration of the housekeeping gene β-glucuronidase (GUSB) in the same cDNA sample.Quantification was performed by the standard-curve method. The mean CThe logarithms of the expression levels were compared with Student's two-sided t-test on each group of tumours; low stage and high stage tumours.2O and stored at -20°C.DNA was phenol extracted with the use of phase lock gel (Eppendorf AG, Hamburg, Germany) according to standard procedure and was, with some minor changes, modified according to previously published papers ,33. Brie) and CpG islands with MethPrimer software , where after 10–30 clones were picked. PCR products were purified with ExoSAP-IT™ (USB Corporation, Cleveland, Ohio) and sequencing was carried out using forward or reverse primer with ABI Prism BigDye™ cycle sequencing Ready Reaction Kit (Applied Biosystems). The samples were analyzed in an ABI 3100 or an ABI 3730 Genetic Analyzer.The putative promoter regions of the genes were predicted using Genomatix Promoter Inspector software (URL: HC participated in the design of the study, carried out the methylation analysis and drafted the manuscript. SF, LH and FL participated in parts of the methylation study. KE and CK carried out the TaqMan runs. KE also contributed to drafting the manuscript. RMS participated in the cloning. TM coordinated the study. All authors read and approved the final manuscript.


Dialysis disequilibrium syndrome (DDS) is the clinical phenomenon of acute neurologic symptoms attributed to cerebral edema that occurs during or following intermittent hemodialysis (HD). We describe a case of DDS-induced cerebral edema that resulted in irreversible brain injury and death following acute HD and review the relevant literature of the association of DDS and HD.A 22-year-old male with obstructive uropathy presented to hospital with severe sepsis syndrome secondary to pneumonia. Laboratory investigations included a pH of 6.95, PaCO2 10 mmHg, HCO3 2 mmol/L, serum sodium 132 mmol/L, serum osmolality 330 mosmol/kg, and urea 130 mg/dL (46.7 mmol/L). Diagnostic imaging demonstrated multifocal pneumonia, bilateral hydronephrosis and bladder wall thickening. During HD the patient became progressively obtunded. Repeat laboratory investigations showed pH 7.36, HCO3 19 mmol/L, potassium 1.8 mmol/L, and urea 38.4 mg/dL (13.7 mmol/L) (urea-reduction-ratio 71%). Following HD, spontaneous movements were absent with no pupillary or brainstem reflexes. Head CT-scan showed diffuse cerebral edema with effacement of basal cisterns and generalized loss of gray-white differentiation. Brain death was declared.Death is a rare consequence of DDS in adults following HD. Several features may have predisposed this patient to DDS including: central nervous system adaptations from chronic kidney disease with efficient serum urea removal and correction of serum hyperosmolality; severe cerebral intracellular acidosis; relative hypercapnea; and post-HD hemodynamic instability with compounded cerebral ischemia. Acute renal failure requiring hemodialysis (HD) is a common clinical problem in critically ill patients that is independently associated with increased mortality. DialysiA 22-year-old homosexual male presented to hospital with progressive dyspnea, productive cough, generalized malaise and fever. He had a known history of intravenous cocaine abuse and recent serology in prior 3 months was negative for human immunodeficiency virus (HIV). Results of a physical examination showed signs of tachypnea, tachycardia, accessory muscle use and left lung base crackles. Tympanic temperature was 34.7°C. The remainder of the examination was unremarkable except for urethral meatus stenosis.2 10 mmHg, PaO2 109 mmHg, HCO3 2 mmol/L, and lactate 0.6 mmol/L consistent with high anion gap metabolic acidosis with respiratory compensation. Serum creatinine and blood urea nitrogen were 587 μmol/L and 46.7 mmol/L, respectively. Toxicology and drug screen was negative. The metabolic acidosis was partially accounted for by acute renal failure with retained unmeasured anions and ketonemia. Urinalysis showed pyuria. Electrocardiogram (ECG) showed normal sinus rhythm.Initial laboratory investigations are presented in Table Staphylococcus aureus, blood cultures were positive for S. aureus, Escherichia coli, and Group B Streptococcus. Urine cultured greater than 108 CFU/L of multiple gram positive and negative organisms.Chest radiograph revealed right middle lobe and lingular patchy opacification. An abdomino-pelvic CT scan demonstrated moderate to severe bilateral hydronephrosis, bladder wall thickening with multiple diverticuli, and retroperitoneal streaking consistent with acute infection. A provisional diagnosis of severe sepsis was made with multiple potential foci of infection. The patient was given empiric ceftriaxone, metronidazole and vancomycin. Sputum specimen cultured heavy methicillin-sensitive 2 and KUf 50 mL/hr/mmHg), dialysate sodium 136 mmol/L, potassium 3 mmol/L, calcium 1.25 mmol/L, bicarbonate 40 mmol/L, and QD 500 mL/min, QB 250–300 mL/min via a 25 cm left femoral double-lumen Uldall catheter. The patient had 71.5 L of blood processed over four hours with no fluid removal. Although the patient was alert and appropriate  with tachycardia and stable normal range blood pressure before the initiation of dialysis, he was demonstrating an increased work of breathing and oxygen requirements suggestive of worsening sepsis syndrome. Approximately 2.5 hrs after start of dialysis the patient became rapidly unresponsive prompting intubation for airway protection. At completion of HD and over the subsequent 4 hours the patient's neurologic status deteriorated with evidence of loss of all brainstem reflexes. Head CT-scan is shown in Figure The patient was admitted to the intensive care unit (ICU). The metabolic acidosis persisted (pH 7.00) a despite 100 mEq of 8.4% sodium bicarbonate bolus and infusion of three liters of normal bicarbonate solution (150 mEq of 8.4% sodium bicarbonate in 1000 mL D5W). The patient had a suprapubic bladder catheter inserted by angiography. However, due to concern the patient remained oliguric following 4 L crystalloid resuscitation, hemodialysis was organized. Hemodialysis parameters included: F160 membrane   with an increased serum osmolality would have resulted in adaptive changes in the central nervous system. Ensuing hemodialysis correction of the plasma metabolic acidosis may have eclipsed a more severe cerebral intracellular acidosis. Further, urea clearance by hemodialysis was efficient at approximately 70% and probably generated a sufficient plasma-to-brain urea gradient for development of cerebral edema, intracranial hypertension and DDS. A less efficient initial course of hemodialysis would have diminished the osmolar gradient of urea across the central nervous system reducing the likelihood of symptoms of DDS.2 10 mmHg); however, initial post-intubation PaCO2 was 42 mmHg. Rapid elevations in PaCO2 can alter cerebral autoregulation resulting in exacerbated intracranial hypertension[Other variables may have contributed. The patient was compensating for the severe metabolic acidosis by hyperventilation , or initiation of continuous renal replacement therapy (CRRT) with more gradual and stable clearance of urea-28. As aIn summary, the precise epidemiology and pathophysiology of DDS remain unclear. Although DDS usually presents in end-stage renal disease patients undergoing initial therapy, critically ill patients may represent a unique population where co-existing illnesses such as sepsis, brain injury or other central nervous system disease, multiorgan dysfunction, and need for sedation can present obstacles for prompt diagnosis of DDS. Furthermore, for similar reasons, critically ill patients may have increased susceptibility to DDS conditions.None declared.SMB wrote and revised the manuscript. ADP, MH, PJEB, KBL and CJD provided critique of successive drafts of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


A wide range of childhood illnesses are accompanied by fever,, including malaria. Child mortality due to malaria has been attributed to poor health service delivery system and ignorance. An assessment of a mother's ability to recognize malaria in children under-five was carried out among the Bwatiye, a poorly-served minority ethnic group in north-eastern Nigeria.A three-stage research design involving interviews, participatory observation and laboratory tests was used to seek information from 186 Bwatiye mothers about their illness-related experiences with childhood fevers.Mothers classified malaria into male (fever that persists for longer than three days) and female (fever that goes away within three days) and had a system of determining when febrile illness would not be regarded as malaria. Most often, malaria would be ignored in the first 2 days before seeking active treatment. Self-medication was the preferred option. Treatment practices and sources of help were influenced by local beliefs, the parity of the mother and previous experience with child mortality.The need to educate mothers to suspect malaria in every case of febrile illness and take appropriate action in order to expose the underlying "evil" will be more acceptable than an insistence on replacing local knowledge with biological epidemiology of malaria. The challenge facing health workers is to identify and exploit local beliefs about aetiology in effecting management procedures among culturally different peoples, who may not accept the concept of biological epidemiology. Malaria is a major cause of death among children in many parts of the world, despite the availability of simple and effective treatments  and is oThe study was carried out during the malaria season (May-September) of 1999 among the Bwatiye, a minority group living in six villages along the Benue valley in Adamawa State of north-eastern Nigeria. A multiple-stage approach consisting qualitative, quantitative and laboratory techniques was used.In order to orient the data collection process, informal conversations were held with women and lay people in markets and health facilities about malaria illness experience within communities, the method of recognition and help-seeking behaviour. On the basis of received information, key informants were identified and selected  and formally interviewed about local knowledge and practices regarding malaria in children. The information was used for designing a cross-sectional household survey questionnaire. Households with children between 9 and 60 months in six Bwatiye villages were identified with the assistance of community health personnel. The study objectives and procedures were explained to household heads and mothers of children that were eligible participants in the study. It was emphasized that anyone was at liberty to decline participation and that those participating were also free to withdraw at any stage of the study. The pre-tested cross-sectional survey questionnaire was administered in the Hausa language to 186 mothers in their homes. Information on the mother's demographic, educational and parity status was collected. Mothers were also asked about symptoms used for recognizing and classifying malaria and to describe their help-seeking practices when a child has malaria. Previous history of child mortality was also recorded. The interviews were complemented with observations of fifteen mothers  identified during the interview process. The results were analysed and used for developing a group discussion guide.Six group discussions, each consisting of eight carefully selected mothers of different ages. family settings (polygamous or monogamous) and religions were held.Culturally appropriate explanations were further obtained from four key informants when the data had been analysed and a draft report written.zazzabi, meaning "hot body" with intermittent episodes of cold shivers ("when the child wants to stay in the sun") accompanied by headache as the main symptom of malaria in their children. Diarrhoea and teething were also important (44.6%) besides dehydration, sweating and vomiting (33.3%), loss of appetite (30.6%) and persistent crying (29.6%). Only very few mothers (6.5%) use faint spells, restlessness, moodiness and withdrawal as symptoms. Only 9.7% mentioned convulsion as an important symptom of malaria in children. Several symptoms were combined, although "child feels cold but has hot body" is an omnipresent clue to presuming that a child has zazzabi (Table Most mothers (88%) refer to malaria as bi Table .zazzabi as the major symptom (100%), while 79.0% of those with more than three child-births were content to presume malaria on noticing "hot-body with cold shivers" without any additional symptom. Mothers with more than three children (28.4%) and mothers with two or three children (81.0%) differed widely with respect to enteric symptoms.While the number of childbirths and previous experience with child mortality were important factors in the recognition of malaria (Chisquare test, P > 0.05), age and education (Chisquare test, P < 0.05) were not. Association between age and number of childbirths on the one hand and age and child mortality on the other were significant (Chisquare test, P < 0.05). Young mothers had fewer children and fewer child mortality rates than the older mothers. Among first time mothers, 47.6% identified fever with persistent crying, an association unique to this group and used A history of child death in the family plays an important role in a mother's approach to recognition of malaria. Surprisingly, education played a minor role in the recognition of childhood malaria. For mothers who had experienced a child dying, emphasis was placed on diarrhoea.zazzabi and "evil doers" are suspected to be using zazzabi to hide a more severe ailment.Malaria was classified into two categories; the female and the male. The female form goes away after a couple of days without any particular treatment other than that provided by the household or in some cases the mother. When illness defies home remedies after several days, or deteriorates, it is considered to be the male form. However, should extra-community consultants become necessary, the illness is no longer regarded as boka) rather than the health facility could handle.Convulsions or any form of complications including hallucinations were not regarded as symptoms of malaria but manifestations of other problems that the local healer  for most mothers  is strong, especially among the older mothers. The local belief is that convulsions are not curable in health facilities and a sick child taken to another village, out of reach of the local enemies, is likely to survive. Most child deaths occur at this stage. In some cases, the child is taken to the health facility, often too late to be helped. This experience, which seems common, has created a strain between the health personnel and mothers, one accusing the other of negligence. The delay action of first-time mothers is interpreted as stemming from fear of rebuke by their husband, while third-time mothers being more confident, would rather wait to be sure it was not an ordinary illness. Third-time mothers are often older, more experienced, but also more entrenched in the local beliefs about malaria aetiology than the young mothers. Although they have more responsibilities, they are economically more independent and are able to take vital decisions with respect to their children with little reference to the husbands, not the case with the younger, inexperienced mothers. Unlike the younger ones, older and multigravid mothers are assisted by their older children who serve as child-minders and help with domestic and economic tasks. When drugs are used, the habit of sharing health resources in times of illness signifying solidarity contributes to non-compliance with management regulations. A mother stops administering drug as soon as the child seems to have improved in health and the remaining drugs are kept for another episode. The act of sharing has been exploited for the development of community management of childhood malaria in Uganda. The Bwatiye and other similar societies could benefit from this management approach.As in other cultures, an attempt to differentiate between the severe and the mild forms of malaria has led to classification in order to determine at which stage a particular remedy would be required. While the Yoruba and Igbo are reported to have three classifications for malaria or fever episodes ,7 the BwThe logical categorization of malaria as female  and male (severe), the interpretation of the class of ailment and its consequences, provide useful clues in the search for healing within the local milieu. Local consultants are useful allies in appropriately managing malaria or other febrile illnesses among the Bwatiye and perhaps other ethnic groups in north-eastern Nigeria.zazzabi to cover up their evil deeds until it is too late for the affected person to seek a potent cure. This belief may not be ignored when drafting health education material. During post-analysis discussions, the women saw the logic of promptly administering antimalarial drugs at the onset of any febrile illness as a means of eliminating malaria and exposing any underlying evil that may not be malaria. The belief that malaria is incapable of killing a child without enemy intervention is a challenge to health educationists in this and other parts of Africa, where such beliefs prevail. These findings raise a number of points about what is appropriate in health education provision and empowerment for community health-care delivery. Should the development of educational materials be based on promoting biomedical aetiological theory of disease and should we insist that others accept it first, or should we work within the traditional understanding of disease transmission, which includes concepts of evil and ill intent?The Bwatiye have an all-pervading belief that enemies often use The health authorities need to consider working within cultural epidemiological parameters if the programme objective is to reduce morbidity and mortality due to malaria. Insistence on acceptance of biomedical models of disease transmission belongs to another goal that may not be directly linked to reduction in morbidity and mortality.Until mothers are empowered with the capability to recognize and treat malaria, its impact on child mortality will continue unabated. The need is to educate mothers to suspect malaria first in every case of febrile illness and take appropriate action to rid the child of malaria to expose the underlying "evil". This is a more acceptable form of education than an insistence on replacing local knowledge with biological epidemiology of malaria. The challenge before health personnel is to identify and exploit local beliefs about aetiology in effecting management procedures among culturally different peoples irrespective of their acceptance of biological models of disease transmission.OBA conceived, designed, coordinated the implementation of the protocol, analysed and developed the manuscript while KKJ led the data collection team.


Tuberculosis continues to be an important health problem in the world. Besides pulmonary involvement extrapulmonary involvement becomes an affair in developing countries, even in developed countries.A thirty-six year old male patient was admitted with abdominal pain, diarrhea, nausea, vomiting and fever which had started one week before. The patient had been followed up with predialisis Chronic Renal Failure(CRF) diagnosis for 4 years and receiving continuous ambulatory peritoneal dialysis (CAPD) treatment for 4 months. In peritoneal fluid, 1600/mm3 cells were detected and 70% of them were polymorphonuclear leukocytosis. The patient begun nonspesific antibiotherapy but no benefit was obtained after 12 days and peritoneal fluid bacterial cultures remained negative. Peritoneal smear was positive for Asid-fast basilli (AFB), and antituberculosis therapy was started with isoniazid, rifampicine, ethambutol and pyrazinamide. After 15 days his peritoneal fluid cell count was decreased and his symptoms were relieved. Peritoneal fluid tuberculosis culture was found positive.Considering this case, we think that in patients with CAPD catheter and peritonitis; when peritoneal fluid leukocytes are high and PMNL are dominant, AFB and tuberculosis culture must be investigated besides bacterial culture routinely. In addition to pulmonary involvement, extrapulmonary involvement becomes an issue in most developing countries. Extrapulmonary tuberculosis, because of several factors, has greatly contributed to the total tuberculosis mortalities during the 20 century . Risk of century ,4. Tuber century . Our casThe 36 year- old male patient, after receiving CAPD treatment for 4 months, consulted our clinic because of stomachache, diarrhea, nausea, vomiting and continous fever. The patient had been diagnosed with chronic renal deficiency and had been followed up with diagnosis of predialysis CRF for 4 years. The patient was referred to us because of his symptoms such as of nausea, vomiting, weakness, and a general condition of fatigue. Immediate care involved an urgent hemodialysis followed by CAPD and planning for renal replacement therapy.Through a physical examination, the patient's blood pressure was 110/70 mmHg. The general condition was bad and pulmonary sounds in the respiratory system were diminished slightly in lower zones. On CAPD catheter's entering segment, infections were not seen.3, the platelet count was at 203000/mm3, and the erythrocyte sedimentation rate was at 22 mm/h. In biochemical findings, furthermore, serum creatinine was at 6.05 mgr/dl [ref. 0,5–1,4], urea nitrogen was at 38 mgr/dl [ref.5–20], protein was at 3.8gr/dl [ref:6–8.5], albumin was at 1.2 gr/dl [ref:3,5–5], and lactic dehidrogenase was at 565 iu/L. Serum sodium, potassium, glucose, bilirubin, alkaline phosphatase, aspartate and alanine aminotransferase, gamma-glutamyl transpeptidase, amylase, triglyseride, and cholesterol were normal. A coagulation factor protrombin time was found to be 18.7 sn. C-reactive protein was 9.54 mgr/dl. Bilateral costofrenic angles were blunted in posteroanterior pulmonary graphy.In a laboratory investigation Hg was at 9.1gr/dl, Htc was at %27.6, WBC was at 5200/mm3 cell were detected and it was seen that 70 percent of these cells were polymorphonuclear leukocytosis (PMNL). The patient was given ceftazidime (IV), cephazol, and amikacin , but no benefit was noticed after 12 days of antibiotherapy and there was no growth in peritoneal fluid cultures. There were PMNL present but no microorganism could be detected. Acid-fast basilli (AFB) was found to be positive in the gram staining of peritoneal fluid in the remaining follow up periods, and the patient had begun antituberculosis therapy in fours(with isoniazid, rifampin, ethambutol and pyrazinamide). Tuberculin skin test was anergical. On the 15th day of anti tbc therapy, peritoneal fluid cell count decreased to 300/mm3 . Peritoneal fluid bacterial culture, blood cultures, throat culture and urine culture were negative but peritoneal fluid tbc culture was found to be positive, in Lowenstein- Jensen medium in 24 days. The patient was followed up with the treatment for recovery with an anti-tbc treatment.No parasites and cystes were found in fecal examination due to diarrhea. No pathogenic agent was detected in stool cultures. In peritoneal cell counting, 1600/mmThe peritoneal fluid of the patient was sent to be examined with Gram staining and Ziehl Neelsen staining. The peritoneal fluid was centrifuged at 3,000 × g for 15 minutes and the sediment was stained by Gram and Ziehl-Neelsen staining. The Gram staining showed PMNL presence but no microorganisms. The Ziehl-Neelsen staining(AFB) was positive.The peritoneal fluid was transferred to 10 ml sterile glass tube and centrifuged at 3,000 × g for 15 minutes. The concentrated sediment was inoculated onto Lowenstein Jensen (LJ) medium without prior decontamination. LJ medium was incubated at 37°C. Two specimens were later sent to be examined with Ziehl Neelsen staining on two different days. Both of them were detected to be positive for Ziehl Neelsen staining.LJ medium was examined for growth twice weekly for the first two weeks and once a week thereafter until the eighth week. After 24 days, the colonies were able to be seen on LJ medium. Positive growth was confirmed by Ziehl Neelsen staining.CRF increases the risk of tuberculosis. In patients receiving hemodialisis, the risk of tbc increases within twelve months after the occurrence of extrapulmonary tbc. The risk in these patients is ten times more for extrapulmonary tbc than in any other population. Peritoneal tuberculosis is rarely seen but remains a very important complication in CAPD patients,6. MortaIn English literature the most common complaints of tbc peritonit are as follows: fever 78 percent), stomachache (92 percent), misty dialisat (90 percent) and PMNL are dominant in peritoneal fluids in 76 percent of the cases and in 73 percent of the cases AFB and culture are positive [ percent,th–57th days of antituberculosis treatment (on average 30 day) [Abraham et al. have reported tbc peritonit in 4 of 155 CAPD patients and tuberculin test were found anergical in all patients . In our  30 day) . We had It was reported in the literature; that in tbc peritonitis treatment, removing peritoneal catheter has no apparent benefit and does not increase efficacy of the treatment ,6,9.Considering this case, we think that in patients with CAPD catheter and peritonitis; when peritoneal fluid leukocytes are high and PMNL are dominant, AFB and tuberculosis culture must be routinely investigated along with bacterial culture.


An aggressive trimodality approach from the Minnie Pearl Cancer Research Network [carboplatin AUC = 6, days 1 and 22; 5-fluorouracil 225 mg/m2 continuous infusion, days 1–42, paclitaxel 200 mg/m2, days 1 and 22; 45 Gy] has resulted in remarkable pathologic response rates but notable toxicity. This trial was designed to mitigate this toxicity by starting with a lower carboplatin dose, AUC = 4, and by adding subcutaneous amifostine.This phase II trial included patients with locally advanced, potentially resectable esophageal cancer. All were to receive the above regimen with modifications of carboplatin AUC = 4 and amifostine 500 mg subcutaneously before radiation. All were then to undergo an esophagectomy. A planned interim toxicity analysis after the first 10 patients was to determine whether the carboplatin dose should escalate to AUC = 6.Ten patients were enrolled, and all required dose reductions/omissions during neoadjuvant therapy. One patient died from paclitaxel anaphylaxis. Six patients manifested a complete pathologic response.With this regimen, carboplatin AUC = 4 for patients with locally advanced esophageal cancer is appropriate. Combined modality treatment is often administered for locally advanced esophageal cancer ,2. PartiHowever, this high complete response rate occurred at the expense of substantial treatment-related toxicity. During neoadjuvant therapy, grade 3 or worse leukopenia occurred in 65% of patients. Twenty-two percent of patients required hospitalization for neutropenic fevers. Grade 3 or worse esophagitis occurred in 31%. Over half of patients suffered weight loss of 10 pounds or more during neoadjuvant therapy. Finally, although there were no deaths during the administration of neoadjuvant therapy, the postoperative death rate was 9%.These toxicity data, coupled with these high response rates, underscore the need to test such novel, aggressive treatments in conjunction with methods to mitigate toxicity. This report describes the planned interim toxicity analysis from an ongoing North Central Cancer Treatment Group (NCCTG) trial that was undertaken with this goal in mind. The NCCTG trial employed two modifications to the Minnie Pearl Regimen in an attempt to mitigate toxicity: 1) carboplatin dosing was reduced from AUC = 6 to AUC = 4 in the first 10 patients with the possibility of dose escalation thereafter; 2) amifostine 500 mg subcutaneously to be given before radiation, as prompted by data from Koukourakis and others  as well This trial was initiated and conducted within the NCCTG. The Institutional Review Boards at each site approved the protocol before patient enrollment, and all patients provided signed informed consent at the time of enrollment.9/L, platelet count ≥ 100 × 109/L, total bilirubin ≤ 1.5 times the upper limit of normal, serum creatinine ≤ 1.5 times the upper limit of normal, asparatate aminotransferase ≤ three times the upper limit of normal.Eligibility criteria consisted of the following: 1) age ≥ 18 years; 2) histologic or cytologic evidence of squamous cell carcinoma or adenocarcinoma of the esophagus; 3) surgically resectable tumor, as deemed by a surgeon; 4) Eastern Cooperative Oncology Group (ECOG) performance score of 0–2; 5) anticipated life expectancy of ≥ 12 weeks; 7) the laboratory parameters < 14 days prior to registration of absolute neutrophil count ≥ 1.5 × 10Exclusion criteria consisted of the following: 1) uncontrolled infection; 2) prior chemotherapy for esophageal cancer; 3) pregnancy or unwillingness to utilize contraception if pregnancy was a possibility; 4) New York Heart Association classification III or IV; 5) other severe underlying illness that, in the opinion of the treating oncologist, would make the patient inappropriate for study entry; 6) prior radiation that would overlap anticipated radiation fields; 7) antihypertensive or diuretic medications that could not be safely discontinued, if necessary, for several days during study treatment.The neoadjuvant treatment regimen consisted of both chemotherapy and radiation. Chemotherapy consisted of carboplatin with an area under the curve (AUC) of 4 to be given intravenously on days 1 and 22, paclitaxel 200 mg/m2 to be given intravenously on days 1 and 22, and 5-fluorouracil 225 mg/m2/day to be given by continuous intravenous infusion on days 1 through 42. In addition, amifostine was to be administered as a 500 mg flat dose subcutaneously immediately before each radiation treatment.For evaluation purposes, a treatment "cycle" is defined by the administration of carboplatin and paclitaxel, where the initiation of these agents heralded the start of a new chemotherapy "cycle." In effect, cycle 1 occurred during days 1–21 of the treatment, and cycle 2 between days 22–42.The protocol was written to allow for a dose increase of carboplatin to an AUC of 6 in the event that a planned interim toxicity analysis after the first 10 patients deemed this increase could be undertaken safely.Radiation was prescribed at a total dose of 4500 centigray. Each fraction size was 180 centigray, and a total of 25 fractions were to be given.9/L for greater than 2 days and/or platelet count ≤ 25 × 109/L) prompted a 25% dose reduction of both paclitaxel and carboplatin on day 22. Similarly, on day 22, the protocol mandated that paclitaxel and carboplatin be held until the absolute neutrophil count was ≥ 1.5 × 109/L and the platelet count ≥ 100 × 109/L. For grade 3 or worse esophagitis, the paclitaxel and carboplatin were held until the esophagitis resolved to grade 1 or less. Paclitaxel was also held for grade 3 or worse neuropathy. Re-treatment was permitted with a 30% dose reduction in the event the neuropathy resolved to grade 2 or better. For any grade 3 or 4 event attributable to amifostine, the amifostine was to be decreased to 300 mg and subsequently discontinued if the event recurred at the lower dose. Finally, radiation was to be held for myelosuppression (absolute neutrophil count < 1.0 × 109/L and/or the platelet count < 50 × 109/L) or for grade 4 esophagitis.Chemotherapy dose reductions were initiated based on toxicity. As determined by the National Cancer Institute's Common Toxicity Criteria (NCI CTC), version 2, Grade 3 or worse stomatitis, esophagitis, or diarrhea prompted holding 5-fluorouracil until symptoms were grade 2 or better. If treatment was held for diarrhea, the protocol called for resuming the continuous infusion 5-fluorouracil at 80% of the prior dose. 5-Fluorouracil that was held was not to be made up. Severe myelosuppression on weekly blood counts ; 15 grade 4 events (myelosuppression (11); mucositis (1); dyspnea (1); neutropenia (1); and cerebral vascular accident (1); and 55 grade 3 events of various types  prior to each dose of radiation. Preliminary analyses show that this modified regimen resulted in a substantial rate of severe toxicity: one death from a paclitaxel allergic reaction, several episodes of grade 4 neutropenia/leukopenia, one episode of grade 4 mucositis as well as several grade 3 events – all of which were attributable to the study regimen. At the same time, however, this regimen resulted in 6 of 10 patients manifesting a complete pathologic response. The results of this planned analysis led to the NCCTG's decision to reopen this trial with a carboplatin dose of AUC = 4. In effect, the results of this interim analysis suggest that oncologists who might choose to treat patients with the Minnie Pearl Regimen should consider treating with this lower carboplatin dose, especially should they choose to include amifostine for its purported cytoprotective effects.In fact, a major modification of the Minnie Pearl Regimen in the NCCTG trial described here is the addition of subcutaneous amifostine. It remains unclear whether the addition of amifostine compounded or mitigated the toxicity observed in this trial. Certainly, the inclusion of amifostine into this regimen did not permit a dose escalation of carboplatin to an AUC of 6, as tested in the original Minnie Pearl Regimen. Although amifostine is considered cytoprotective, it also carries toxicity in its own right including nausea, vomiting, and hypotension. Preliminary data suggest that subcutaneous administration of amifostine might be better tolerated than intravenous administration, but these conclusions are not based on large, comparative studies. The fact that the amifostine, carboplatin, 5-fluorouracil, paclitaxel, and radiation were all given in combination as part of the phase II study presented here makes it impossible to sort out attribution as it pertains to an individual study agent, such as amifostine. Should the results of this trial continue to appear promising, a larger phase III trial would be necessary to provide a definitive answer on the contribution of subcutaneous amifostine to the efficacy and toxicity profile of this regimen.In short, the preliminary toxicity assessment of this trial suggests that the Minnie Pearl Regimen should include a carboplatin dose with an AUC of 4 rather than 6, especially if subcutaneous amifostine is included as part of the regimen.


In 2003, the United States Food and Drug Administration (FDA) released a guidance document on the scope of "Part 11" enforcement. In this guidance document, the FDA indicates an expectation of a risk-based approach to determining which systems should undergo validation. Since statistical programs manage and manipulate raw data, their implementation should be critically reviewed to determine whether or not they should undergo validation. However, the concepts of validation are not often discussed in biostatistics curriculum.This paper summarizes a "Plan, Do, Say" approach to validation that can be incorporated into statistical training so that biostatisticians can understand and implement validation principles in their research.Validation is a process that requires dedicated attention. The process of validation can be easily understood in the context of the scientific method. The United States Food and Drug Administration (FDA) has defined validation as "confirmation by examination and provision of objective evidence that computer system specifications conform to user needs and intended uses, and that all requirements can be consistently fulfilled" . ValidatPlan what you are going to do,Do what you planned,AndSay what you did.As an introduction to the application of this mantra, consider the following. Clinical trial operations are detailed in numerous manuals and procedural documents with the protocol being the most notable. A clinical trial protocol serves as the master research plan. Great efforts will be employed, both by researchers and by external advisory boards, to ensure that the protocol is sound and is designed to support the clinical hypothesis. Conducting the protocol at clinical sites allows for the execution of the research methodology in a reproducible and orchestrated manner, and in the end, the final report or manuscript summarizes the work performed. If any one of these components is missing from the research plan, the scientific integrity of the research would be questioned or directly refuted. Associating systems validation to this well developed paradigm is at the heart of the "Plan, Do, Say" approach to validation. There are many good references available that detail the process of validation. Stokes' two books are excellent resources for investigators new to validation ,3. The FWhen revisiting the FDA definition of validation in the context of the scientific method, it becomes clear that "confirmation by examination and provision of objective evidence that the computer system specifications conform to user needs and intended uses, and that all requirements can be consistently fulfilled" is essentially applying the scientific method to the life cycle of computerized systems. The definition implies that a validated system is one in which processes are specific and clearly defined, reproducible and consistently applied, and result in a measurable and observable level of quality in the delivered product. Validation of applicable systems is viewed as the best way to ensure that research objectives can be consistently met.All clinical trials need to collect and deliver results in an efficient and accurate manner while ensuring the integrity of the data. The CONSORT Statement provides guidance to researchers on the format and content of clinical trial reporting . ComputeIn addition, validation should be considered as a part of the complete life cycle of a computerized system . This liJust as the "Plan, Do, Say" mantra illustrates, there are three main validation deliverables . First, The scope and magnitude of the delieverable will vary from computerized system to computerized system, and it is the responsibility of the management team to decide upon the level. The following section provides an outline to serve as an aid to assembling the validation deliverables for statistical programs.1. Planning activities(a) Develop a validation planState how the validation process will be conducted, including personnel roles, test parameters, and decision points on what constitutes acceptable test results. Management prior to the development of the detailed specifications should approve the validation plan. The plan may be amended with time.(b) Utilize standard operating procedures (SOPs)SOPs should be available to formalize the procedures used in the validation process as well as establish statistical programming standards. Incorporation of standards will aid in producing reproducible deliverables. The following is a partial list of applicable SOPs:i. Validation of statistical programsii. Statistical programming standardsiii. Statistical program archival: Outlines the necessary steps to archive the analysis program, data sets, and, if necessary, computer hardware so that the results may be reconfirmed at a future date.(c) Document training on SOPsWritten SOPs are not useful unless they are incorporated into practice. In order to do this, applicable individuals need to be orientated to the procedures. This orientation session should be documented for regulatory inspection.(d) Develop detailed specificationsi. Data management planA. Annotated database structureB. List of coding conventions for dataC. List of procedures used to process the dataD. Merging criteria (database keys)E. System environment: analysis package (with version), system hardware, input data structure, output data structure, long-term storage environmentii. Analysis objectivesThe analysis objectives may vary according to the application; however, for the primary clinical trial report, the protocol or a statistical analysis plan may be sufficient to detail the requirements of the analysis.(e) Develop a test plan and/or test scripti. Mock tablesii. Expected results of testsiii. Programming standardsiv. Testing procedures2. Execution of the plan(a) Retain raw test resultsRecord individual pass/fail for each step outlined in the test script. A pass occurs when the observed result matches the expected result.(b) Note variances and deviations to the test plan(c) Document location, time and individuals involved in the testing process3. Summary report(a) Summarize validation process(b) Summarize the variances and deviations(c) Summarize test results and provide interpretation when necessary(d) Approve by managementThe definition of a computerized system encompasses both the hardware and software. For analytical applications, the key components of the system also will include the individual programs written to perform the analysis. The use of validated macros aides in reducing the burden introduced by the validation process for an individual system. For example, suppose for the clinical reporting of a trial's results, a table is desired that reports the mean and standard deviation or count and percentage for all putative covariates. A SAS macro  could beThe validation of the macro begins in the planning stage. For the macro to "conform to user needs", the needs need to be clearly identified. The following may describe its user requirements:1. Export a word-processing compatible table consisting of three columns;2. The table columns must be appropriately labeled;3. Column 1 must be the labeled "Characteristic";4. Column 2 must be the labeled "Control Group";5. Column 3 must be the labeled "Intervention Group";6. For categorical covariates, calculate the count and percentage for each level of the variable;7. For levels of a categorical variable, the table should indent the formatted description; and8. For interval-scaled continuous covariates, calculate the mean and standard deviation.This set of requirements would then be discussed from a technical perspective. Issues such has how to best calculate the summary statistics could be a topic of discussion. The use of PROC TABULATE and the Output Delivery System (ODS)  could beOnce this plan has been approved, a revision log should be kept to ensure traceability of changes that may occur during development.Once the macro has been developed, the validation system sponsor should work either independently or in conjunction with appropriate staff to review the programming and develop test cases. The test cases are assembled into a test that specifies the system input and expected output. To ensure all user requirements are addressed, a traceability matrix can be utilized. A simple traceability matrix for this validation exercise would list each of the requirements and cross reference the applicable test.The test script is then implemented under the direction of the validation system sponsor. Documentation of the event should be recorded so that it can be compiled into the validation report. Any discrepancies between the observed and expected results need to be addressed, and, if applicable, documentation on the corrective actions employed to resolve the issue(s) needs to be compiled into the validation deliverables. A revision log of programming changes is desirable; however, in practice, this may prove difficult to document completely. Once the testing has been completed, the computer system owner and validation system sponsor can determine what additional steps are required prior to release. For SAS macros, it is critical to document the parameters, default values of the parameters and system dependencies, then, perhaps include illustrative examples of the macro's use. Finally, a written statement by the computer system owner and the validation system sponsor should be issued stating that the macro has been validated and has been deemed acceptable for use.The above example illustrates a full-scale implementation of a validation process. However, it is acknowledged that individuals and organizations must decide the level of validation that is required. In many cases, more traditional approaches of program verification (code review, double programming, etc.) may be sufficient. The importance of the "Plan, Do, Say" mantra is that procedures used for validation are specified and operated on.The "Plan, Do, Say" approach presented addresses the key deliverables an auditor expects to see in validated systems. By organizing validation activities into a plan, an action or a summary, one begins to gain efficiencies through proper planning. However, individual researchers and support organizations need to evaluate their particular needs for validation and develop a set of procedures to support their needs. The intent of this article is not to provide an ironclad approach to validation that will ultimately meet all needs. However, this design for validation has been conveyed successfully to a variety of audiences. The analogy of the scientific method helps break the technology and nomenclature barrier associated with a more computer science-driven approach to systems validation by associating the importance of validation to the process of designing a research plan.The author(s) declare that they have no competing interests.The pre-publication history for this paper can be accessed here:


As development proceeds the human embryo attains an ever more complex three dimensional (3D) structure. Analyzing the gene expression patterns that underlie these changes and interpreting their significance depends on identifying the anatomical structures to which they map and following these patterns in developing 3D structures over time. The difficulty of this task greatly increases as more gene expression patterns are added, particularly in organs with complex 3D structures such as the brain. Optical Projection Tomography (OPT) is a new technology which has been developed for rapidly generating digital 3D models of intact specimens. We have assessed the resolution of unstained neuronal structures within a Carnegie Stage (CS)17 OPT model and tested its use as a framework onto which anatomical structures can be defined and gene expression data mapped.Resolution of the OPT models was assessed by comparison of digital sections with physical sections stained, either with haematoxylin and eosin (H&E) or by immunocytochemistry for GAP43 or PAX6, to identify specific anatomical features. Despite the 3D models being of unstained tissue, peripheral nervous system structures from the trigeminal ganglion (~300 μm by ~150 μm) to the rootlets of cranial nerve XII (~20 μm in diameter) were clearly identifiable, as were structures in the developing neural tube such as the zona limitans intrathalamica (core is ~30 μm thick). Fourteen anatomical domains have been identified and visualised within the CS17 model. Two 3D gene expression domains, known to be defined by Pax6 expression in the mouse, were clearly visible when PAX6 data from 2D sections were mapped to the CS17 model. The feasibility of applying the OPT technology to all stages from CS12 to CS23, which encompasses the major period of organogenesis for the human developing central nervous system, was successfully demonstrated.In the CS17 model considerable detail is visible within the developing nervous system at a minimum resolution of ~20 μm and 3D anatomical and gene expression domains can be defined and visualised successfully. The OPT models and accompanying technologies for manipulating them provide a powerful approach to visualising and analysing gene expression and morphology during early human brain development. Brain development, particularly in human, involves complex changes in shape and structure over time. During a period of approximately 4 weeks  the major subregions of the human brain are established and development proceeds from a simple neuroepithelial tube to a highly complex three dimensional (3D) structure . MRI is [The OPT models are visualised and manipulated using MAPaint, a suite of software programmes developed as part of the Edinburgh Mouse Atlas Project -10). The. The[10]OPT reconstructions were made of human embryos for a number of different stages of development. In all cases, reconstructions were made from autofluorescent imaging – in other words, the intrinsic fluorescence from the fixed specimens was used as the basis for the histological contrast seen in the 3D model. One of the models  was examined in detail in order to test the resolving power of the OPT technology on these unstained embryos, in relation to structures in the developing nervous system and to determine the feasibility of using the model as a framework for mapping gene expression patterns. Digital OPT sections were compared with corresponding histological sections stained in 3 different ways: standard haematoxylin and eosin stain to visualise cell nuclei, cytoplasm and connective tissue  and immuAn OPT model was generated from an intact, unstained embryo which had been staged according to the Carnegie staging protocol  modifiedFollowing OPT the embryo was embedded in paraffin wax and sectioned using standard methods and then every fifth section was either stained with haematoxylin and eosin or immunocytochemically with antibodies against GAP43 or PAX6. The actual plane of sectioning was identified in the OPT model by manipulating the model in MAPaint. This permitted a matched series of digital and physical sections to be compared  to CS23 (approximately 56 days of development) is important because during this time all the major regions of the developing brain are established [), which Many structures within the developing nervous system can be identified in the CS17 OPT model with a minimum defined resolution of approximately 20 μm. The CS17 model also acted as a framework onto which anatomical domains were easily painted and gene expression patterns mapped. OPT models have been successfully generated from CS12 to CS23, and these models will provide a means of analyzing and relating changes in anatomy and gene expression both within individual developmental stages and across developmental time.[In the long-term, our aim is to link the 3D models to an anatomical database and embed both of them within a custom-designed gene expression database in order to create an Electronic Atlas of the Developing Human Brain .Human embryos were collected from termination of pregnancy material, with appropriate written consent, approval from the Newcastle and North Tyneside NHS Health Authority Joint Ethics Committee and following national guidelines . EmbryosIntact specimens were rehydrated through a graded series of ethanol and embedded in a block of 1% low melting point agarose. They were then dehydrated through a graded series of methanol before being cleared using a mixture of benzyl alcohol and benzyl benzoate. 400 digital images were then captured while the now almost transparent specimens were rotated in a full circle, with 0.9° steps between each image. The signal corresponded to the weak autofluorescence originating from the paraformaldehyde-fixed tissue and was detected using a wideband FITC filter with excitation at 465–500 nm and emission from 515–560 nm. The images were then assembled to recreate the 3D shape of the embryo, using modified tomography algorithms [th section was stained with haematoxylin and eosin, following standard procedures.After OPT scanning the CS17 embryo N365 was rehydrated through a graded series of methanol and was then removed from the agarose block by incubation in a 0.29 M sucrose solution at 55°C. It was then processed for paraffin wax embedding and 5 μm microtome sections were cut. Every 5The remaining N365 sections were used for immunohistochemistry. Alternate one-in-five section series were stained with antibodies against GAP43 (GAP-7B10, Sigma), or PAX6 (PRB-278P, Covance) using standard techniques. The reaction was visualised with diamino benzidine and the sections lightly counterstained with toluidine blue.Images of the stained sections were captured through a ×2.5 objective (as viewed down the microscope at 25× magnification) using the Zeiss Axiovision system. For the PAX6 and GAP43 data, a modified warping interface of the MAPaint software was used to match each stained, physical section to the corresponding digital OPT section. Correspondences between the physical (source) and digital (target) images were identified and manually tie-pointed. The source image was then transformed to the shape of the target section, and the image transformation saved. The interface uses interactive thresholding to extract the expression signal from the source image and then applies the image transformation to map this signal into the space of the 3D OPT model. This was carried out for approximately 190 sections through the head of the CS17 embryo until the full 3D expression pattern was built up for each of the GAP43 and PAX6 data sets.JK reconstructed the OPT data, carried out the immunocytochemistry, mapped the GAP43 gene expression patterns to the CS17 model and drafted the manuscript. MS optimised the OPT methodology for human specimens, scanned the embryos, carried out the sectioning and mapped the PAX6 gene expression. JS invented the OPT technique and established the methodology in Newcastle. LP, MdlT and JLF identified and painted anatomical regions of the CS17 neural tube. GF developed the Java Atlas Viewer. RB designed/authored the MAPaint software and with DD heads the Edinburgh Mouse Atlas Project. TS, SCR and SL co-ordinate and oversee the Human Developmental Biology Resource which provided the human material. SL and TS conceived of the study and participated in its design and coordination. All coauthors participated in drafting or refining the text.3D-three dimensional4D-four dimensionalbv-blood vesselCNS-central nervous systemCS-Carnegie StageD-diencephalonD/M-midbrain/ diencephalon boundarydrg-dorsal root ganglionEADHB-Electronic Atlas of the Developing Human Brainfp-floor plateGAP43-Growth Associated Protein 43h-heartH&E-haematoxylin and eosinH-hindbrainIII-oculomotor nervel-liverM-midbrainnh-neurohypophysisOPT-optical projection tomographyov-otic vesicleth ventricler-collapsed roof of 4sc-spinal cordT-telencephalonv-developing vertebraeVg-trigeminal ganglionX-Vagus nerveXg-Vagus ganglionXIIroot-hypoglossal rootletsZli-zona limitans intrathalamicaCS17 OPT model.mpg. Mpeg movie of 3D CS17 OPT model.Click here for fileCS17 painted anatomy.mpg. Mpeg movie of anatomical domains painted on the CS17 model. Sagittal and transverse views of the painted model are shown, together with a representation of the 3D domains.Click here for filePAX6 3D expression.mpg. Mpeg movie of the 3D PAX6 expression domain.Click here for fileJAtlasViewer request form.pdfClick here for fileAcademic Licence Agreement.pdfClick here for file