The multiple drug resistance protein (MDR1/P-glycoprotein) is overexpressed in glia and blood-brain barrier (BBB) endothelium in drug refractory human epileptic tissue. Since various antiepileptic drugs (AEDs) can act as substrates for MDR1, the enhanced expression/function of this protein may increase their active extrusion from the brain, resulting in decreased responsiveness to AEDs.Human drug resistant epileptic brain tissues were collected after surgical resection. Astrocyte cell cultures were established from these tissues, and commercially available normal human astrocytes were used as controls. Uptake of fluorescent doxorubicin and radioactive-labeled Phenytoin was measured in the two cell populations, and the effect of MDR1 blockers was evaluated.Frozen human epileptic brain tissue slices were double immunostained to locate MDR1 in neurons and glia. Other slices were exposed to toxic concentrations of Phenytoin to study cell viability in the presence or absence of a specific MDR1 blocker.MDR1 was overexpressed in blood vessels, astrocytes and neurons in human epileptic drug-resistant brain. In addition, MDR1-mediated cellular drug extrusion was increased in human 'epileptic' astrocytes compared to 'normal' ones. Concomitantly, cell viability in the presence of cytotoxic compounds was increased.Overexpression of MDR1 in different cell types in drug-resistant epileptic human brain leads to functional alterations, not all of which are linked to drug pharmacokinetics. In particular, the modulation of glioneuronal MDR1 function in epileptic brain in the presence of toxic concentrations of xenobiotics may constitute a novel cytoprotective mechanism. Failure to respond to therapeutic concentrations of antiepileptic drugs (AEDs) is the usual basis for defining multiple drug resistant epilepsy, but the mechanisms underlying resistance to AEDs are still largely unknown. It is generally believed to be a multifactorial phenomenon, depending on both pharmacodynamic and pharmacokinetic mechanisms. The electrical and synaptic properties of neurons in epileptic human tissue are reported to undergo changes that may result in decreased susceptibility to some AEDs . MultidrRecent findings suggest that the molecular mechanisms of clinically defined multiple drug resistance involve drug-efflux transporters such as the ATP-binding cassette subfamily B member 1 (ABCB1), also known as MDR1 or P-glycoprotein (P-gp) -4. SpeciThe link between MDR1 overexpression and drug resistance in epilepsy is still poorly understood ,11. ThusIn this study we have evaluated the relationship between neuronal and astrocytic MDR1 expression and the capacity of the cells to survive cytotoxic insults, using drug resistant epileptic human specimens. Our results suggest that overexpression of MDR1 leads to functional alterations in the CNS that may be linked to both drug pharmacokinetics and neuroglial survival in injured brain.Human subjects were used as donors of cortical tissue samples. The investigation conformed to the principles outlined in the Declaration of Helsinki. Patient consent was obtained as per Institutional Review Board instructions before collection of the specimens. All the experiments involved small portions of human neocortical or hippocampal tissue, which were excised for therapeutic reasons from patients with pharmacoresistant epilepsy .To investigate MDR1 protein expression and cellular distribution in human epileptic tissue, slide-mounted sections (10 μm thickness) from frozen brain tissue  were treated overnight with 1μM XR9576, a specific MDR1 blocker [2) were randomly analyzed and optical density measurements were averaged to obtain representative data. To quantify the doxorubicin in the cells, the fluorescence in each sample was compared with standard solutions of doxorubicin (10 nM-10 μM). Data were analyzed using Origin Lab 7 software.Astrocytes from epileptic tissue (Table  blocker ,18, or f blocker ,20. Furt blocker  was then14C-Phenytoin uptake was measured in cultured astrocytes from epileptic tissue (Table 6 cells) and incubated at 37°C with 1 ml of T3 cell buffer (Tris-HCl 50 mM, NaCl 120 mM, KCl 50 mM, pH 7.4) containing 10 μM 14C-Phenytoin (Specific activity = 9.57 μCi/μmol). The incubation was terminated after 10 s, 30 s, 1 min, 5 min or 10 min by adding 1 ml of ice-cold T3 buffer. The cells were washed with 1 ml of ice-cold T3 buffer and solubilized with 1% (w/v) Triton X-100 for 1 h at 37°C. Intracellular radioactivity was measured using a scintillation cocktail (Packard Ultima Gold, ECN, Costa Mesa, CA, USA).in vitro as previously described [2 1; MgCl2 1.4; NaHCO3- 26; KH2PO4 1.25; glucose 10, with or without 3 μM XR9576. A toxic concentration (375 μM) of Phenytoin (PHE) was then added for 5 h [2) within each slice. These values were averaged for each slice and expressed as percentages of the average number of DAPI-positive cells in the chosen fields.Brain slices (500 μm) were cut using a vibratome from dysplastic human cortex and temporal lobe epilepsy brain specimens , 10 μm slices from 11 patients . Differences with p < 0.05 were considered significant.Results were obtained from 14 patients  affected by intractable seizures. The mean patient age was 16.4 years . For details, see Table thin arrows in A) were observed in epileptic cortex, as well as immunopositive parenchymal and perivascular astrocytes (arrowheads in A) double-labeled with GFAP, a specific glial marker. Out of 107 GFAP-positive astrocytes, 91 (85%) showed MDR1 staining . The difference was abolished in the presence of 1 μM XR9576, indicating MDR1-mediated efflux of PHE in epileptic astrocytes. The MDR1 blockers did not affect doxorubicin or Phenytoin uptake in control astrocytes (not shown). Neither Phenytoin nor doxorubicin treatment induced toxicity in cell cultures under our experimental conditions.Fig. MDR1 is involved in detoxification mechanisms that protect cells from xenobiotics , apoptosFigure The main finding was that MDR1 might have different roles depending on the location in the brain where it is expressed. Thus, in addition to overexpression in endothelial cells, which is likely to affect the penetration of AEDs into the brain, MDR1 is also overexpressed in the parenchyma, where it might have a cytoprotective role, extruding otherwise toxic concentrations of xenobiotics from the intracellular compartment.In agreement with previous findings, we report here that MDR1 is highly expressed in vessels of the BBB and in parenchymal cells  in drug refractory epilepsy of different etiologies -9,11,27.perivascular astrocytes impinging on blood vessels may act as an additional barrier to drug penetration into the brain in regions where BBB permeability is transiently altered, for example during epileptic activity [We found that PHE and doxorubicin uptake by astrocytes from human epileptic tissue is reduced in comparison with normal astrocytes. The difference is abolished when MDR1 function is blocked, indicating that these drugs are efficiently extruded by "epileptic" astrocytes by a P-gp mediated mechanism. We suggest that this might contribute to decreasing the AED brain levels only if it occurs in perivascular astrocytes. Thus, activity ,33 (Fig.We show in this paper that MDR1 is expressed in immunocytochemically identifiable neurons and astrocytes in brain slices from refractory human epilepsy patients. We have previously reported that basic apoptotic mechanisms may be defective in glia from epileptic tissue, since the pro-apoptotic proteins p53 and p21 could not be detected in "epileptic" astrocytes ,9,16. ThFinally, we found a positive correlation between neuronal and astrocytic expression of MDR1 and lack of nuclear condensation, a marker of apoptosis and irreversible cell damage. Thus, expression of MDR1 in glia and neurons may protect the cells against toxic xenobiotics or against endogenous compounds that enter the brain in pathological conditions.In conclusion, as summarized in figure AEDs: anti-epileptic drugsBBB: blood-brain barrierDOX: doxorubicinGFAP: glial fibrillary acidic proteinMDR1: multiple drug resistance proteinNeuN: neuronal nucleiNF: neurofilamentPHE: PhenytoinThe authors declare that they have no competing interests.NM and LC carried out the pharmacological experiments and data analysis.KLH and KMK obtained surgical resections, established astrocytic cell cultures and performed the immunohistochemical and cell counting studies.GM performed the acute in vitro toxicity experiments.WB provided the surgical specimens.GD provided additional input and editing.AV and DJ designed and co-ordinated some of the experiments.They also contributed equally to the preparation of the manuscript.DJ, the PI of the study, co-ordinated and supervised most of the personnel involved in this project.The pre-publication history for this paper can be accessed here:


The CXCL1 chemokines, macrophage inflammatory protein-2 (MIP-2) and cytokine-induced neutrophil chemoattractant (KC), have been shown to play a role in a number of pathophysiological disease states including endotoxin-induced inflammation and bacterial meningitis. While the expression of these chemokines has been identified in a variety of cell types in the mouse, little is known about their expression with murine B-lymphocytes.Here, we demonstrate that highly purified murine splenic B cells are capable of expressing both MIP-2 and KC protein and mRNA upon activation with lipopolysaccharide (LPS) but not in response to anti-μ and anti-CD40 in combination with interleukin-4 (IL-4) stimulation. Moreover, these chemokines are expressed at higher levels in B cells derived from young (4 m) compared to old (24–29 m) mice. Upon fractionation into distinct B-cell subsets, we found that the expression of MIP-2 and KC by aged follicular (FO) B cells is significantly decreased when compared to the same cells from younger mice, while only MIP-2 production was found to be diminished in aged marginal zone (MZ) B cells. Interestingly, MIP-2 and KC production by newly formed (NF) B cells did not significantly differ with age. Moreover, the potential relevance of these findings is supported by the poor ability of LPS-activated aged B cells to specifically mediate CXCL1-dependent leukocyte recruitment when compared to younger B cells.Overall, the decreased expression of CXCL1 chemokines by aged B cells in response to LPS may have potential implications on the secondary recruitment of leukocytes to sites of microbial infections and inflammation possibly contributing to the increased susceptibility of older subjects to pathogen challenge. One oin vivo . These l in vivo -7. These in vivo ,9. MIP-2 in vivo .It has been well established that endotoxin and various proinflammatory cytokines (e.g., TNF-α and IL-1) stimulate the expression of MIP-2 by macrophages, neutrophils and epithelial cells -13. NumeAge-related dysfunction of the immune system has often been attributed to a variety of measurable changes in the functional activity of many immunomodulatory factors. This immune deterioration with age is believed to contribute to the morbidity and mortality in humans, possibly due to the greater incidence of infection, autoimmunity and cancer in the elderly. Dysregulation of lymphocyte function is thought to play a critical part in these processes. Many factors are believed to contribute to age-associated immunodeficiencies including defects in cellular signaling, stem cell and bone marrow defects, thymus involution, alterations in hormone and growth factor production, and replicative senescence. Chemokines are believed to play a pivotal role in the complex communication network between different cell types that enable the selective trafficking of many immune effector cells to the necessary sites at the appropriate times. Several reports have also demonstrated increased expression of inflammatory cytokines and chemokines in the circulation and by peripheral blood cells with age suggesting that uncontrolled inflammation may contribute to the increased susceptibility to infection and injury in certain aged cohorts. For example, IL-8 chemokine production by human T, NK and monocytes is altered with age in response to bacteria products -27. AlteThe objective of the current study is to explore both young and aged murine splenic B cells and B cell subsets for their ability to express the CXC chemokines, MIP-2 and KC, in response to LPS and other stimuli. Three B cell subpopulations were studied here including: (1) Marginal zone (MZ) B cells, which are uniquely positioned in the marginal sinus where they interact with efficient Ag trapping circulating cells and are thought to be involved in the early response against thymus-independent (TI) blood-borne Ags ,29; (2) +) following 24 h incubation with LPS, but was less evident in non-stimulated splenic B cells. Similar results were also obtained examining cytoplasmic KC (data not shown). In agreement with the above findings, MIP-2 and KC mRNA was only detected in LPS- . The+ B cells were sorted by FACS based on their surface IgM, CD21, and CD23 markers  Ags derived from a number of encapsulated bacteria, including fluenzae ,45. MZ B antigen . The rem antigen , the dimMurine MIP-2 and KC exhibit similar expression patterns and functional activities to that of human IL-8 in inflammatory response. For example, it has been previously shown that a significant increase of IL-8 or MIP-2 in human and mice, respectively, occurs in the grain dust-induced inflammation of the lower respiratory tract . AlthougIt should also be noticed that human IL-8 has been shown to be a potent chemoattractant for human B cells ,24. In tin vivo studies will be necessary to assess these various possibilities and the significance of this CXCL1 production defect by aged B cells.In summary, we demonstrate for the first time that murine splenic B cells are highly efficient in producing ELR-positive CXC inflammatory chemokines, in particular MIP-2, upon activation by LPS stimulation. Moreover, MIP-2 production, particularly by MZ B cells, was found to decline with age. Our finding suggests a possible linkage between functional activity of MZ B cells in production of neutrophil-attracting inflammatory chemokines and host defense. However, detailed and well-controlled ad libitum. All mice with evidence of disease (e.g., enlarged spleen, gross tumors) were not utilized in these studies.Specific pathogen-free 3–5 months (young) and 24–29 months (aged) C57BL/6 mice were purchased through the Office of Biological Resources and Resource Development of the National Institute on Aging (Bethesda, MD). All mice were maintained in an AAALAC-certified barrier facility and were acclimated for 2 weeks prior to use. All mice were fed autoclaved food and water Splenic B cells were negatively selected via depletion of non-B lineage cells from spleen cells using a MACS system. Briefly, spleen cells were incubated with magnetic microbeads coated with anti-CD43 antibody and anti-CD11b antibody (Miltenyi Biotec, Bergisch Gladbach, Germany) at 4°C for 15 min, after which the cells were passed over a MACS apparatus. The purity of splenic B cells was consistently >95% as routinely checked by FACS analysis.Supernatants were collected from cultures and were then frozen at -80°C. The frozen supernatants were thawed at room temperature and chemokine levels were measured with commercial ELISA assay kits for MIP-2 and KC (R and D Systems, Minneapolis, MN) and immunoglobulins (Igs) (Bethyl, Montgomery, TX) according to the manufacturers' instructions.2. After culture, the cells were harvested, washed, fixed and permeabilized using 3.7% paraformaldehyde and 0.1% Triton X-100 for 15 min. After thoroughly washing these cells, non-specific binding sites were blocked using a 2% BSA solution containing 1% goat, rabbit serum and normal mouse IgG for 15 min at room temperature. Post incubation, these cells were incubated overnight at 4°C in presence of biotinylated mouse anti-MIP-2 and -KC antibodies (1 μg/ml) (R & D Biosystems, Minneapolis, MN). Streptavidin-conjugated Oregon green-488 (Molecular Probes, Eugene, OR) was utilized to label these cells the following day at a concentration of 1:250 for a period of 45 min at room temperature. After washing, cells were then labeled with PE-conjugated anti-IgM antibody  for 30 min., followed by the stains of cellular nuclei with DAPI (Molecular Probes, Eugene, OR) at concentration of 1 μg/ml for 10 min. These cells were subsequently placed into cytospin funnels and spun onto glass slides using a cytospin centrifuge (Shandon, Pittsburgh, PA) at 1200 rpm for 5 minutes. After being mounted in Immuno Fluor medium , images were acquired by Spot Advanced software on a Zeiss Axiovert S100 microscope under 100X objective (Carl Zeiss, Thornwood, NY).Purified splenic B cells derived from young and aged C57BL/6 mice were cultured in the presence of absence of LPS (10 μg/ml) for 24 h at 37°C in 5% COFor conventional RT-PCR analysis, total RNA was extracted from cells using the RNeasy Mini kit  and cDNA was prepared from 1 μg of total RNA transcribed by the SuperScript First-strand Synthesis system for the RT-PCR procedure (Invitrogen, Carlsbad, CA) according to the manufacture's instructions. The mouse MIP-2 and KC primers (Sigma Genosys, Woodlands, TX) utilized in these studies were: MIP-2 sense 5'-TGCCGGCTCCTCAGTGCTG-3' and MIP-2 antisense 5'-AAACTTTTTGACCGCCCTTGA-3'; KC sense 5'-CGCTCGCTTCTCTGTGCA-3'and KC antisense 5'-ATTTTCTGAACCAAGGGAGCT-3' as described previously . The cycCt) values normalized for GAPDH expression and then normalized to the value derived from controls.Approximately 1 μg of total RNA was reverse transcribed by using SuperScript First-strand Synthesis system (Invitrogen, Carlsbad, CA) as described above. Real-time primers for murine MIP-2 and KC were designed using Primer Express software (Applied Biosystems) using the sequences from GenBank . Primers were constructed as follows: MIP-2 , KC , and GAPDH . Reverse-transcribed cDNA was amplified with primer sets for murine MIP-2, KC and GAPDH as indicated above using SYBR Green PCR core reagents and the GeneAmp 5700 Sequence Detection System (PE Applied Biosystems) following the manufacturer's instructions. No PCR products were generated from genomic versus cDNA template. Fold induction of mRNA was determined from the threshold cycle (4 irradiated (5,000 cGy) murine EL-4 thymoma cells (clone B5) in 200 μl of RPMI 1640 medium supplemented with 10% FCS, 10-5 M 2-mercaptoethanol (2-ME), 25 mM HEPES buffer, penicillin (100 U/ml), streptomycin (100 μg/ml), 10 μg/ml LPS, and 10% supernatant from culture of J774A.1 macrophage cells . MACS purified splenic B cells or FACS sorted B cells were seeded directly onto a feeder layer of irradiated EL-4 cells and cultured at 37°C in 5% CO2. Moreover, we have previously shown that approximately 97% of wells contained sorted single cells using the same outfitted FACStar Plus. These results were verified based on the resulting sequencing histograms demonstrating evidence of only one V6 light chain sequence in the amplified cDNA [The splenic B cell culture was performed using the EL-4-based B cell culture system as previously described . Brieflyied cDNA . Therefo6 cells were pre-incubated with the blocking antibodies, the anti-Fc receptor (24G2), for 30 minutes on ice and then incubated with a mixture of mAbs for an additional 15 min on ice. Post washing, the cells were subsequently incubated with PE-streptavidin for 15 min on ice after which the cells were washed with 5% FCS/HBSS. These stained cells were subsequently analyzed on a Becton Dickinson FACScan flow cytometer using CellQuest software.The monoclonal Abs utilized for the cell surface staining were FITC-anti-CD21 , -anti-IgM , PE-anti-CD23 (PharMingen), APC-anti-IgM , and/or biotinylated anti-IgG1 (PharMingen). Biotin-labeled antibody binding was visualized using UltraAvidin-R-Phycoerythrin (Linco Technologies, St. Louis, MO). For each staining, 10+ cell population were sorted based on their differential expression of CD21 and CD23 using a FACStar Plus (Becton Dickinson, San Jose, CA). The purity of each sorted population was consistently >95%.The B cell subsets, NF, FO and MZ, were isolated through the use of cell sorting using the combination of anti-IgM-APC, anti-CD23-FITC and anti-CD21-PE mAbs. Single spleen cell suspensions were stained with the aforementioned Abs and the MZ, FO and NF B cells within the gate of IgM5/well) were cultured with 10 μg/ml of LPS for 1 and 3 days or FACS-sorted distinctive B cell subpopulations (1000/well) were cultured in EL-4 culture system for 3 and 5 days at 37°C in a 5% CO2. Cultures were pulsed with 1 μCi [3H] thymidine (NEN, Boston, MA) for the final 18 h. Cells were harvested on fiberglass paper. [3H] thymidine uptake was measured in a liquid scintillation counter (Beckman, Fullerton, CA).MACS-purified splenic B cells (1.25 × 106/ml purified splenic B cells derived from young and aged mice suspended in 600 μl of RPMI 1640 medium supplemented with 2% FCS, 10-5 M 2-mercaptoethanol (2-ME), penicillin (100 U/ml), streptomycin (100 μg/ml) were cultured in the presence and in the absence of LPS (10 μg/ml) and incubated at 37°C in 5% in CO2 for 24 hr. After incubation, the cells and supernatants in the lower chambers were assessed for chemotactic activity using young splenocyte responder cells. These splenocytes were isolated from 4 month old C57Bl/6 mice after which they were preincubated with 10 μM Hoechst 33342 (Molecular Probes, Eugene, OR) in RPMI 1640 supplemented with 10% FCS for 30 min at 37°C. Subsequently, the Hoechst-stained splenocytes (1 × 106) were washed with RPMI 1640 supplemented with 2% FCS, resuspended in 100 mL of medium, and then added to the upper chamber, containing a 6.5-mm diameter polycarbonate Transwell culture insert with 5 μm size pore. Each expected group was performed in duplicates with supernatants in the lower chambers for 8 to 10 h at 37°C. In certain assays, neutralizing antibodies specific for MIP-2 and KC at (1 μg/ml) were added to the low chamber containing the LPS-stimulated B cell supernatants. The transmigration of the Hoechst-labeled cells into the lower chamber were measured in a fluorescent spectrophotometer at 350 nm (excitation)/460 nm (emission). The results are expressed as fluorescent units or as the percentage inhibition of Hoechst-labeled splenocyte migration.In the lower chambers of Transwell plates (Costar, Cambridge, MA), 1.5 × 10Statistical evaluation of significance between the experimental groups was determined by Student's t test.Ag, antigen; MIP-2, Macrophage inflammatory protein-2; ELR, glutamate-leucine-arginine; GRO, growth-related oncogene; HSV-1, herpes simplex virus type 1; KC, cytokine-induced neutrophil chemoattractant; MZ, marginal zone; FO, follicular; NF, newly formed; TI, Thymus-independent; TD, Thymus-dependentLH, VMC, and VDD performed the experiments. LH and DDT prepared the figures and wrote the paper. DDT also supervised the work and edited the manuscript. All authors have read and approved the final manuscript.


The Science and Development Network (SciDev.Net) is a source of online news and analysis about the role of science and technology in meeting the needs of the developing world Developing countries today face a wide range of needs, from more secure food supplies to cheap and effective medicines. One factor that almost all these needs have in common is that satisfying them adequately will not occur without the use of modern science. These same countries also face a range of political dilemmas—such as whether to accept the growing of genetically modified crops or how to adapt to the impact of climate change—that also require a knowledge of science, albeit of a slightly different nature. From both points of view, development can be characterised as the process of putting scientific and technical knowledge into practice. Conversely, it is important that building the capacity to absorb and make use of scientific and technical knowledge must be placed at the heart of the development aid efforts if these are to be successful in achieving their goals.But knowledge will not reach those who can benefit from it unless it has been effectively communicated to individuals with the power and skills to put it into practice, whether those are government officials and decision-makers, community groups and their representatives, or even nongovernmental organisations. Formal education, of course, has a key function here. But so does the informal education provided through the media. Furthermore, information and communications technologies (ICTs) have an important role to play in this process by reducing or eliminating the transactional (nonproduction) costs of communicating knowledge about science and technology.At the same time, it is important that the practice of science communication reflects the fact that it takes place in social context. In other words, it is not just a question of conveying information, but also of engaging the potential users of that information. The need is to encourage dialogue and eventually to empower those to whom the information is being provided so that this information can be applied in a practical and useful way.www.scidev.net), of online news and analysis about the role of science and technology in meeting the needs of the developing world.It was with this in mind that the Science and Development Network (SciDev.Net) was launched in December 2001 as a source, through its Web site  makes up 20%.For those with an interest in science activities in a particular geographical region, we link together coverage into so-called regional gateways on the Web site. At present, there are four of these—covering Latin America, the Middle East, South and East Asia, and sub-Saharan Africa. Each gateway therefore has a strong regional identity, and in some cases this is reinforced by the translation of material—either in full text or as a summary—into local languages. The Latin American gateway, for example, carries three languages, and material can appear in one, two, or all three of these, with summaries in the others.Reflecting a commitment to the idea that scientific knowledge will only be effectively communicated if individuals are adequately trained to carry out this task, we are engaged not only in presenting information on our Web site, but also in helping to generate a capacity within developing countries to improve channels of communication for this knowledge.One way in which we do this is through workshops on science communication. Some of these have a broad focus, seeking to help stimulate a national or regional debate on the importance of science communication, how it can be best carried out, and the challenges it faces. Such, for example, was the goal of two roundtables on the theme of “science communication and sustainable development” organised in August 2002 during the World Summit on Sustainable Development in Johannesburg, South Africa. Similarly, a meeting launching our activities in Latin America was held in Sao Paulo in May this year under the title “Science Communication and Development in Latin America.”Other events to encourage scientific communication have been more specific. In particular, we organised  a five-day workshop for African women communicators in Kampala, Uganda, in April 2003 on the use of ICTs to report on the science of HIV/AIDS . A similBuilding the communication channels that will allow science to be fully integrated into the development process at all levels—from governments down to communities—is a major challenge that involves many participants. SciDev.Net does not pretend to be more than one actor and is keen to establish partnerships with others in this field, convinced both of the synergies that can be gained through collaboration and of the contribution that ICTs can make to this (for example, by sharing information and links between Web sites).One of the major challenges that we face is to build up our role as a platform for the voice of the developing world on the issues that we cover. We already do this to a certain degree through our correspondents, our regional networks of contributors, and the participation of scientists from developing countries who are on the advisory panels for our dossiers. But we are very aware that more effort is needed. Despite this, we already feel that we are beginning to make a mark. A staff member of the United States' National Academy of Sciences recently informed us: “We hosted a group of visitors from seven African scientific academies here in Washington last week, and they spoke highly of the SciDev project … you are becoming a truly global newsletter!”


Lys3, is selectively packaged into the virus during its assembly, and annealed to the viral genomic RNA. The ribonucleoprotein complex that is involved in the packaging and annealing of tRNALys into HIV-1 consists of Gag, GagPol, tRNALys, lysyl-tRNA synthetase (LysRS), and viral genomic RNA. Gag targets tRNALys for viral packaging through Gag's interaction with LysRS, a tRNALys-binding protein, while reverse transcriptase (RT) sequences within GagPol (the thumb domain) bind to tRNALys. The further annealing of tRNALys3 to viral RNA requires nucleocapsid (NC) sequences in Gag, but not the NC sequences GagPol. In this report, we further show that while the RT connection domain in GagPol is not required for tRNALys3 packaging into the virus, it is required for tRNALys3 annealing to the viral RNA genome.The primer tRNA for reverse transcription in HIV-1, tRNA Lys isoacceptors in mammalian cells, tRNALys1,2 and tRNALys3, are selectively incorporated into the virus [Lys3 is the primer for initiating minus-strand cDNA synthesis, and its annealing to the 18 nucleotide primer binding site (PBS) region in the 5' part of the viral genome via the 3' 18 nucleotides in tRNALys3 complementary to the PBS, is a key step in viral replication [During assembly of HIV-1, the major tRNAlication .Lys3 and sites of annealing in viral RNA contain double stranded regions which may require denaturation for annealing to proceed efficiently. Nucleocapsid protein (NC) has been shown to facilitate tRNALys3 annealing both in vitro [in vivo [in vitro, and that the protein only has very subtle tertiary structural and helix destabilization effects on tRNALys3 alone [Both tRNAin vitro ,6 and in[in vivo , primaris3 alone -11.Lys3 annealing to genomic RNA in vitro, the annealing of primer tRNA onto the genomic RNA within HIV-1, murine leukemia virus, and avian retrovirus occurs independently of precursor protein processing [Lys3 is annealed efficiently in protease-negative HIV-1 (about 80% that found in wild-type virions), optimal placement on the viral genome to achieve efficient initiation of reverse transcription requires exposure of the viral genome to mature nucleocapsid protein [Lys3 annealing, while mutations in NC sequences within GagPol do not, indicating the importance of Gag NC sequences in the annealing [In vitro, Gag has been reported to facilitate tRNALys3 annealing to viral RNA as efficiently as mature NC [Although processed nucleocapsid proteins have been shown to facilitate tRNAocessing -14. Howe protein . In thesnnealing . In vitrature NC .Lys3 annealing onto the viral RNA, independent of its role in the packaging of tRNALys3 into the virion. We present data herein indicating that the RT connection domain, while non-essential for tRNALys3 incorporation into virions, is required for tRNALys3 annealing to the viral RNA genomeNevertheless, we will present evidence in this report that GagPol still plays an important role in tRNALys incorporation into virions, but is required for the annealing of tRNALys3 to the viral genomeThe RT connection domain within GagPol is not required for tRNA.Lys3, to determine the tRNALys3/genomic RNA in each viral variant. These results are shown graphically in Figure Lys incorporation into virions is not dramatically affected until GagPol sequences including the thumb domain of RT are deleted (Δ581 and Δ715).293T cells were transfected with protease-negative HIV-1 proviral DNA coding for either full length, protease-negative, GagPol (BH10.P-) or C-terminally deleted GagPol species. The different constructs are shown in Figure Lys3 annealed in vivo to the viral RNA genome, total viral RNA was used as the source of primer/template in an in vitro reverse transcription reaction, using exogenous HIV-1 RT, dCTP, dTTP, α-32P-dGTP, and ddATP. This assay measures the amount of extendable tRNALys3 placed onto the viral genome. It is not known if all annealed tRNALys3 is extendable. Since the sequence of the first six dNTP's incorporated is CTGCTA, annealed primer tRNALys3 will be extended by 6 bases, and the extended tRNALys3 can be resolved and detected by one dimensional polyacrylamide gel electrophoresis (1D PAGE). These results are shown in Figure Lys incorporation, but do severely reduce the ability of tRNALys3 to be functionally annealed to the viral RNA genome.To measure the amount of tRNALys3 as BH10P-, but cotransfection of mutant DNA with DNA coding for GagPol gives a small increase in tRNALys3 packaged to over 90% of BH10P- .Attempts were also made to rescue tRNAd to Vpr , but unlIn vitro studies of the interaction between purified RT and tRNALys3 have indicated an interaction between the RT thumb domain and the tRNA [In vivo studies also indicate an important role of the RT thumb domain in GagPol in tRNALys3 viral packaging. tRNALys3 incorporation into HIV-1 is not affected by deletion of the IN domain in GagPol, nor by further deletion of the RNaseH and connection domains in RT, but is severely inhibited by further deletion of the thumb domain as well [Lys3 interacts with the RT thumb domain during incorporation into virions, and Gag nucleocapsid plays a role in promoting tRNALys3 annealing to viral RNA [the tRNA -22. In v as well . Thus tRiral RNA -7, presuLys3 annealing? One possibility, suggested by in vitro studies, is that RT plays a direct role in tRNALys3 annealing. Early work indicated that the in vitro annealing of primer tRNATrp to AMV genomic RNA was promoted by the addition of AMV reverse transcriptase [Lys3, HIV-1 RT was also shown facilitate the in vitro annealing of tRNALys3 to the PBS sequence [in vitro works suggest that RT alone can directly promote tRNALys3 annealing to viral RNA. Whether the RT sequences in GagPol can function similarly in vivo is not known.What then is the role the RT connection domain sequence in GagPol in facilitating tRNAcriptase . In a lasequence . These iLys3 bound to the thumb domain in RT closer to either NC in Gag or to the genomic RNA that is bound to Gag NC. Recent work has indicated that that Pol sequences alone can bind to Gag p6 through the RT sequences in Pol [Lys incorporation into the virus and tRNALys3 annealing to the viral genome at levels approximately 35% those achieved using full-length GagPol. Thus, in addition to the interactions which probably occur between Gag and homologous sequences in the Gag part of GagPol, the interaction of RT sequences in GagPol with Gag p6 could place the RT-bound tRNALys3 closer to Gag NC sequences and viral RNA in the packaging complex. It remains to be determined which sequences within RT bind to Gag p6, but if it were those of the connection domain, this could explain how these sequences could promote tRNALys3 annealing through altering the configuration of GagPol.Alternatively, the RT connection domain may undergo interactions with Gag that may result in placing the tRNAs in Pol . Pol proLys3, and its annealing to HIV-1 RNA. One also finds two separate domains in Gag involved in these same processes. Evidence has been presented supporting the role of lysyl-tRNA synthetase (LysRS) in targeting tRNALys for viral incorporation, through a specific interaction of Gag capsid sequence with LysRS in a tRNALys/LysRS complex [Lys3 annealing [Lys3 annealing, and LysRS may be required to dissociate from tRNALys3 so as to free this tRNA for annealing to the viral RNA.Thus, two separate RT domains (thumb and connection) appear to be involved, respectively, in the viral incorporation of tRNA complex , while onnealing ,16,17. IBH10 and BH10P- are protease-positive and protease-negative strains of HIV-1, respectively . All delTransfection of COS7 cells with wild type and proviral DNA was performed using the calcium phosphate method as previously described . BrieflyViral particles were washed with 1X TNE and cellular or viral proteins were extracted with 1X RIPA buffer . Western analysis was performed using 300 mg cellular protein or 10 μg viral protein, as determined by the Bradford assay [Lys3 into virions, hybridization to dot-blots of viral RNA was carried out with DNA probes complementary to tRNALys3 [Lys3 annealed to genomic RNA, tRNALys3-primed initiation of reverse transcription was measured using total viral RNA as the source of primer tRNA/template in an in vitro HIV-1 reverse transcription reaction, as previously described [Lys3 is extended by 6 bases, and this product can be resolved by 1D PAGE, and quantitated by phosphorimaging, as previously described [Total viral RNA was extracted from viral pellets by the guanidinium isothiocyanate procedure , and distRNALys3  or to getRNALys3 . To measescribed . The seqescribed .SC carried out the molecular genetic studies, assisted by MJ. LK conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.


The pathological hallmarks of chronic obstructive pulmonary disease (COPD) are inflammation of the small airways (bronchiolitis) and destruction of lung parenchyma (emphysema). These forms of disease arise from chronic prolonged infections, which are usually never present in the normal lung. Despite the fact that primary hygiene and defense of the airways presumably requires a well controlled fluid environment on the surface of the bronchiolar airway, very little is known of the fluid and electrolyte transport properties of airways of less than a few mm diameter.We introduce a novel approach to examine some of these properties in a preparation of minimally traumatized porcine bronchioles of about 1 mm diameter by microperfusing the intact bronchiole.-, the bionic Cl- diffusion potentials  were as large as -90 mV. TEP diffusion potentials from 2:1 NaCl dilution showed that epithelial Cl- permeability was at least 5 times greater than Na+ permeability. The anion selectivity sequence was similar to that of CFTR. The bionic TEP became more electronegative with stimulation by luminal forskolin (5 μM)+IBMX (100 μM), ATP (100 μM), or adenosine (100 μM), but not by ionomycin. The TEP was partially inhibited by NPPB (100 μM), GlyH-101* (5–50 μM), and CFTRInh-172* (5 μM). RT-PCR gave identifying products for CFTR, α-, β-, and γ-ENaC and NKCC1. Antibodies to CFTR localized specifically to the epithelial cells lining the lumen of the small airways.In bilateral isotonic NaCl Ringer solutions, the spontaneous transepithelial potential  of the bronchiole was small , but when gluconate replaced luminal Cl- conductance that is most likely due to CFTR.These results indicate that the small airway of the pig is characterized by a constitutively active Cl Most, if not all, forms of chronic obstruction pulmonary disease (COPD) as well as asthma begin in the small airways. While the pathogenesis of small airway diseases is poorly understood ,2, it is- selective conductance that dominates the electroconductive properties of this epithelium, that is most probably duo to CFTR.At the same time, almost nothing is known with certainty about the transport properties of distal airway epithelia or how fluid movements help maintain hygiene. No doubt, the paucity of understanding is due to the inaccessibility and the fragility of the tissue. Most concepts of the mechanisms and functions at this level have been taken from findings in the upper respiratory tract or from the larger cartilaginous ringed structures of the trachea and bronchi -7. More 2O. The assembly was wrapped in a plastic bag and transported from the abattoir to the laboratory (<60 min) in an insulated box chilled with ice. In the laboratory, small pieces of about 0.5 cm3 were cut from the peripheral lung parenchyma, usually from along the costal diaphragmatic ridge of the lower lobes. In general, the freshest tissue gave the best responses although some tissue responded well after 6–8 hours of storage in a cooled environment.Lungs were excised intact immediately after sacrifice of young pigs (30–60 kg). Lungs were maintained inflated through a ligated plastic tube connected to an aquarium air pump (~1 L/min) to maintain a positive airway pressure of 10–14 cm-HUnder a dissecting microscope, the opening to a small airway was visualized on the proximal cut surface of a small block of lung tissue. The airway opening was then cannulated with a system of two concentric micropipettes -17 with intact and therefore remained embedded in the mass of connective tissue and air filled alveoli that normally surround the bronchi in vivo. The surrounding parenchymal tissue effectively prevented changing the solution in contact with the serosal surfaces of the airway epithelium, which in vivo is the extracellular fluid and in the intact preparation could not be readily removed. NaCl Ringer solution is designed to mimic mammalian extracellular fluid. Therefore, we used NaCl Ringer in the bath to establish electrical continuity with the serosal surface of the bronchiolar epithelium during the entire experimental period. The Ringer solution contained in (mM): Na+ (~155), K+ (4.5), Mg2+(1.2), Ca2+ (1.0), PO43- (3.5), Cl- (152), SO42- (1.2), Glucose (5) buffered to pH 7.4 with NaOH. For ion diffusion studies, 150 mM of Cl- was replaced with an equivalent amount of gluconate (taken as impermeant), HCO3-, NO3-, I-, or Br-. Luminal solutions perfusing the bronchiolar airway were rapidly changed as needed via a manifold distributing stores of the above solutions through a needle tube to the tip of the perfusing pipette  [Inh-172 and GlyH-101 were generous gifts from Dr. A. Verkman, University of California, San Francisco, CA.).The perfused airways were M): Na+ ~55, K+ (4-172 (5) ,20, GlyH-172 (5)   of the system, which includes the summed resistances of the epithelium  plus the core resistance of the lumen plus the extracellular fluid resistance, the resistance of the epithelium relative to Rt was small (even after floating the current passing circuit), and therefore changes in the epithelial resistance were obscured. Consequently, the response of TEP's was taken as the primary indication of the permeability properties of the epithelium.Although we recorded the total resistance . RNA was reversely transcribed using Sensiscript RT Kit (QIAGEN Inc. CA). The resulting first-strand cDNA was directly used for PCR amplification (TaqPCR Core Kit, QIAGEN Inc. CA). The conditions for PCR reactions were as follows: 3 min at 94°C ; 35 cycles of 1 min at 94°C, 1 min at 55–60°C, 1 min at 72°C and then 72°C 10 min . For the negative control, RT-PCR was performed in the absence of RT. The PCR products were analyzed by agarose gel electrophoresis stained with ethidium bromide.The primers were constructed on the basis of the published cDNA sequence of CFTR, ENaC, NKCC1 and β-Actin from GenBank. Since the pig gene sequence was not complete, primers were obtained from the human accordant gene, in which highly conserved regions were selected. The pairs of primers for CFTR (accession no. NM_000492) were sense 5'-TCCTAAGCCATGGCCACAA-3' and antisense 5'-GCATTCCAGCATTGCTTCTA-3'; sense 5'-GCCTGGCACCATTAAAGAAA-3' and antisense 5'-CTTGCTCGTTGACCTCCACT-3', which generated a 197-bp and 171-bp CFTR PCR product respectively; for α-ENaC (Z92978) were sense 5'-CAACAACACCACCATCCAC-3' and antisense 5'-TAGGGATTGAGGGTGCAGA-3', which generated a 225-bp PCR product; for β-ENaC (NM_000336) were sense 5'-TGCTGTGCCTCATCGAGTTTG-3' and antisense 5'-TGCAGACGCAGGGAGTCATAGTTG-3', which generated a 277-bp PCR product; for γ-ENaC (X87160) were sense 5'-TCAAGAAGAATCTGCCCGTGA-3' and antisense 5'-GGAAGTGGACTTTGATGGAAACTG-3', which generated a 237-bp PCR product; for NKCC1 (U30246) were sense 5'-TCCAGGTAATGAGTATGGTGTCAG-3' and antisense, 5'-GTTAAGATGTAGCCACGAAGAGGT-3', which generated a 205-bp PCR product; and for β-Actin (BC004251) were sense, 5'-TTCAACTCCATCATGAAGAAGTGTGACGTG-3' and antisense, 5'-CTAAGTCATAGTCCGCCTAGAAGCATT-3', which generated a 312-bp PCR product. All primers showed products closely corresponding to the predicted size for expression of RNA transcripts for these genes.The bronchioles were dissected and then fixed in ice-cold 4% formaldehyde buffered in phosphate at 4°C for 3 hours, infiltrated with cryoprotectant (30% sucrose in PBS) overnight, and frozen in OTC medium  at -35°C. Sections of 5 μm thickness were cut on a cryostat microtome (Thermo Electron) and mounted on glass slides (Fisher Superfrost Plus). Antigen retrieval was performed using a pressure cooker (10 min in 10 mM citrate buffer, pH 6). To reduce autofluorescence, sections were treated for 20 min with 1.5% sodium borohydride in PBS. Sections were incubated sequentially with blocking solution (30 min), primary antibody (overnight at 4°C), and secondary antibodies conjugated to Alexa Fluor-488 and/or -546 (Molecular Probes). Confocal images were acquired with a Zeiss LSM-510 microscope and assembled using Adobe Photoshop. CFTR was labeled with rabbit antibody R3194 (courtesy of C. Marino), and ENaC with a rabbit antibody against the β-subunit of human ENaC (kindly courtesy of C. Fuller and D. Benos). Tight junctions were labeled with a mouse antibody against the junction-associated protein zonula occludens-1 (Zymed). Nuclei were stained with TO-PRO-3 (Molecular Probes).Differences in mean measurement were assayed by applying the Student T test to paired or unpaired means as appropriate. A probability  of ≤ 0.05 was taken as significantly different.- gradient. Despite the fact that this small Cl- gradient should render the lumen positive, there was a small spontaneous lumen negative potential of about -3 mV , the TEP decreased slightly, but without statistical significance  to serosa (110 mm) Cl mV Fig. ; Table 145 mM to nce Fig. ; Table 1- with the impermeant anion, gluconate, the TEP hyperpolarized to as much as -90 mV , GlyH-101 (50 μM) and CFTRInh-172 (5 μM) significantly depolarized the TEP by 36.7, 20.0 and 8.7 mV .Under conditions of symmetrical [Cl mV Fig. ; Table 2- in NaCl Ringer. Amiloride (10 μM) was added in order to block Na+ transport. For luminal Cl-, Br-, I-, NO3-, HCO3- and gluconate, the mean estimated Px/PCl were, respectively, 1.0, 0.92, 0.79, 0.65, 0.33 and 0.28 , but in the presence of the Cl- gradient, the TEP hyperpolarized significantly to all agonists; the response appeared to be increased when both ATP and adenosine were added together  was present in all luminal perfusates to block ENaC Na+ conductance.When we added forskolin (5 μM) plus IBMX (100 μM), adenosine (100 μM), ATP (100 μM) or ATP (100 μM)+adenosine (100 μM) to the perfusate to activate CFTR gClher Fig. ; Table 4Two sets of different primers for CFTR as well as primers for α-, β-and γ-ENaC, NKCC1, and β-Actin all produced products of predicted sizes for each of the corresponding mRNAs Fig. .Immunoreactive CFTR antibody (gift of C. Marino) was detected in the apical domains of the bronchiolar epithelia in a continuous border of the bronchiole. The tight junctions were labeled simultaneously and appeared as punctate areas within the border staining for CFTR consistent with the expected location for these structures Fig. .The airways are extraordinarily specialized conduits for air to and from the alveoli for gas exchange. They must remain moist in order to remain flexible and to effectively filter air before it enters the delicate tissues of the alveoli. Hence, a principal function of the airway epithelia is to provide and service a continuous layer of aqueous fluid on the airway surfaces. For decades, the upper airways, trachea, and large bronchi have served as models for the entire tracheobronchial epithelial function ,22,23, a- free solution of about -16 mV [- free Ringers [in vivo. We, too, found similar, relatively small TEP voltages in microdissected porcine airways.The functions of the lower peripheral airway epithelium is poorly defined and understood because this tissue is inaccessible and relatively unamenable to standard physiological techniques for study. Nonetheless, a few courageous attempts to unravel these mysteries have been made. About ten years ago, Ballard ,14 and A Ringers . In all - free solutions in the lumen was as much as -90 mV , we found that the bi-ionic TEP with Clin vivo electrical properties in vitro, it is fundamental that the integrity of the epithelial sheet be conserved because TEP measurements depend on separation of charge. Breaks, holes, or tears in the barrier inescapably create electrical shunts, which, by allowing simultaneous back leak of epithelial current, prevent separation of charge. Even though the individual cells or groups of cells of the epithelia function and respond physiologically, the transepithelial voltage signals will be erroneously muted or lost through such shunts. In the present case, it appears that perfusion of the bronchiole without dissection from surrounding parenchyma, minimizes trauma induced shunts and allows detection of a much more complete and robust electrical signal. Unfortunately, the price for this preservation of signal is the inability to control the contra luminal solution. Keeping in mind that the undissected bronchiole is embedded in a mass of air filled alveoli together with numerous other elements of the tissue at this level, it is easy to see that changing the bath solution can have no acute effects on the composition of the contra luminal solution at the basilateral membrane of the epithelium. Consequently, we did not change the bath solution and assumed that normal Ringer solution is sufficiently similar to extracellular fluid in vivo to avoid introducing significant free solution diffusion potentials or altering the physiological composition of the native fluid present in the extracellular compartment of the in tact preparation.In order for an epithelium to reflect its - in the lumen spontaneously created a large lumen negative transmural potential , suggesting that since CFTR is not sensitive to Ca2+ mediated activation either, a.) the conductance is due to CFTR, b.) other Ca2+ activated Cl- conductances must be fully constitutively activated or not present, or c.) luminal application of the ionophore drug does not increase intracellular Ca2+ effectively. Third, CFTR is known to be inhibited by NPPB, CFTRInh-172, and GlyH-101[- conductance in the airway epithelium. However, the fact that none of the inhibitors appeared to completely inhibit the transepithelial potential might argue that another anion channel conductance is present. Two points diminish this argument. First, if the tissue is actively transporting, anion channel inhibitors will not completely ablate the TEP because that component of potential generated by the basilateral K+ emf and the apical Na+ emf should not be blocked and should be reflected across the epithelium in the TEP. Secondly, even when the CFTR Cl- channel has been isolated from active transport components, anion channel blockers do not usually completely block its conductance in native tissue [- conductance of pig nasal airways [Since obstructive airway disease in Cystic Fibrosis arises in the small airways -34 and Cluconate -37. Secoluconate -40. In c GlyH-101,21. These tissue . Moreove airways . It is n- channel that responds to activation of PKA, but both are also apparently insensitive to ionomycin. They both show equally large bionic diffusion potentials for Cl- and are several fold more permeable to Cl- than to Na+, and yet both have very low transmembrane potentials in bilateral isotonic Ringer solutions [Not only does the bronchiole resemble the sweat duct with respect to exhibiting a constitutively active Clolutions . Both arolutions ,41,44, aWe have found that the epithelium of terminal airways of the pig appears to express an anion permeability that constitutively dominates the electroconductive properties of this zone of the airway. Its anion selectivity sequence is similar to that expected for CFTR, and its activity can be enhanced by forskolin/IBMX or decreased by anion channel blockers known to inhibit CFTR. RT-PCR amplification products and specific antibodies identify CFTR in this tissue. The small airway appears to share a number of properties with the human sweat duct and may, by analogy, belong to a class of highly absorptive epithelia.CFTR: Cystic Fibrosis Transmembrane Conductance Regulator+ ChannelENaC: Epithelial Na+-K+-2Cl- CotransporterNKCC: NaTEP: Transepithelial PotentialPKA: Protein Kinase A


We conclude that the contribution of neurons to behavior can depend strongly upon tuning to stimulus dimensions that appear to be irrelevant to the current task, and we suggest that these findings are best explained in terms of the strategy used by animals to perform the task.Cortical neurons are frequently tuned to several stimulus dimensions, and many cortical areas contain intercalated maps of multiple variables. Relatively little is known about how information is “read out” of these multidimensional maps. For example, how does an organism extract information relevant to the task at hand from neurons that are also tuned to other, irrelevant stimulus dimensions? We addressed this question by employing microstimulation techniques to examine the contribution of disparity-tuned neurons in the middle temporal (MT) visual area to performance on a direction discrimination task. Most MT neurons are tuned to both binocular disparity and the direction of stimulus motion, and MT contains topographic maps of both parameters. We assessed the effect of microstimulation on direction judgments after first characterizing the disparity tuning of each stimulation site. Although the disparity of the stimulus was irrelevant to the required task, we found that microstimulation effects were strongly modulated by the disparity tuning of the stimulated neurons. For two of three monkeys, microstimulation of nondisparity-selective sites produced large biases in direction judgments, whereas stimulation of disparity-selective sites had little or no effect. The binocular disparity was optimized for each stimulation site, and our result could not be explained by variations in direction tuning, response strength, or any other tuning property that we examined. When microstimulation of a disparity-tuned site  Greg DeAngelis and Bill Newsome use microstimulation to activate small populations of neurons with different combinations of response properties in monkey visual cortex Determining how information is “read out” of sensory maps in the cerebral cortex is of fundamental importance for understanding how neural activity gives rise to cognitive processes such as perception, planning for action, and working memory. A substantial portion of our knowledge about sensory read-out comes from studies of the middle temporal (MT) visual area, an extrastriate area known to play important roles in processing visual motion information  Do MT columns that possess or lack disparity tuning contribute differentially to direction judgments? We used electrical microstimulation to test the hypothesis that neurons in the nondisparity-selective regions of MT contribute to motion perception, whereas those in the disparity-selective regions are mainly involved in depth perception. Our hypothesis was confirmed for two of the three monkeys in this study: microstimulation of nondisparity-selective sites produced strong direction biases, whereas stimulation of disparity-selective sites had little or no effect. For the third monkey, microstimulation biased direction judgments when it was applied at either disparity-selective or nonselective sites. For disparity-tuned sites that did yield effects on direction judgments, we also asked a second question. (2) Does the influence of a disparity-tuned column on direction judgments vary as a function of the actual disparity of the motion display? We found that stimulation effects were stronger when the disparity of the visual stimulus matched the preferred disparity of the stimulated column.We conclude that tuning for task-irrelevant stimulus dimensions can exert dramatic effects on the contribution of cortical neurons to a particular perceptual judgment. In extreme cases, columns tuned for an irrelevant dimension (disparity) fail to contribute at all to perceptual judgments of the task-relevant dimension (direction). In less extreme cases, the contribution of a column is modulated by tuning along the task-irrelevant dimension, so that microstimulation effects are obtained primarily when the visual stimulus possesses the right conjunction of properties (direction and disparity) to excite the column optimally. We discuss our findings in terms of the strategies employed by animals to solve the task.Microstimulation experiments were performed at 102 recording sites in area MT of three rhesus monkeys (38 sites in monkey S, 36 sites in monkey T, and 28 sites in monkey R) during the performance of the direction discrimination task illustrated in We have previously shown that disparity-selective neurons tend to occur within discrete patches of MT . Given tp << 0.001), with no significant change in the slope of the curve (logistic regression, p > 0.5). This effect is qualitatively similar to those obtained previously in our laboratories (e.g., es e.g., .p > 0.5 for shift, p > 0.25 for slope). Thus, the activity of neurons at this stimulation site did not appear to contribute to direction discrimination.p < 0.05), whereas open symbols indicate nonsignificant effects. The filled and open triangles correspond to the examples shown in r = −0.69, n = 38; monkey T, r = −0.52, n = 36; p << 0.001 for both animals). An analysis of covariance that included monkey identity as a coregressor revealed no significant difference between regression slopes for the two animals (ANCOVA, p > 0.6). Note that microstimulation almost always produced a significant effect on direction judgments in experiments for which the DTI was less than 0.5. In contrast, significant effects of microstimulation occurred much less frequently when the DTI exceeded 0.5.The result in r = −0.025, p > 0.9, n = 28). We often observed significant effects of microstimulation at sites with strong disparity tuning. It is worth emphasizing that all of the data in We obtained quite different results in a third animal, monkey R B. For thn = 51) was significantly lower than the mean psychophysical thresholds for monkey S (21.5% ± 0.9% SE, n = 89) and monkey T (22.8% ± 1.0% SE, n = 70) (Student's t-test, p < 0.0005 for both comparisons). In contrast, the average slope of the psychometric functions did not differ between the three animals (ANOVA, p > 0.7). We shall consider these issues further in the Discussion.The individual differences between monkeys in the data of r = 0.45, p = 0.01, n = 32) as a function of the DTI. The data reveal a clear inverse relationship between the two effects. Columns with low DTIs produce large effects on direction discrimination performance and little or no effect on depth discrimination. In contrast, columns with large DTIs show the converse pattern. In this monkey, therefore, the functional segregation of MT columns according to the strength of disparity tuning is particularly clear.Monkeys T and R were subjects both in the current set of experiments and in a separate study in which we showed that stimulation of disparity-tuned columns influences perceptual judgments of depth . For theIt is important to note that the differences between animals seen in The striking result in n = 162) and in three additional monkeys (n = 409). Across this unbiased sample of 571 recordings, we find no significant correlation between Disparity Tuning Index (DTI) and Direction Tuning Index  between direction-tuning bandwidth and DTI across our sample of 571 MU recordings, indicating that the sharpness of direction tuning also does not covary with disparity selectivity. These observations, combined with the fact that we only performed microstimulation experiments in the portions of MT with the strongest direction tuning , we computed the standard deviation (SD) of directional preferences within a 400-μm region around each microstimulation site. We find no significant correlation between the strength of microstimulation effects and the SD of preferred directions . Correspondingly, there is no significant correlation between the strength of the microstimulation effects and the peak MU response (r = 0.17, p = 0.08), and all of the microstimulation effects in Systematic variations in responsiveness as a function of disparity tuning (point 3 above) can be excluded as a possible explanation for our findings because there is no correlation between the peak response of MU activity and the DTI , indicating that variations in these parameters are also unlikely to account for the results shown in Finally, using a dataset of 409 MU recordings and a multiple regression analysis, we also tested for correlations between DTI and several other response properties, including preferred speed, Speed Tuning Index, RF eccentricity, optimal stimulus size, and percentage of surround inhibition. None of these variables was significantly correlated with DTI (Although significant microstimulation effects were rare at sites with strong disparity tuning in monkeys S and T, significant effects occurred at a good number of sites with moderate disparity tuning (i.e., DTI > 0.4). At these sites, and at many sites in monkey R, we could ask whether the efficacy of microstimulation varied when the random-dot stimulus was presented at different points along the disparity-tuning curve of the stimulated column.The logic of this experiment is illustrated for a disparity-selective site in p < 0.001). In the second block of trials, we presented dots at the nonpreferred disparity (−0.5°), and microstimulation exerted no effect whatsoever on the monkey's choices (p > 0.5). To be certain that this effect did not result from some nonstationarity in electrode position, cell responsiveness, etc. (p < 0.001). At this stimulation site, therefore, we were able to switch the result from a very substantial effect to no effect and back again simply by manipulating the disparity of the random-dot stimuli.ss, etc. , we collp = 0.025). We chose three different disparities at which to perform the direction discrimination task: 0°, 0.6°, and −0.6°. p < 0.0001), with no corresponding change in slope (p > 0.4).p < 0.05) for at least one of the two disparities. We computed a Microstimulation Selectivity Ratio (MSR) as follows:The individual example sites in Figures PE is the effect of microstimulation when dots are presented at the preferred disparity, and NPE is the effect when dots are presented at the nonpreferred disparity. This index is a standard contrast measure, except that the quantities in the denominator are absolute values. This formulation was necessary to keep the index bounded between −1.0 and 1.0.where r = 0.37, F(1,61) = 9.9, p < 0.005), with no significant differences between the three monkeys (F(2,61) = 0.14, p > 0.8).Thus, as hypothesized see , microstUsing microstimulation to probe the link between neuronal activity and behavior, we have tested whether the contribution of MT neurons to direction discrimination depends on their disparity selectivity. This work addresses the general question of how neurons that are tuned to multiple stimulus dimensions contribute to behavior in situations where one or more of these stimulus dimensions are task-irrelevant. Relatively little is currently known about how the responses of sensory neurons are pooled by decision mechanisms see  and how How can we explain the finding see A that reA second possibility is that the output connections of disparity-selective and nonselective regions of MT have different targets, such that decision mechanisms for motion receive input from nondisparity-selective portions of MT whereas decision mechanisms for depth receive input from disparity-tuned regions. Experiments have not been done to test this hypothesis, so we cannot rule it out. One argument against this idea, however, is that one of the three monkeys (monkey R) did not show a dependence of microstimulation effects on disparity selectivity see B. Thus, A third possibility, which we favor, is that our findings reflect the strategy that each monkey adopted for reading out motion signals from MT during the extended period of training on the task. In this scenario, all regions of MT could project to decision mechanisms for both motion and depth, but the relative weights of the connections would vary with the animal's task strategy. This would allow the read-out strategy to be altered rapidly based on the demands of the task. In our experiments, one strategy for performing the task would be to extract motion signals from all MT columns with the appropriate direction selectivity and spatial RF, regardless of their disparity selectivity. This strategy would entail pooling signals from many columns, including those with unfavorable signal-to-noise ratios due to their poor responsiveness to stimuli of nonoptimal disparity.A second strategy, which could yield better performance, would be to monitor primarily columns that are maximally activated by the stimulus, but this would entail pooling responses from columns with different disparity preferences when the stimulus disparity changed. Thus, some sort of complex “switching” would be required to route information to the decision process from the set of columns optimal for each experiment. A third, and perhaps the simplest, strategy would be to monitor motion signals only from the nondisparity-selective portions of MT; these columns would respond well to all stimulus disparities, providing a good signal-to-noise ratio for all stimulus sets on which the monkey was trained. This strategy offers the further advantage that one can monitor the same set of columns for all stimulus conditions in our task. Given that correlated noise among neurons limits the benefits of pooling across large populations of neurons , this laIf monkeys were to adopt the simple strategy of monitoring only the nondisparity-selective regions of MT, then the microstimulation results shown in r = 0.55, p < 0.01) than for monkey R (r = 0.36, p = 0.15).We found a statistically significant, but relatively weak, dependence of microstimulation effects on the difference between the preferred disparity of MT neurons and the stimulus disparity see . What acAnother possible source of variability in Considering these likely sources of variability, the fact that we see a significant overall effect in Many of the standard experimental approaches in systems neuroscience  find their utility in exposing correlations between neuronal activity and external stimuli or behavioral states. Of course, finding signals that are correlated with behavior does not prove that those signals underlie the behavior. The value of electrical microstimulation, reversible inactivation, and lesion techniques is that they can establish causal links between neural activity and behavior. In this study, we only microstimulated at sites in MT that had strong directional selectivity; thus, one might assume that all sites would be equally likely to contribute to performance of the direction discrimination task. The central finding of this study is that the contribution of MT direction columns to task performance is modulated by the tuning of the neurons to a stimulus variable that is irrelevant to completion of the task. Thus, even within a single area of the brain, the causal linkage between neurons and behavior may depend on uncontrolled stimulus dimensions, and may be determined by unexpected factors such as task strategy. This result highlights the importance of causal techniques for studying the neural basis of behavior, and suggests that microstimulation studies may be able to reveal how high-level task strategies modulate the read-out of neuronal signals from topographic maps in the brain.(Macaca mulatta) are described elsewhere , all of which had previously been subjects in other studies in the laboratory. Each animal had a scleral search coil implanted in at least one eye (monkey S had coils in both eyes) to allow monitoring of eye position. In addition, each subject was equipped with a head restraint post and a stainless-steel recording chamber that was positioned over the occipital cortex. Electrodes were introduced into the visual cortex through a transdural guide tube that was positioned within a square array of grid holes at 1-mm intervals .All visual stimuli used in this study were dynamic random-dot patterns presented on a standard 21-in. color display (Sony 500PS, Sony Corporation, New York, New York, United States). The display subtended 39° × 29° at the viewing distance of 57 cm and was refreshed at a rate of 100Hz. The visual stimuli were generated by a Cambridge Research Systems VSG2/3 board (Cambridge Research Systems Ltd., Rochester, United Kingdom) that was housed in a dedicated PC. Stereoscopic presentation was achieved through the use of ferroelectric shutters (Displaytech, Inc., Longmont, Colorado, United States) that were switched in antiphase for the two eyes. Left and right half-images were presented on alternate video frames, and the shutters were synchronized to the vertical refresh, thus exposing each eye to the appropriate visual stimulus on alternate frames. With this technique, the quality of stereo separation is limited mainly by phosphor persistence. Thus, random-dot stimuli were always presented using the red gun only, since the red phosphor has a much faster decay than either the green or blue phosphors. We achieved a contrast ratio of approximately 40:1 (“open” eye:“closed” eye) using this approach, and “ghosting” artifacts were barely visible, even under dark-adapted conditions.2-s) for the central patch and 8 dots/(deg2-s) for the background.Monkeys performed two separate tasks in these experiments: a visual fixation task, and a direction discrimination task. In the visual fixation task, a small, yellow fixation point (FP) appeared to begin each trial, and the monkeys were required to maintain fixation within a 2° × 2° or 3° × 3° electronic window, centered on the fixation target, until the fixation target was extinguished. The monkeys received a liquid reward for successful fixation, typically 0.1–0.15 ml of water or juice. If the monkey broke fixation before the end of a trial, the trial was aborted, the data were discarded, and the monkey was not rewarded. During the fixation period, a bipartite random-dot stimulus was presented for 1.5 s. It consisted of a central, circular patch of coherently moving dots that could be presented with variable binocular disparity, and which covered the receptive field of the MT neurons under study. To assist the monkey in maintaining binocular convergence on the FP, we filled the remainder of the visual display with zero-disparity dots that were randomly repositioned every fourth video frame (25 Hz), thus producing a twinkling, zero-disparity background. Each dot was approximately 0.1° in size. Dot density was 32 dots/ operating in constant-current mode. The current was a train of biphasic pulses with a frequency of 200 Hz and an amplitude of 20 μA. Each biphasic event consisted of a 200-μs cathodal pulse followed by a 200-μs anodal pulse, with a 100-μs gap between the two. Microstimulation parameters were chosen to elicit robust perceptual biases but were well below the current and frequency levels at which stimulation has been shown to flatten the slope of the psychometric function . MicrostWe searched for candidate microstimulation sites by examining the tuning properties of MU activity at regular intervals of 100 μm along electrode penetrations through MT. At each recording site, we rated the strength of direction selectivity on a scale from 1 to 3 (3 = strongest tuning), and we carefully estimated the preferred direction of motion (see p > 0.3). Whenever possible (e.g., see At each identified microstimulation site, we performed the following battery of tests. (1) First, we carefully mapped the MU RF of the MT neurons by dragging a small patch of moving dots (100% coherence) through the RF with a pointing device. Spike densities were plotted on a Cartesian map of visual space during this process to facilitate visual mapping of the RF. In addition, we mapped the direction and speed selectivity of the neurons by moving a cursor throughout a polar direction-speed domain while spike densities were again plotted on the screen. From this procedure, we determined the location and size of the MU RF, as well as the preferred direction and speed of motion. We also estimated the range of disparities over which the neurons were selective, and these parameters were then used in subsequent quantitative tests. 2) We next measured a disparity-tuning curve for MU activity at the identified stimulation site, while the monkey performed a block of fixation trials. Nine evenly spaced disparities were typically tested within the disparity range determined from our initial qualitative probing . Neural signals were amplified, filtered (0.5–5.0 kHz), and discriminated using conventional electronics (Bak Electronics, Inc.), and event times were stored on magnetic disk with 1 ms resolution. To record MU activity, we simply set the threshold level of our window discriminator to approximately 1–2 SD above the noise level. Thus, a MU event was defined as any deflection of the analog signal that exceeded this threshold. Since the absolute frequency of the MU response depends heavily upon the event threshold, we attempted to achieve a consistent response magnitude from site to site by adjusting our event threshold such that the spontaneous activity level was in the range from 50 to 100 events/s. This setting typically yielded peak MU responses in the range of 300–500 events/s (mean 378.5 ± 78.3 SD).Horizontal and vertical eye-position signals were low-pass filtered with a cutoff frequency of 250Hz, sampled at 1 kHz, and stored to disk at 250 Hz.To construct disparity-tuning curves, we computed the firing rate for each trial during the 1-s stimulus presentation, and we plotted the mean firing rate (± SE) as a function of the horizontal disparity. Smooth curve fits to disparity-tuning curves were achieved using a cubic spline interpolation. To quantify the strength of disparity tuning at each stimulation site, we computed the DTI as follows:Rmax denotes the response to the preferred disparity, Rmin denotes the response to the antipreferred disparity, and S indicates the spontaneous activity level. Values larger than unity can occur if Rmin is less than S. For the quantification of direction-tuning strength  in which we obtained quantitative measurements of both direction tuning and disparity tuning. There is no significant correlation between Direction Tuning Index and Disparity Tuning Index (DTI) across the sample. Note also that the data from monkey S overlap completely with the data from the other animals, indicating that monkey S was not unusual.(358 KB EPS).Click here for additional data file.Figure S2Monkey S is shown in black; monkey T in red.(A) The strength of the microstimulation effect is plotted against the SD of direction preferences within a 400-μm window centered on each stimulation site (five recording sites, 100 μm apart). There is no significant correlation between these variables, indicating that variability in direction preferences (within the observed range) did not determine the efficacy of microstimulation. Note, however, that all stimulation sites were chosen to have a small range of preferred directions; we did not apply microstimulation at locations in MT where the direction preference changed rapidly over short distances.(B) There is also no significant correlation between the DTI of MU activity at each stimulation site and the SD of direction preferences. This shows that disparity-selective microstimulation sites did not have larger variations in direction preferences.(216 KB PS).Click here for additional data file.Figure S3See (320 KB EPS).Click here for additional data file.


Rural Indian tribes are anthropologically distinct with unique cultures, traditions and practices. Over the years, displacement and rapid acculturation of this population has led to dramatic changes in their socio-cultural and value systems. Due to a poor health infrastructure, high levels of poverty and ignorance, these communities are highly vulnerable to various health problems, especially, communicable diseases including HIV/AIDS. Our study sought to assess knowledge, attitudes and practices regarding sexuality, and the risk factors associated with the spread of HIV/AIDS and STDs among these communities.A nested cross sectional study was undertaken as part of the on going Reproductive and Child Health Survey. A total of 5,690 participants age 18–44 were recruited for this study. Data were obtained through home interviews, and focused on socio-demographics, knowledge, attitudes and behaviors regarding sexuality, HIV/AIDS and other STDs.The study revealed that only 22% of adults had even heard of AIDS, and 18 % knew how it is transmitted. In addition, only 5% knew that STDs and AIDS were related to each other. AIDS awareness among women was lower compared to men (14% vs.30 %). Regarding sexual practices, 35% of the respondents reported having had extramarital sexual encounters, with more males than females reporting extramarital affairs.Lack of awareness, permissiveness of tribal societies for premarital or extra-marital sexual relationships, and sexual mixing patterns predispose these communities to HIV/AIDS and STD infections. There is a dire need for targeted interventions in order to curtail the increasing threat of HIV and other STDs among these vulnerable populations. India is the second most populous nation in the world and has changing sociopolitical and demographic characteristics as well as varied morbidity and mortality patterns . These cAs a direct result of these challenges, India has begun to assess and monitor the impact of HIV/AIDS throughout the country's various states and regions with the assistance of several international health organizations. According to 2003 estimates from UNAIDS, approximately 5.1 million individuals in India are infected with the HIV virus . This trFor effective control of the spread of HIV/AIDS, it is crucial to have data on knowledge, attitudes and behavioral practices for specific population as research has shown that socio-cultural influences, traditional lifestyles, societal norms, and traditions influence HIV/AIDS transmission rates ,6. BecauThe rapid spread of HIV/AIDS in rural Indian communities has been attributed to the country's poor health infrastructure, poverty and lack of awareness ,10. DespThroughout India, approximately 8% of the population lives within rural tribal communities, which are collectively referred to as 'Tribes'. These communities are geographically distinct; with each tribe having its own unique customs, traditions, beliefs and practices. Even within a particular tribal entity, differences in dialect, health practices, unique customs, values, and traditions are apparent. In rural Indian communities indices of reproductive health are typically very poor: maternal mortality rate is about 230 per 100,000 live births and 61.2% of the women suffer from at least one gynecologic pathology . BecauseWe conducted a cross-sectional study nested within an existing enumerative study referred to as the Reproductive and Child Health Survey (RCHS). The RCHS was initially designed to enumerate and ascertain basic demographic and health profiles for all tribal members. Data collection for this particular study was done in two phases. Phase one involved adding additional questions to the original RCHS study to assess risk factors  associated with the transmission of HIV/AIDS and other communicable diseases.The study population comprised of tribal communities living in the southern region of Karnataka Figures ,2. MembeBecause of the enumerative nature of the RCHS, all persons within the age group 18–44 years who participated in the RCHS study were included for phase one of this study. This age group was selected as the focus of the initial study was on reproductive health issues.A semi-structured questionnaire with both open- and close-ended questions was developed to collect information on knowledge, attitudes and behaviors regarding HIV/AIDS, as well as other relevant demographic information not included in the RCHS. The instrument was developed in English, translated to the native language, and subsequently back translated to English for content and language verification. The survey instrument was field-tested for validity purposes and modified accordingly.A team of ten interviewers from the local tribal communities with a minimum of high school education were selected and trained for two weeks to ensure uniform and high-quality data collection. All adults were interviewed separately to ensure confidentiality. Each interviewer read out each of the questions and response choices (where appropriate) to the interviewees and recorded all answers directly on the questionnaire. Verbal informed consent was obtained from each respondent prior to starting the interview.Data were entered into an electronic database using Sybase Central Software (Sybase, Inc. Dublin, CA). To ensure confidentiality, all respondent identifiers were expunged to create a secondary data set that was used for the final analysis. Frequency tables were generated for selected demographics and health related categorical variables. In addition, univariate analysis was performed on relevant continuous variables. The findings are presented below.Ethical clearance was obtained from ethical boards within each of the tribal communities, and appropriate government agencies were informed about the study objectives. Permission was also obtained from the Institutional Review Board (IRB) at the University of South Florida in Tampa, Florida. Verbal informed consent was obtained from the local leaders, and the individual participants. Because of the high levels of illiteracy, it was not feasible for us to obtain written consent.A total of 11,379 individuals from the 5 tribal communities had been enumerated as part of the RCHS. Of these, 5,690 were within the study age range (18–44 years) and formed the basis for this analysis.Table The findings revealed that these tribal communities did not have a structured marital system; instead members practiced a form of serial monogamy in which they change partners and remarry every four to five years. Regarding sexual practices, 35% of the respondents reported either premarital affairs or extramarital affairs Table . HoweverAmong these communities, there was a low level of knowledge on HIV/AIDS; only 22 % of all study participants (n = 1,252) had heard of AIDS Table . Among tIn today's modern world, it is difficult to imagine societies that are still socially and culturally isolated from the rest of civilization; however, they do exist. The tribal societies throughout India have remained socially and culturally alienated from mainstream Indian society until developmental and conservation activities in tribal areas forced interactions between them. Displacement of the tribal people of southern Karnataka has led to a complex process of rapid acculturation and loss of cultural identity; as they struggle to maintain their traditional social structure, they must adopt new skills, beliefs, and practices necessary for success in their new environment. During this acculturation process, they have been faced with a myriad of public health challenges complicated by poverty, ignorance, and reluctance to abandon traditional beliefs and practices that would allow them to assimilate successfully.To date this is the first health related study among the displaced tribal communities of southern Karnataka that has attempted to assess risk threshold for the transmission and spread of HIV/AIDS and other STDs. It is not surprising that knowledge and awareness about HIV/AIDS and STDs was very low among tribal communities compared to the national figures given the degree of isolation, low literacy rates, and minimal access to information.The high level of poverty, inadequate health resources, ignorance and high-risk beliefs and practices among the tribal communities has contributed to the vulnerability of this population. As such it has created a highly susceptible population for the rapid spread of HIV/AIDS and other STDs' as well.Because tribal members are forced to migrate outside of their communities in search for work and increased wages, this may contribute to the spread of HIV/AIDS as many engage in extramarital affairs, seek commercial sex partners, or are under the threat of sexual harassment . While there has been limited scientific research exploring the cultural context of extramarital sexual behaviors, it is generally noted that in these communities, extramarital affairs are condoned and widely practiced especially during periods when women are pregnant or nursing or during period of travel for work . This kiIt is evident from this study that the Indian tribal community is experiencing a latent phase that is potentially a precursor for an HIV/AIDS epidemic. There is a high prevalence of behavioral risk factors, coupled with ignorance, and inadequate health infrastructure thus creating a potential risk for rapid spread of HIV/AIDS, as well as other related diseases. In a country that is struggling to contain the spread of HIV, it is particularly important for concerned parties to pay attention to this population. Currently, virtually no resources are allocated toward the treatment of those infected with HIV/AIDS; the main stay of management is through education and preventive measures to control the spread of the scourge as they represent the most practical and cost effective strategies in this developing nation. It therefore becomes imperative and urgent to address the health concerns revealed in our study in order to formulate effective, culture-sensitive and appropriate intervention programs so that an imminent disaster (i.e. HIV/AIDS epidemic) in this remote and isolated communities can be averted.The author(s) declare that they have no competing interests.All the authors were involved in the design of the study, analysis, interpretation and development of the manuscript.The pre-publication history for this paper can be accessed here:


Dedifferentiated liposarcomas represent heterogeneous tumors with lipomatous and nonlipomatous elements starkly juxtaposed. It is thought that the high grade nonlipomatous elements of the tumor portend a worse prognosis.A 19.8 kg heterogeneous retroperitoneal tumor was successfully and completely resected. Because of its extent, no additional treatment modalities were practicable. The tumor soon recurred. The recurrent tumor differed from the primary tumor in that it was more homogeneous, consisting mainly of nonlipogenic, calcific tissue.Dedifferentiated liposarcomas are known to have a very high recurrence rate. The biological behavior of dedifferentiated liposarcomas is likely dictated by the most aggressive element of these heterogeneous tumors. Sarcomas arising from the retroperitoneum are rare tumors, accounting for 10–15% of all soft tissue sarcomas . These fA 65-year-old male presented with a three-week history of progressively worsening abdominal distension. He denied any abdominal pain but stated that he noticed an increased frequency of bowel movements. His past medical history was unremarkable. On examination, he was afebrile and had a hugely distended abdomen with an immobile, nontender mass occupying all four quadrants of the abdomen. Computed tomographic (CT) scan revealed a large, heterogeneous lobulated mass occupying most of the abdomen Figure . The perWhile neoadjuvant chemotherapy and radiation comprise a frequent approach for retroperitoneal sarcomas at our institution, the extent of the tumor made this approach unfeasible. Resection was therefore planned unless an intraoperative biopsy revealed lymphoma. Resection was accomplished through a T-type incision Figure , and entThe tumor was submitted for histological examination in two parts measuring, 30.0 × 10.0 × 6.5 cm and 32.0 × 20.0 × 10.0 cm, weighing 19.8 kg in total Figure .The patient was discharged from hospital on the seventh postoperative day. He was followed in the surgical oncology outpatient clinic monthly. Four months after resection the patient had a follow-up CT scan, which demonstrated an intra-abdominal recurrence consisting almost completely of a calcified, nonlipomatous tumor Figure . The patde novo, although secondary dedifferentiation can occur with multiple recurrences of a well differentiated liposarcoma [The patient in our case manifested the dedifferentiated variant of liposarcoma. The term "dedifferentiated liposarcoma" refers to the development of a high grade nonlipogenic sarcoma juxtaposed to a well differentiated liposarcoma . The majosarcoma . CT and osarcoma . Calcifiosarcoma ,6. Otherosarcoma ,7. A furosarcoma .Among liposarcomas, the presence of features of the dedifferentiated variant strongly portends a worse prognosis. The overall 5-year survival of dedifferentiated liposarcomas is 20%; the 5-year survival of well differentiated liposarcomas is 83% . DediffeWhile generally the phenotype of the nonlipogenic component does not impact prognosis of dedifferentiated liposarcomas, the presence of calcifications has been identified as an adverse prognostic factor . In the Complete resection of the tumor is perhaps the most important factor determining long-term survival. Unfortunately, the rate of complete respectability is only about 53% . As illuDedifferentiated liposarcomas represent aggressive variants of liposarcomas. Each morphological element of these heterogeneous tumors may manifest completely different biology. The overall biological behavior of dedifferentiated liposarcomas is likely dictated by the most aggressive element, which typically resides in the nonlipomatous portion of the tumor.None declared.SK, HB, WT, and OB made substantial contributions to the intellectual content of the paper, in the interpretation of data, and in drafting the manuscript. All authors read and approved the final manuscript.