Commercially available diagnostic test kits for detection of dengue virus (DENV) non-structural protein 1 (NS1) and anti-DENV IgM were evaluated for their sensitivity and specificity and other performance characteristics by a diagnostic laboratory network developed by World Health Organization (WHO), the UNICEF/UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR) and the Pediatric Dengue Vaccine Initiative (PDVI). Each network laboratory contributed characterized serum specimens for the panels used in the evaluation. Microplate enzyme-linked immunosorbent assay (ELISA) and rapid diagnostic test (RDT formats) were represented by the kits. Each ELISA was evaluated by 2 laboratories and RDTs were evaluated by at least 3 laboratories. The reference tests for IgM anti-DENV were laboratory developed assays produced by the Armed Forces Research Institute for Medical Science (AFRIMS) and the Centers for Disease Control and Prevention (CDC), and the NS1 reference test was reverse transcriptase polymerase chain reaction (RT-PCR). Results were analyzed to determine sensitivity, specificity, inter-laboratory and inter-reader agreement, lot-to-lot variation and ease-of-use. NS1 ELISA sensitivity was 60–75% and specificity 71–80%; NS1 RDT sensitivity was 38–71% and specificity 76–80%; the IgM anti-DENV RDTs sensitivity was 30–96%, with a specificity of 86–92%, and IgM anti-DENV ELISA sensitivity was 96–98% and specificity 78–91%. NS1 tests were generally more sensitive in specimens from the acute phase of dengue and in primary DENV infection, whereas IgM anti-DENV tests were less sensitive in secondary DENV infections. The reproducibility of the NS1 RDTs ranged from 92-99% and the IgM anti-DENV RDTs from 88–94%. Dengue virus (DENV) infection occurs throughout tropical and sub-tropical regions of the world where dengue is a major public health problem. Laboratory diagnosis of dengue with a single serum specimen obtained during the acute phase of the illness requires tests to detect IgM antibodies to DENV or the virus genome. A previous evaluation of available tests for IgM anti-DENV showed wide variability. The present study examined newly available commercial tests that detect the virus protein NS1, as well as new tests for IgM anti-DENV in microplate or rapid diagnostic test formats. This analytic study used specimens from laboratory confirmed dengue patients worldwide, which makes the results widely generalizable. The study found variability among the microplate ELISAs for both analytes but some tests performed with sensitivity and specificity acceptable for routine dengue diagnostics. The RDT's for both analytes had variable sensitivity that could be considered acceptable for routine clinical diagnostics. There is the need to maintain a network of dengue reference laboratories to conduct similar evaluations as additional dengue diagnostic tests become commercially available in order to guide the use for surveillance, clinical diagnosis and research. Dengue is a major public health problem with more than 2.5 billion people at risk for DENV infection and an estimated 96 million cases occur annually in over 100 tropical and sub-tropical countries Timely and accurate laboratory diagnosis of dengue performed on a single serum specimen must rely on detection of DENV RNA or NS1 antigen during the period from fever onset until 5–6 days later, or detection of anti-DENV IgM beginning 3–5 days after fever onset until 6 weeks later Rapid diagnostic tests (RDTs) for dengue have become increasing available over the last 5 years because of the need for point-of-care diagnosis in resource limited settings. However, previously available RDTs for IgM anti-DENV did not have acceptable sensitivity compared to anti-DENV IgM microplate ELISAs which considerably limited their utility To determine the analytic performance and reproducibility of commercially available NS1 antigen tests and newly available IgM anti-DENV RDTs and microplate ELISAs, a network of laboratories established by WHO/TDR and the PDVI developed specimen panels from dengue patients infected with all DENV serotypes from both the Asian and American continents, and with primary and secondary DENV infections. In addition, the evaluation included specimens from patients with other dengue-like illnesses and other conditions that can produce false-positive test results.The present study was conducted by the seven laboratories of the WHO/TDR/PDVI dengue diagnostic network that previously evaluated commercially available IgM anti-DENV diagnostic tests in 2007 This study was reviewed by the WHO Ethics Review Committee and considered exempt. All patient samples used for this study were de-linked from personal identifiers and renumbered with a study identifier.The mission of the Accessible Quality Assured Diagnostics program within the UNICEF/UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (WHO/TDR) is to promote and facilitate development, evaluation and deployment of diagnostic tools for the control of the tropical diseases. PDVI was a product development partnership established to accelerate development, evaluation and introduction of dengue vaccines for children in endemic countries.The previous study by the WHO/TDR/PDVI dengue diagnostic laboratory network resulted in recommendations to included three commercially available IgM anti-DENV ELISA tests in the WHO bulk procurement scheme . Case-patient specimens were classified as dengue fever (DF), dengue hemorrhagic fever (DHF), or dengue shock syndrome (DSS) based on data from clinical records using the 1997 WHO case definition.Lipemic, hemolyzed or bacterially contaminated specimens were not included in the panels. Only specimens with ≤2 freeze-thaw cycles were included, all were heat-inactivated at 56°C for 30 minutes, lyophilized in aliquots, coded by the reference laboratories, assembled into the respective panels which were shipped to each evaluation laboratory. Written standard operating procedures were developed and used by participating laboratories to assure uniformity in sample handling, storage and testing. Because some commercial tests can be highly sensitive to storage and shipping conditions, data on transit time was recorded for later analysis.Letters requesting study participation were sent to 20 dengue diagnostic kit manufacturers; seven companies agreed to participate and provided the tests shown in Ten specimens known to be NS1 positive using the Platelia ELISA  were tested following lyophylization, heat inactivation and one freeze-thaw cycle and showed no change in sensitivity by any of these treatments (data not shown).Specimens from each panel were tested in duplicate in the same analytical run to measure within-run precision; inter-run precision was evaluated by comparing results across evaluation laboratories. RDTs were tested in duplicates, with two readers evaluating each result independently. Inter-reader agreement was considered an important variable since RDTs required subjective judgment. All testing was conducted with strict adherence to the manufacturer's protocol described in the package insert. Each evaluation laboratory recorded testing data on standardized data sheets, which were submitted to WHO/TDR for analysis. Information was also collected from each performing technician regarding the ease-of-use of these products. This study was initiated in 2011 and completed within the same year.This panel consisted of a total of 390 dengue patient serum specimens in 3 sub-panels: 192 dengue patients who were DENV positive by virus isolation and/or RT-PCR for DENV RNA, 146 DENV RNA or culture negatives and 52 challenge specimens from other illnesses or conditions that may result in false positive results to determine the specificity of these tests  and 4. Det al., 1992 Network laboratories tested a limit of detection panel to ensure comparable sensitivity of their RT-PCR assay as a reference standard. The methods of RNA extraction and RT-PCR used in the network laboratories included: nested RT-PCR by Lanciotti DENV positive specimens included patients with primary and secondary DENV infections and represented all 4 DENV serotypes. Primary and secondary infections were distinguished according to criteria established by WHO using hemagglutination-inhibition assay described by Clark and Casals, 1958 adapted to 96-well microtiter plate This panel also included paired, acute and convalescent specimens from patients who were DENV RNA and/or virus isolation positive and anti-DENV IgM negative in the acute phase specimens but DENV RNA and/or virus isolation negative and anti-DENV IgM positive in the convalescent specimens. These specimens were used to determine the duration of NS1 antigen detection over the course of the illness. The NS1 panel was used to test microtiter ELISAs and RDTs. All tests were performed in duplicates including the RDTs. Since each RDT represents a single test the number of specimens in the table are including the duplicate and is exactly twice as many specimens number as the ELISA tests.The subpanels of DENV negatives and challenge specimens were obtained from normal, healthy persons living in dengue non-endemic areas and were negative for DENV by RT-PCR and/or virus isolation, and IgM and IgG anti-DENV negative. The challenge specimens included individuals with non-DENV flavivirus illnesses, febrile illnesses of other etiologies, or systemic conditions known to give rise to false positive results in immunoassays  and someThis panel consisted of 3 subpanels with 527 patient serum specimens: 228 anti-DENV IgM positive paired specimens from patients with dengue  and 155 DENV IgM negative specimens were obtained from healthy individuals living in dengue non-endemic areas and were negative for DENV RNA and/or virus isolation, and IgM and IgG anti-DENV. Challenge specimens were obtained from patients with febrile illness of other etiologies, non-DENV flavivirus illnesses or systemic conditions known to give rise to false positive results in immunoassays . ChallenSensitivity and specificity were assessed against reference test by analyte evaluated (NS1 or IgM). Kappa is a measure of the degree of non-random agreement between observers or measurements of the same categorical variable. Agreement is considered as good if kappa is between 0.60 and 0.80, and very good if greater than 0.80.95). P-values lower than 0.05 were considered as statistically significant. The statistical program used was STATA .The McNemar paired test was used to compare the difference of agreement between operators. Proportions were compared using the chi-square test or the Fischer exact test, whichever was appropriate. Confidence intervals were set at 95% (CIThe sensitivities of the NS1 ELISAs in specimens from dengue patients during the acute phase of illness ranged from 60–75% . Based o95 21–40 p = <.001) and the lowest positive rate was Panbio E 19% , , . All of 95 62–88), and the least sensitive was Bio-Rad Platelia 60% (CI95 46–75); these results were significantly higher than observed among dengue cases with secondary infections: Standard Diagnostics 46% , BioRad Platelia 42% . This analysis included both acute and convalescent specimens  was most sensitive and CTK Biotech 40% (CI95 31–49) the least (95 51–66); and the lowest test positive was the Panbio 12% (CI95 7–17); With the exception of SD BIOLINE Dengue Duo RDT, all of these were lower than the rates observed in the acute phase specimens.The sensitivities of NS1 RDTs in the acute phase specimens (DPO 0–5) ranged from 40 to 59%: Panbio 60% (CIhe least . Among s95 60–81) vs. 55% (CI95 50–61), (p = .016); Bio–Rad RDT 59% (CI95 48–70) vs. 31% (CI95 25–36), (p<.001); CTK Biotech 47% (CI95 36–59) vs. 21% (CI95 16–27), (p<.001), and Panbio 38% (CI95 28–49) vs. 38% (CI95 32–43) (p = .915)  .All four RDTs had similar specificities in the DENV negative specimens (76–80%) . Against95 94–99). Comparison of acute versus convalescent specimens demonstrated 98% (CI95 95–100) sensitivity in acute phase specimens and 97% (CI95 94–100) sensitivity in convalescent specimens. When analyzed according to DENV infection status, the sensitivity was 97% (CI95 92–100) in primary infections compared to 96% (CI95 94–99) in secondary DENV infections (95 80–89) against the DENV negative panel. False positives reactions were observed against other flavivirus infections - St Louis Encephalitis virus (SLEV), Japanese Encephalitis virus (JEV) and West Nile virus (WNV), as well as Chikungunya virus (CHIKV) and Hantavirus. False positives rates were also observed for malaria, leptospirosis and scrub typhus , and the least sensitive was CTK Biotech 46% (CI95 36–55)  and SD BIOLINE Dengue Duo IgM = 98% . The following tests had lower sensitivities in the convalescent panel when compared to the acute panel: Orgenics 82%  and Abon 56% .The overall sensitivities of the IgM RDTs ranged from 52–95%. In the acute phase panel, the most sensitive test was Orgenics 95% (CI5 36–55) . In the 95 91–100) in primary infections compared to 84% (CI95 80–88) for secondary infections (p = .009) and the Abon test had a sensitivity of 75% (CI95 65–86) for primary infections compared to 55% (CI95 50–60) for secondary infections (p = .002). RDTs with statistically significant increase in sensitivity in secondary infections, included: Orgenics 84% (CI95 76–93) compared to 97% (CI95 96–99) (p<.001) and CTK Biotech 30% (CI95 19–41) compared to 55% (CI95 50–60) (p = <.001) for primary and secondary, respectively  Since NS1 is a viral antigen, the only available reference standard has been the presence of DENV as determined by RT-PCR. However, viral antigens may persist in serum longer than viral nucleic acid detected by molecular amplification or virus detected by cell culture. Thus, the interpretation of NS1 ELISA and RDT results obtained from specimens in the convalescent panel is not straightforward. All specimens in our panel were anti-DENV IgM positive but negative for DENV by RT-PCR or virus isolation. By strict definition, we were not able to determine the sensitivity of these tests in convalescent phase specimens because there was no reference standard for comparison. One interpretation is these are false-positive results since the specimens were DENV RNA negative. However, it has been shown in longitudinal studies of dengue patients that NS1 antigen remains positive after DENV RNA amplicons disappear Our study showed higher sensitivity in primary than secondary DENV infections for both NS1 ELISAs and RDTs, with the exception of the Panbio RDT which had equal sensitivity . The anaAlthough generally the NS1 RDTs had reduced sensitivities compared to the NS1 ELISAs, they had similar specificities, and the RDT's good agreement (>.80) between operators. However, there were still differences noted between laboratories as observed previously in the evaluation of the IgM anti-DENV RDTs As might be expected, there were significant differences in sensitivity between acute and convalescent specimens for the IgM anti-DENV RDTs because anti-DENV IgM peaks at DPO 10, except for the Abon RDT which had essentially similar sensitivity in the both periods. Anti-DENV IgM titers have been shown to be higher in primary compared to secondary infections, with as many as 20% of dengue cases with secondary infection having undetectable levels of anti-DENV IgM in convalescent specimens The utility of using both IgM anti-DENV and NS1 antigen for dengue diagnostic testing has been evaluated in recent studies, suggesting an added diagnostic benefit to this testing combination. This study had several limitations as well as strengths. Test performance was compared to the evaluation panels, which were only characterized by the two reference laboratories using the reference methods. Additionally, the NS1 and IgM anti-DENV test sensitivity could not be evaluated by DENV serotype because of an insufficient number of specimens to allow for this sub-analysis. In addition, the small number of samples in some of the challenge panel categories did not provide strong point and confidence interval estimates similar to those derived for the DENV positive panels, and only identified potential issues. Lastly, the infectious agents represented in the challenge panels may not be of epidemiologic importance in all populations where dengue occurs. The convalescent specimens used for the NS1 evaluation were not characterized with the same reference tests as the acute specimens and no further testing was performed to determine if a positive result was a true or false positive. The strength of the study was that a high proportion of specimens in the panels were from persons with secondary DENV infections which, reflects the situation in most dengue endemic countries. In addition, most tests tended to perform better for primary DENV infections, which would mitigate any concerns about test performance in patients with this infection status .Results from this evaluation have been provided to the manufacturers and WHO member states and provide benchmarks for the evaluation, procurement and use of dengue diagnostic tests at the country or regional level. This study indicates that most of the NS1 microplate ELISAs performed adequately for routine, clinical diagnostic use in dengue endemic countries. However, the clinical and epidemiologic consequences of the lower sensitivity of NS1 tests compared to DENV RNA detection should be considered when using these tests for routine use.The microplate anti-DENV IgM ELISA continues to show good performance and this evaluation indicates that the one test evaluated had good sensitivity in the early phase of the illness, an area that has been problematic in the past. Whether these tests should be combined with NS1 microplate ELISAs for routine testing or in a testing algorithm based on when a patient presents for clinical care, needs to be evaluated in large-scale clinical studies.Unfortunately the RDTs for both NS1 and anti-DENV IgM continue to have relatively poor performance profiles; a problem for resource poor countries. Since the combination NS1/anti-DENV IgM RDTs was only evaluated for each analyte individually, no statement can be made regarding its performance as a combination test. This study did not address the issue of how RDTs, with their lower sensitivity, might be used in resource poor areas. Even with their lower sensitivity but comparable specificities compared the ELISAs, they could be used to identify dengue outbreaks. However, large studies are needed to determine the most effective way to use them for case management, disease surveillance or whether their present shortcomings can be overcome by a combination testing algorithm.Checklist S1STARD checklist and STARD flowchart.(DOC)Click here for additional data file.Table S1Number of dengue virus (DENV) positive specimens in the non-structural protein 1 (NS1) panel  by infec(DOCX)Click here for additional data file.Table S2Commercial test characteristics of: A) NS1 ELISAs B) NS1 rapid diagnostic tests (RDTs) C) anti-DENV IgM ELISA and D) anti-DENV IgM RDTs.(DOCX)Click here for additional data file.


B cell hybridomas are an important source of monoclonal antibodies. In this paper, we developed a high-throughput method to characterize mouse IgG antibodies using surface plasmon resonance technology. This assay rapidly determines their sub-isotypes, whether they bind native antigen and their approximate affinities for the antigen using only 50 μl of hybridoma cell culture supernatant. Moreover, we found that mouse hybridomas secreting IgG antibodies also have membrane form IgG expression without Igα. Based on this surface IgG, we used flow cytometry to isolate rare γ2a isotype switched variants from a γ2b antibody secreting hybridoma cell line. Also, we used fluorescent antigen to single cell sort antigen binding hybridoma cells from bulk mixture of fused hybridoma cells instead of the traditional multi-microwell plate screening and limiting dilution sub-cloning thus saving time and labor. The IgG monoclonal antibodies specific for the native antigen identified with these methods are suitable for in vivo therapeutic uses, but also for sandwich ELISA assays, histology, flow cytometry, immune precipitation and x-ray crystallography. B cell hybridomas have been an important source of mouse monoclonal antibodies (mAbs) since the initial production in the 1970s [Over the years, many useful mouse monoclonal antibodies have been successfully made by this fusion and screening method. However, it has two major shortcomings. First, mice hyperimmunized with soluble proteins not only produce antibodies specific for the native protein, but also those specific for epitopes unique to the denatured protein, especially when adjuvants such as Freund’s adjuvant or alum are used in the immunization ,3. WhileTo overcome these shortcomings, we have developed a rapid method to characterize mouse IgG antibodies and single cell sort antigen specific IgG hybridomas cells. We captured potential antigen-specific mAbs from hybridoma culture supernatants within flow cells of a BIAcore BIAsensor chip, each containing an immobilized anti-Fc antibody specific for an individual mouse IgG isotype, with the surface plasmon resonance (SPR) signal identifying the mAb isotype. The captured mAb was tested for its ability to bind the native antigen, following the binding kinetics with SPR from which the mAb affinity was estimated. We also found that mouse hybridoma cells secreting IgG antibodies have a surface form of IgG that lacks Igα but binds antigen normally. We used fluorescent antigen bound to this surface IgG to single cell sort hybridoma cells secreting mAbs specific for native antigen.The Biacore CM5 chip was purchased from GE Healthcare. Goat anti-mouse IgG, Fc specific and IgGγ1, 2a, 2b and 2c specific antibodies were bought from Jackson ImmunoResearch. AKP conjugated anti-mouse IgGγ2a or IgGγ2b antibodies were from BD Pharmingen. Actin antibody was from Cell Signaling Technology. Rabbit polyclonal Igα antibody was a gift from Dr. John Cambier lab. Ovalbumin (OVA) was purchased from Sigma. Native OVA protein was obtained by dissolving OVA in PBS and collecting monomeric OVA peak from a superdex 200 size column using fast protein liquid chromatography (FPLC). Alexa fluor 647 conjugated OVA protein was purchased from Life technology. Anti-Alexa Fluor 647 MicroBeads, anti-CD43 microbeads and LS column were from Meltenyi Biotec. BCIP was from Promega. LANAC adjuvant was provided by Dr. Steven Dow. Spike protein of SARS-CoV was made by Dr. Chengyu Jiang lab.Mice were purchased from Jackson lab and kept in a specific pathogen free environment at Naitonal Jewish Health. All mice experiments were carried out in strict accordance with the guides for the Care and Use of Laboratory Animals of National Institutes of Health. The protocol AS2517-07-13 was approved by the National Jewish Health Institutional Animal Care and Use Committee.H22, H71 and M1 hybridoma cells against Maltose Binding Protein (MBP) were made by fusion of MBP immunized mice spleen cells with Sp2/0 in our lab. S212 hybridoma cell against the spike protein of SARS-CoV was made by Xia Hong and Janice White . 2JLO-5 Goat anti-mouse IgG, Fc subclass 1, 2a, 2b or 2c specific antibodies were immobilized in flowcells of a BIAcore CM5 as per manufacturer’s instructions. The antibodies were dissolved at 50μg/ml in 50mM sodium acetate pH5.0. The surface of CM5 was activated by N-hydroxysuccinimide (NHS)/ethyl (dimethylaminopropyl) carbodiimide (EDC). Each one of four antibodies was then injected into a different channel of a CM5 chip and the conjugation was achieved by covalent coupling of antibody to the surface of chip. Around 8000 RU of antibody was immobilized on each channel. The unoccupied activated sites of the chip were then blocked by injection of ethanolamine.3-month old BALB/c mice were immunized intraperitoneally three times with 20 μg OVA protein mixed with LANAC (Liposomes and unmethylated DNA) with one week interval. OVA antibody titer in mouse serum was confirmed by ELISA using OVA coated plate. 20 μg OVA in PBS was then injected intraperitoneally for boosting three days before sacrificing the mice.Mouse hybridoma cell lines, 2JLO-5, M1, S212 and AL1-4A2 which secreted mouse IgGγ1, IgGγ2a, IgGγ2b and IgGγ3 antibodies respectively, were stained with 2 μg/ml goat anti mouse heavy chain Fc region isotype specific antibody or control antibody. Staining was carried out on ice for 15 minutes. Cell surface staining was detected by FACScan.Splenocytes were fused with Sp2/0 myeloma cells. Instead of traditional 96-well plates, fused cells were cultured in T225 flask. HAT was added the following day. 8 days after fusion, dead cells were removed by PI staining and live cells were stained with Alexa Fluor 488 conjugated goat anti-mouse IgG antibody and Alexa Fluor 647 conjugated OVA. 2JLO-5 cell which secretes IgGγ1 antibody against native OVA was also stained as a positive control for gating purpose. Moflo XDP was used to single cell sort double positive hybridoma cells.mRNA from hybridoma cells was extracted in Trizol. The first stand cDNA was made using oligo-dT as primer. A common forward primer and different reverse primer targeting the end of secreted or membrane form of IgG heavy chain were used for RT-PCR using Taq polymerase. The PCR product was separated with agarose gel and verified by sequencing. The primers used were listed below in 2) at 37°C.ELISA plate was coated with SARS-CoV spike protein (expressed from HEK293 cells ) overnig+ IgGγ2b- cells from this enriched population into wells of a 96-well micro-culture plate.10 million S212 hybridoma cells were stained with Alexa Fluor 647 conjugated goat anti-mouse IgGγ2a antibody, washed and then incubated with Anti-Alexa Fluor 647 MicroBeads. Microbeads bound cells retained on the LS column in the magnetic field were recovered by the removal of the magnetic field. The recovered cells were then stained with Alexa Fluor 488 conjugated goat anti-mouse IgGγ2b antibody. The Moflo XDP flow cytometry instrument was used to single cell sort IgGγ2a+ B cells were used as Igα positive control. Cell lysate was loaded on to SDS-PAGE, transferred to nitrocellulose membrane and probed with anti-Ig α antibody. Actin was also probed as a loading control.Hybridoma cells or CD43 bead-purified B cells were washed twice with ice-cold PBS and lysed in RIPA buffer. Purified CD43To discriminate between mAbs that bind native vs. denatured forms of protein antigens, we used Surface Plasmon Resonance (SPR) technology with a BIAcore instrument, which follows the increase in refractive index that occurs as protein binds to the surface of a flowcell . We covaSince the mAb is captured via its Fc region, its two Fab arms are free to interact with the antigen. By injecting the native immunizing protein, we can tell whether the captured mAb recognizes native protein. For example, HO14 and HO1 are both IgGγ1 mAbs produced from OVA-immunized mice. They were previously scored to bind to OVA protein coated on standard ELISA plates. However, when these mAbs were captured via their Fc within the anti-IgGγ1 Fc flowcell , subsequAnother useful variation of this experiment is shown in In vivo therapeutic uses of mAbs to block receptor-ligand interactions has been very successful. To be useful these mAbs must bind to a receptor or ligand epitope that lies within the interface of the interaction. The robustness of the goat anti-IgG Fc reagents allows the flowcells in these experiments of be stripped of the captured mAb’s and antigen with a low pH  wash without significant damage to the immobilized anti-Fc reagent. Thus a single BIAsensor chip can be used for hundreds of experiments.The heavy chain secreted and membrane forms of immunoglobulins are produced from a single RNA transcript through differential splicing. In non-activated B cells this splicing results in virtually exclusive production of the membrane form. In fully differentiated antibody secreting cells the mRNA expressing the secreted form predominates, but some of surface form is still produced . This haWe performed additional experiments to assure that there was a tight association between hybridoma cells with surface expression of IgG and those secreting IgG. A previously single cell cloned hybridoma cell line, S161, described above, began to lose IgG secretion in culture over time. We found that not all of the cells had expression of surface IgG . HoweverFor many purposes, e.g. viral protection, complement fixation, preparation of Fab fragments, the physical properties and effector functions of mouse IgGγ2a (or γ2c) mAbs are preferred over other genetically upstream isotypes. It has been reported that switching of heavy chains to downstream isotypes in mouse hybridoma cell lines occurs at an extremely low, but detectable rate, allowing the use of heavy chain isotype specific antibodies to isolate these switched variants with several rounds of selection –15.-6/cells (+/IgGγ2b─ cells could now be identified with flow cytometry and single-cell sorted to produce clones (H and VL sequence as the parent antibody (Data not shown).We used the finding that the presence of surface IgG on hybridomas correlated with their secreted IgG, to improve methods for isolating IgG class switched variants of MAbs. We started with the mouse hybridoma cell line, S212, that produced IgGγ2b mAb against the SARS-CoV Spike protein. Analyzing these cells using an ELISPOT assay, we found that IgGγ2a switched variants existed in this cell line at the rate of ~1 per 3x10-6/cells . These ce clones . Startine clones . Also, tUsing SPR with initial hybridoma culture supernatants can greatly speed up the characterization of the mAbs, but the most time consuming process in making mAbs is selection and cloning hybridoma cells secreting the desired antigen-specific mAb. Normally, the initially fused cells are distributed into multiple microculture plates at a dilution estimated to produce less than one HAT resistant hybridoma per well. Since the fusion to the myeloma cells is not selective, many of the resulting HAT resistant hybridomas do not produce mAb specific for the immunizing antigen. Therefore all wells with growing hybridomas must be screened for the mAb specificity. Also, problems can arise when a miscalculation of the cell dilution results in wells with multiple HAT resistant hybridoma cells, only one of which is secreting the desired mAb. Furthermore, since after fusion initial daughter cells of the fused hybridoma cell tend to lose chromosomes, progeny in a well arise that is no longer secreting the mAb. Therefore the hybridomas cells in a positive culture well must be cloned and rescreened before the overgrowth of the desired mAb producing hybridoma cells by other HAT resistant cells in the culture well.We performed a set of experiments to see if surface IgG on the hybridoma cells could be used to solve these problems and further reduce the time and effort to produce cloned stable clonal hybridoma cells producing only the useful mAbs. We developed a method to use fluorescent anti-mouse IgG antibodies with fluorescent antigen to single cell sort antigen-specific hybridoma cells from a fusion bulk mixture directly targeting native antigen specific hybridomas.H and VL sequencing. Five of the six clones had the same VH and VL sequence suggesting at least some clonal outgrowth during the bulk culture (data not shown). As a negative control we also single cell sorted one plate of surface IgG positive, but OVA negative clones. All 40 of the resulting colonies secreted IgG, but only one secreted antibody binding the native OVA. This hybridoma cell was later found to be positively stained by OVA. The sorting of this hybridoma cell might be due to a system error. Thus, this selection method greatly improved the detection and selection of hybridomas producing mAb binding to the native form of the immunizing antigen prior to initial screening for antibody specificity.First we showed that fluorescently labelled OVA stained a hybridoma cell line producing an anti-OVA specific IgG mAb . Then weIn this paper, we optimize two methods to greatly streamline the production and characterization of mAbs, especially those specific for epitopes present on native proteins. The first uses SPR with a small amount of primary hybridoma supernatant to identify quickly the IgG isotope of the mAb, its ability to bind native antigen and its affinity for the antigen. The second uses flow cytometry to take advantage of the strong correlation between hybridoma secreted and surface IgG in order to isolate either rare cells in hybridoma cultures that have switched to useful genetically downstream isotypes or rare antigen-specific hybridomas in a heterogeneous mixture of hybridoma cells grown in bulk immediately following fusion. Taken together these methods greatly reduce the time and effort required to go from the initial fusion to the isolation of hybridoma clones producing mAbs of the appropriate specificity and functions for downstream applications.These methods can be used to find appropriate mAbs for research, commercial and therapeutic uses. We have shown examples here of how they can be used to identify IgGγ2a switch variants, rapidly identify mAbs suitable for sandwich ELISAs and identify mAbs capable of preventing virus/receptor interactions. However, there are other potential uses as well. For example our laboratories have used these antibodies successfully to improve co-crystallization trials with the antigens that have been difficult to crystallize on their own. It is thought that these antibodies work by shielding unstructured or highly mobile regions of the proteins from disrupting internal crystal contacts . For exaAlthough we did not explore this use here, one could imagine how the ability to sort hybridoma cells based on the surface binding of fluorescent antigen might be used to screen IgG V-region mutant libraries in order to evolve mAbs with higher affinity IgG. These types of in vitro affinity maturation experiments are most often done via IgG surface display on phage or yeast . While tWe and others have found that the surface form of IgG receptor on hybridomas lacks the signaling Igα co-receptor . This maS1 FigDetection of Igα expression in fusion partner cell SP2/0, IgG hybridoma cells and spleen B cells by western blotting.(TIF)Click here for additional data file.S1 Document(DOCX)Click here for additional data file.


Alcoholic liver disease has various manifestations: asymptomatic steatosis, alcoholic hepatitis and alcoholic cirrhosis, which substantially increase the risk for developing hepatocellular carcinoma. Transforming growth factor (TGF-β) signaling pathway is a major regulator in chronic liver diseases contributing to all liver disease progression from liver injury, inflammation and fibrosis to HCC. With the aim of generating a mouse model of alcoholic liver disease that would rapidly develop steatosis, inflammation as well as fibrosis, we formulated a regimen that combined chronic injections of low dose (2mg/kg) lipopolysaccharide (LPS) with Lieber DeCarli-based diet containing 6.7% ethanol feeding to mice with impaired TGF-β signaling through constitutive disruption of β2-spectrin and/or Smad3. Unexpectedly, the mice treated with chronic low dose LPS and fed the alcohol-containing diet developed very aggressive T-cell lymphomas to which the TGF-β mutant mice succumbed more rapidly than the wild type mice. In contrast, their liver phenotype was mild as they only developed steatosis but not hepatitis or significant fibrosis. To our knowledge, this is the first report of a mouse model of aggressive T- cell lymphoma based on chronic challenge with low dose LPS and TGF-β disruption. Chronic alcohol abuse may lead to the development of alcoholic liver disease (ALD), a major cause of morbidity and mortality worldwide. ALD has various manifestations: asymptomatic steatosis, alcoholic hepatitis and alcoholic cirrhosis, which substantially increase the risk for developing HCC. The oxidative metabolism of ethanol in the liver has been extensively studied as it is the main cause of alcohol-induced liver toxicity. The production ofacetaldehyde, a toxic metabolite, the alteration of NADH:NAD+ ratio in hepatocytes and thesubsequent changes in lipid and glucose metabolism as well as the production of reactive oxygen species (ROS) are at the core of the damage that chronic alcohol consumption causes to the liver tissue . Intereset al. have previously demonstrated that disruption of the Smad adaptor protein β2-spectrin (gene Sptbn1) leads to disruption of TGF-β signaling mediated by Smad proteins. This effect is primarily due to mislocalization of Smad3 and Smad4 and the subsequent loss of the TGF-β-dependent transcriptional response  received an isocaloric diet without alcohol (Bioserv #F1259SP) and 3 weekly injections of normal saline solution. The animals were closely monitored for symptoms of physical distress or illness.All experimental procedures involving mice were approved by the Institutional Animal Care and Use Committee (IACUC) of MD Anderson Cancer Center. Wild type (n=15), ce C57BL/ of 8-10 Tissues were fixed in 10% buffered formalin, and embedded in paraffin. Five micron paraffin sections were stained with the CD3 antibody  or B220 antibody , then DAB staining was carried out according to manufacturer instructions . Hematoxylin and eosin staining were used for counterstaining. Sirius Red staining was performed with Direct Red 80 (Sigma-Aldrich). After the slides were stained, they were scanned and analyzed by an ACIS III image analyzer .


Lymph node metastasis indicates poor prognosis in esophageal cancer. To understand the underlying mechanisms, most studies so far focused on investigating the tumors themselves and/or invaded lymph nodes. However they neglected the potential events within the metastatic niche, which precede invasion. Here we report the first description of these regulations in patients on transcription level. We determined transcriptomic profiles of still metastasis-free regional lymph nodes for two patient groups: patients classified as pN1  or pN0 . All investigated lymph nodes, also those from pN1 patients, were still metastasis-free. The results show that regional lymph nodes of pN1 patients differ decisively from those of pN0 patients – even before metastasis has taken place. In the pN0 group distinct immune response patterns were observed. In contrast, lymph nodes of the pN1 group exhibited a clear profile of reduced immune response and reduced proliferation, but increased apoptosis, enhanced hypoplasia and morphological conversion processes. DKK1 was the most significant gene associated with the molecular mechanisms taking place in lymph nodes of patients suffering from metastasis (pN1). We assume that the two molecular profiles observed constitute different stages of a progressive disease. Finally we suggest that DKK1 might play an important role within the mechanisms leading to lymph node metastasis. In esophageal cancer (ESC), lymph node metastasis is associated with poor prognosis. During the migration of tumor cells through lymph vessels and during their homing in lymph nodes, these tumor cells get into close contact to endothelial cells and lymph node sinus. Tumor expansion currently forms the main basis for therapy selection. But despite the high prognostic relevance of lymphogenic metastases, the molecular mechanisms underlying this metastatic pathway are still mostly unknown.A number of studies investigated the molecular profiles of the tumorsYet, to the authors' best knowledge there is no published work specifically examining the early molecular mechanisms in this metastatic niche in patients – mechanisms within the non-affected lymph nodes, which could propagate or suppress early metastasis in patients suffering from ESC.The aim of this study therefore was to specifically investigate these early alterations in lymph nodes prior to histopathologically detectable metastasis. The work has been driven by the hypothesis that before tumor cells metastasize to regional lymph nodes, important changes must have already taken place propagating or suppressing the homing process. To get insight into this process, we collected tumor regional and distant lymph nodes from patients suffering from ESC staged as pN0 (no metastatic nodes exist) or pN1 (metastatic nodes exist). All lymph nodes included in the analysis were metastasis-free – irrespective of the patient staging. We performed microarray analyses and investigated the mechanisms linked to the genes differentially expressed between regional and distant lymph nodes of the same patient.Key to our work here are two features. First, all analyzed lymph nodes without exception, even those of the pN1 patients, were still free of metastasis. Second, we used samples of patients who did not undergo any neoadjuvant radio-chemotherapy and established further procedures that ensured very high sample quality.The included patients suffered from esophageal cancer and underwent a radical thoraco-abdominal en-bloc esophagectomy including lymph node dissection of the upper and lower mediastinum (R0 resection) without neoadjuvant radio-chemotherapy. We included only lymph nodes from patients staged as pN0  or pN1  and verified by histopathology that the lymph nodes selected for our analysis were all tumor cell free. We also confirmed by immunohistology that these lymph nodes were free of micro-metastasis. We isolated from each lymph node total RNA for microarray expression analysis. A paired T-test was used for the identification of genes that were differentially expressed between regional and distant lymph nodes and a functional enrichment and network analysis was performed. Subsequently major candidates were validated via real time PCR and DKK1 was validated in addition via immunohistological staining.The aim of the work presented here was to identify potential factors facilitating and driving metastasis in lymph nodes of patients suffering from esophageal carcinoma. To enable such understanding the experiment design  incorporFirst, samples were classified according to the principal status of the patients exhibiting (pN1 group) or non-exhibiting (pN0 group) metastasis in regional lymph nodes. The comparison of pN1 and pN0 patients should highlight the general differences in regulations that might lead to or prevent from metastasis.Second, two lymph node samples were collected from each patient, one node located close to the tumor  and the second node distant to the tumor. While the distant nodes served as reference sample the regional nodes were investigated for transcriptional regulations. The pairwise comparison of the tumor distant samples with the patient specific regional nodes should reveal the early regulations potentially resulting from the location close to the tumor. In this publication the term “up-regulation” is used for those genes and functions that exhibit a higher expression in the regional lymph nodes in comparison to the distant lymph nodes. Conversely, those genes and functions that are decreased in the regional lymph nodes are termed “down-regulated”.prior to metastasis homing.It is important to note that all nodes investigated were made sure to be still metastasis free, independent of the patients' pN1/pN0 classification and of the location relative to the tumor. This should allow the identification of early changes occurring This study was performed in accordance with the applicable regulations and was approved by the Hamburg Ethics Committee, Germany, under vote number PV3548. Patients written consent to participate in the study was collected in accordance with the applicable regulations.Overall samples from 22 patients with esophagus carcinoma were analyzed of which 14 sample pairs exhibited an adequate sample quality. For each patient sample pairs of tumor close and tumor distant lymph nodes were collected to reduce inter-individual differences that might occur due to diverging genetic background. Lymph nodes were immediately dissected upon extraction in the operation room. The segments that were dedicated for the microarray expression analysis were directly transported in liquid nitrogen to the laboratory for RNA extraction. The other part of the lymph nodes was embedded in paraffin for classification and diagnosis.After histological diagnosis of an esophageal carcinoma, patients with early stage lesions (pT1a) were referred to surgery either if the endoscopic resection was incomplete or the tumor was staged as pT1b. Patients were directly referred to surgery if the preoperative staging was suspicious for either muscular wall involvement (cT2) or lymph node involvement (cN1). No patient was submitted to neoadjuvant therapy.All patients underwent median T-shaped laparatomy and right-sided thoracotomy. For patients with collar anastomosis an additional left-sided cervicotomy for collar anastomosis was performed. Every patient received a radical en bloc esophagectomy with resection of the esophagus, thoracic duct, azygos vein, ipsilateral pleura, and all periesophageal tissue in the posterior mediastinum. Reconstruction was performed by gastric tube or colon interposition usually with a high intrathoracic or cervical anastomosis.Additionally, a standardized two-field lymphadenectomy was performed. All lymph nodes were systematically sampled from eight locations: regional mediastinal, both in the vicinity of the tumor and distant from it; infraclavicular; diaphragmatic ; perigastric lymph nodes ; common hepatic artery lymph nodes; and lymph nodes at the celiac trunk. All nodes were mapped by the surgeon according to the scheme of the American Thoracic Society as modified by Casson et al.Postoperative follow-up was carried out as part of the usual aftercare. Patients were seen at the outpatient clinic at 3- to 4-month intervals for the first 2 years and at 6-month intervals thereafter. Follow-up evaluation included physical examination, plain chest radiography, abdominal ultrasonography, endoscopy, endosonography, CT of the chest and abdomen, with PET-CT after January 2006 in selected cases, CEA and CA 19-9 tumor marker studies, and bone scans. Whenever relapse was suspected, additional radiologic, endoscopic, or histologic confirmation was sought.Recurrence was diagnosed if proven by biopsy or by unequivocal evidence of tumor masses  with a tendency to grow during further follow-up and/or follow-up until death. Recurrent disease was defined as either locoregional (occurring in the upper abdomen or mediastium) or as distant metastasis.Hematoxylin and eosin staining was used for tumor cell detection in a first diagnostic step. Histologically “tumor-free” lymph nodes were then screened immunohistochemically using anti–cytokeratin antibody AE1/AE3  diluted 1∶50 (v/v) by the biotin-streptavidin method as described previously.Formalin fixed and paraffin embedded sections of 5 to 6 µm thickness were cut at three different levels in each node and deparaffinized according to standard histological techniques and transferred onto glass slides treated with 3-triethoxysilylpropylamin . One section of the sample obtained at each level was stained using alkaline phosphatase–anti-alkaline phosphatase technique combined with the new fuchsine stain  for the visualization reactionSections of normal colon mucosa served as positive staining controls and isotype-matched, irrelevant murine monoclonal antibodies served as negative controls .The slides were evaluated in a blinded procedure by two independent investigators. In case of incongruent findings a third investigator was consulted and a consensual decision was made. Minimal tumor cell involvement in a lymph node that was considered to be tumor-free by conventional histological staining was defined as the presence of one to ten positive cells in the body of the node.Isolation of total RNA from liquid nitrogen frozen lymph node samples was performed with the Trizol from Invitrogen according to the manufacturers protocol (Rev. date 12. June 2007) and was purified afterwards with the Qiagen RNeasy-Mini-Kit. Concentrations were determined with a PeqLab NanoDrop photometer and RNA quality by capillary electrophoresis using an Agilent Bioanalyzer 2100 System. Out of the 22 sample pairs 9 pairs of patients with observed far metastasis and 5 sample pairs of patients with no far metastasis observed showed an RNA quality sufficient for the following analysis.Microarray experiments were performed using Affymetrix human whole genome GeneChips U133 Plus 2.0  according the manufacturers protocol. To prepare the RNA for the expression arrays cDNA-synthesis with preceding photometric determination of the RNA concentration was performed. The following steps were performed using 250ng of total RNA for the first strand synthesis. Sample preparation incorporated steps for cDNA synthesis, purification, synthesis of the biotin-labled cRNA, in vitro transcription, fragmentation, hybridization as well as washing and staining. The preparation was performed according to the One Cycle Protocol using the GeneChip 3' IVT Express Kit from Affymetrix. For Hybridization 12.5 µg fragmented cRNA were used. The arrays were incubated for 16h in the Affymetrix Hybidization Oven 640 at 45°C. Washing and staining steps were performed semi-automatic in the Affymetrix Fluidics Station 450, the single steps following the Affymetrix One Cycle Protocol for standard arrays. After washing and staining the arrays were scanned using the Affymetrix GeneChip Scanner 3000 7G and the Command Console software at a wavelength of 570 nm and a pixel size of 1.56 µm.Lymph nodes from both patient groups (pN0 and pN1) were analyzed independently. As the focus of this work were early changes preceding metastasis, only metastasis free lymph nodes were analyzed. The tumor distant lymph node samples were used as reference for the paired regional lymph nodes.Background correction and normalization were performed using RMA procedure and quantile normalization. A paired T-test within the two groups  was performed and a bootstrapping procedure was performed to adjust for false positive regulations. As cutoff values a corrected p-value of 0.05, a raw p-value of 0.01 and a signal-log-ratio (SLR) of ±0.7 were used to determine the top regulated genes.Following differential gene expression analysis we performed gene set enrichment and network analysis with Ingenuity Pathway Analysis software . For the analysis the corrected p-value cutoff was set to a less stringent value of ±0.1 to allow for the incorporation of more subtle changes.Twelve of the determined candidate genes were validated via RT-PCR using three technical replicates per sample. The RT-PCR was performed with 96-well plates on the StepOne Plus real Time PCR System from Applied Biosystems. For each sample 2 µl with a concentration of 5 ng/µl were used. To account for fluctuations in sample concentration Beta-2-microglobulin (B2M) was taken as housekeeping reference. For determination of the regulations on expression level the mean value of the three technical replicates' delta-Ct-values was calculated. Significance of the changes was determined using a paired t-test between tumor close and tumor distant mean delta-Ct-values within each group .Immunohistochemistry was performed on paraffin sections of the investigated lymph nodes using polyclonal antibody against DKK1 . For immunohistochemical detection sections were deparaffinized, rehydrated, and pre-treated with 0.1% pronase, for 10 min for enzymatic antigen unmasking. After incubation in 3% hydrogen peroxide for 15 min to block endogenous peroxidase activity and incubation with 5% BSA for 30 min to block non-specific antibody binding primary antibody was applied. Immunohistochemical staining was performed over night at 4°C. A biotinylated secondary goat anti-rabbit IgG  was used, followed by incubation with a streptavidin/HRP  as a third antibody. Peroxidase activity was detected using DAB as chromogenic substrate (Dako Cytomation). Sections were counterstained with hematoxylin, dehydrated, and mounted. Images were collected on a Zeiss Mirax MIDI slide scanner (Zeiss) and images were captured using the Pannoramic Viewer software.We asked if a) the metastatic niche in lymph nodes might be affected even before metastasis has taken place and if b) still metastasis-free lymph nodes of pN1 patients might be more on the verge of metastatic invasion than those of pN0 patients and if c) therefore those “pN1” lymph nodes might exhibit distinct transcriptional deregulations associated with the niche preparation.We included tumor regional lymph nodes from 14 esophageal carcinoma patients  to investigate gene expression in the potential metastatic niche. In addition we collected a paired tumor distant lymph node from each patient to serve as reference for the regional nodes . Afterwaprior to metastasis homing. Information about patients' age, gender, histology or classifications is provided in Key to the work presented here is that all nodes investigated were confirmed to be histopathologically and immunohistologically metastasis and micrometastasis-free, independent of patients' pN1/pN0 classification and of the location relative to the tumor. This should allow identification of early changes occurring To identify the potential pre-invasive changes we compared the regional lymph nodes with the distant lymph nodes using a paired T-test within the two groups . The tumor distant lymph nodes served as reference for the regional nodes, such that up-regulations depict those genes with a higher expression level in the regional nodes and down-regulations respectively those genes with a lower expression level. The T-test was followed by a bootstrapping procedure to account for multiple testing. As cutoff values a corrected p-value of 0.05, a raw p-value of 0.01 and a signal-log-ratio (SLR) of ±0.7 were used to determine the top regulated genes. The most significant candidates identified are displayed in FPR1 and FPR2,IGHG1, IL1R2, CCL23, MSR1, C5AR1, LILRA5,LILRB2L1CAM (CD171),EREG,NOVA1, HPR,LAMC2Applying these thresholds we identified 173 transcripts (128 genes) significantly regulated between regional and distant lymph nodes of pN0-classified patients. Several of these genes were associated with immune response  in the regional lymph nodes of pN1-classified patients. Among those genes pro-proliferative and anti-apoptotic molecules were down-regulated – such as IGF1 and LTF which act through regulation of the AKT signaling pathwayIn contrast we identified 134 dysregulated transcripts . The gene was clearly down-regulated in the regional lymph nodes of pN1 patients. DKK1 is a WNT pathway inhibitor that has been shown to be of prognostic significance in diverse tumor entities such as breast cancer, lung cancer, myeloma but also in esophageal carcinoma.Notably ISL1, LAMC2, NOVA1, EREG, L1CAM) the regulation tendencies and strengths were confirmed though the p-values determined in the PCR analysis ranged between 0.08 and 0.15, probably due to the small sample size. Seven genes  could be validated successfully showing a significant p-value below of 0.05. Highest significance  was observed again for DKK1  and in pN0 samples (128 genes) in one common list and performed network analysis. Interestingly, despite the clear difference in molecular mechanism profiles between pN1 and pN0 samples, the analysis revealed that several of the involved genes  seemed to be tightly connected  across bDKK1 regulation via the WNT/β-catenin pathway seems more plausible. This being said, the molecular networks underlying these different patterns might be connected. In ESC patients lymph node metastasis is only a question of time unless a timely tumor resection is performed. Therefore it seems plausible that the observed transcriptional patterns do not correlate with two totally different mechanisms but rather depict two time points of a progressing process.Taken together these observations suggest that metastasis-free regional lymph nodes of pN1 patients are already subject to molecular processes different to those of pN0 patients – though metastasis has not yet taken place in these nodes. In addition an involvement of DKK1 in our results and because DKK1 has already been shown to be prognostic for the outcome of a number of malignant tumor entitiesBecause of the significance of While several cancer incidence rates dropped in the United states and other Western countries over the last decades the incidence rate of esophageal adenocarcinoma has increased.Our scope was to investigate this pre-metastatic niche in patients suffering from ESC and to determine the potential factors involved in early metastatic spread of tumor cells to lymph nodes. We hypothesized that the regional lymph nodes (adjacent to the tumor) might be more prone to be invaded first than the far lymph nodes. In consequence we used microarray technology to analyze these regional lymph nodes of ESC patients whom either suffered (pN1) or did not suffer (pN0) from lymphogenic metastasis at the time point of surgical intervention.As there is evidence that squamous cell carcinoma (SCC) and adenocarcinoma (AC) of the esophagus might differ in their tumor progression one could imagine that these differences might have an impact on our observations. While SCCs are mainly observed in the proximal esophagus and are ascribed to smoking or alcohol abuse, ACs usually occur in the distal esophagus and are frequently preceded by Barrett's disease or gastroesophageal reflux disease. There are hints, that the tumor type might affect the location of lymph node metastasis.The sample size in our work  is small and the authors are aware that these numbers are below the power necessary for solid prognostic studies. This sample size goes back to the stringent quality selection process that the samples underwent before being included in our study. Only patients that did not undergo neoadjuvant radio-chemotherapy were included in the study. The lymph node segments dedicated for expression analysis were immediately dissected upon extraction in the operating room and directly transported in liquid nitrogen to the laboratory for RNA extraction. The other part of the lymph nodes was embedded in paraffin for diagnosis. And finally and foremost only metastasis- and micro-metastasis-free lymph nodes were included in the study, even for pN1 patients. The power of the work presented here is therefore the quality assurance process and the novel experimental design see .The results of this study suggest that regional lymph nodes of ESC patients might undergo alterations prior to metastatic invasion. This assumption is derived from the observation that lymph nodes of pN0 and pN1 patients exhibit clearly distinct transcriptional patterns . While tWe assume that the enhanced immune response in the pN0 lymph nodes might result from a lymph node's exposure to circulating tumor cells or signaling processes of the primary tumor. An effective immune response might prevent these lymph nodes from metastatic invasion. The reduction of the immune response pattern in the pN1 lymph nodes might indicate a loss of this protective shield. This loss might provide circulating tumor cells an enhanced chance of survival. The observed reduction of proliferation and cancer & tumor morphology patterns as well as the enhanced apoptosis is primarily surprising and intriguing in terms of carcinogenesis. However, it has to be considered that at this point the investigated tissue constitutes the metastatic niche – not the metastasis itself or invaded lymph nodes. Increased apoptosis and reduced proliferation would suggest a morphological remodeling process in the lymph nodes that could facilitate invasion.Approximately all ESC patients tend to develop metastasis if not treated in time. Therefore, we assume that these two different patterns do not reflect completely different molecular pathways but rather two different stages of a progressing disease. We assume that key molecules might mark the transition from one state to the next and that DKK1 might be one of these molecules.An up-regulation of DKK1 has already been described in the context of diverse malignant tumors such as breast cancer, lung cancer, myeloma or even esophageal cancer.DKK1 expression in epithelial ovarian cancer cells in vitro and that siRNA silencing of DKK1 expression significantly enhanced invasion. Reis et al.Notably our immunohistochemical staining  showed tMSX1, HOXA10, DKK1, MET and CCND1) and by prediction of a β-catenin inactivation  help improving classification by better defining the intermediate stage between metastasis-free and invaded lymph nodes and b) help better identify patients with an increased metastatic risk that might need closer post-surgical observation or treatment, and c) help find therapies, which could reduce the risk of the “upcoming” metastatic invasion by restoring the lymph nodes' protective shield and finally d) raise awareness that targeted treatment of the metastatic niche alongside targeting the tumor cells might improve treatment outcome – a concept that potentially might be called “Assistance to Self-assistance”. The current paradigm in cancer treatment is targeting the tumor or metastatic cells. If a barrier is breached, however, or the environmental conditions in the niche are fit for metastatic invasion, then assisting classic targeted therapy by as well targeting these alterations within the metastatic niche might increase therapy effectiveness. Understanding the underlying mechanisms might help find solutions for such a niche-targeted therapy.Until then our results should be validated in a bigger study setting and the precise role of DKK1 within lymph nodes remains to be investigated.Table S1Detailed sample information.(XLSX)Click here for additional data file.Table S2Complete microarray differential regulation statistics.(XLSX)Click here for additional data file.Table S3Detailed PCR analysis results.(XLSM)Click here for additional data file.


ELOVL2, GLRA1, MYOD1 and PDE4C genes) and quantified DNA methylation across these regions in four tissues  from male and female C57BL/6J mice, ranging in age from fetal (embryonic day 17) to 630 days. We observed tissue-specific age-associated changes in DNA methylation that was directionally consistent with those observed in humans. These findings lend further support to the notion that changes in DNA methylation are associated with chronological age and suggest that these processes are often conserved across tissues and between mammalian species. Our data highlight the relevance of utilizing model systems, in which environmental and genetic influences can be carefully controlled, for the further study of these phenomena.Epigenetic disruption has been implicated in many diseases of aging, and age-associated DNA methylation changes at specific genomic loci in humans are strongly correlated with chronological age. The aim of this study was to explore the specificity of selected age-associated differentially methylated positions (aDMPs) identified in human epidemiological studies by quantifying DNA methylation across multiple tissues in homologous regions of the murine genome. We selected four high-confidence aDMPs (located in the vicinity of the  Aging, the progressive decline in physiological and psychological functioning that occurs across the lifespan, involves a complex suite of molecular changes  includinELOVL2, GLRA1, MYOD1 and PDE4C  that have been previously associated with chronological age  from fetal (embryonic (E) 17, -4 days old) to elderly (630 days old) individuals . Targeted assays were designed to quantify DNA methylation across regions of the murine genome homologous to four robustly-associated human aDMPs in the vicinity of the genes ical age . BrieflyP = 0.01; GLRA1, P = 3.86E − 05) was found to be significantly associated with age in the same direction as reported in human data, with individual CpG units within each amplicon being strongly associated with age , multiple CpG units within both amplicons were significantly correlated with age in the direction predicted from human studies . Together, these data provide evidence that human blood aDMPs are also associated with chronological age in mouse.Average and CpG site-specific DNA methylation across the four amplicons in each tissue is shown in Tables C.3–C.6 of Supplementary material. Age-associated changes in DNA methylation were identified using a linear model for each of the four tissues , although not in cerebellum or hippocampus . In contrast, cerebellum-specific associations with age were observed for amplicon-average DNA methylation across the two other amplicons  .We next examined changes in DNA methylation with age at these four loci in three additional tissues dissected from the same individual animals. Amplicon average DNA methylation was associated with age in lung across both the ELOVL2 and GLRA1 amplicons, reflecting the patterns seen in whole blood (ELOVL2, These findings lend further support to the notion that changes in DNA methylation are associated with chronological age and suggest that these processes may often be conserved across tissues and between species . CharactJM and HS conceived the project. CF, SW and BP performed mouse work. HS performed DNA methylation quantification and analysis with advice from EH. HS and JM wrote manuscript. All authors approved the manuscript before submission.We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript.


Plasmodium falciparum multidrug resistance highlights the urgency to discover new targets and chemical scaffolds. Unfortunately, lack of experimentally validated functional information about most P. falciparum genes remains a strategic hurdle. Chemogenomic profiling is an established tool for classification of drugs with similar mechanisms of action by comparing drug fitness profiles in a collection of mutants. Inferences of drug mechanisms of action and targets can be obtained by associations between shifts in drug fitness and specific genetic changes in the mutants. In this screen, P. falciparum, piggyBac single insertion mutants were profiled for altered responses to antimalarial drugs and metabolic inhibitors to create chemogenomic profiles. Drugs targeting the same pathway shared similar response profiles and multiple pairwise correlations of the chemogenomic profiles revealed novel insights into drugs’ mechanisms of action. A mutant of the artemisinin resistance candidate gene - “K13-propeller” gene (PF3D7_1343700) exhibited increased susceptibility to artemisinin drugs and identified a cluster of 7 mutants based on similar enhanced responses to the drugs tested. Our approach of chemogenomic profiling reveals artemisinin functional activity, linked by the unexpected drug-gene relationships of these mutants, to signal transduction and cell cycle regulation pathways.The spread of  Plasmodium falciparum led to significant reductions in deaths and disease, but resistance to this most potent class of antimalarial treatments has emerged and is spreading1235Plasmodium, whole-cell chemical-genetic methods that are scalable to identify high value, druggable genes from critical pathways of the parasite life cycle are still nascent7101113P. falciparum piggyBac mutants can potentially speed up the antimalarial drug discovery process similar to other programs . Functional profiling creates a chemogenomic profile of drug fitness changes in which drugs with similar mechanisms of action lead to similar fitness profiles of various mutants. Comparison of chemogenomic profiles to each drug across a set of mutants can help classify lead compounds with unknown mechanisms of action relative to well-characterized drugs with established mechanisms of action.The widespread use of artemisinin derivatives (ART) in combination therapies to treat 50, IC90) against the same collection of 60 different cancer cell linesThe National Cancer Institute operated the NCI60 program for years and screened new compounds for efficacy  validated by sequence analysisP. falciparum formed a piggyBac library of disruptions in genes of diverse Gene Ontology (GO) functional categoriesThe piggyBac insertions; these genes were previously proposed to interact with a group of six cyclophilins and two peptidyl-prolyl-cis-trans isomerasespiggyBac mutants with CsA but not FK-506 is consistent with the expectation that CsA interferes with cyclophilin activity while FK-506 does not, in spite of the ability of both drugs to inhibit calcineurin. Hence, these observations validate our approach in predicting MOA, even for two highly related compounds that affect the same pathway but through distinct molecular processes. These results indicate that chemogenomic profiling of the piggyBac mutant library could also reveal unexpected drug relationships and connect them to gene functions, including hypothetical genes in the malaria parasite. Consequently, we profiled 71 piggyBac mutants for altered responses to standard antimalarial drugs and inhibitors of known metabolic pathways (50s) of the parental clone NF54 and each of the mutants to a library of antimalarial drugs and inhibitors of metabolic pathways  on parasites in which loci of two hypothetical RNA binding proteins (PF3D7_1360100 and PF3D7_0812500) were disrupted by pathways .50 growth responses of piggyBac mutants to a wide range of inhibitors (Table S3) normalized to that of wild type NF54 parasites (Table S4) allowed assessment of genotype-phenotype associations among inhibitors and piggyBac mutants. These chemogenomic profiles were visualized by two-dimensional hierarchical clustering to discern chemogenomic interactions  (Table S5). In all but one of the pathways considered, at least one drug pair with the same mechanism of action is predicted to interact in the drug-drug network  represent the strength of the Spearman correlation between drug pairs across all mutants to define distinct ‘drug sensitivity’ clusters . As a co network . Binary 1 complex inhibitor atovaquone are positively associated with lumefantrine . Conversely, similarity in chemogenomic responses between drugs with unknown mechanisms of action and those with well-characterized targets might lead to identification of unexpected interactions of drugs with targets. For example, we observed that responses to the iron-sulphur cluster inhibitor rotenone and the bcfantrine , a drug e.g. inhibitors of hemoglobin digestion; 12Network analysis can robustly capture these and other important drug-gene interactions related to each drug sensitivity cluster, by using the same data used to define drug:drug relationships. For example, inhibitors acting on related parasite biosynthetic pathways grouped together based on their drug response profiles, which is similar to grouping of compounds targeting the same organelles . Drugs with the highest correlations to ART compounds also target pathways of genes directly connected to K13-propeller in the co-expression network analysis (p < 0.05) containing the other mutants in the ART sensitivity cluster from the original analysis (data not shown). Identification of these genes provides new candidates for monitoring the spreading resistance to ART, which is especially important as evidence emerges of alternative resistance mechanisms not linked to known mutations in the K13-propeller2Further analysis of the 7 mutants contained in the ART sensitivity cluster  identifianalysis  and reprP. falciparum using a random piggyBac insertional mutant library. Chemogenomic profiles from P. falciparum mutants provided unique signatures that mapped molecular structures of drugs to their targets demonstrating this technique can be an important tool to annotate mechanism of action of drugs and for validating novelty of compounds to vulnerable pathways of malaria parasites. The necessary addition of many more mutants and drugs will more precisely define the chemical-genomic spaces of the cell-drug interface, which will significantly augment the sensitivity and predictive capacity to define mechanisms of action, functionally annotate hypothetical genes, and identify interactions among metabolic pathways. Thus this approach provides a robust addition to the set of experimental tools for antimalarial drug research to improve target identification and understanding of mechanism of action.This study presents the first chemogenomic screen of Plasmodium. These inhibitors are known to kill Plasmodium by its association with a specific gene product, were tested for their growth inhibition in the wild type P. falciparum NF-54 strain in a 384 well plate format at 12 fold 1:3 serial dilutions (Table S1). After initial calibration assays only 53 inhibitors  were filtered out and added to the library for chemical-genetics analysis. All inhibitors were purchased from Sigma-Aldrich  unless otherwise mentioned except for Erythro and Threo-PPMP from Matreya, LLC , cycloguanil and atovaquone was a gift from WRAIR, USA.Growth inhibitors belonging to diverse chemical scaffolds were identified to cover all critical biochemical pathways in P. falciparum clone NF54 and all piggyBac single loci mutants were generated from previously reported transfections using pXL-BACII-HDFR/BSD and pHTHpiggyBac parasites were cultured in 4% hematocrit  and 1% Albumex II in RPMI 1640 medium (Invitrogen) supplemented with 50 μg/ml hypoxanthine  and 25 mM HEPES (Invitrogen) which is in accordance to the standard method described by Trager and Jensen, 19762 and 5% O2 respectively).The wild type P. falciparum functional genetics study using a high-throughput forward genetic screen. A library of 53 inhibitors, at least 2 per pathway targeting at different levels Plasmodium metabolism were assayed. In all chemical-genetic screens, compound dilutions and mutant culture dispensing to assay plates was handled robotically with a Beckman-Coulter liquid handling system . All assays were conducted in a 384 well culture plate with a total assay volume of 50 μl where compound stock was added in 10× to the final assay volume.We developed an automated platform for mutant piggyBac mutant assays were accompanied with wild type NF54. The growth inhibition concentration affecting 50% and 90% (GI50 and GI90) parasite growth was calculated by plotting the relative fluorescence unit values (RFU) of the SYBRGreen I added to culture plates. Statistically, each data point was converted to fit a non-linear logistic dose response function . The assay values Mean±SD of least three assays, R2 (coefficient of determination) >0.9 was considered significant and considered for chemogenomic correlations.In each assay, highly synchronous (5% sorbitol treated) ring-stage culture with >80% confluence were considered for growth assays. Briefly, the parasitemia and hematocrit was 0.5 and 1.5% respectively and the cultures were grown in a humidified mixed gas saucer for 72 hours. The growth response for each inhibitor dilution was obtained by reading the fluorescence generated by DNA intercalating dye SYBRGreen IpiggyBac mutants and to derive chemotype-phenotype associations, three parameters were considered out of chemogenomic assays for each inhibitor: 1) GI50, 50% growth inhibitory concentration; 2) Relative Phenotype Response (RPR): [GIpB50 over GI50WT (GIpB50/GI50WT)]. RPRs are described either as a shift towards resistance  or sensitivity . The RPR for each mutant is scored from the wild type assayed along with the mutants except for piggyBac PF3D7_0812500 and PF3D7_1360100.To generate profiles of the et al., 1998p for each branch point in the piggyBac and drug dendrograms, the package was modified to allow a Spearman correlation based distance metric. The output is displayed graphically, conveying the clustering and the essential similarity in RPR of the gene functions in presence of an inhibitor. Thus patterns like increased resistance is positive shift in GI50 and vice-versa for sensitivity. A distinct subset of Gene Ontology (GO) functional annotations relevant to the pathways indicated in the study was used to annotate each gene in the chemogenomic interaction dataset for color coding in cluster analysis. Any gene not falling into the defined category has been designated as ‘other’. The genes falling under multiple annotations, we choose to define it as a most probable on the basis of reviews and similarities published concerning the gene. Information on all genes can be found in the Plasmodium genome resource like (http://plasmodb.org/plasmo/), GeneDB  and KEGG GENES Database (http://www.genome.jp/kegg/genes.html).A genotype-phenotype association was made by cluster analysis of RPRs. Clustering was executed using a standard agglomerative algorithm described by Eisen Correlation between chemogenomic profiles of various drugs was determined using the spearman correlation coefficient. For each drug pair, a permutation test was conducted, in which a random distribution of 1000 correlation coefficients was obtained by permuting the chemogenomic profile of a given drug pair 1000 times, followed by computation of a correlation coefficient in each instance of permutation. Correlation between any drug pair was regarded as significant if the observed correlation coefficient was greater than any of the 1000 correlation coefficients observed in the permutations of the drug pair’s profiles. Drug pairs whose correlation met this criterion were regarded as interacting. Visualization of the drug-drug network interactions was performed in cytoscapeseryl-tRNA synthetase (PF07–0073) and actin (PFL2215w)A comparative CT method was used and for every plate a standard curve was set with 5 dilutions for target gene and reference gene. A comparison of all the time point samples with NF54 Time point 1 (2 hour) was made against a pooled reference comprised of NF54 RNA samples from 5 time points. Genes used in previous studies were used as endogenous control genes as reference, http://www.beiresources.org/About/MR4.aspx).The data reported in this paper are tabulated in the How to cite this article: Pradhan, A. et al. Chemogenomic profiling of Plasmodium falciparum as a tool to aid antimalarial drug discovery. Sci. Rep.5, 15930; doi: 10.1038/srep15930 (2015).