The Exploratorium in San Francisco offers museum visitors the opportunity to use and manipulate state-of-the-art microscopes to visualize an array of living specimens The Exploratorium, based in San Francisco, is a “hands on” science museum filled with interactive science and art exhibits, as well as a laboratory for the research and development of innovations in science education. In the summer of 2004, the Exploratorium launched the most ambitious microscope facility ever created for general public use. This initial phase of the project gives visitors the ability to image living specimens, as well as control the instruments themselves. Visitors can select among various specimens, move over them, change the magnification and focus, and, where appropriate, change the lighting to illuminate through the specimen, or use reflected light and fluorescence to dramatically change how it looks. They can image and explore tiny zebrafish embryos from the first stages of development to two-day-old fry with beating hearts and circulating blood cells, as well as a host of other organisms and cells from crawling amoebas to human blood cells.Below the surface, all living things share common features. The primary goals of this facility are to open a door on the wonder of the microscopic world to a diverse range of museum visitors and allow them to explore it, and to allow them to make connections to science and biomedical research. By empowering visitors with the instruments to explore this unfamiliar universe, the Exploratorium seeks to recreate some of the excitement and wonder that the earliest researchers found as they discovered another world all around them .The light microscope falls amongst the greatest inventions of human history. Images from it in the 17th century literally revolutionized our understanding of life, providing first-hand evidence of a previously unseen or unsuspected world of organisms and cells all around us. This knowledge profoundly shaped our view of life, and of our placement in the universe. Robert Hooke used a primitive early microscope to see the walls between cells in a piece of cork , and Anton van Leeuwenhoek's simple scope revealed a previously unknown world of microorganisms living inside his own mouth. Swimming sperm were observed in semen, changing our fundamental understanding of conception. Since that time our world has become populated by marvelously beautiful and intriguing images and movies created by scientists using precise lighting and optics. Most recently, computer-controlled image-capturing techniques and digital technologies capture events and processes too small, slow, or fast for our unaided eyes to see.Van Leeuwenhoek's first microscopes were probably about as powerful as a simple water-drop scope see ; he usedBringing a first-hand, high-quality microscope experience to a wide range of visitors and students has been a major challenge for many science museums and classrooms.In the expanded, frenetic classroom of the science museum exhibition floor, microscopes themselves are too delicate and precise for operation without assistance and supervision: the optics can be easily damaged, all but the most robust of specimens are easily destroyed, and the user interface with lighting, positioning, and focus compose a world unfamiliar to most novice users. Over the years, we had collected our share of failed and broken microscopes in various projects. For these reasons most microscopes in museums have been armored, stripped-down, single-magnification instruments.Problems related to the operation of the microscope hardware, however, represent only some of the challenges created by bringing uninitiated visitors to the microscopic world. The experience has to be repeatable. It has to be engaging. It has to be both simple and complex. It needs to work for individuals and groups. It needs to support investigation as well as provide essential information.An array of research scientists joined us as we began work to redefine and renew our existing presentation on biology, most notably Christian Sardet, a cell biologist with a passion for visualization imagery. During the summer and fall of 1999, we engaged in a series of conversations directed at defining and developing a publicly accessible microscope imaging station, and the more we explored, the more technically feasible the project became.At the same time, research-grade microscopes with high-quality optics underwent a revolution of their own. Many manufacturers planned to introduce fully automated, computer-controllable microscopes in their next models, and the prospects of having an off-the-shelf remotely controllable microscope seemed to solve a major hardware problem. It seemed as if software programs might be used to address many other user interface issues. In 2000, we applied for and received funding from the National Institutes of Health  and from the David and Lucile Packard Foundation to support the development of a microscope imaging facility.Seven months later we obtained the first microscope and its associated imaging equipment. But the software to control the microscopes proved dauntingly problematic. Programs that worked well in the laboratory proved difficult to adapt for our educational uses. Instead, we wrote our own programs to control stage movement (for specimen positioning), focus, specimen selection, magnification, and lighting. Each of these controls needed to be selectable and limited so that visitors might operate the microscope within a defined range.Caenorhabditis elegans attracted attention with its earthworm-like movements. Overall, these biomedically relevant specimens provided a treasure trove of potential educational activities for visitors.On a parallel track, with the generous help of numerous biomedical researchers and their laboratories across the United States, we explored specimens for their visitor attraction and interest. Not surprisingly, we found that familiar structures and organisms provided the best entry points. A zebrafish embryo with a beating heart, circulating blood, and twitching tail movements rated more popular than zebrafish or sea urchin embryos at earlier stages of development. The tiny transparent roundworm Given a range of wonderfully attractive specimens and potential activities, we then aimed to create an educational experience that combined the observable features in the specimens, supporting information, and further activities to be chosen as desired by the visitor. To achieve this, we adopted a multimedia approach . The incVisitors can pick and choose specimens of their choice, refine their selection, change magnification, and engage in suggested activities at their own pace with integrated appropriate information. Individuals and groups engage with the microscope imagery and companion information for relatively long periods of time, ranging from several minutes to upwards of 20 minutes, a long time for a museum exhibit. To expand the use of the microscope imagery beyond that of one or two visitors, we incorporated large plasma-screen presentations in to the Microscope Imaging Station facility.Escherichia coli) and a roundworm (C. elegans) and found that the genetically modified organisms emitted an eerie green glow under certain conditions. Other scientists built on this technique to create a powerful tool that makes hidden structures and processes easier to study. The coupling of vital staining with the green fluorescent protein gene allows scientists to observe events inside developing cells and detect the presence of diseased structures and environmental toxins with extreme sensitivity. This newly created sensitivity has sparked new insights and discoveries, re-revolutionizing the capabilities of the light microscope.In addition to using some of the latest devices in microscope technologies, the Imaging Station also provides a window on revolutionary research techniques. For example, in 1994, Columbia University's Martin Chalfie inserted a gene for a fluorescent jellyfish protein into a bacterium (http://www.exploratorium.edu/imaging_station).At the Imaging Station, visitors can take advantage of this technique to observe specific types of cells—such as the brain cells, sex cells, or muscle cells of roundworms. Visitors can also peer inside the developing embryo of a tropical zebrafish whose circulatory cells have been made visible by the protein made from the transplanted green fluorescent protein gene. In the immediate future, visitors will be able to closely examine living human blood cells and fruit flies that researchers use to study the genetics of a wide range of human disorders. Over the next year, we intend to add a major component on mouse stem cells and the process of differentiation. At the Imaging Station, visitors have access to seamless video footage of events that take place in a fraction of a second or occur slowly over weeks or months . Using tVideo S1Collected at the Microscope Imaging Station Many of the cells and organisms found in these movies are available via visitor-accessible microscopes on a daily basis. At the station, visitor-directed, microscope-based observations form the basis for informal education on basic and biomedically relevant research. Video microscopy and production by Kristina Yu.(5.4 KB MOV).Click here for additional data file.Materials. You'll need the clear plastic part of a CD case; if you can't find one, any thin sheet of rigid, clear plastic will work. Remove any paper or packaging so that you can see through the plastic. (Take the CD out, too!)What to do. (1) Put the object you want to examine—a bug, a tiny leaf or plant, the small lettering on a coin or newspaper—on a flat surface. (2) Put one large drop of water on the center of the clear plastic. (An eyedropper makes this easy.) (3) Pick up the plastic and hold it horizontally so that the water drop is directly over the object. Move the plastic slowly back and forth to center the object, and up and down to focus. The water will form a curved shape that magnifies the object!


With chronic infection, hepatitis C virus (HCV) RNA can be detected in B cells and associated with B-cell disorders, but these are not well defined.+ T cells, CD8+ T cells, CD19+ B cells, and CD56+ NK cells were quantified by flow cytometry using cryopreserved peripheral blood mononuclear cells of 24 hemophilic patients in each of five age-matched groups [uninfected; chronic HCV with or without human immunodeficiency virus (HIV); and cleared HCV with or without HIV].The relationship between HCV infection and lymphocyte subpopulations was evaluated rigorously in 120 asymptomatic hemophilic patients, randomly selected from a prospective cohort study. CD4+ and increased CD8+ T cells. Irrespective of HIV, patients with chronic HCV infection had approximately 25% fewer CD19+ B cells than those without chronic HCV infection.As expected, patients with HIV had significantly reduced CD4These data support the hypothesis that asymptomatic patients with chronic HCV infection have an altered B-lymphocyte population. The natural history of hepatic C virus (HCV) infection may include B-cell diseases. HCV can be detected in peripheral blood mononuclear cells (PBMCs) and may also replicate in these cells -3. Type + T cells [+ T cells, CD8+ T cells, B cells, and natural killer (NK) cells in PBMCs of carefully selected, well characterized hemophilic patients.One study reported that hemophilic patients co-infected with HCV and human immunodeficiency virus (HIV) had reduced B cells and CD4 T cells . To furtth percentile of all HCV-positive MHCS patients (described below), all of whom had received HCV-contaminated plasma products many years earlier and thus were considered to have chronic HCV infection. All samples were taken before 1996, the year when highly active antiretroviral therapy (HAART) became available for treatment of HIV infection.Patients with hemophilia or another congenital coagulation disorder (herein referred to as "hemophilia") enrolled in the Multicenter Hemophilia Cohort Study ,7 were cHIV status was defined by antibody testing with licensed immunoassays and immunoblot confirmation. HCV antibody status was determined with a commercially available second- or third-generation enzyme immunoassay, with most of the reactive samples confirmed by recombinant immunoblot assay (HCV RIBA2.0 or 3.0, Chiron Corp., Emeryville CA). HCV RNA was detected and viral load was determined with branched-DNA technology [Quantiplex HCV RNA 2.0 Assay (bDNA), Chiron Corp., Emeryville CA] with a lower limit of sensitivity of 200,000 genome equivalents/mL, which is 31,746 international units (IU)/mL. HIV-1 viral load was determined with the HIV Amplicor Monitor (Roche Molecular Diagnostics, Branchburg, NJ).2 until testing, was analyzed by flow cytometry for proportions of CD4+, CD8+, CD19+, and CD56+ cells. In a biological safety cabinet, vials were thawed in a 37°C water bath with agitation. To each, 300 units of DNase I  was added. The contents were transferred to a 15 ml polypropylene tube. Thawed cells were slowly diluted with RPMI-1640 media supplemented with 20% fetal calf serum, mixed frequently, then pelleted at 1000 rpm in a Sorvall RT6000 refrigerated centrifuge. The liquid was decanted. Cells were mixed in residual volume by gentle shaking, washed a second time with 4.0 ml of thawing media, then resuspended in 2.0 ml calcium- and magnesium-free Dulbecco's phosphate buffered saline (PBS) containing 4% heat-inactivated human AB serum to block high affinity Fc receptors. After 10 minutes the cells were pelleted in the centrifuge, then resuspended in 0.6 ml of PBS/2% BSA. 100 μl of cell suspension was added to each of five 12 mm × 75 m polypropylene tubes containing three-color combinations of fluorescently tagged monoclonal antibodies (CD45/CD14/CD3, CD45/CD4/CD3, CD45/CD8/CD3, CD45/CD19/CD3 and CD45/CD56/CD3). The lymphocyte gate was defined with light scatter properties and CD45/CD14/CD3. A Coulter XL flow analyzer was used with stops set to collect 5,000 CD3+ lymphocytes in each sample, if possible. Listmode files were analyzed offline using Coulter System II software (version 3.0). Quality control criteria included purity of gated lymphocytes; percentage recovery of lymphocytes in the gate; reproducibility of CD3 between tubes (range <4%); and sum of CD3+, CD3-/CD19+, CD3-/CD56+ (range 90–110%).A vial of 5 million cryopreserved PBMCs, stored in vapor phase LNThe primary analysis was performed on proportions, rather than absolute levels, of each PBMC subset, to reduce variance that results from counting total lymphocytes in peripheral blood . The Kru6 IU/mL) than with HCV only (median 2.4 × 106 IU/mL, P < 0.01). Among 48 HIV-infected subjects, 6 (25%) with cleared and 7 (29%) with chronic HCV had clinically defined AIDS (P = 0.75) when their PBMCs were collected for this study. HIV viral load did not differ between the nine with cleared and the eight with chronic HCV who were tested on the date PBMCs were isolated (P = 0.89). Ten of 120 subjects were chronically infected with hepatitis B virus, and the prevalence of chronic HBV infection did not differ among 5 groups (P = 0.12). Only two subjects, both HIV-infected but one with cleared HCV infection and one with chronic HCV infection, had developed ascites, a manifestation of hepatic decompensation, at the time that their PBMCs were collected for this study.Of the 120 patients, 78 had hemophilia A (factor VIII deficiency). Because patients with hemophilia A generally required more intensive clotting factor replacement therapy as well as product (Factor VIII concentrate) that was more infectious for HIV than was Factor IX concentrate used for patients with hemophilia B, hemophilia A was significantly more common in the two groups with HIV co-infection (n = 19 with chronic HCV and n = 21 with cleared HCV) than the three groups without HIV co-infection (n = 11 to 14, p = 0.01). By design, the five groups had similar ages (mean 26.5 years, range 3.4 – 54.8 years, Table + T cells whether they had cleared HCV (12.4% vs. 40.1%) or chronic HCV .As expected, subjects with HIV had lower proportions of CD4+ T cells differences mirrored those of CD4+ T cells (Table + T cells whether they had cleared HCV (52.3% vs. 26.0%) or chronic HCV (55.1% vs. 29.1%, P#0.0001). Regardless of HIV, subjects with cleared and with chronic HCV had similar proportions of CD8+ T cells (P∃0.30).CD8ls Table . Subject+ B cells differed significantly among the five groups . CD19+ B cells also were lower, albeit not significantly, with chronic HCV than with cleared HCV infection in both HIV-uninfected (13.3% vs. 17.8%, P = 0.09) and HIV-coinfected (11.4% vs. 15.4%, P = 0.08) groups. CD19+ B-cell levels did not differ between uninfected subjects and those with cleared HCV infection (P = 0.63).Proportions of CD19+ B-cell proportions did not correlate with HCV viral load (Spearman R = 0.06, P = 0.78).CD19+ NK cells did not differ significantly among the five groups /15.5 = 0.26]. We found subjects with chronic HCV without HIV co-infection had a reduction of the same magnitude, albeit of marginal statistical significance. HIV infection was more frequent with hemophilia A than with other coagulation disorders [Among HIV-infected subjects, we found that CD19isorders . OverallInfection of B cells by HCV is controversial. Both B cells and hepatocytes express CD81, a possible receptor for HCV . If HCV + NK cells with HCV infection, but this does not negate possible impairment of NK cell function with HCV infection. In vitro, binding of HCV E2 to CD81 inhibits the functions of cross-linked NK cells [+ NK cell proportion was reduced among our subjects who cleared HCV despite HIV co-infection, an observation that has no ready explanation and may have appeared by chance.We found no differences in proportions of CD56NK cells ,15. CD56+ and CD8+ T cells with HIV infection. These findings, our careful flow cytometry methods, and our rigorous study design, with frequency matching on age and random selection of subjects from a large, well characterized cohort, probably offer a reliable "snapshot" of PBMCs in hemophilic patients infected with HCV alone or co-infected with HIV.Our study is limited by its cross-sectional design and analysis of PBMCs collected long after primary HCV and HIV infections had occurred. We cannot define sequential PBMC changes in relation to critical virologic events, such as spontaneous clearance of HCV. Furthermore, the cells that we detected in cryopreserved PBMCs may not be representative of those in fresh PBMCs. Still, we did observe the expected associations of CD4+ or CD8+ T cells, although this does not negate the likely functional importance of HCV-specific T-cell subpopulations in clearance of HCV [+ B-cell levels. Further studies need to clarify the temporal sequence of B-cell changes with HCV chronicity and associated clinical conditions.We found no association of cleared or chronic HCV infection with altered levels of CD4e of HCV ,17. SubjM. Zhang, J.J. Goedert, T.R. O'Brien, P.S. Rosenberg, C.S. Rabkin, E.A. Engels, D. Whitby ; M.E. Eyster ; B. Konkle (Cardeza Foundation Hemophilia Center, Philadelphia PA); M. Manco-Johnson ; D. DiMichele, M.W. Hilgartner ; P. Blatt ; L.M. Aledort, S. Seremetes ; K. Hoots (Gulf States Hemophilia Center, University of Texas at Houston, Houston TX); A.L. Angiolillo, N.L.C. Luban ; A.Cohen, C.S. Manno ; C. Leissinger ; G.C. White II (Comprehensive Hemophilia Center, University of North Carolina, Chapel Hill NC); M.M. Lederman, S. Purvis, J. Salkowitz (Case Western Reserve University School of Medicine, Cleveland OH); C.M. Kessler ; A. Karafoulidou, T. Mandalaki ; A. Hatzakis, G. Touloumi ; W. Schramm, F. Rommel (Medizinische Klinik Innerstadt der Maximilian, Universitaet Muenchen, Munich, Germany); P. de Moerloose ; S. Eichinger ; K.E. Sherman ; and B.L. Kroner (Research Triangle Institute, Rockville MD)."The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organization imply endorsement by the U.S. Government."The author(s) declare that they have no competing interests.MZ and JG conceived of the study and drafted the manuscript. MZ performed the random sampling and statistical analyses. JG provided advice on the sampling and statistical analyses, obtained funding, managed the parent cohort, and supervised the virologic testing. TOB provided advice on the design. WK provided advice on and performed the FACS analyses. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Hypersensitivity pneumonitis (HP) is an interstitial lung disease caused by repeated inhalations of finely dispersed organic particles or low molecular weight chemicals. The disease is characterized by an alveolitis sustained by CD8(+) cytotoxic T lymphocytes, granuloma formation, and, whenever antigenic exposition continues, fibrosis. Although it is known that T-cell migration into the lungs is crucial in HP reaction, mechanisms implicated in this process remain undefined.Using flow cytometry, immunohistochemistry, confocal microscopy analysis and chemotaxis assays we evaluated whether CXCL10 and its receptor CXCR3 regulate the trafficking of CD8(+) T cells in HP lung.in vitro migratory capability in response to CXCL10. Alveolar macrophages expressed and secreted, in response to IFN-γ, definite levels of CXCL10 capable of inducing chemotaxis of the CXCR3(+) T-cell line. Interestingly, striking levels of CXCR3 ligands could be demonstrated in the fluid component of the BAL in individuals with HP.Our data demonstrated that lymphocytes infiltrating lung biopsies are CD8 T cells which strongly stain for CXCR3. However, T cells accumulating in the BAL of HP were CXCR3(+)/IFNγ(+) Tc1 cells exhibiting a strong These data indicate that IFN-γ mediates the recruitment of lymphocytes into the lung via production of the chemokine CXCL10, resulting in Tc1-cell alveolitis and granuloma formation. Hypersensitivity pneumonitis (HP) is an interstitial lung disease (ILD) caused by the inhalation of and sensitization to a variety of environmental organic antigens. The immune mediated nature of the disorder is testified to by the characteristic sequel of events taking place in the lung after antigenic inhalation: an acute pulmonary neutrophilia occurs early followed by an interstitial T-cell infiltration of CD8 T-cell showing a limited expression of the T-cell receptor . (Mig/CXCL10), interferon-inducible T-cell α-chemoattractant (I-TAC/CXCL11) bind to the CXCR3 receptor molecule which is expressed by activated T lymphocytes and natural killer cells [Saccharopolyspora rectivirgula (SR) and successive granuloma formation [A number of data point to chemokines as orchestrators of inflammatory disorders which are characterized by a massive accumulation of immunocompetent cells within affected organs, including the lung . Chemokier cells ,8. We haer cells ,10, provin vitro migration of CXCR3(+)T cells. The ligand CXCL10 can be detected in pulmonary macrophages and is released by these cells.Using immunohistochemical studies of tissue sections and a flow cytometry evaluation of cells recovered from the bronchoalveolar lavage (BAL), we studied the role of CXCR3/CXCL10 interactions in the regulation of T-cell migration into the lung of patients with hypersensitivity pneumonitis. We have shown that CXCR3 is expressed by T cells accumulating in the lower respiratory tract of patients with this hypersensitivity disorder. In addition, we found that signalling of CXCR3 with CXCL10 induces the 12 HP patients were included in the study . The majority of the patients had farmer's lung disease (10 patients); 1 patient had bird fancier's lung, 1 patient had mushroom worker's lung. The following criteria for HP diagnosis were used: a) history of exposure to HP antigens, b) a symptomatic acute episode with chills, fever, cough, breathlessness 4 to 8 hours after exposure to specific antigens, c) radiological features (mainly diffuse reticular pattern) and/or a functional pattern of interstitial lung disease, and d) evidence of antibodies against S. rectivirgula in all except one case (bird fancier's lung). Each patient underwent bronchoscopy for transbronchial biopsy (TBB) and BAL analysis. BAL was performed according to the technical recommendations and guidelines for the standardization of BAL procedures [Five healthy controls were selected . They showed normal physical examinations, chest X-rays, lung function tests and BAL cell numbers.+ lymphocytes were removed using high-gradient magnetic separation columns (Mini MACS, Miltenyi Biotec, Germany) [Alveolar macrophages (AMs) and T cells were enriched from the BAL cell suspensions by rosetting with neuraminidase-treated SRBC followed by F/H gradient separations and residual CD3Germany) . FollowiThe commercially available conjugated or unconjugated mAbs used belonged to the Becton Dickinson and PharMingen series and included: CD3, CD4, CD8, isotype matched controls. Anti-IL-4 and anti-IFNγ mAbs were purchased from PharMingen (San Diego, CA). Purified rabbit anti-human CXCL10 polyclonal antibody (R&D Systems Inc, Minneapolis, MN) and anti-hCXCR3 mAb (R&D Systems Inc) were also used.Open lung biopsies from 8 patients with clinical and histological diagnosis of hypersensitivity pneumonitis were studied by immunohistochemistry for the immunophenotype characterization of inflammatory cells and for CXCR3 and CXCL10 expression.Immunohistochemistry for the characterization of inflammatory infiltrate was carried out using the following antibodies (Dako Glostrup, Denmark): CD45 (1:20), CD43 (1:40), CD45RO (1:100), CD20 (1:100), CD3 (1:50), CD68 (1:50), CD4 (1:100), and CD8 (1:100). The immunoreaction products were developed using the avidin-biotin-peroxidase complex method. Immunostaining for CXCR3 was performed as previously described. Briefly, after the microwave antigen retrieval procedure and neutralization of endogenous peroxidase activity, the slides were incubated with primary antibody for 1 hr in a humidified chamber at 37°C (anti-hCXCR3 mAb 1:100). Immunoreactivity was detected using biotinylated secondary antibodies incubated for 45 min followed by a 30 min incubation with avidin-peroxidase and visualized by a 7 min incubation with the use of 0.1% 3,3'-diaminobenzidene tetrahydrochloride as the chromogen. Parallel control slides were prepared either lacking primary antibody or lacking primary and secondary antibodies, or stained with normal sera to control for background reactivity. The intensity of antibody staining was classified in three groups: strong, weak, negative.Paraffin sections were prepared for immunofluorescent labelling. Briefly, primary antibodies against CD3 and CXCR3  and secondary antibodies (goat anti-mouse IgG and donkey anti-goat IgG) conjugated with TEXAS red or ALEXA 488 (Sigma) were used. Double labelling using both antibodies on the same section was performed. Primary antibodies and secondary antibodies were incubated for 1 h at room temperature. Nuclear staining was carried out with DAPI (Sigma) in PBS. Slides were stored at 4°C and analysed within 24 h. As a control, the primary antibody was omitted.Immunofluorescence was evaluated with a confocal microscopy , We used an argon laser at 488 nm in combination with a helium neon laser at 543 nm to excite the green (CD3) and red (CXCR3) fluorochromes simultaneously. Emitted fluorescence was detected with a 505–530 nm band pass filter for the green signal and a 560 nm long pass filter for the red signal. Images were analyzed using the Adobe Photoshop 7.0 program.® analyzer (Becton Dickinson), and data were processed using the Macintosh CELLQuest software program (Becton Dickinson). The expression of cytoplasmic cytokine was evaluated following permeabilization of cell membranes using 1:2 diluted PermeaFix (Ortho, Raritan, NJ) for 40 min. After permeabilization procedures anti-IL-4, anti-IFN-γ and anti-CXCL10 antibodies were added. Since pulmonary cells bear cytoplasmic cytokines in a unimodal expression pattern, indicating that the entire cell population exhibits relatively homogeneous fluorescence, the percentage of positive cells does not represent the most accurate way of enumerating positive cells. Mean fluorescence intensity (MFI) was used to compare the positivity of these specific antigens on different cell populations. To evaluate whether the shift of the positive cell peak was statistically significant, the Kolmogorov-Smirnov test for analysis of histograms was used according to the Macintosh CELLQuest software user's guide (Becton Dickinson).The frequency of BAL cells positive for the above reagents was determined by overlaying the flow cytometry histograms of the samples stained with the different reagents as previously reported . Cells wFor immunofluorescence analysis, control IgG1 and IgG2a and IgG2b were obtained from Becton-Dickinson; control rat antiserum consisted of ascites containing an irrelevant rat IgG2b; control rabbit antiserum consisted of rabbit IgG (purified protein) purchased from Serotec (Serotec, U.K.); goat-anti-rabbit IgG and goat F(ab')2 anti-rat IgG were obtained from Immunotech (Marseille, France).Each PCR product was analysed and quantitated by Bio-Rad's Image Analysis System Gel Doc using Quantity One software (Bio-Rad, Hercules, CA). Briefly, the images of the gels were acquired from the Gel Doc system densitometer and saved in digitised forms to perform volume analysis. The intensity of each band was differentiated by the intensity of the background, whose value was subtracted from each individual band and the resulting PCR product value was expressed in mm*mm*intensity of the pixels of the specific band in the gel.6/ml) were isolated from the BAL of HP patients, resuspended in RPMI medium and cultured for 24 hr in 24-well plates at 37°C in 5% CO2. In separate experiments AMs were stimulated with IFN-γ (100 U/ml), PMA (10 ng/ml) and LPS . Following the incubation period, supernatants were harvested, filtered through a 0.45 μm Millipore filter and immediately stored at -80°C. At the end of the culture time AM viability was always greater than 95%. Chemotactic activity of supernatants was determined as reported below.To verify the ability of AMs to release CXCL10, AMs (1 × 10T-cell migration was measured in a 48-well modified Boyden chamber (AC48 Neuro Probe Inc., USA). The chamber is made of two sections: different chemotactic stimuli were loaded in the bottom section while cells were added in the top compartment. Polyvinylpyrrolidone-free polycarbonate membranes with 3 to 5 μm pores (for lung T cells obtained from HP patients and the CXCR3+ and CXCR3- T-cell lines, respectively) (Osmonics, Livermore, CA) and coated with fibronectin were placed between the two chamber parts. Only the bottom face of filters was pretreated with fibronectin; the fibronectin pretreatment maximizes attachment of migrating cells to filters, avoiding the possibility that they may not adhere. Using this procedure in preliminary experiments we demonstrated that only a trivial number of cells may be recovered in the bottoms of the wells. To avoid the shedding of fibronectin, fibronectin-treated filters were extensively washed. In preliminary experiments, fibronectin-treated filters did not induce spontaneous chemotaxis in absence of chemokines.6 cells/ml T cells or CXCR3-/+ cells resuspended in RPMI 1640 were added to the top wells. The chamber was incubated at 37°C with 5% CO2 for 2 hrs. The membranes were then removed, washed with PBS on the upper side, fixed and stained with DiffQuik (Dade AG, Düdingen, Switzerland). Cells were counted in three fields per well at 800× magnification. All assays were performed in triplicate. In blocking experiments, cell suspensions were preincubated before chemotaxis assay for 30 min at 4°C with anti-human CXCR3 mAb at a concentration of 20 μg/ml.To evaluate the migratory properties of pulmonary T lymphocytes rhIP-10/CXCL10 (200 ng/ml) were used. The CXCR3- and CXCR3+ cell lines (300-19, kindly provided by Dr. B. Moser, Theodor-Kocher Institute, University of Bern, Switzerland) were used as negative and positive controls. 30 μl of chemokines or control medium were added to the bottom wells, and 50 μl of 5.0 × 10The CXCR3(-) and CXCR3(+) cell lines were also used to evaluate both the chemotactic activities of macrophage supernatants and the fluid component of BAL samples. Supernatants from cell cultures and the fluid components of BAL were obtained as reported above and used undiluted; different concentrations of CXCL10 were utilized as a positive control. Chemotactic assays were performed as reported above. In blocking experiments, anti-CXCL10 was added to the cell supernatants before chemotaxis assay at a concentration of 20 μg/ml.Data were analysed with the assistance of the Statistical Analysis System. Data are expressed as mean ± SD. Mean values were compared using the ANOVA test. A P value <0.05 was considered as significant.In all cases typical pathological examination showed features with poorly formed non-necrotizing granulomas and widespread thickening of the alveolar walls by a diffuse lymphocytic infiltrate. Pleural lymphoid aggregates were seen in a few cases and pleural lymphoid aggregates were seen in a few samples.Diffuse interstitial lymphocytic infiltrates were characterized by an accumulation of T cells and a few B-lymphocytes. Sub-pleural and peri-bronchiolar nodules consisted mostly of T lymphocytes mainly represented by CD8 cytotoxic T lymphocytes which strongly stained for CXCR3 in all cases Figure  and 1B. Confocal microscopy analysis of lung biopsies confirmed that lymphocyte infiltrates were formed by T cells coexpressing CXCR3  cell line; the CXCR3(-) cell line did not migrate in the presence of supernatants (data not shown). The addition of an anti-CXCL10 neutralizing antibody inhibited chemotactic activities of supernatants. The inhibitory activity shown by the neutralizing antibody was not complete, suggesting that other CXCR3 ligands (CXCL9 and CXCL11) are likely to be present in supernatants.in vivo in the lung microenvironment, the fluid component of BAL obtained from 10 HP patients was evaluated for chemotactic activity on CXCR3(+) cell lines  T cells might play a role not only in the pathogenesis of T-cell alveolitis but also in favouring granuloma formation since T cells surrounding the macrophagic core of the HP granuloma expressed this chemokine receptor. This mechanism is likely to be shared by various ILDs since we and others have previously demonstrated the presence of a significant infiltrate of CXCR3(+) Th1 cells in other ILDs characterized by T cell alveolitis and granuloma formation, such as sarcoidosis and tuberculosis ,14.Saccharopolyspora rectivirgula (SR) (i.e., the etiologic agent involved in the immunopathogenesis of HP reaction in the majority of our patients) [in vivo role of IP-10/CXL10 in preventing or favouring pulmonary fibrosis in HP before proposing this strategy.Our data provide definitive confirmation of the recent findings obtained in an animal model of IFN-γ-knockout (GKO) mice exposed to the particulate antigen atients) . While WThe relationship between CXCL10 release and the local production of other chemokines involved in HP immune response is another important aspect that deserves further investigation. In a murine model it has recently been shown that monocyte chemotactic protein-1 (MCP-1/CCL2) is increased in the BAL of mice challenged with SR . Furtherin vivo in HP lungs.Thus, our findings and the data of Pardo et al.  suggest Our findings clearly indicate the effects of CXCR3/CXCL10 interactions on hypersensitivity reaction to SR antigens. Considering the importance of CD8 T cells in mediating granuloma formation and lung damage, further studies are needed in animal models to explore the therapeutic potential of CXCR3 antagonists with the ultimate goal of offering new clues for immune intervention in subjects continuously exposed to inhaled antigens and thus at risk of developing HP-related lung fibrosis.AM: alveolar macrophage; BAL: bronchoalveolar lavage; CXCR3: Receptor 3 for CXC chemokines; HP: Hypersensitivity pneumonitis; IP10/CXCL10: IFN-γ-inducible protein-10; I-TAC/CXCL11: interferon-inducible T-cell αlfa-chemoattractant; Mig/CXCL10: monokine induced by IFN-γ; TBB: transbronchial biopsy.CA: planned the experimental design, coordinated the research group and drafted the manuscriptFC: participated in the study design and in immuohiostochemical analysisVP: participated in the study design and in the bronchoalveolar lavage executionGM: participated in the study design and in the clinical evaluation of the patientsMF: participated in the study design and performed migration assays studiesMM: participated in the study design and performed cellular studiesAC: participated in the study design and performed flow cytometry studiesIB: participated in the study design and performed flow cytometry studiesRZ: participated in the study design and helped the draft of the manuscriptLT: participated in the study design and helped the draft of the manuscriptGS: participated in the study design and coordination of the research group


Saccharomyces cerevisiae, commonly known as baker's yeast, measures just 2 microns—it takes about 4 billion to fill a teaspoon. But as a eukaryote (its cells have nuclei), its genes function in much the same way a human's do. For a gene to function, its DNA sequence must first be transcribed into RNA , whose sequence can then be translated into a specific string of amino acids to form the unique protein that the gene encodes.The single-celled The population of mRNA transcripts in each cell (its “transcriptome”) is dynamic—the genome uses its vocabulary of genes to write an ever-evolving script for the cell as its life unfolds and its environment changes. By binding to specific sequences of DNA, proteins called transcription factors process signals from the cell's sensory and information-processing systems to control which genes are transcribed in each cell, under what conditions, and at what rate. While the steps and regulatory programs that govern gene expression at this level are reasonably well known, much less is known about the orchestration of the later steps in the gene expression program—where in the cell each mRNA molecule goes when it leaves the nucleus, at what rate and under what conditions it is translated into protein, and how long it survives.RNA-binding proteins (RBPs) have been implicated in diverse aspects of post-transcriptional gene regulation. Hundreds of RBPs are encoded in the eukaryotic genome, but because few have been studied in detail and few of their mRNA targets are known, the nature and extent of an RBP-mediated post-transcriptional program has been obscure. Now a systemic analysis of a specific family of RBPs and their mRNA targets in yeast by André Gerber, Daniel Herschlag, and Patrick Brown, of Stanford University, suggests that such a program may exert detailed control over the life history of every mRNA. By selectively binding and regulating specific classes of mRNAs, RBPs may provide a mechanism to coordinate the collective fate of these transcripts and serve as an integral part of the global transcriptome.Gerber, Herschlag, and Brown focused on the binding targets of a family of RBPs called Pumilio-Fbf (Puf) proteins, which are defined by the presence and configuration of an amino acid domain that mediates RNA-binding. Little is known about the physiological function of the five yeast Puf proteins the researchers studied here . After using “affinity tags” to snag each of the five Puf proteins from yeast cells, together with their bound mRNA targets, the researchers identified the associated mRNAs with microarray analysis. They found more than 700 mRNAs bound by at least one Puf protein, with each Puf RBP targeting a distinct group of mRNAs. The group of mRNAs associated with each Puf protein turned out to encode proteins with strikingly similar functions and locations in the cell. Many of the mRNA sets encode proteins that reside in the same cellular location, are part of the same protein complexes, or act in the same signaling pathway. Some Puf proteins target mRNAs that encode membrane proteins while others preferentially bind to mRNAs that encode proteins involved in cell division. The most pronounced bias occurs with Puf3p, which overwhelmingly binds mRNAs that encode proteins destined for the mitochondria, the cell's power generators.This selective tagging of functionally related mRNAs by specific RBPs suggests a mechanism for coordinated global control of gene expression at the post-transcriptional level. Just as transcription factors regulate transcription by binding to specific DNA sequences, RBPs may mediate regulation of the subcellular localization, translation, and degradation of the set of specific mRNAs they target. Noting the striking themes in the subcellular localization of the proteins encoded by the mRNAs bound by each Puf protein, Gerber, Herschlag, and Brown propose that RBPs may play important roles in the subcellular localization and efficient assembly of protein complexes and functional systems by ensuring that the location in the cell at which mRNAs are translated “is not left to chance.” Since the number of RBPs encoded in eukaryotic genomes approaches that of transcription factors, the regulatory program that controls the post-transcriptional fate of mRNAs—their localization, translation, and survival—may prove to be nearly as diverse and complex as the regulation of transcription itself.


A mutation in the abnormal limb mutant 5 (ALI5) mouse in the region coding for the hydrophobic ridge loop 3 (HRL3) of the phospholipaseCγ2 (PLCγ-2) gene, corresponding to human PLCγ-2 exon 27, leads to acute and chronic inflammation and granulomatosis. For that reason, we screened exons 11, 12 and 13 coding for the hydrophobic ridge loop 1 and 2 (HRL1 and 2, respectively) and exon 27 of the PLCγ-2 protein by single strand conformation polymorphism (SSCP), sequencing and PCR/ restriction fragment length polymorphism (RFLP) analyses. In addition, we screened indirectly for disease association via 4 microsatellites with pooled DNA in the PLCγ-2 gene.Wegener Granulomatosis (WG) is a multifactorial disease of yet unknown aetiology characterized by granulomata of the respiratory tract and systemic necrotizing vasculitis. Analyses of candidate genes revealed several associations, Although a few polymorphisms in these distinct exons were observed, significant differences in allele frequencies were not identified between WG patients and respective controls. In addition, the microsatellite analyses did not reveal a significant difference between our patient and control cohort.PLCγ-2 gene in the pathogenesis of WG in our case-control study.This report does not reveal any hints for an involvement of the  Staphylococcus aureus is a risk factor for disease exacerbation in WG [HLA-DPB1 alleles or rather an extended haplotype, respectively, in the MHC class II region has been reported [e.g. with α(1)-antitrypsin, and proteinase 3 [[Wegener granulomatosis (WG) is a systemic inflammatory disease of unknown aetiology characterized by granulomata of the respiratory tract and systemic necrotizing vasculitis . There ion in WG . Recentlreported . In addiinase 3 [ and 9]]]StaphyloPLCγ-2, was proposed on the basis of a novel animal model system. A mutation in the abnormal limb mutant 5 (ALI5) mouse [PLCγ-2 mutation leads to acute and chronic inflammations with a phenotype comparable to WG. Therefore, human PLCγ-2 is a good candidate gene for seeking predisposing genetic factors for WG. The human gene is a member of the PLC family comprising 12 closely related molecules involved in signal transduction from numerous receptors [2+ mobilization, which is required for activation of B-cells [2+ entry in cell lines [PLCγ-2 gene is localized on chromosome 16 (16q24.1) spanning 179 kb of genomic DNA. The 4.2 kb mRNA consists of 33 Exon coding for a Mr 140,000 protein with 1265 amino acids [PLCγ-2 is highly polymorphic [A primary candidate gene, 5) mouse  causes aeceptors . The pro B-cells . PLCγ-2  B-cells . The pro B-cells . It was ll lines . The humymorphic .Here, we report on an indirect association screen by microsatellite analysis and pooling of DNA in a case-control study design. Furthermore, we screened exons 11, 12 and 13, partly coding for the hydrophobic ridge loop 1 and 2 of PLCγ-2 protein, by single stranded conformation polymorphism (SSCP), sequencing and the PCR/RFLP method. As the ALI5 mutation is located in the region corresponding to the human exon 27, this exon was also screened by SSCP.PLCγ-2 gene with pooled DNAs from WG patients compared with those from controls  at position 3030 in one WG patient.Our study revealed 4 single base substitutions, 2 of which were reported before . A furthPLCγ-2 with WG. Altogether 4 microsatellites spread in the PLCγ-2 gene were analysed. The approach using pooled DNA and ad hoc designed markers intragenic or in the immediate vicinity of a distinct gene has proven to be a reliable and efficient method to detected predisposing loci in WG before [The indirect microsatellite based approach did not reveal any association of G before . Here, nPLCγ-2 gene did not reveal an association of PLCγ-2 with WG. In contrast to ALI5 mice, where a single mutation leads to distinct symptoms of inflammatory autoimmunity, human WG depends on a more complex genetic background. Further analysis of all exons of PLCγ-2 might yield an association with WG but our microsatellite analysis strongly suggests that predisposition for WG is not due to variations in the PLCγ-2 gene.In conclusion, our analysis of the human + status were included in present study. Diagnosis of WG was established according international standards [[175 well-characterized patients of German origin with a clinical diagnosis of WG and a defined PR3-ANCAandards [ and 20]+ status Pooling of DNA was performed as reported before . PatientIn this study 3 intragenic microsatellites as well as one in the immediate vicinity of the gene were included (table 2, 0.2 pmol tailed F, 1.5 pmol reverse primer, 0.25 U Qiagen Hot Start Taq (Qiagen) and 50 ng DNA.For PCR we used the 'tailed primer PCR' as described before . For ampElectrophoreses were run using ABI standard protocols. Raw data were analyzed by the Genotyper software (ABI) producing a marker-specific allele image profile (AIP, see  and 7]))7]). AIPStatistics for comparisons of allele frequencies of patients and controls was performed as described before [ and 7]]]7]]. CasExons 11, 12, 13 and 27 were analysed by the SSCP method. PCR was performed using oligonucleotides reported before (, exon 27PJ and SW carried out the molecular genetic studies, performed the statistical analysis and drafted the manuscript. PY participated in study design and helped to draft the manuscript. EC and WLG provided the samples and performed diagnostics of the patient group. JTE conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.


Metallothionein (MT) protein expression deficiency has been implicated in carcinogenesis while MT over expression in tumors is indicative of tumor resistance to anti-cancer treatment. The purpose of the study was to examine the expression of MT expression in human renal cell carcinoma (RCC) and to correlate MT positivity, the pattern and extent of MT expression with tumor histologic cell type and nuclear grade, pathologic stage and patients' survival.The immunohistochemical expression of MT was determined in 43 formalin-fixed and paraffin-embedded RCC specimens, using a mouse monoclonal antibody that reacts with both human MT-I and MT-II. Correlation was sought between immunohistochemical (MT positivity, intensity and extension of staining) and clinico-pathological data .Positive MT staining was present in 21 cases (49%), being mild/moderate and intense in 8 and 13 cases, respectively. The pattern was cytoplasmic in 7 cases and was both cytoplasmic and nuclear in 14 cases. MT expression in a percentage of up to 25% of tumor cells (negative MT staining included) was observed in 31 cases, in a percentage 25–50% of tumor cells in 7 cases, and in a percentage of 50–75% of tumor cells in 5 cases. There was no significant correlation of MT intensity of staining to histological type, stage and patients' survival, while it was inversely correlated to higher tumor nuclear grade. MT extent of staining did not correlate with histological type, nuclear grade, and pathologic stage while a statistically significant association was found with patients' survival.The inverse correlation between MT staining intensity and tumor nuclear grade in RCC suggests a role of MT in tumor differentiation process. Since extent of MT expression is inversely correlated with survival it may be possibly used as a clinical prognostic parameter. Metallothioneins (MTs) were firstly discovered by Margoses and Valle in 1957  documentMT-I and MT-II isoforms are usually expressed in low levels, but are inducible by a variety of metal ions, hormones, inflammatory cytokines and xenobiotics -12. InduIn organs such as kidney, colon and liver, normally implicated in metal ions homeostasis, MT protein is apparently expressed. It would be of great scientific importance to elucidate the pattern of MT expression in tumors developed from these organs, since it could not only delineate the role of MT in carcinogenic transformation but could also provide prognostic information for patients' outcome. The aim of the present study was to examine the expression of MT in human renal cell carcinoma (RCC) and to correlate the MT positivity, the pattern and extent of MT expression with tumor histological cell type and nuclear grade, pathologic stage and patients' survival.Forty three consecutive patients, 31 men and 12 women, who underwent nephrectomy for RCC comprised the group of our study. Their age ranged from 33 to 85 years (mean age 59.6 ± 11.1). Tumors were histologically classified as clear cell type in 32 cases, papillary type in 2 cases, chromophobe cell type in 4 cases and sarcomatoid type in 5 cases. The 8, 2, 16, 6 and 9 tumors were pathologically staged as T1 or T2N0M0, T2N+M0, T3N0M0, T3N+M0 and T4N+M+, respectively as per the TNM classification . The graSections of 5 μm thickness were deparaffinized in xylene and rehydrated in graded alcohol series. To remove the endogenous peroxidase activity, sections were treated with freshly prepared 0.3% (v/v) hydrogen peroxide in methanol in dark, for 30 min, at room temperature. Non-specific antibody binding was then blocked using normal rabbit serum (Dakopatts, Glostrup, Denmark) diluted 1:5 in phosphate buffered saline (PBS), for 20 minute. A mouse (IgG1k) monoclonal antibody that reacts with both human MT-I and -II isoforms  was used in this study. The sections were then incubated for 1 hour, at room temperature, with the primary antibody diluted 1:50 in PBS. After three washes with PBS, sections were incubated for 30 minute at room temperature with rabbit, peroxidase conjugated, anti-mouse serum (Dakopatts) diluted 1:200 in PBS and rinsed three times with PBS. Sections were then incubated with swine, peroxidase conjugated, anti-rabbit serum (Dakopatts) diluted 1:100 in PBS and rinsed three more times with PBS. The resultant immune peroxidase complexes were developed in 0.5% (v/v) 3,3'-diaminobenzidine hydrochloride  in PBS containing 0.03% (v/v) hydrogen peroxide. Sections were counterstained with Harris' hematoxylin and mounted in gelatin (Sigma). Control slides included in MT immunostaining procedure consisted of specific tissues previously shown to express MT (lung cancer) as positive controls, whereas the primary antibody was replaced by PBS in the case of negative controls.The stained sections were independently assessed by the pathologists (A.K., S.T., E.M.) without prior knowledge of the clinical data as previously described . SpecimeCorrelation between immunohistochemical data (MT intensity and extent of staining) and clinicopathological data (histologic cell type, nuclear grade, pathologic stage) was assessed using the Chi-square test. The association of intensity and extent of MT immunohistochemical staining with survival was determined by comparing Kaplan-Meier survival curves constructed for different patient groups, and were compared using the log-rank test.Tubular cells but not glomeruli and interstitial cells of normal autologous renal tissue stained positive (both in the nucleus and the cytoplasm) for MT, although the intensity and extent varied significantly. Positive MT expression was prominent in 21 out of 43 cases (49%), while 22 out of 43 ones (51%) were MT negative. As far as the intensity of staining is concerned, low/moderate intensity was observed in 8 cases, while intense staining was evident in 13 cases. The pattern of positive MT immunostaining observed was either cytoplasmic (7 out of 21 cases, 33.3%) or cytoplasmic and nuclear (14 out of 21 cases, 66.6%). In certain cases of clear cell RCC membranous staining was also observed. Nuclear pattern of staining only, was not observed in any of the RCC cases examined . No statistically significant differences were also observed between MT intensity of staining and stage (χ2 = 9.24, p = 0.32), or patients' survival (log rank test: 4.75, p = 0.09) in the cases of RCC examined . However, no such correlation was found in the case of clear cell RCC (χ2 = 4.75, p = 0.57).There was no significant difference between MT intensity of staining and the histological types of RCC cases examined (χ2 = 2.54, p = 0.86), stage (χ2 = 7.12, p = 0.52) and grade (χ2 = 6.24, p = 0.39) of RCC cases examined. Statistically significant inverse correlation was found between MT extent of staining and patients' survival (log rank test: 6.59, p = 0.037) (Figure Non statistical correlation was found among MT extent of staining and histological types (χMT expression has been observed in different types of human tumors ,22, inclet al [et al [Developing and adult kidneys consistently express MT-I and MT-II mRNA  and the et al , using tet al  used anol [et al  who usedet al [The cytoplasm and nucleus of normal and malignant cells may both express MT but there is no conclusive data on the functional significance of their subcellular distribution. The majority of the cases in our study expressed MT in both cytoplasm and nucleus, the expression being cytoplasmic only in one third of the cases. Analogous pattern has been reported in other studies -25 whileet al  pointed et al . The funet al , as MT iIn certain tumors MT over expression has been associated with unfavorable prognostic characteristics such as advanced stage and poor differentiation . In our MT concentrations might also prove useful in predicting the efficacy of a particular cancer treatment protocols. Several types of transformed cells enriched for MT have been shown to exhibit greater resistance to chemotherapeutic agents . In thisThe current data on the expression of MT in RCC cases examined emphasize the necessity to investigate larger numbers of patients with RCC comparing the staining profile of different MT isoforms with other clinico-pathological parameters and survival status of patients. Currently, it is unknown whether the presence of MT in renal carcinoma cells is related to the induction or inhibition of apoptosis or plays an active role in cell proliferation. Since cytokines may also induce MT expression and immunotherapy is the only, albeit with limited efficacy, available treatment for RCC, the intensity and extent of MT immunostaining should be studied in correlation to the immune status of RCC patients before and after immunotherapy. Thus, it will be possible to elucidate the potential role of MT in renal cell carcinogenesis, as well as its clinical usefulness as a tumor marker and as a tool for selecting patients for adjuvant immunotherapy.


A novel tyrosine kinase receptor EphA2 is expressed at high levels in advanced and metastatic cancers. We examined whether vaccinations with synthetic mouse EphA2 (mEphA2)-derived peptides that serve as T cell epitopes could induce protective and therapeutic anti-tumor immunity.671–679, mEphA2682–689) and CD4+ (mEphA230–44) T cells. Splenocytes (SPCs) were harvested from primed mice to assess the induction of cytotoxic T lymphocyte (CTL) responses against syngeneic glioma, sarcoma and melanoma cell lines. The ability of these vaccines to prevent or treat tumor  establishment/progression was then assessed.C57BL/6 mice received subcutaneous (s.c.) vaccinations with bone marrow-derived dendritic cells (DCs) pulsed with synthetic peptides recognized by CD8+  peptides, prevented the establishment or prevented the growth of EphA2+ or EphA2-negative syngeneic tumors in both s.c. and lung metastasis models.These data indicate that mEphA2 can serve as an attractive target against which to direct anti-tumor immunity. The ability of mEphA2 vaccines to impact EphA2-negative tumors such as the B16 melanoma may suggest that such beneficial immunity may be directed against alternative EphA2+ target cells, such as the tumor-associated vascular endothelial cells. EphA2 is a member of Eph family of receptor tyrosine kinases comprised of two major classes (EphA and EphB), which are distinguished by their specificities for ligand (ephrin-A and ephrin-B, respectively -3). Rece+ or CD4+ T-cell responses, respectively [These data suggest that EphA2 may serve as an attractive target for cancer vaccines. In this regard, we have identified five human leukocyte antigen (HLA)-A2 binding and three HLA-DR4-binding peptides derived from EphA2 that are capable of inducing specific, tumor-reactive CD8ectively . A more ectively .These observations and findings support our rationale for near future implementation of EphA2-targeted vaccine clinical trials. For pre-clinical evaluation of EphA2-targeted vaccines, however, there is little information on immune responses against EphA2 in mouse models. Therefore, we hypothesized that identification of mouse T-cell epitopes in mEphA2 would allow us to determine the effect of EphA2-targeted vaccinations in mice bearing tumors. In this study, we examined whether novel T-cell epitope peptides identified in the mEphA2 protein sequence could elicit protective and therapeutic anti-tumor immune responses in murine models. Our results indicate that DC-based vaccines incorporating these peptides elicit effective CTL responses that can inhibit the growth both EphA2+ and EphA2-deficient tumors.Female 6–8-week-old C57BL/6 mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Animals were handled under aseptic conditions in microisolator cages within the Central Animal Facility at the University of Pittsburgh per an Institutional Animal Care and Use Committee-approved protocol, and in accordance with recommendations for the proper care and use of laboratory animals.Nf1 and Trp53 [b) cell lines were cultured in complete medium (CM) [RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin, and 10 mM L-glutamine ] in a humidified incubator in 5% CO2 at 37°C.Glioma cell lines KR129, KR130, KR233 and KR158D were derived from spontaneously arising gliomas in F1 between B6 × CBA (KR129), B6 × SJL (KR130), and C57BL/6 (KR233 and KR158D)-background NPcis mice that express mutations in two tumor-suppressor genes, 2 incubator for 7 days. DCs were then isolated at the interface of 14.5% (w/v) metrizamide (Sigma, St. Louis, MO) in CM discontinuous gradients by centrifugation. DCs typically represented >90% of the harvested population of cells based on morphology and expression of the CD11b, CD11c, CD40, CD54, CD80, CD86, and class I and class II MHC antigens as assessed using flow cytometry (data not shown).The procedure used to generate DCs has been previously described . Brieflyb-, H-2Db-, and I-Ab-binding binding motifs using BIMAS, and a proteosomal cleavage site prediction system [b-binding mEphA2671–679 (FSHHNIIRL), H-2Kb-binding mEphA2682–689 (VVSKYKPM), and I-Ab-binding mEphA230–44 (LLDFAAMKGELGWLT) epitopes were synthesized using an automated solid-phase peptide synthesizer (Applied Biosystems, Foster City, CA) by the protein synthesis facility at the University of Pittsburgh Cancer Institute, purified (to greater than 95%) by reverse phase HPLC, and characterized for amino acid sequences by mass spectrometry.The protein sequences of mEphA2 was obtained from GenBank and analyzed for H-2Kn system , respectn system . The H-2Day 7 DCs were pulsed with 10 μM each of the indicated peptides for 4 hours at 37°C, as previously described . Cells w5 B16-BL6 tumor cells on day 0, and they subsequently received s.c. vaccinations of peptide-loaded DCs on days 3, 10 and 17. Animals were sacrificed on day 28 post-tumor injection and analyzed for the assessment of pulmonary metastases by enumerating the number of surface tumor-nodules.In the s.c. model, animals were injected with the indicated numbers of tumor cells in the right flank. Anti-tumor responses were assessed based on comparative longitudinal measurements of tumor area. In the lung metastasis model, animals were injected i.v. with 2 × 10Protein lysates isolated from normal mouse brain, spleen, liver, lung, heart, skeletal muscle, and mouse tumor lines were separated by SDS-PAGE, blotted onto nitrocellulose and analyzed for expression of mEphA2 using EphA2 monoclonal antibody . Blots were imaged on Kodak X-Omat Blue XB-1 film (NEN Life Science Products, Boston, MA) after using horseradish peroxidase (HRP)-conjugated goat anti-rabbit Ig (Biorad, Hercules, CA) and the Western Lighting™ chemiluminescence detection kit (Perkin Elmer, Boston, MA).6 cells/ml with 2 μg/ml mEphA2671–679 or mEphA2682–689 in presence of 10 U/ml human IL-2 (Chiron, Emeryville, CA), 50 μM 2-mercaptoethanol (Sigma), and 50 μM NG mono-methyl-L-arginine  in 24 wells plates (Corning, Corning, NY) for 5 days. Specific CTL activity was determined in 4 h 51Cr release assays against the indicated target cells, as previously described [Single cell suspensions of SPCs were cultured at 2 × 10escribed .671–679/30–44) or OVA-derived peptides (OVA 257–264/265–280). Each animal then received 400 μl of Matrigel (BD Biosciences) supplemented with 400 ng/ml vascular endothelial growth factor (VEGF) in the dorsal area [Eight-week-old mice were injected s.c. twice (days -14 and -7) with DCs loaded with either EphA2-derived peptides . We immunized syngeneic C57BL/6 mice twice s.c. with syngenic DCs loaded with the Kb-binding and the I-Ab-binding epitopes, or DCs loaded with the Db-binding and the I-Ab-binding epitopes on a weekly basis. Control animals received DCs loaded with the cancer-irrelevant Kb-binding OVA257–264 and the I-Ab-binding OVA265–280 epitopes [in vitro with the MHC class I peptide that was used for in vivo immunization, in the presence of 10 U/ml hIL-2, for 7 days. SPCs from control animals immunized with OVA peptides were stimulated in vitro with the mEphA2671–79 to provide an index for a control, "primary" level of specific CTL induction. CTL activity against mEphA2-expressing, and H-2b-syngeneic MCA205 sarcoma, GL261 glioma and KR158D glioma cells were assessed by standard 51Cr-release assays  for the CTLs raised against EphA2-epitopes. B16 melanoma and YAC cells, which were used as negative control target cells, displayed constantly less than 10% of specific lyses in all groups (data not shown). These data support the conclusion that these mEphA2-derived peptides may serve as effective in vivo immunogens for anti-tumor T cell activation.We have previously characterized the immunogenicity of the H-2Depitopes . One weeb or H-Db presented) + Th epitope peptides on days 3 and 10 following tumor inoculation . Control animals received DCs loaded with the OVA-derived CTL + Th epitopes. As displayed in Figure b or H-Db presented) + Th epitope peptides significantly inhibited the growth of tumors in comparison to control vaccinations with irrelevant (OVA)-derived T cell epitopes.We evaluated the efficacy of protective and therapeutic immunizations with mEphA2-derived peptides in s.c. tumor models. These included the mEphA2-positive MCA205 sarcoma and the mEphA2-negative B16 melanoma models. First, animals bearing established s.c. MCA205 tumors received therapeutic vaccinations with mEphA2-derived CTL . The control group received irrelevant OVA257–264 instead of mEphA2-derived peptides. Tumor size and the number of animals that had measurable tumors are assessed on day 21 .We next examined whether vaccination with mEphA2-derived peptides could induce anti-tumor effects against mEphA2-negative tumors, expecting that this would not be the case. Since s.c. B16 melanomas grow aggressively, we employed a pre-immunization model to assess the anti-tumor response. Syngeneic C57BL/6 mice were pre-immunized with either mEphA21 Figure . All cono Figure , these d5 B16-BL6 melanoma cells via tail vein injection. The animals subsequently received s.c. immunizations with 1 × 106 DCs loaded with mEphA2 CTL (H-2Kb or H-Db presented) + Th on days 3, 10 and 17 . Control animals received DCs loaded with the OVA CTL + Th epitopes. On day 28, the animals were sacrificed, and the lungs harvested from each animal. The number of surface pulmonary metastases and lung weight were measured and compared between the groups. As demonstrated in Figure We next evaluated a lung metastasis model using B16-BL6 melanoma cell line. Syngeneic C57BL/6 mice received 2 × 10VEGF has been implicated to be a major mediator of tumor-angiogenesis ,20, and in vivo. Of major note, the anti-tumor response was not restricted to mEphA2-postive models, but was also observed in the case of both s.c. and metastatic B16 melanoma models.In this report, we have demonstrated that mEphA2-derived peptide epitopes can elicit specific CTL responses and anti-tumor response in vitro as assessed by Western Blotting, it was conceivable that they had become EphA2+ after in vivo injection, which could more easily explain the efficacy of the mEphA2 peptide-based vaccinations. However, we were unable to detect mEphA2 expression even in the in vivo grown B16 tumors (data not shown), suggesting that the T-cell immunity against mEphA2-derived epitopes suppress the in vivo tumor-growth not only through the direct anti-tumor cell mechanisms but also through indirect mechanisms, such as inhibition of tumor-angiogenesis.While the melanoma cell lines were negative for EphA2 expression The Eph kinases and ephrins play a crucial role in vascular development during embryogenesis and tumor formation . In two in situ, our careful observation of EphA2-vaccinated animals at autopsy did not reveal any evidence for tissue infiltration by inflammatory leukocytes or the destruction of EphA2+ tissues.As expected, based on our Western Blotting, several normal organs examined expressed EphA2 including the spleen, liver and lung. This could provide concern for the potential clinical translation of EphA2-based vaccines, given concerns for autoimmune pathology that might occur in these anatomic sites. However, despite the effective induction of antigen-specific CTLs that are capable of mediating DTH-type reactivity (unpublished data) and anti-tumor responses 671–679, mEphA2 682–689 and mEphA2 30–44 could induce anti-tumor response. It was noteworthy that mEphA2 30–44, which was found to bind I-Ab and induce DTH and specific CD4+ T cell responses, also suppressed the growth of s.c. (MHC class II-deficient) B16 melanomas as a single agent. This may suggest that neovessels in the B16 microenvironment are MHC class II+ either constitutively or under inflammatory conditions under which IFN-γ is elaborated, that B16 melanomas become class II+ after in vivo transfer or upon provision of IFN-γ in situ, or that the mEphA2 30–44 Th peptide also contains an embedded CTL epitope. We are currently assessing each of these possibilities.In our assessment of CTL and anti-tumor response, in most cases, we employed both MHC class I and II associated peptides in our treatment regimens based on our previous study demonstrating that the combination of MHC class I and II-restricted OVA-immuno-epitopes induced higher levels of anti-tumor efficacy than MHC class I-restricted OVA-epitopes solely . With reIt is also intriguing to determine whether there is a correlation between expression levels of mEphA2 in the target cells and their susceptibility to CTLs raised against EphA2-derived peptides. Our preliminary data indicated EphA2 expression levels did not simply correlate with the CTL susceptibility, suggesting other factors, such as MHC class I expression and intracellular anti-apoptotic proteins, may contribute in the CTL-mediated cytotoxicity (data not shown). To address this issue, our ongoing studies with human glioma cells employ small inhibitory RNA for EphA2 to determine whether specific inhibition of EphA2-expression affects the CTL response.Although the precise mechanisms that underlie the anti-tumor efficacy of EphA2-based vaccines remain to be elucidated, our results demonstrate that the mEphA2-derived epitopes may represent important vaccine candidates for the development of clinical trials for the treatment of (both EphA2+ and EphA2-negative) cancers.s.c., subcutaneous; i.v., intravenous; CTL, cytotoxic T lymphocyte; Th, T helper; DCs, dendritic cells; APCs, antigen-presenting cells; OVA, ovalbumin; CM, complete medium; HBSS, Hank's balanced salt solution; PBS, phosphate buffered saline; SPCs, splenocytes; HLA, human leukocyte antigen; ELISPOT, enzyme-linked immuno-spot; DTH, delayed-type hypersensitivity; VEGF, vascular endothelial cell growth factorThe author(s) declare that they have no competing interests.NK carried out the Western Blotting and immuno-assays. NK, JED, TT, MH and FH carried out assessment of in vivo anti-tumor response and preparation of peptide-pulsed DCs. WJS participated in peptide identification, the design of the study and critical review of data and the manuscript. HO conceived of the study, and participated in its design and coordination. All authors have read and approved the final manuscript.


Drosophila demonstrate that regulation is also critical. The SOCS  proteins in mammals are regulators of the JAK pathway that participate in a negative feedback loop, as they are transcriptionally activated by JAK signaling. Examination of one Drosophila SOCS homologue, Socs36E, demonstrated that its expression is responsive to JAK pathway activity and it is capable of downregulating JAK signaling, similar to the well characterized mammalian SOCS.The Janus kinase (JAK) cascade is an essential and well-conserved pathway required to transduce signals for a variety of ligands in both vertebrates and invertebrates. While activation of the pathway is essential to many processes, mutations from mammals and Drosophila genome, there are three identifiable SOCS homologues in flies. All three are most similar to mammalian SOCS that have not been extensively characterized: Socs36E is most similar to mammalian SOCS5, while Socs44A and Socs16D are most similar to mammalian SOCS6 and 7. Although Socs44A is capable of repressing JAK activity in some tissues, its expression is not regulated by the pathway. Furthermore, Socs44A can enhance the activity of the EGFR/MAPK signaling cascade, in contrast to Socs36E.Based on sequence analysis of the Drosophila SOCS proteins have some overlapping and some distinct capabilities. While Socs36E behaves similarly to the canonical vertebrate SOCS, Socs44A is not part of a JAK pathway negative feedback loop. Nonetheless, both SOCS regulate JAK and EGFR signaling pathways, albeit differently. The non-canonical properties of Socs44A may be representative of the class of less characterized vertebrate SOCS with which it shares greatest similarity.Two  The vertebrate JAK signaling pathway is an essential component of cellular response to a wide array of cytokines and growth factors. The JAK cascade is reutilized for signaling events in numerous tissues and at multiple stages of mammalian development [reviewed by -3]. ManyThough the JAK pathway is vital to many developmental processes, strict control of JAK signaling is equally important. As with other signaling pathways, mechanisms must be in place to balance the activation of JAK pathway activity. Regulation serves to "reset" the pathway so that it will be responsive to subsequent signals and it restricts the level or duration of the signal so that it is properly interpreted by the cell. Inappropriate JAK activation is the direct cause of a specific form of acute lymphocytic leukemia (ALL) -6. In adBecause of the need to limit JAK activity, it is not surprising that there are several conserved protein families that regulate JAK activation [reviewed by ,14,15]. Drosophila, the JAK pathway is involved in embryonic patterning, sex determination, blood cell development, patterning of adult structures, planar polarity of photoreceptor clusters, maintenance of stem cells in spermatogenesis, and follicle cell patterning and function [see reviews [Drosophila. Two dominant gain-of-function alleles of hopscotch result in hypertrophy of the larval lymph glands, the hematopoietic organ, and melanotic masses [Use of the JAK signaling pathway for developmental processes is not restricted to mammals. Indeed, the JAK cascade is evolutionarily conserved, and can be found as an intact signaling pathway even in insects ,19-21. Ic masses -24. Excec masses ,25-27.Drosophila Socs44A. It contains the same modular domain architecture as mammalian SOCS and shows greatest sequence similarity to the relatively uncharacterized SOCS6 and SOCS7. We show that, unlike the previously studied Drosophila Socs36E [Socs44A expression in embryogenesis is independent of JAK pathway activity. However, Socs44A is able to regulate the JAK cascade in embryogenesis, but not in oogenesis. Finally, Socs44A genetically interacts with and upregulates the EGFR/MAPK pathway. The characteristics of Socs44A that distinguish it from the canonical Socs36E may be representative of features that are shared with the class of less-defined mammalian SOCS genes.Of the eight mammalian SOCS, four have been studied extensively (CIS, SOCS1-3). These genes have been shown to respond to JAK pathway activation and subsequently are able to downregulate its activity as described above, completing a classical negative feedback loop. In comparison, very little is known of the remaining four. Here we present the identification and characterization of  Socs36E ,29, SocsDrosophila Genome Project (BDGP) database [Socs16D (overlapping with CG8146), Socs36E (overlapping with CG15154), and Socs44A (CG2160), based upon their cytological location. Comparison of these three fly SOCS genes with vertebrate SOCS reveals that Socs36E is most similar to the mouse SOCS5, while Socs16D and Socs44A are less similar to specific mouse SOCS  was isolated by the BDGP, has been completely sequenced and encodes the expected SH2 and SOCS domains at the carboxyl terminus (gb AF435923). To determine whether Socs44A is similarly regulated by JAK pathway activity, in situ hybridization to embryos and ovaries was performed. No specific expression of Socs44A was detected until very late in embryogenesis. The only striking staining pattern observed was in the trachea of late embryos  technique was used to generate females that fail to produce hop in the germline [hop germline clone embryos using Socs36E as probe demonstrates a strong reduction in Socs36E expression in the mutant embryos as compared with wild-type . We conclude that Socs44A expression is not responsive to JAK pathway activity, therefore cannot function via a traditional auto-regulatory feedback loop.Activity of the JAK pathway is both necessary and sufficient for the expression of pattern [ and dataSocs44A was misexpressed using the GAL4/UAS system. Similar experiments performed with Socs36E have demonstrated that expression in the developing wing reproducibly results in the production of ectopic wing vein near the posterior crossvein [Fig. hop or Stat92E [Fig. Socs36E misexpression may cause a reduction in JAK signaling in the wing. But unlike observed JAK mutations, the anterior crossvein was also completely missing from Socs36E misexpression wings, perhaps suggesting an additional role for Socs36E that is independent of the JAK pathway. Callus and Mathey-Prevot [The lack of transcriptional regulation by JAK signaling does not preclude a role for Socs44A in the control of JAK activity. To test whether it can attenuate JAK signaling, ein Fig. ]. This p92E Fig. ], suggesy-Prevot  demonstrSocs44A in the posterior compartment of the wing caused mild venation defects similar, but not identical, to Socs36E , and Egfr  as well as both cross-veins , indicating that the suppression by UAS-Socs44A was not merely a consequence of titrating GAL4.In mammals, there are multiple points of cross-talk between the JAK and EGFR/MAPK signaling pathways ,38-40. Evenation ,42. As mhe wings . To detegfr Fig. . Consistype Fig. . In contins Fig. . Concurr L4 Fig. . The ressay Fig.  or what nts Fig. . Finallying Fig. . These dSocs44A in the argos misexpression background. Engrailed-GAL4 misexpression of argos produces a range of phenotypic classes in which parts or all of L4 and/or the posterior cross-vein are missing , there is a loss of pnt-lacZ expression, indicating failure to specify the posterior terminal follicle cells.Evidence presented here and elsewhere indicates that Socs36E and Socs44A can downregulate JAK signaling in the wing . Howeverogenesis ,33,44-47[pointed . In clonity Fig.  or egfr Socs36E at high levels in posterior cells of the developing egg chamber, there was a dramatic loss of the pnt-LacZ marker  wing and thorax [Socs44A reduced JAK activity in the imaginal wing, as illustrated by the enhancement of that phenotype by reduction of endogenous hop. Furthermore, misexpression of Socs44A rescued wing vein loss resulting from misexpression of hop. Perhaps most importantly, introduction of deficiencies that remove Socs44A rescued a hop wing vein phenotype. Taken together, these data strongly suggest that Socs44A downregulates JAK pathway activity during normal wing development. However, misexpression of Socs44A had no effect on expression of a marker for JAK pathway activity during oogenesis. This indicates that there is context specificity to SOCS action in Drosophila, a phenomenon that has been observed in the study of mammalian SOCS [Socs36E was able to downregulate expression of the pnt-lacZ marker in follicle cells, although it cannot be distinguished whether this is due to reduction of signaling through JAK or EGFR. However, because Socs36E is expressed in the pattern of JAK activation in follicle cells, it is likely that it has a function in regulating JAK signaling in the ovary.Despite the difference in expression of the two SOCS genes, both are able to downregulate JAK activity in some tissues. Misexpression of d thorax . Similarian SOCS . In contDrosophila SOCS was in their abilities to regulate signal transduction cascades in addition to JAK/STAT. Precedence for such additional roles for vertebrate SOCS include regulation of Tec, Vav, TCR, c-kit, and FAK mediated signaling [argos in a dose-dependent manner. Furthermore, mutations in EGFR pathway components rescued Socs44A misexpression phenotypes. Importantly, a reduction of endogenous Socs44A activity enhanced the argos phenotype. Taken together, these data suggest that a normal function for Socs44A is to enhance the EGFR pathway. A potential mechanism for this genetic interaction can be found in a recent report describing physical interaction between SOCS3 and the p120 RasGAP [Another distinction we noted between the ignaling -60. It hignaling . Socs44A0 RasGAP . p120 RaDrosophila SOCS, all of which have greatest homology to the two classes of vertebrate SOCS that are least well characterized. One of these, Socs36E, is a member of the vertebrate SOCS4/5 class and has been previously characterized [Socs36E, activation of the JAK pathway was neither necessary nor sufficient for the expression of Socs44A. We conclude that Socs44A is unlike classical SOCS because it does not participate in a JAK pathway negative feedback loop. Still, Socs44A was capable of repressing JAK signaling, but that activity was limited to certain tissues. This context specificity is a feature that is shared with classical SOCS. Finally, Socs44A and Socs36E had opposite effects on EGFR/MAPK signaling. The enhancement of MAPK signaling that was seen for Socs44A is reminiscent of the influence of SOCS3 on this pathway, which is exerted through physical interaction of SOCS3 with p120 RasGAP. Perhaps a similar mechanism explains the enhancement of MAPK activity due to Socs44A. The differences observed here between Socs36E and Socs44A strongly suggest that they have distinct functions in the fly. Furthermore, the differences between Socs44A and the well studied class of canonical vertebrate SOCS may be representative of undiscovered distinctions amongst the three classes of vertebrate SOCS.There are three cterized ,29. It iDrosophila SOCS genes were identified using a simple tBLASTn 2.0 query with a consensus sequence for the vertebrate SOCS domains [Putative  domains  used to Socs36E coding region, including the SOCS box and SH2 domain. Two independent clones (Genbank accessions AF435838 and AF435839) were recovered, with the former being structurally similar to an EST from the BDGP (clot #7147). The BDGP also recovered two cDNA clones representing socs44A which have been designated as clot #8463. We have determined the complete sequence of the longer clone, LP02169 (Genbank AF435923).A cDNA library constructed from RNA of 12–24 hr old embryos  was screSocs36E and Socs44A were generated from the 5' ends of the respective cDNAs and did not include the coding region for the conserved SH2 and SOCS domains. Germline clone mutants for the hopc111 null allele were generated using the ovoD1 dominant female sterile technique [upd in a specific pattern were generated by crossing females carrying a UAS-upd transgene with males heterozygous for paired-GAL4, which expresses GAL4 in the seven stripe pair-rule pattern of the paired gene. Progeny were collected and hybridized as above. Trachea in germline clone-derived hopc111 embryos were visualized with the trh10512 enhancer trap [In situ hybridizations to embryos were performed as previously described . Digoxigechnique . Embryoscer trap  using ancer trap .Socs36E and Socs44A under control of GAL4, the full-length cDNAs described above were cloned into the pUAST vector [engrailed-GAL4 (e16E-GAL) was used to drive expression of the transgenes in the posterior compartment. Wings were dissected and mounted in Hoyer's medium [To express T vector . GermlinT vector  and trans medium  for phothopc111, were generated by hsFLP mediated mitotic recombination as previously described [Socs36E and Socs44A were generated using a GAL4 flip-out cassette [w [hsFLP]1; [Act5C>y>GAL4] [UAS-GFP.S65T]/ [UAS-socs36E]11.2 and w [hsFLP]1; [Act5C>y>GAL4] [UAS-GFP.S65T]/+; [UAS-socs44A]/ pnt-LacZ, respectively. For each, ovaries were fixed and stained with anti-β gal and anti-GFP as previously described [Ovarian clones of the null allele, escribed ,33. Misecassette . Genotypescribed ,33.in situ hybridization and wing images were acquired using a Spot Camera (Diagnostic Instruments) on a Nikon E800 microscope using differential interference contrast (DIC). A Leica TCS-SP laser scanning confocal microscope was used to capture all fluorescence micrographs. All images were then exported to Adobe Photoshop for manipulation and annotation.All JR performed experiments with Socs44A and participated in drafting the manuscript. GR performed most experiments with Socs36E. SH performed some experiments with Socs36E. RX performed immunofluorescence experiments in ovaries, except for Socs44A. DH conceived of the study, participated in its design and coordination, and participated in drafting the manuscript. All authors read and approved the final manuscript.