The geminivirus and nanovirus families of DNA plant viruses have proved to be a fertile source of viral genomic sequences, clearly demonstrated by the large number of sequence entries within public DNA sequence databases. Due to considerable conservation in genome organization, these viruses contain easily identifiable intergenic regions that have been found to contain multiple DNA sequence elements important to viral replication and gene regulation. As a first step in a broad screen of geminivirus and nanovirus intergenic sequences for DNA segments important in controlling viral gene expression, we have 'mined' a large set of viral intergenic regions for transcriptional enhancers. Viral sequences that are found to act as enhancers of transcription in plants are likely to contribute to viral gene activity during infection.Agrobacteria-infused Nicotiana tobacum leaf tissue. Of the 105 elements tested, 14 were found to reproducibly elevate reporter gene activity , while 91 elements failed to increase luciferase activity. A previously described "conserved late element" (CLE) was identified within tested repeats from 5 different viral species was found to have intrinsic enhancer activity in the absence of viral gene products. The remaining 9 active elements have not been previously demonstrated to act as functional promoter components.DNA sequences from the intergenic regions of 29 geminiviruses or nanoviruses were scanned for repeated sequence elements to be tested for transcription enhancing activity. 105 elements were identified and placed immediately upstream from a minimal plant-functional promoter fused to an intron-containing luciferase reporter gene. Transient luciferase activity was measured within Arabidopsis promoter regions. These data provide a useful starting point for virologists interested in undertaking more detailed analysis of geminiviral promoter function.Biological significance for the active DNA elements identified is supported by repeated isolation of a previously defined viral element (CLE), and the finding that two of three viral enhancer elements examined were markedly enriched within both geminivirus sequences and within  A relatin planta promoter activity using isolated or modified geminivirus or nanovirus intergenic sequences [To test the enhancer mining approach we chose to examine a collection of geminivirus and nanovirus intergenic sequences obtained from GenBank. There are a relatively large number of available sequences for these DNA viruses and due to conserved genomic organization they contain easily identifiable intergenic regions . Additioequences -21. Althequences , very feequences .in planta, any elements identified using this approach are likely to contribute to regulation of in vivo viral gene expression during plant infection. By allowing relatively large numbers of viral sequences to be examined using a defined system, the approach has the potential of generating data useful in comparing positively acting viral promoter elements within and between viral families. In addition, identification of elements that are active in planta in the absence of viral infection provides results pertinent to understanding virus-host interactions at the level of gene control. Finally, the resulting list of active and inactive viral sequences provides a valuable starting points for subsequent, more detailed, analysis of transcription regulation of individual viruses.The described enhancer mining of viral sequences is not intended to be a comprehensive analysis of viral promoter structure since by design it is limited to identification of promoter elements that up-regulate gene expression and that make use of endogenous plant transcription factors available within the un-infected test plant. However, based upon their iteration, location within intergenic regions, and ability to enhance transcription et al [The initial search for sequence repeats was performed on the major intergenic regions of 29 different geminivirus or nanovirus genomic sequences Figure . The seaet al , the resin vivo assay were subsequently tested using an in vitro dual-luciferase® system from Promega Corp. and produced levels of enhancement very similar to those obtained using the in vivo assay . The observed enhancement of promoter activity (~2 fold) is relatively modest compared to other viral transcriptional enhancers that have been isolated and tested  and, as controls, several well defined plant promoter element consensus sequences  and may, therefore, be similar or identical to endogenous plant promoter elements. In order to test for enhancement of viral enhancer sequences within higher plant promoters, the PatMatch page of the TAIR web site  was used factors ,34, and  factors ) and abs factors ).Arabidopsis genome sequence and PatMatch. As an alternative, screens were performed to determine frequencies of occurrence for viral enhancers (and control plant elements) within a sequence database consisting of all geminivirus or nanovirus GenBank entries as of May 13, 2004 [Arabidopsis PatMatch datasets. The searched viral sequence database has the potential for bias due to the existence of a numerous entries containing only coding regions or only intergenic sequences, as well as some duplication of sequences in separate entries. Any such bias should, however, similarly affect the baseline frequency values resulting from searches using the 18 matched random oligonucleotides  versus 'coding sequence' frequency comparisons that were possible using the fully annotated 13, 2004 , and theArabidopsis upstream databases (-3000 to -1001, -1000 to -501, and -500 to -1 bp) were normalized relative to the value obtained for each element's occurrence within the A. thaliana coding sequence database . In addition to the predicted frequency values, in each case, the element's observed frequency was also compared to a value generated using the average of 18 random oligomers having the same length and base composition as the element tested . Results for the DRE element were less convincing (2.13 vs 1.46 in the -1 to -500 dataset) and likely reflect lower functional usage of this element within the Arabidopsis genome [The results of the searches are displayed in Table Arabidopsis promoters is less obvious (CLE = 3.9 in the -1 to -500 database vs 2.79 for random sequences). The observed relatively small CLE enrichment is consistent with reports of a low frequency of occurrence for a CLE-like "TCP domain" binding consensus sequence (Gt/cGGNCCC) within Arabidopsis promoters [Arabidopsis promoters containing the TCP domain consensus binding element were found to function in transgenic tobacco and to show reduced activity after mutation of the element's core sequence [As expected, the CLE consensus sequence (GTGGNCCC) was found to be markedly enriched within the viral database, occurring 6 times more frequently than the mean of 18 random 8-mers of identical base composition . This frequency is similar to that found (17.11 vs 3.42) using a short sequence of identical base composition and length that matches a highly conserved replication stem-loop sequence (CGCGNCCA), a component that is evolutionarily conserved within the geminivirus population . Enhanceromoters . It is psequence .The test sequences for plant element occurrence within the viral database (ABRE-like = 2.4 vs 1.28 and G-box = 3.81 vs 1.43, DRE = 1.09 vs 0.85) provide further indication of the technique's utility. The G-box viral frequency is consistent with a previous report that a G-box element contributes to transcriptional regulation from the major intergenic region of Tomato Golden Mosaic Virus {TGMV, . Similar TC-rich regions have been reported within plant promoter regions [Of the remaining viral elements tested against the es Table , only th regions ,41, but Except for the CLE elements, none of the active elements identified in this work have been experimentally reported as regulatory components of viral promoters. This is likely a reflection of both the limited number of geminivirus and nanovirus promoters that have been examined in detail ,27,42,43Arabidopsis upstream sequences for at least the CLE and TC-rich (DR08/13) classes of elements, strongly supports participation of the enhancers in viral gene expression.This screen of viral intergenic repeats was undertaken to specifically identify general transcriptional enhancing elements contained within intergenic regions of a subset of geminivirus and nanovirus genomes. The screen was successful in demonstrating transcriptional enhancer activity from one proven viral promoter element and several previously unidentified elements. The occurrence of the repeated elements within intergenic regions, combined with the clear enrichment within viral sequences and The technique of testing isolated elements represents an alternative to normal promoter-by-promoter dissection and provides a useful tool for screening promoter regions for potential functional elements that have been implicated by any number of possible criteria (e.g. copy number, evolutionary conservation, comparison of promoters with similar function, microarray data, etc.). Although the number of elements tested is relatively small and, so far, only representative of promoters from the geminiviruses and nanoviruses classes of plant viruses, there is a clear trend suggesting that directly repeated elements  are more likely to play significant roles in the enhancement of transcription than inverted repeats. This work represents one of the first attempts to directly screen for individual plant promoter elements that are isolated from their native promoter context. It is therefore, difficult to gauge the actual contribution of any of the elements identified to viral gene regulation and biological activity. These results do, however, provide a useful starting point for more detailed analyses of not only geminivirus and nanovirus promoters, but also overall plant promoter structure-function relationships.The search for repeated DNA sequences was performed by visual inspection of computer-generated dot matrix comparisons (criteria: ≥ 66% identity, 10 base window, GeneWorks v2.5.2, Oxford Molecular Group Inc.). Dot matrices generated using each viral plus strand plotted against itself were used to identify direct repeats while inverted repeats were found by plotting each plus strand against its complement.Agrobacteria tumefaciens strain, EHA105 [Agrobacteria strains each contain, in addition to the test plasmids, a second, compatible, binary transformation vector expressing an intron-modified version of the Renilla reniformis luciferase gene (RiLUC) [The identified repeats were synthesized as DNA cassettes containing the duplicated elements in their original orientation, either directly repeated with spacer sequence ('DR', 41 elements), inversely repeated with spacer sequence 'IR', 45 elements), or palindromic inverted repeats without spacer ('PAL', 20 elements). In order to limit the tested component to only the repeated elements themselves, any sequence occurring between the viral repeats (ranging from 0 to 146 bp, median separation = 9 bp) was replaced with a 10 bp randomized stuffer sequence (GAAGATAATC). The resulting cassettes were inserted immediately upstream from a minimal promoter (-46 to +1 relative to transcription start, 35S min) reporter system derived from the cauliflower mosaic virus (CaMV) 35S promoter fused to an intron-modified firefly luciferase (FiLUC) gene   under co (RiLUC) . The FiLAgrobacteria harboring the test and normalization binary plasmids were grown at 28°C in LB media containing the appropriate antibiotic selection (25 μg/mL kanamycin sulfate or 100 μg/mL spectinomycin) until an OD600 of 0.8 was achieved. The resulting cultures were centrifuged at 3000 rpm for 15 minutes, washed and re-suspended in an equal volume of infiltration media (50 mM MES, 0.5% glucose, 2 mM NaPO4, 100 μM Acetosyringone) before being mechanically infused (5 ml syringe) into multiple individual tobacco (N. tobacum, cv. SR1) leaves (2–4 leaves per test construct). Assays were performed in groups of 4–8 constructs and the resulting luciferase activities (both FiLUC and RiLUC) determined after 3–4 days using an in vivo floating leaf-disk assay developed for this enhancer screen [in vivo assays were each measured for light production in separate wells of a white-walled 96 well microtiter plate (FLUOstar Optima luminometer® from BMG Lab Technologies Inc.) and all elements that tested positive in the in vivo assay were subsequently confirmed using the in vitro dual-luciferase® from Promega Corp (assays performed according to the manufacturers instructions, separate leaf disks from the same leaf infusions were used for the in vivo assays). Each test group included an infusion containing the 35S min construct . In order to compare the various assay systems, all activities were normalized to the activity of the 35S min construct included within each assay set (35S min activity arbitrarily set to 1.0).r screen . Test coA. thaliana genomic sequence at the TAIR, PatMatch web site [Arabidopsis coding sequences {"GI CDS (- introns, - UTRs)"}, and various lengths of upstream regions {"Locus Upstream Sequences", -1 to -500, -1 to -1000 and -1 to -3000}. Results from the -500 search were subtracted from the -1000 results, to generate hits from -501 to -1000 and -1000 results subtracted from the -3000 data to calculate hits from -1001 to -3000. In order to allow direct comparison between searches in different databases, using sequence elements of differing length and base composition, the number of database hits was corrected for the size of the database (number of hits divided by the database size in mega-base pairs {Mbp}) and base composition (hits/Mbp divided by the predicted number of hits per Mbp using upon the element sequence and base composition of each search database). The dataset base compositions were determined from downloaded sequence files and are: A. thaliana CDS: A/T = 55.8%, G/C = 44.2%; A. thaliana upstream (-1 to -500): A/T = 67.43%, G/C = 32.57%; A. thaliana upstream (-501 to -1000): A/T = 66.24%, G/C = 33.76%; viral: A/T = 56.2%, G/C = 43.8%. The resulting frequency of occurrence is a corrected number of hits per mega-base pairs (cHits/Mbp). For ease of comparison between elements, all of the cHits/Mbp values have been normalized to the corresponding cHits/Mbp number from the A. thaliana CDS database (set arbitrarily to 1.0). Correction of the element's frequency using the calculated random probability of occurrence does not account for the possible impacted by intrinsic base-order bias that may occur within each sequence database, specifically the coding region database. These biases can potentially shift cHits/Mbp numbers markedly from those calculated using simple random base composition frequencies. To help confirm the significance of any observed enhancement in an elements frequency, mean cHits/Mbp values for 18 randomly generated sequences that match each test sequence for base composition and length were determined to provide a baseline value for comparison to that of the test element (shown in parenthesis, Table Since the element enrichment approach works best when applied to relatively short, core consensus sequences , databasweb site . The gemA patent application is being considered for synthetic plant promoters containing some of the elements described in this article.Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.in vivo assay used to quantify enhancer activity. All authors read and approved the final manuscript.JV conceived of the study, participated in its design and coordination and drafted the manuscript. KM performed much of the search for short repeats within viral sequences and contributed to development of protoplast-based reporter gene assays. CIC generated and tested all the elements examined and developed the Excel worksheet listing viral elements that fail to enhance expressionClick here for file


It has been shown that transthyretin (TTR) exists in different molecular variants. Besides point mutations associated with different diseases such as amyloidosis, other posttranslational modifications occur that might be of diagnostic interest.TTR levels as determined by ELISA in plasma and urine of healthy individuals were 489 ± 155 μg/ml plasma and 46 ± 24 ng/g creatinine, respectively. Average levels in urine of pregnant women were 45 ± 65 μg/g creatinine. The molecular heterogeneity of TTR was analyzed using a high-throughput mass spectrometric immunoassay system. TTR was extracted from plasma or urine onto an antibody-coated (via protein A) affinity chip surface (PS20) using the surface-enhanced laser desorption/ionization (SELDI) technique. Subsequently samples were subjected to time-of-flight mass spectrometry (TOF-MS). In healthy individuals, TTR in plasma occurred rather consistently in two variants of 13732 ± 12 and 13851 ± 9 Da for the native and S-cysteinylated forms and at a smaller signal of 14043 ± 17 Da for the S-glutathionylated form. In urine of pregnant women, various signals were observed with a dominant signal at 13736 ± 10 Da and a varying number of smaller immunoreactive fragments. These fragments are possibly the consequence of metabolism in plasma or kidney.This chip-based approach represents a rapid and accurate method to characterize the molecular variants of TTR including protein or peptide fragments which are either related to TTR or have resulted from its catabolism. These molecular variants may be of diagnostic importance as alternative or novel biomarkers due to their predominant relation to the TTR metabolism both in healthy and diseased individuals. Transthyretin  belongs to a group of proteins including thyroxine-binding globulin and albumin which bind and transport thyroid hormones in the blood. It is a single polypeptide chain of 127 amino acids 14 kDa) and is present in the plasma as a tetramer of non-covalently bound monomers. The major sites of TTR synthesis are the liver and choroid plexus . Under p kDa and The TTR variants described thus far have mostly been associated with variable degrees of cardiac and/or neural tissue amyloid deposits ,8. ThereWith regard to nutrition, TTR is a so-called visceral protein that is synthesized in the liver in response to nutritional supply. TTR plasma levels have thus been proposed as sensitive biochemical parameters of subclinical protein malnutrition, because both the adequacy and levels of protein as well as energy intakes are reflected in plasma levels. Plasma levels of TTR, however, are as well affected by acute and chronic diseases associated with an acute-phase response. Under these conditions, liver activity is converted to the synthesis of acute-phase response proteins, resulting in a dramatic drop in visceral proteins, despite nutritional support -3.This study was conducted to establish a sensitive and reproducible high-throughput SELDI-TOF-MS immunoassay for characterizing TTR variants in plasma and urine arising from amino acid substitutions, posttranslational modifications and/or products of protein degradation or proteolysis.TTR levels in plasma determined by ELISA in 10 healthy individuals were 489 ± 155 μg/ml. TTR levels in urine of the healthy individuals were one thousand times lower than in the 40 pregnant women with 46 ± 24 ng/g creatinine and 45 ± 65 μg/g creatinine, respectively.In Figure SELDI-TOF-MS immunoassays showed that in healthy non-pregnant and pregnant females as well as in males TTR in plasma occurred rather consistently in two major variants of 13732 ± 12 and 13851 ± 9 Da, of which generally the 13851 Da variant is the dominant one. The mass difference between these two variant was 120 ± 9 Da. In addition to the two major signals a minor one at 14043 ± 17 Da was observed in plasma. The mass difference to the signal at 13851 Da was 192 ± 11 Da. In urine, traces of TTR were detected in samples from non-pregnant females and from males. In the urine of pregnant women however strong mass signals were observed. As shown in Figure The SELDI platform can be readily adapted for developing an immunoassay format. This approach, using antibodies as an affinity capture device has been successfully used to detect and quantify different proteins by MS in complex biological mixtures . Unlike The results support and confirm previous studies with regard to molecular variants of TTR in plasma and urine -15. As iApart from the case of absence, some modifications might not be detected with this method due to limitations in resolution. Both methods, ELISA and SELDI immunoassay are sensitive enough to observe only trace amounts of TTR in body fluids as in the case of urine of healthy individuals. These amounts were not detected using immunoblotting. Despite the small signal it was obvious that additional variants to the ones in plasma are present in urine. Contrary to a pervious study using a different method  we were No study is available in which TTR has been described quantitatively in the urine of healthy individuals. In this study we are able to show for the first time that levels of TTR in urine of pregnant women are 1000-fold higher compared to the levels in urine of healthy non-pregnant individuals. The excretion of substantial amounts of TTR has only been described in individuals with different kidney diseases indicating disturbed glomerular filtration and/or insufficient tubular reabsorption . The invInterestingly, as shown by us also for the first time, TTR in the urine of pregnant women is present in different molecular variants. In contrast to plasma, the S-cysteinylated form dominant in plasma was if at all present only in trace amounts. It remains to be determined if this is limited to the excretion of TTR in pregnancy or if under other circumstances such as in different kidney diseases similar molecular variants occur as well. The lower molecular weight immunoreactive variants found in urine of pregnant women may arise from limited proteolysis. At this point it is not possible to determine location and extent of proteolysis within the molecule. The immunoreactive fragments might arise from a breakdown within the plasma or the kidney structures.The SELDI immunoaffinity-isolation of TTR in combination with mass spectrometry offers a rapid, highly reproducible and cost-effective system for the determination of molecular variants of microheterogenous proteins and peptides. This is of importance in "second phase" proteomics which is characterized by the repetitive investigation of the same protein to validate the protein phenotype in large population based studies. This provides the basis for a substantial progress in the diagnostic with regard to personalized medicine ,21. Withg, 10 min, 4°C) within 1–3 h after acquisition. Additionally, urine samples of the ten individuals and of healthy pregnant women (n = 40) undergoing routine medical examination, were collected and immediately mixed with protease inhibitors (Sigma, Deisenhofen, Germany). Hematuria was tested using a routine dipstick method (Combur 9 Test, Roche, Basel, Switzerland). Cells and other non-soluble material were cleared from the sample by brief centrifugation (1500 × g, 2 min). Aliquots of centrifuged urine were stored at -80°C and processed as soon as possible. The study protocol was approved by the hospitals and University of Potsdam Ethics Committee. Informed consent was obtained from each participant.Paired plasma and urine samples from 10 healthy individuals were obtained. The plasma was prepared by centrifugation of the blood . TTR in plasma and urine was quantitatively determined by an enzyme-linked immunoassay (ELISA) method developed in our laboratory. To further assess the presence of TTR quantitatively and qualitatively, we performed a SDS-polyacrylamide gel electrophoresis (PAGE) immunoblot analysis as has been described .Protein A (Sigma, Deisenhofen, Germany) at 0.1 mg/ml in PBS was added (5 μl) to the spots of a pre-activated ProteinChip® Array . The PS20 array consists of a surface with epoxy groups that dock proteins by covalently reacting with their amine groups. The arrays were incubated for 1 h at 25°C in a humidity chamber. After blocking residual active sites with 5 μl blocking buffer (0.5 M ethanolamine in PBS at pH 8.0) for 25 min, the array was washed three times in a 15 ml conical tube with PBS (pH 7.4). TTR antibody (4.1 μg/μl) was added to individual spots (2 μl) and incubated in a humidity chamber for 1.5 h with mixing. The unbound antibodies were removed by washing the array three times in a 15 ml conical tube with PBS (pH 7.4).2O. After drying, 0.6 μl of a saturated energy-absorbing molecule (EAM) solution (5 mg sinapinic acid dissolved in 75 μl acetonitrile and 75 μl 1% trifluoroacetic acid) was applied to the spot surface, and the sample was allowed to dry.Before incubation urine was diluted 1:1 (v:v) in PBS and plasma 1:100 (v:v) in PBS. A total volume of 10 μl was applied to each spot and incubated for 1.5 h at 25°C in a humidity chamber. Samples were washed on the spot with 5 μl PBS three times and finally with 5 μl HAfter insertion of the ProteinChip Array into the ProteinChip Reader, a laser pulse was focused on the sample under vacuum. The array was analyzed under the following settings: laser intensity 250/220, detector sensitivity 8, mass focus, molecular mass range 10 to 18 kDa, position 20–80 and a 150–200-shot average per sample.CytochromeC , myoglobin , GAPDH , albumin  and β-galactosidase  were used as calibrators.Mass resolution (defined as m/Δm) is routinely achieved below 300 and mass accuracy was within 0.1%. Peaks with amplitudes at least 3 times greater than the average background noise level were considered. The reproducibility was tested with different aliquots of the same sample on eight different spots of the protein chip array.None declared.FJS participated in the conception, design, data analysis and the writing of the manuscript, KW in data analysis and writing of parts of the manuscript. JR participated in the conception, data analysis and the writing of the manuscript. All authors have read and approved the last version of the manuscript.


Of the 200-plus different types of cells that form the mammalian body, most have a finite life span. Like nearly everything in biology, there are exceptions—neurons and muscle cells, for example, can last a lifetime—but the vast majority of cells eventually wear out and must be replaced. Among the most short-lived cells, blood cells are generated continuously, mainly in the bone marrow of an adult, recharging the bloodstream as their depleted predecessors are efficiently dispatched and removed from circulation every 120 days. Some 2.5 million new red blood cells are generated every second from a small pool of stem cells.Blood cell development, called hematopoiesis, passes through discrete stages in specific tissues in the developing embryo before converging in the bone marrow, where it continues throughout adulthood. Some researchers have proposed that hematopoietic stem cells (HSC) flood the bloodstream during short, precise intervals to build the developing hematopoietic system (which includes the liver, bone marrow, spleen, tonsils, and lymph nodes). Presenting an alternative model for HSC migration, Julie Christensen and her colleagues in Irving Weissman's lab at Stanford University report that HSC in mice gradually leave the fetal liver to colonize the developing spleen and bone marrow as the organs acquire the means to support them.In mouse embryos, HSC precursors develop first in the yolk sac and a region called the aorta-gonad-mesonephros (AGM), then they migrate to the liver, and later to the spleen, before finally settling into the bone marrow just before birth. It was thought that this migration occurs in distinct waves of HSC production because HSC numbers decrease in one region just before increasing in newly forming hematopoietic sites. Analyzing the concentration and activity of HSC, Christensen et al. found the cells in the blood at low but fairly constant levels during much of late fetal development, when they migrate from the liver to the spleen and bone marrow. Although the HSC population decreases in the liver at 15.5 days after conception, the authors propose that this drop occurs primarily because the HSC have differentiated into mature blood cells, not because they've exited the liver en masse to help build the spleen and bone marrow. On the other hand, the slight decrease in circulating HSC, which also occurs around this time, may be attributed to their recruitment from the bloodstream to these developing tissues.Christensen et al. also examined the impact of intercellular signaling proteins called chemokines, which help regulate fundamental developmental processes, on HSC migration. To effectively “seed” developing tissues, HSC must first be recruited from the blood, guided to the appropriate nascent tissue, then corralled and sustained. The chemokine SDF-1 attracts and retains HSC in the bone marrow but was thought to have a lesser effect on fetal liver HSC. Christensen et al. demonstrate not only that liver HSC migrate in response to this chemokine, but that their migratory response increases dramatically when both SDF-1 and a signaling protein called steel factor (SLF) are present. While adult marrow HSC respond to SDF-1, they do not respond to SLF alone and do not show improved migration in the presence of both SLF and SDF-1.Bone marrow transplants have become increasingly common for a number of hematological disorders, including leukemia and aplastic anemia. Since hematopoiesis occurs primarily in the bone marrow in both mice and humans after birth, these findings offer valuable insights into the migratory behavior of these stem cells and suggest how HSC migration might be applied to bone marrow transplants and other clinical therapies.


Sooner or later, a gardener looking for “nontoxic” ways to control the inevitable attack on a favorite plant will discover the nicotine remedy. Steep a cup of loose tobacco in a gallon of water, let it sit overnight, strain, and spray away. Caterpillars, aphids, and a diverse array of insects predisposed to devouring plants will soon abandon your vegetables and flowers in search of less disagreeable forage.The ultimate sitting duck, plants rely on an arsenal of chemical metabolites to fend off predators. Many of these chemicals harbor anti-herbivore properties, which have been exploited for commercial use. Nicotine, it turns out, is so toxic that it was one of the first chemicals used in agricultural insecticides. It's not clear, though, whether these toxic metabolites are really defending plants against hungry herbivores in their natural environment, especially since many insects can tolerate various plant chemicals and sometimes even incorporate them into their own defenses. Though scientists have cataloged a long list of these presumed resistance traits, there's no evidence that they offer plants a competitive advantage against their leaf-covetous foes in nature.With plant and plant-eater engaged in an ever-escalating battle of evolutionary one-upmanship and with plants capable of producing an array of defensive responses, teasing out the predator-resistant effects of individual plant metabolites has proved challenging. Theoretically, one could track down a resistance gene by breeding plants that are genetically identical save for the gene that controls expression of a particular resistance trait. In practice, however, traditional breeding techniques aren't that precise and tend to generate additional variations in genomic regions that are linked to the target gene and that might affect resistance as well.The tools of genetic engineering have largely overcome such limitations, allowing scientists far greater control and specificity. Following this approach, Ian Baldwin and colleagues use transgenic silencing (which introduces gene “constructs” into an organism to inactivate a gene of interest) to investigate a single resistance trait, nicotine production. Even though nicotine is one of the best-studied putative resistance traits, its specific role has been unclear.Nicotiana attenuata tobacco plant. Focusing on an enzyme, called putrescine methyl transferase (PMT), central to nicotine biosynthesis, the authors used two techniques that interfere with PMT production by silencing the gene, pmt, that encodes the enzyme. One of the techniques  proved far more effective at silencing pmt, producing 29 out of 34 plant lines with only 3%–4% of normal nicotine levels.To isolate the resistance effects of nicotine from possible confounding factors, Baldwin and colleagues blocked nicotine production in the N. attenuata's native habitat. A subset of the plants was also treated with a chemical known to increase both nicotine content and resistance to herbivore attack. Predictably, several of the plant's natural insect enemies made an appearance. Untreated nicotine-deficient transgenic plants fared the worst, losing twice as much foliage to herbivores as nonmutant plants. Transgenic plants treated with the chemical boost performed much better, showing about the same amount of damage as the nonmutants. Interestingly, tobacco hornworms—which, as their name implies, feed primarily on tobacco—preferred nicotine-free plants when given the choice. Though the worms have evolved strategies for coping with nicotine's deleterious effects, these adaptations come at a price: worms feeding on nicotine-deficient tobacco grew bigger and faster than those feeding on plants with normal nicotine levels.With suitably nicotine-deprived plants, Baldwin and colleagues could directly test nicotine's role in tobacco fitness. They transplanted the transgenic plants, along with nonmutant cultivated plants, in southwestern Utah, These results clearly show that nicotine protects tobacco plants in their native habitat, the authors conclude, and that tobacco-chewing insects “prefer low nicotine diets.” Removing nicotine from the equation reveals the relentless pressure that plants face from herbivores. Without such defenses, plants would be unceremoniously eliminated posthaste, leaving a world without greenery, not to mention oxygen. But these experiments also demonstrate the unprecedented power of transgenic tools to peel back the obfuscating layers inherent in ecological interactions to reveal the fundamental properties of those interactions. And that scientists working to unravel the tangled web of ecological interactions would do well to take advantage of the longest running experiment on earth—the natural environment.


We examined the association and interaction between maternal viral load and antibodies in vertical transmission of HIV in a non-randomized prospective study of 43 HIV-1 infected pregnant women who attended the San Juan City Hospital, Puerto Rico, and their 45 newborn infants. The women and infants received antiretroviral therapy.A nested PCR assay of the HIV-1 envelope V3 region and infant PBMC culture were performed to determine HIV status of the infants. Maternal and infant plasma were tested for HIV neutralization or enhancement in monocyte-derived macrophages.-1 dilution compared with HIV negative plasma. Infant plasma neutralized HIV (p = 0.001) at 10-2 dilution but maternal plasma lost neutralizing effect at this dilution. At 10-3 dilution both maternal and infant plasma increased virus replication above that obtained with HIV negative plasma but only the increase by maternal plasma was statistically significant (p = 0.005). There were good agreements in enhancing activity in plasma between mother-infant pairs, but there was no significant association between HIV enhancement by maternal plasma and vertical transmission.Twelve (26.7%) infants were positive by the HIV V3 PCR assay and 3 of the 12 were also positive by culture. There was a trend of agreement between high maternal viral load and HIV transmission by multivariate analysis (OR = 2.5, CI = 0.92, p = 0.0681). Both maternal and infant plasma significantly (p = 0.001 for both) reduced HIV replication at 10Although not statistically significant, the trend of association between maternal viral load and maternal-infant transmission of HIV supports the finding that viral load is a predictor of maternal-infant transmission. Both maternal and infant plasma neutralized HIV at low dilution and enhanced virus replication at high dilution. The antiretroviral treatments that the women received and the small sample size may have contributed to the lack of association between HIV enhancement by maternal plasma and vertical transmission. The rate of HIV-1 infection has been increasing rapidly among women of childbearing age. At the end of 2003 women accounted for 50% of adults living with HIV/AIDS worldwide ,5. HowevAlthough some studies show no association between the presence of HIV neutralizing antibodies in maternal sera and the risk of perinatal transmission ,7, otherin vitro by either Fc gamma receptor- or complement receptor-mediated endocytosis (FcγR-ADE or C'-ADE) have been identified in sera from HIV-1-infected individuals [in vitro study of antibody dependent enhancement (ADE) of HIV-1 infection in human term syncytiotrophoblast cell cultures suggested that both FcγR-ADE and C'-ADE may contribute to maternal-infant transmission of HIV-1 [In contrast to neutralizing antibodies, non-neutralizing antibodies may enhance HIV infection by binding to the virus and facilitating its uptake by cell types that carry immunoglobulin (Fc) or complement receptors. Antibodies that enhance HIV replication ividuals -25 and fividuals ,20. An iof HIV-1 . Pancinoof HIV-1 , reporteof HIV-1 , observeof HIV-1  and the Maternal plasma virus load has been shown to be strongly associated with perinatal transmission of HIV ,30,31 anHowever, the association of both viral load and enhancing activity (presumably by FcγR-ADE) in maternal plasma and vertical transmission of HIV has not formerly been examined. Thus, we examined the association of these factors (independently and combined) in vertical transmission using samples from mother-infant pairs from San Juan, Puerto Rico, previously described by Melendez-Guerrero et al . NeutralA cohort of 43 HIV-1 subtype B infected pregnant women attending antenatal clinic at the San Juan City Hospital, Puerto Rico, was enrolled into a prospective study from their first antenatal visit until delivery . Eleven 3 of blood with a mean count of 425 cells/mm3. Twenty-six of the 43 women delivered their babies by normal vaginal delivery; the remaining women had cesarean sections  of 45 infants were HIV V3 positive indicating that HIV transmission had occurred (Table 6.7% of 43, median = 508 cells/mm3) compared to non-transmitting mothers . Maternal CD4 cell counts have been shown to be a less effective predictor of transmission of HIV than viral load [3 level which is used to define AIDS. Only one of the three women whose infants were HIV culture positive had AIDS (CD4 = 149 cells/mm3). The other two women had CD4 counts of 510 and 538 cells/mm3.The mothers of HIV V3 positive infants had a non-significantly higher mean CD4+ T count  from the mothers (transmitters and non-transmitters) and infants (HIV V3+ and HIV V3-) on HIV replication was examined in the neutralization/enhancement assay (Table -1 dilution, over 90% of plasma samples from both groups of mothers (transmitters/non-transmitters) and infants (HIV V3+/ HIV V3-) neutralized HIV. At 10-2 dilution the percentages of plasma that neutralized HIV dropped to approximately 75% for both groups of mothers and to 58% and 70% for HIV V3+ and HIV V3- infants respectively. The percentages again dropped at 10-3 dilution to ≤ 67% for mothers and ≤ 33% for infants (Table The effect of plasma (at 10-1) dilution when compared with HIV negative sera  reduced virus replication, but the maternal plasma lost neutralizing activity  above seronegative plasma and the infant plasma showed a non-significant increase (88%) in HIV replication . Examination of enhancing activity in plasma of mothers and infants showed that there were good agreements (60% or greater) in enhancement status between mother-infant pairs. For example, at 10-4 dilution, the plasma of 7 infants of 9 mothers (78%) whose plasma enhanced HIV replication also exhibited enhancement, and at 10-6 dilution, the plasma of 8 infants of 10 mothers (80%) whose plasma enhanced HIV replication also exhibited enhancement (data not shown). Comparison of p24 antigen between transmitter and non-transmitter mothers or their infants at 10-4 to 10-6 dilutions showed no significant difference between the two groups of mothers or infants. The lack of association of enhancing activity and HIV transmission in this study is similar to the findings for C'-ADE by Gras, et al. [in vivo by most HIV-1 variants [Sixty-nine percent (31/45) of plasma from mother-infant pairs were also tested in the neutralization/enhancement assay at higher dilutions  and low negative associations at higher dilutions (10-4 – 10-6). There were low positive associations between maternal viral load and the p24 antigen values of their infants at low dilutions (10-1 – 10-3) and low negative correlations at higher dilutions except at 10-5 dilution  and the infants' p24 antigen values. A correlation matrix showed a positive association between mothers' p24 antigen values and those of their infants . The combined effect of maternal viral load and enhancing antibodies as potential risk factors for the vertical transmission of HIV-1 was examined in a multivariable model. Log viral load was treated as a continuous variable in the model and enhancement was categorized as enhancement versus neutralization. There was a trend of association between maternal viral load and transmission of HIV-1 so that the higher the viral load, the more likely mothers were to transmit HIV-1 to their infants (OR= 2.5, CI= 0.93 – 6.67, p = 0.0681). This is in agreement with other studies discussed earlier that show viral load as a predictor of maternal-infant transmission ,30,31. T-1) dilution and enhanced virus replication at higher dilution (10-3). Neutralizing and enhancing antibodies can occur together in the blood of HIV positive individuals and the neutralizing effect can be lost at high plasma dilution.In agreement with published data, multivariate analysis showed a trend of association between maternal viral load and maternal-infant transmission of HIV. The non-significant difference in the mean log viral load of transmitting and non-transmitting mothers is probably due to the small sample size. No significant associations were found between HIV antiretroviral treatment protocols, classification of HIV disease, method of infant delivery and CD4+ T cell count of the mothers and vertical transmission of HIV. Both maternal and infant plasma significantly neutralized HIV infection at low  were stored frozen at -85°C. The HIV status of the infants was determined using a nested PCR assay of the HIV-1 envelope variable (V3) region as previously described [Blood samples were taken from the mothers during each trimester of pregnancy and during delivery. Blood samples from the infants were collected from the umbilical cord at birth and at 1–2, 3–4 and 5–12 months. Blood was collected in vacutainer tubes containing ACD anticoagulant and centrifuged at 2500 rpm for 15 minutes. The plasma was stored frozen (-20°C) for use in maternal viral load and HIV neutralization/enhancement assays. The remaining blood was diluted in phosphate-buffered saline (PBS) pH 7.4 and processed by Ficoll-hypaque density gradient for cell isolation. For virus isolation, one million cells from each mother or infant were co-cultured with HIV seronegative donor cells previously stimulated by PHA as described in the ACTG virology manual . The remescribed ,56. Thisgag gene was amplified by RT-PCR for viral load determination. The mean log viral load was calculated and compared between transmitting and non-transmitting mothers to determine the effect of maternal viral load on HIV vertical transmission.HIV RNA copies in maternal plasma was determined by the amplicor HIV monitor test (Roche Diagnostics, Branchwater, NJ, USA) at the Puerto Rico ACTG-certified laboratory . A 142 b4.6 TCID50/ml) obtained from the NIH AIDS Research and Reference Reagent Program was used to inoculate fresh cultures of macrophages grown on 75 ml tissue culture flasks. The cultures were washed 24 hours later and incubated with fresh media. Supernatant fluids were harvested at 7 and 14 days post-infection, clarified by centrifugation at 1800 rpm for 10 minutes and used as stock virus for these studies. The stock contained 5 × 105 TCID50/ml.HIV-1BaL stock was prepared in primary macrophages as reported previously by Jolly . Briefly3 TCID50/ml of virus and pre-incubated at 37°C for 30 min. The mixtures were then inoculated into replicate cultures of monocyte-derived macrophages as prepared previously [2 incubator. The inocula were removed and the cells washed, and incubated with fresh media for up to 8 days. Culture supernatant fluids were then collected on days 2, 4, 6, and 8 and tested for p24 antigen using the Coulter assay (Coulter, Miami, FL, USA). Cultures treated with HIV-negative sera were used as controls. All maternal and infant plasma samples were tested at three 10-fold dilutions; 69% of samples were tested up through six 10-fold dilutions. Two independent assays were conducted for each maternal or infant plasma sample and each sample was run in duplicate on each assay (4 replicates).A neutralization/enhancement assay was conducted using maternal or infant samples and HIV-1BaL. Maternal plasma samples collected during the third trimester of pregnancy were used in these assays since most prenatal HIV infections occur in the third trimester . Brieflyeviously  in 8 welDescriptive statistics such as mean, median, range were calculated to summarize maternal characteristics such as viral load, CD4+ T cell count and p24 antigen levels. The number of HIV vertical transmission among infants was summarized and the univariate association of maternal characteristics, such as, treatment , stage of disease based on the 1993 CDC classification , method Correlation between maternal versus infant p24 antigen levels and between maternal viral load and maternal or infant p24 antigen levels was evaluated using the Spearman's correlation coefficient. Enhancement, decrease , or no change in p24 levels compared to seronegative control was assessed for mother-infant pairs. The distribution of virus p24 antigen was compared between transmitting vs. non-transmitting mothers and between HIV V3+ infants and HIV V3- infants using Fisher's exact test.The author(s) declare that they have no competing interests.LMG and PEJ were involved in conception and design of the study, collection of the samples, interpretation of the data and drafting and critical review of the manuscript. PK and MA were involved in performing the laboratory experiments that resulted in the acquisition of the data, in data entry, and in drafting and revising the manuscript with LMG and PEJ. HLW was responsible for data analysis and interpretation along with PEJ, LMG and PK. All authors have read and approved the final manuscript.


Fluoxetine and olanzapine combination therapy is rapidly becoming an effective strategy for managing symptoms of treatment-resistant depression. Determining drug-drug interactions, drug metabolism and pharmacokinetics is of particular interest for revealing potential liabilities associated with drug augmentation in special patient populations. In the current studies, we chronically administered fluoxetine and olanzapine in non-stressed rats to extend our previous findings regarding body weight dynamics.Chronic fluoxetine (10 mg/kg) and olanzapine (5 mg/kg and 0.5 mg/kg) treatment decreased weight gain irrespective of olanzapine dosing. At the 10 mg/kg and 5 mg/kg dose, respectively, fluoxetine and olanzapine also significantly reduced food and water consumption. This pharmacodynamic event-related effect, however, was not observed at the 10 mg/kg and 0.5 mg/kg dosing paradigm suggesting differences in tolerability rates as a function of olanzapine dose. The decrease in weight gain was not associated with apparent changes in glucose metabolism as vehicle- and drug-treated rats showed undistinguishable serum glucose levels. The combination of fluoxetine and olanzapine in rats yielded drug plasma concentrations that fell within an expected therapeutic range for these drugs in psychiatric patients.These data suggest that fluoxetine and olanzapine treatment decreases weight gain in rats; a pharmacodynamic event-related effect that differs considerably from what is observed in the clinical condition. The possibility of mismatched models regarding body weight changes during drug augmentation therapy should be seriously considered. Treatment-resistant depression is a serious issue in psychiatry as a significant number of affected individuals show an inadequate response to single antidepressant therapy. An emerging strategy to achieve maximum mood stabilization for treatment-resistant depression, bipolar illness and depression with psychotic features is the augmentation of fluoxetine (Prozac) with novel anti-psychotic agents such as olanzapine (Zyprexa). Indeed, a number of clinical trials have suggested that such an augmentation strategy offers superior efficacy for treating resistant major depression when compared with either fluoxetine or olanzapine alone -3. Despi2A), dopamine , acetylcholine muscarinic  and histamine (H1) receptors distributed widely in limbic neural circuits may somehow account for the pharmacological basis of olanzapine-induced weight gain [We have previously shown that sub-chronic (i.e., 7 days) administration of fluoxetine plus olanzapine results in a significant reduction of weight gain in rats . This fight gain . Needlesght gain . In thisnecrotising vasculitis within the site of injection. The necrotic properties of the above antidepressant have previously been reported [All rats tolerated the fluoxetine plus olanzapine regimen well. There were no mortalities as a result of 18 days of drug administration in any of the rat groups tested. The only apparent untoward side effect was tissue necrosis in the peritoneum of rats injected with fluoxetine plus 5 or 0.5 mg/kg olanzapine  when compared with the vehicle-treated group Fig. . This we10 = 5.5, P ≤ 0.001) than vehicle-treated animals  when compared with cyclodextrin-treated animals  or water (t10 = -1.2, P ≥ 0.05) consumption during the dark cycle when compared with cyclodextrin-treated rats  or the lower 0.5 mg/kg olanzapine dose (t10 = -1.4, P ≥ 0.05). Thus, changes in glucose metabolism are not the proximate cause affecting the differential body weight dynamics, nor the differential consumption of food and water among vehicle- and drug-treated rats. Along the same lines, levels of the hormone leptin did not differ between cyclodextrin- and drug-treated animals (data not shown), thus suggesting that chronic fluoxetine and olanzapine drug therapy does not affect leptin messages under fasted conditions in male rats.In contrast to the above findings, animals injected first with fluoxetine and then 15 min later with 0.5 mg/kg olanzapine did not show differences in food . For the chronic drug regimen, rats were injected intraperitoneally (IP) first with fluoxetine (10 mg/kg) followed 15 min later by olanzapine (5 or 0.5 mg/kg) to decrease potential pharmacokinetic interactions. Fluoxetine was dissolved in 5% γ-cyclodextrin, whereas olanzapine was dissolved in 12% γ-cyclodextrin (cyclodextrin was used to improve the stability and bioavailability of poorly soluble drugs). Dosages of the two drugs were chosen according to each drug's in vivo potencies for affecting 5-HT, DA, ACh and H systems [Guide for the Care and Use of Laboratory Animals and with approval from the NYIT IACUC.Adult male Long-Evans rats (Harlan, Indianapolis, IN) were used in all experiments described herein. Prior to any drug treatment, all rats were handled for 5 days to minimize non-specific stress. Rats were then randomly assigned to the various experimental groups and cage mates received the same drug treatment. Animals were group-housed, 2–3 per cage under a 12 hr light:dark cycle (light on 0700) and allowed  systems ,34, and  systems ,11,35,36Rats were injected with fluoxetine plus olanzapine or their respective vehicle-solutions for 18 consecutive days and body weights recorded before (day 0) and after (day 18) drug treatment. In addition, body weights were also recorded 7 and 14 days after drug treatment to adjust for dosage. To determine average food intake over a 12 hr period, control-vehicle and experimental rats were given pre-measured food pellets (25 g/rat) on day 10 and subsequent consumption was recorded on the next day. To keep track of food spillage, cage bedding was randomly separated to assess degree of unconsumed food. A similar procedure was instituted to assess average water intake over a 12 hr period: both groups were given pre-measured tap water (100 mL/rat) on day 12 and subsequent consumption was recorded on the next day. On the last day of injections (day 18), rats were either decapitated or perfused under deep chemical anesthesia (ketamine/xylazine/acepromazine, 60 mg/kg) with 4% paraformaldehyde. Trunk blood or blood collected from cardiac puncture was collected in centrifuge tubes containing either no EDTA or a 0.3 M EDTA (pH 7.4) solution. Blood samples with 0.3 M EDTA were centrifuged at 10,000 RPM for 10 min and the collected serum frozen at -80°C until determination of glucose levels. Blood collected without EDTA was also frozen at -80°C until determination of drug plasma concentrations by GC-MS.To determine relative glucose levels, all animals were fasted for 12 hr on the last day of injections (i.e., day 18). Glucose serum levels were determined using the Life-Scan One Touch Basic Meter (Johnson & Johnson, New Brunswick, NJ). In brief, 10 μl of serum was pipetted as a free-flowing drop onto each One Touch Test Strip and read in the glucose meter for 45 sec. Fasting glucose levels are presented as means ± SEM in mg/dL.All solvents used for drug analyses were HPLC grade. Chloroform and 1-chlorobutane were obtained from Burdick & Jackson (Muskegon, MI). Ethyl Acetate and acetonitrile were obtained from EMD (Gibbstown, NJ). Ammonium Hydroxide, ACS certified, was obtained from Fisher (Fairlawn, NJ). Ascorbic acid was obtained from Sigma (St. Louis, MO), whereas trifluoroacetic anhydride was obtained from Pierce (Rockford, IL).Working solutions containing olanzapine, fluoxetine and norfluoxetine at 10 ng/μL, 1.0 ng/μL, and 0.1 ng/μL, respectively were prepared in methanol. The working solutions used to prepare the calibration standards and controls were derived from different sources of reference material (e.g., fluoxetine) or different weighing of the same reference material (e.g., olanzapine). Calibration standards and controls were prepared by adding appropriate amounts of working solutions to clean, separate silanized 16 × 100 mm culture tubes that contained 1 mL of blank bovine blood and 0.2 mL of 2.5% ascorbic acid. The calibration standards ranged from 1 ng/mL to 1000 ng/mL. The controls were prepared at 35 ng/mL, 100 ng/mL, and 650 ng/mL.2O was added to each tube. The samples were therefore a 4-fold dilution compared with the standards and controls. A 0.2 mL volume of 2.5% ascorbic acid was added to each sample in a preparation tube. Sample, standards, and controls were extracted by a liquid/liquid procedure. Eighty ng of fluoxetine-d6 (80 μL of 1 ng/μL fluoxetine-d6 in Milli Q H2O) and 80 ng of clozapine (80 μL of 1 ng/μL clozapine in methanol) were added to each tube and the tubes were then briefly vortexed. A 0.1 mL volume of the concentrated ammonium hydroxide and a 4 mL volume of 1-chlorobutane: acetonitrile (4:1) was then added to each tube. A clean teflon-lined screw cap was placed on each tube. The tubes were mixed 20 min using a reciprocating shaker and centrifuged at 2000 rpm for 10 min using an IEC centrifuge (Needham, MA). Using clean, separate glass Pasteur pipettes, the upper organic layer from each tube was transferred to clean, separate 13 × 100 mm culture tubes. The organic layer was evaporated to dryness under a stream of air at 40°C using a Turbo Vap evaporator (Zymark Corporation, Hopkinton, MA).A 0.25 mL volume of each blood sample was transferred to clean silanized 16 × 100 mm culture tubes. To bring the sample preparation to a volume of 1 mL, a 0.75 mL volume of Milli Q HFor derivatization, a 0.1 mL volume of chloroform and a 0.1 mL volume of trifluoroacetic anhydride were added to each tube. Clean teflon-lined screw caps were then placed on each tube. The tubes were heated for 20 min at 70°C using a dry block heater. After heating, the tubes were removed from the heater and allowed to cool at room temperature. The caps were then removed and the tubes evaporated using the same conditions as described above. Derivatized extracts were reconstituted with 100 μL of ethyl acetate and were then transferred to clean, separate auto-sampler vials.6: 429, olanzapine: 409, clozapine: 423. For fluoxetine, norfluoxetine, and fluoxetine-d6, the protonated ammonia adducts of the molecule ions were monitored. For olanzapine and clozapine, the protonated molecule ions were also monitored. Fluoxetine-d6 was used as the internal standard for fluoxetine and norfluoxetine. Clozapine was used as the internal standard for olanzapine. The limit of quantification for fluoxetine, norfluoxetine and olanzapine was 5 ng/mL. Data are presented as the means ± SEM.The GC-MS system consisted of an Agilent 6890 gas chromatograph and an Agilent 5973 MSD mass spectrometer . The data system consisted of a Hewlett-Packard X A 6/400 computer and Agilent Chemstation software. For chromatographic separation, a ZB-5, 30 meter × 0.25 mm id, 0.25 μm capillary column (Phenomenex, Torrance, CA) was used. The carrier gas was ultra high purity helium at a flow rate of 1.0 mL/min. The injection port temperature was 260°C and the transfer line temperature was 300°C. The column oven temperature program was 125°C, held at this temperature for 0.2 min, and then increased to 300°C. Positive chemical ionization was used for the mass spectrometry analysis. The ion source temperature was 200°C and ammonia was used as a reagent gas. Selected ion monitoring was used and the following ions (m/z) were analyzed: norfluoxetine: 409, fluoxetine: 423, fluoxetine-dt tests where appropriate. Plasma levels of fluoxetine and olanzapine were analyzed by a Student's t-test. The probability level interpreted as significant was P ≤ 0.05.Statistical comparisons in body weight and glucose levels were carried out using one-way ANOVA or two-tailed JAP and JMC participated in the in vivo studies and in the biochemical assays. BHH and JMH participated in the design of the studies and performed the statistical analysis. GT drafted the manuscript, conceived the study and participated in its design and coordination. All authors read and approved the final manuscript.


Mycobacterium tuberculosis is the major cause of treatment failure in tuberculosis patients, especially in immunosuppressed. We described a young polymyositis patient on immunosuppressive therapy who was started with antituberculosis therapy as a susceptible strain of M. tuberculosis was isolated from a single cutaneous abscess in his neck and from regional lymph nodes.MDR He had non-reactive miliary tuberculosis and multiple cutaneous abscesses 6 months later with the same strain, which was resistant this time to 9 antituberculosis drugs. We described clinical presentation, radiological and laboratory work-up, treatment and follow-up as the patient was cured after 1.5 years with 6 antituberculosis drugs.M. tuberculosis was cured. Intense management and regular follow up were important since the patient was a potent source of MDR M. tuberculosis infection and there was limited choice for therapy.To our knowledge, this is the first reported case where an immunosuppressed patient with suggestive exogenous reinfection within 6 months with the same but MDR strain of  M. kansasii, M. scrofulaceum, rather than M. tuberculosis are the most common etiological agents for cutaneous TB in HIV/AIDS and other immunocompromised patients.World Health Organization published the Global Tuberculosis Control Report 2003) on 'World Tuberculosis Day' where India is ranked number one in the world for high incidence of smear positive cases of pulmonary (about 0.9 million) and extrapulmonary TB (about 0.2 million) every year . Undoubt003 on 'WM. tuberculosis from a patient who has been treated for TB for 1 month or longer [M. tuberculosis with mutations conferring resistance to antitubercular drugs may result from poor management including the prescription of incorrect regimens and non-compliance with treatment.WHO defines acquired drug resistance as the isolation of drug-resistant r longer  and primM. tuberculosis in a polymyositis patient on immunosuppressive therapy resulting in multiple cutaneous abscesses and miliary TB.We report a rare case of reinfection with same but MDR A 23-year-old polymyositis patient with suggestive clinical symptoms, muscle biopsy, enzyme assay, needle EMG and NCV test was on immunosuppressive drugs such as corticosteroids at 1 mg/kg/day for 3 to 4 weeks, tapered gradually over a period of 10 weeks to 1 mg/kg every alternate days and methotrexate at 7.5 mg weekly with gradual increase to 25 mg weekly for a period of 1 year when he experienced slightly improved muscle power.M. tuberculosis as culture was confirmed by niacin and nitrate reduction tests, rapid bacteriophage assay (FAST Plaque Tuberculosis kit, BIOTEC Laboratories Ltd, UK) and RFLP analysis  [He had single cutaneous abscess 6 months later on the right side of the neck with no sinus formation and cervical and axillary lymphadenopathy. He was HIV-negative with normal CXR and there was no AFB in three consecutive (spot-early morning-spot) sputum samples (induced in two occasions). However, the aspirated pus from cutaneous abscess and biopsy of the axillary lymph node showed plenty of s figure  using spnt gene) .devR gene (Gene bank nucleotide sequence accession no. U22037) encodes a response regulator that is part of a two-component signal transduction system of M. tuberculosis. The authenticity of the amplified product was established by hybridization of immobilized PCR products to an internal oligonucleotide, devR1, mapping within the devR gene. The chromosomal DNA was prepared by chloroform-isoamyl alcohol DNA extraction and 4.5 μg of DNA from the isolate was restricted with PvuII. Separation of PvuII-restricted DNA by electrophoresis, Southern blot hybridization with a 513-bp PCR probes for devR  and chemiluminescence detection were done according to the standard method recommended for the DNA fingerprinting of M. tuberculosis [In RFLP analysis, the rculosis . HistopaM. tuberculosis strain by phage assay and RFLP analysis (figure However, the patient returned 6 months later with multiple cutaneous abscesses, mainly on his back, left thigh and left arm and miliary mottling in CXR. He denied any irregularity in antituberculosis therapy. All immunosuppressive drugs (corticosteroids and methotrexate) were discontinued as he was readmitted. Isolates from 2 of 3 consecutive sputum samples and aspirated pus samples from all 5 completely drained abscesses were reconfirmed as the identical s figure . The isoM. tuberculosis during the same episode (multiple infection), in different lesions (multiple infection) or during successive episodes (reinfection) in HIV-positive as well as -negative individuals [M. tuberculosis were isolated at 6 months interval, but contrary to the first, the strain in the second episode was resistant to all the first line and most of the second line antitubercular drugs. As far as could be ascertained, this is the first case of isolation of MDR strain of M. tuberculosis from a HIV-negative but immunosuppressed patient who had an infection with the same, but susceptible strain 6 months before.India is an endemic country for TB with an estimated 20,000 infectious cases and 1–3.3% of new cases of MDR TB every year . Infectiividuals . In thisAs evident from the study, the chance of exogenous reinfection with the same but drug-resistant strain from some undetected source within the endemic community is the most likely explanation. This explains the acquisition of resistance against all those drugs, to which the patient was not exposed earlier. Reinfection though occurs usually after first two to five years in immunocompetent hosts, may progress to active disease at any time after treatment has been discontinued and even during treatment for active tuberculosis . MoreoveEndogenous reactivation which is higher than the rate of exogenous reinfection in endemic countries ,13 with M. tuberculosis from other mycobacteria in M. tuberculosis complex whereas RFLP and phage typing excluded atypical mycobacteria, like M. kansasii and M. scrofulaceum. The overall acuracy of the drug susceptibility test ranges between 84–100%, since mycobacteria often clump [Niacin test (95% positive ) and niten clump . We trieM. tuberculosis invasion of the skin without any muscle involvement and sinus formation as the biopsy of the axillary lymph node showed non-specific necrosis containing disintegrating polymorphonuclear leukocytes and enormous number of AFB. This type is more often seen with severe HIV infection than in patients with immunosuppressive therapy [It was a rare case of miliary TB of non-reactive type  with MDR therapy ,19, wher therapy , granulo therapy ) either This is probably the first reported case of cure where all first and most of the second line drugs  were found to be resistant. However, the 6-drug antituberculosis therapy might not be considered as the only reason for cure, since 4 first line drugs showed high-level resistance and chances of cross-resistance between closely-related aminoglycosides, amikacin and kanamycin and quinolones, sparfloxacin and ciprofloxacin could not be ignored ,22. DiscSevere immunosuppression may lead to disseminated TB such as miliary TB or other rare types of extra-pulmonary TB such as cutaneous abscesses. Follow-up of patients is important, and if response to treatment is poor, adherence to treatment, drug resistance and other possible reasons such as continuation of immunosuppressive therapy should be considered. In this case intervention by drainage of abscesses, discontinuation of immunosuppressive treatment and possibly long-term treatment with additional second line antituberculosis drugs eventually lead to cure.AFB – Acid fast bacilliAIDS – Acquired immunodeficiency disease syndromeCXR – Chest radiographDNA – Deoxyribonucleic acidEMG – ElectromyographyHIV – Human immunodeficiency virusMDR – Multidrug resistantM. tuberculosis- Mycobacterium tuberculosisNCV – Nerve conduction velocityPCR – Polymerase Chain ReactionRFLP – Restriction-fragment length polymorphismTB – TuberculosisUK – United KingdomUSG – UltrasonographyWHO – World Health OrganisationThe author(s) declare that they have no competing interests.plaque assay and molecular identification method and drafted the manuscript.CM conceived the study, carried out the case study and follow-up, in the clinical as well as microbiological aspects, formatted the study design, performed sensitivity of the organism, participated in the FAST plaque assay and molecular identification method.AG carried out FAST AA participated in the design of the study and acted as an overall supervisor.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Upper urothelial cancer (UUC), i.e. transitional cell carcinomas of the renal pelvis and the ureter, occur at an increased frequency in patients with hereditary nonpolyposis colorectal cancer (HNPCC). Defective mismatch repair (MMR) specifically characterizes HNPCC-associated tumors, but also occurs in subsets of some sporadic tumors, e.g. in gastrointestinal cancer and endometrial cancer.We assessed the contribution of defective MMR to the development of UUC in a population-based series from the southern Swedish Cancer Registry, through microsatellite instability (MSI) analysis and immunohistochemical evaluation of expression of the MMR proteins MLH1, PMS2, MSH2, and MSH6.A MSI-high phenotype was identified in 9/216 (4%) successfully analyzed patients and a MSI-low phenotype in 5/216 (2%). Loss of MMR protein immunostaining was found in 11/216 (5%) tumors, and affected most commonly MSH2 and MSH6.This population-based series indicates that somatic MMR inactivation is a minor pathway in the development of UUC, but tumors that display defective MMR are, based on the immunohistochemical expression pattern, likely to be associated with HNPCC. MSH2, the absolute lifetime risk for mutation carriers to develop UUC is <10% [Upper urothelial carcinomas (UUC) represent about 5% of the urinary tract tumors, with transitional cell carcinomas of the renal pelvis and the ureter being the most common . Exogeno is <10% -9.MLH1 (40%), MSH2 (50%) or MSH6 (10%) [MLH1 promoter [HNPCC is caused by a germline mutation in a DNA mismatch-repair (MMR) gene, most commonly affecting either of the genes  or MSH6 % 10,11]MLH1 (40%H6 (10%) . Hence, promoter ,14.Studies of the contribution of defective MMR to the development of urothelial carcinomas, assessed using MSI analysis, loss of MMR protein expression, and MMR gene mutations, have found a low frequency (<10%) of MMR defects in urothelial carcinomas of the urinary bladder , but havIn Sweden a population-based national Cancer Registry was started in 1958 and applies mandatory registration by both clinicians and pathologists in order to achieve maximal coverage (estimated to be 98%). We applied the southern Swedish part of the registry, which currently contains about 300.000 entries, to identify all carcinomas of the upper urothelial tract that had developed between 1992 and 1999. We identified 262 patients with a median age of 70 (range 34–90) years and a male:female ratio of 1.8:1. For further analyses, 27 patients were excluded because of lack of tumor blocks, and 19 because of autopsy-based diagnosis with autolysis that prevented good quality immunostaining. Hence, 216 patients with a median age of 69 (range 34–89) years were analyzed. Tumor location was as follows for the whole material : renal pelvis 173 (154), ureter 75 (60) and an unspecified tumor location in 14 (2) patients. Data on family history of cancer or blood samples for mutation analysis were not available since the study was retrospective and register-based. Ethical approval for the study was obtained from the ethics committee at Lund University.Representative tumor blocks containing at least 20% tumor tissue were selected and DNA was extracted from 3 × 10-μm sections of formalin-fixed, paraffin-embedded tissue through incubation of the samples in EDTA-Tris-buffer with Proteinase K at 65°C for at least 2 hours, followed by boiling, centrifugation, and removal of the aqueous phase, which was stored at 4°C. MSI was assessed with the National Cancer Institute (NCI) panel; BAT25, BAT26, BAT34, BAT40, D2S123 and D5S346 . The priImmunohistochemical staining was performed using 4-μm sections of formaline fixed, paraffin-embedded tissue, which were mounted on DAKO ChemMate Capillary Gap Microscope Slides (DAKO A/S BioTek Solutions, USA) and dried at room temperature overnight followed by incubation at 60°C for 1–2 hours. The tissue sections were deparaffinized in xylol and rehydrated through descending concentrations of alcohol. Antigen retrieval was achieved by microwave-treatment in 1 mM EDTA, pH 9.0, at 900 W for 8 minutes followed by 15 minutes at 350 W. The slides were then allowed to cool for at least 20 minutes in the EDTA-solution. Immunohistochemical staining was performed in an automated immunostainer (TechMate 500 Plus, DAKO), according to the manufacturers instructions. The main steps were as follows: Mouse monoclonal IgG antibodies to MLH1 (clone G168-15, dilution 1:100, PharMingen, San Diego, CA, USA) MSH2 (clone FE-11, dilution 1:100, Oncogene research products, Boston, MA, USA), MSH6 (clone 44, dilution 1:1000, BD Transduction Laboratories) and PMS2 (clone:A16-4, dilution 1:500, BD Pharmingen) were applied and the sections were incubated at room temperature for 25 minutes.Thereafter, the slides were incubated with biotinylated anti-mouse antibody (DAKO) for 25 minutes for MLH1 and MSH2) or with rabbit anti-mouse immunoglobulins (DAKO, dilution 1:400) for 20 min (for MSH6 and PMS2). Endogenous peroxidase activity was blocked in Peroxidase-blocking solution (DAKO) for 3 × 2,5 minutes. This was followed by incubation with streptavidin-horseradish peroxidase for 25 minutes for MLH1 and MSH2, whereas EnVision™/HRP rabbit/mouse (DAKO) incubation for 25 min was used for MSH6 and PMS2. Finally, the tissue sections were treated with diaminobenzidine (DAB) for 3 times 5 min, counterstained with hematoxylin for 1 min, rinsed in running tap water for 10 min, dehydrated in ascending concentrations of alcohol and mounted. After each step, the sections were rinsed in Tris buffered saline, pH 7.4, and Tween-20. In order to block nonspecific protein binding, bovine serum albumin was added to the buffer before the antibody incubation steps in the MLH1 and MSH2 stainings. A detailed protocol is available from the authors upon request. Two of the authors (K.E. and M.N.), who were blinded regarding the MSI status, independently evaluated all stained sections. Sections without nuclear staining in the tumor cells, in the presence of normal nuclear staining in lymphocytes and normal epithelial or stromal cells in the same section, were considered to have a lost expression , and the other patients with synchronous tumors had MSS tumors all of which showed retained expression of all three MMR proteins  patients had been diagnosed with another malignancy, which was bladder cancer in 67 cases . Among the cases with MSI and/or immunohistochemical MMR protein loss, 8 metachronous tumors developed and 5/5 analyzed  displayed MSI and immunohistochemical loss of the concordant MMR protein  of the tumors that occurred in the southern Sweden health care region between 1992 and 1999. A MSI-high phenotype was found in 9/216 (4%) patients and a MSI-low phenotype in 5/216 (2%). In 11/216 cases synchronous tumors occurred within the urothelial tract and 1 patient U2-229) had synchronous MSI-high tumors, all of which displayed a concordant immunohistochemical loss of MLH1. Thus, the vast majority of synchronous UUC does not display MMR defects and does not develop within the HNPCC syndrome. The overall frequency of MSI tumors detected in our study, 4%, is lower than the 13–31% previously reported , with sonogrophy and urinary analysis. None of the patients in this series with MSI and/or IHC loss of MMR protein expression are previously known HNPCC patients in our health care region. Mutation analysis is not planned. A higher frequency of extraintestinal tumors has been reported in families with germline mutations in MSH2, and from the data available, MSH2 seems to play a predominant role also in UUC; loss of MSH2 expression has been reported in 33–60% of MSI-high UUC tumors [The lifetime risk of developing UUC in HNPCC mutation carriers is estimated to be 4–10%, and UUC is in the revised Amsterdam criteria considered to be a HNPCC-associated tumor type, and screening for UUC is generally recommended in HNPCC-families (C tumors ,19,32,33The author(s) declare that they have no competing interests.KE conceived of the study, carried out the MSI analysis, performed immunohistochemical validation and drafted the manuscript. AI also carried out the MSI analysis. BI performed immunohistochemical validation. MN conceived of the study, and participated in its design and coordination and helped to draft the manuscript.The pre-publication history for this paper can be accessed here:


The parenchyma of the brain does not contain lymphatics. Consequently, it has been assumed that arachnoid projections into the cranial venous system are responsible for cerebrospinal fluid (CSF) absorption. However, recent quantitative and qualitative evidence in sheep suggest that nasal lymphatics have the major role in CSF transport. Nonetheless, the applicability of this concept to other species, especially to humans has never been clarified. The purpose of this study was to compare the CSF and nasal lymph associations in human and non-human primates with those observed in other mammalian species.Studies were performed in sheep, pigs, rabbits, rats, mice, monkeys and humans. Immediately after sacrifice (or up to 7 hours after death in humans), yellow Microfil was injected into the CSF compartment. The heads were cut in a sagittal plane.In the seven species examined, Microfil was observed primarily in the subarachnoid space around the olfactory bulbs and cribriform plate. The contrast agent followed the olfactory nerves and entered extensive lymphatic networks in the submucosa associated with the olfactory and respiratory epithelium. This is the first direct evidence of the association between the CSF and nasal lymph compartments in humans.The fact that the pattern of Microfil distribution was similar in all species tested, suggested that CSF absorption into nasal lymphatics is a characteristic feature of all mammals including humans. It is tempting to speculate that some disorders of the CSF system  may relate either directly or indirectly to a lymphatic CSF absorption deficit. The formation of CSF has been studied extensively  and the The experimental evidence would seem to favour the view that a significant portion of CSF is removed from the subarachnoid space by nasal lymphatic vessels. This conclusion is based on experiments that span over 100 years of investigation [reviewed in ] with thThere is also substantial data with various tracers and dyes that this lymphatic CSF transport pathway is a characteristic feature of many mammals . Howeverpost-mortem. Microfil is a coloured, liquid silicone rubber compound which, when combined with a catalytic curing agent, polymerizes within 20 minutes. This agent facilitates the generation of 3 dimensional images of the spaces into which it has been injected. In a previous report, we infused Microfil into the subarachnoid compartment of sheep and noted that it was carried through the cribriform plate into an extensive network of lymphatic vessels in the nasal submucosa of this species [There is no easy way to address the issue of lymphatic CSF transport in humans but recent experience visualizing CSF-lymph connections with Microfil, suggested that it may be possible to examine lymphatic CSF transport pathways in any species  species ,18. DrawAll animal experiments were approved by the ethics committee at Sunnybrook and Women's College Health Sciences Centre and conformed to the guidelines set by the Canadian Council on Animal Care and the Animals for Research Act of Ontario. Studies in the sheep, mouse, rat, rabbit and pig were performed at Sunnybrook and Women's College Health Sciences Centre. Studies on the Barbados green monkey were performed at the Barbados Primate Research Centre and Wildlife Reserve. Investigation of human specimens was approved by the Chief Coroner and the General Inspector of Anatomy for Ontario. These studies were conducted at the University of Toronto, Department of Surgery, Division of Anatomy.The objective of these studies was to infuse a CSF tracer, Microfil (Flowtech, Mass), into the cranial subarachnoid space post-mortem to outline the pathways of CSF outflow. To accomplish this, the specimen was placed in sternal recumbancy with the head fixed in position. A laminectomy was performed on the upper regions of the spinal cord  and the cisterna magna was cannulated with an angiocatheter or a silastic tube of appropriate size. A ligature was placed around the spinal cord and tightened to compress the spinal cord and meninges . This li2O. Mice on the other hand appeared to be very fragile and injection by hand was problematic. In this species, we found that Microfil filling was more successful if this agent was introduced into the CSF compartment using a free-flow pressure system and reservoir as described below.With the exception of studies in the mice, all experiments were performed by infusing Microfil manually using a syringe. We did not routinely monitor infusion pressures post-mortem. However, in several of our initial studies in sheep, we measured infusion pressures between 200 and 300 cm H® (MV-122) and the material catalyzed with 10%  of MV Curing Agent. In several sheep, blue Microfil  was introduced into the vasculature at the same time that yellow Microfil was injected into the CSF compartment. The carotid arteries were catheterized and 20 ml of blue Microfil® (MV-120) was infused into both arteries simultaneously. More specific details pertaining to each species is outlined below.The best results were obtained using a Microfil preparation that was more dilute than that recommended in the product literature. To achieve this, 3 ml of diluent was used for every 1 ml of yellow Microfiln = 21, 16–19 gm; ~4–5 weeks old) were used for this investigation. They were fed lab rat chow (LabDiet 5001) until sacrifice. At the start of the preparation, the mice were euthanized. A laminectomy was performed at the C7 cervical-thoracic level of the vertebral column. Microfil was infused into the cranial subarachnoid space by one of two methods. In some preparations, Microfil was infused into the cranial subarachnoid space over a 5–10 minute interval through manual injection into an angiocatheter. In other experiments, the Microfil was administered using a free-flow pressure system with a reservoir filled with saline regulating the rate of flow into the cisterna magna. To achieve this, the three ports of a 3-way stopcock were attached to (a) a syringe containing Microfil, (b) a cannula attached to an angiocatheter inserted into the subarachnoid space, and (c) a cannula filled with saline hung vertically from a pole. The reservoir height was adjusted such that the saline solution exerted a pressure of approximately 100 cm H20. Microfil was injected via the syringe until the agent reached the hub of the angiocatheter. At this point, the stopcock was adjusted to allow the column of PBS to exert a pressure on the Microfil, which pushed the contrast agent into the subarachnoid space.Randomly bred, wild-type C57/Bl mice  were used for this investigation. They were fed lab rat chow (LabDiet 5001) until sacrifice. The rats were anaesthetized initially by placement in an induction chamber in which 5% halothane was administered for 2 minutes. Immediately after anesthesia, the rats were euthanized. A laminectomy was performed at the C7 cervical-thoracic level of the vertebral column. Microfil was infused into the cranial subarachnoid space over 5–10 minutes.Randomly bred, Fischer 344 rats  were used for this investigation. They were fed rabbit chow (LabDiet 5321) until sacrifice. The rabbits were initially anesthetized with an intramuscular injection of Ketamine (50 mg/kg) and Rompun (5 mg/kg). Prior to surgery, the rabbits were euthanized. A laminectomy was performed at the C7 cervical-thoracic level of the vertebral column. Microfil was infused into the cranial subarachnoid space over a 5–10 minute period.Randomly bred, New Zealand white rabbits  including newborns (2–7 days old) and adult animals (6–8 months old) were used for this investigation. The neonatal lambs were bottle-fed formula (Lamb Replacement, Grober Inc. Cambridge, Ontario) until surgery. The lambs were anaesthetized initially by mask administration of incremental concentrations of halothane from 0.5 to 3%. The adult sheep were anesthetized initially by intravenous infusion of 2.5% sodium Pentothal solution. Subsequently, 1–3% halothane was delivered through an endotracheal tube via an A.D.S.1000 or Narkomed 2 respirator for surgical maintenance. In this species, a laminectomy was performed at the C1-C2 level under anesthesia followed by sacrifice. Microfil was infused into the cranial subarachnoid space over 10–15 minutes. In some animals, blue Microfil  was introduced into the vasculature at the same time that yellow Microfil was injected into the CSF compartment. The carotid arteries were catheterized and 20 ml of blue Microfil® (MV-120) was infused into both arteries simultaneously.Randomly bred sheep  were used for this investigation. They were fed pellets  until sacrifice. The pigs were anesthetized initially with an intramuscular injection of Ketamine (30 mg/kg) and Atropine (0.04 mg/kg). Surgical anesthesia was induced and maintained by 2–5% halothane delivered through an endotracheal tube via a Narkomed 2 respirator. Prior to surgery, the pigs were euthanized. A laminectomy was performed at the C1-C2 level and Microfil was infused into the cranial subarachnoid space over a 10–15 minute period.Randomly bred, Yorkshire pigs (Cercopithecus aethiops sabeus)  were used for this investigation. They were fed twice daily various natural foods until sacrifice. At the start of the preparation, the monkeys were anaesthetized with Xylazine/Ketamine (100 mg/kg) injection. Subsequently, the monkeys were euthanized. A laminectomy between C7 and T1 was performed and Microfil was infused into the cranial subarachnoid space over a 5–10 minute period.Randomly bred, Barbados green monkeys . Access to the cadaveric material was permitted between 6 and 7 hours post mortem. A laminectomy between C7 and T1 was performed and Microfil was infused into the cranial subarachnoid space.Following injection with Microfil, the heads of mice, rats, rabbits, sheep and pigs were stored at 4°C overnight. The next day, the heads were skinned, sectioned in a sagittal orientation and fixed in 10% formalin. In the monkey studies, the Microfil was allowed to set for 2 hours after injection followed by sectioning and fixation with formalin (2–3 weeks) and 70% alcohol (immediately before shipment to Canada). After Microfil infusion in humans, the heads were placed in a freezer for several days (-18°C) after which they were sectioned and fixed with Kaiserling's solution for at least 5 days before dissection.Sectioning of the fixed tissues was made in several orientations including coronal cuts anterior and posterior to the cribriform plate and/or sagittal sections. The various tissues were dissected as appropriate under a dissecting microscope  and images were captured on a Nikon digital camera (Coolpix 995). All images presented in this report are in the midsagittal plane from the cut surfaces of the heads.Filling of the subarachnoid compartment and extension of the Microfil into the lymphatic vessels of the olfactory and respiratory submucosa was dependent on several factors including the size of the species and the time of injection after death. Filling of the CSF space and attendant lymphatic vessels with Microfil was easiest in the larger animals and was increasingly challenging as the species declined in size. Additionally, we were able to achieve successful filling up to 6 hours after death but the best results were obtained when the agent was injected immediately after the animal was euthanized. For reasons unknown, in a few unsuccessful preparations the Microfil did not cure correctly.The number of human specimens was limited to 3. In one attempt, there was incomplete filling of the subarachnoid space due to rupture of the meninges and dura. In another preparation, a large metastatic tumor located in the frontal lobe with accompanying perifocal edema and petechial hemorrhages caused significant dislocation of the brain and prevented filling of the CSF spaces. In a third preparation, where the cause of death was not connected to brain disease (lung cancer), the infusion was successful. Table Microfil was observed throughout the subarachnoid compartment associated with the base of the brain and generally filled the basal cisterns in all species. Microfil was also observed commonly in the subarachnoid space over the convexities of the brain.In all species examined in this report  from that in other mammalian species.In a previous study, we injected yellow Microfil into the subarachnoid space and blue Microfil into the carotid arteries . This prStudies with Microfil provide valuable information on 'potential' CSF transport routes but one must be cautious in applying post-mortem observations to physiological processes. However, in sheep there is considerable quantitative data that supports the concept that the cribriform-olfactory nerve-lymphatic pathway contributes significantly to volumetric CSF absorption ,11,13-16Another reason for the skepticism regarding the role of lymphatics in CSF transport is the generally held view that arachnoid projections function in this role. However, the quantitative evidence supporting a role for arachnoid projections is very limited and unconvincing -39. It m2O, a level of pressure that is clearly pathological. Other groups have failed to observe enrichment of a CSF tracer in the superior sagittal sinus of the rabbit, cat and monkey when the intracranial pressures achieved in the experiments simulated physiological conditions more realistically [The route by which Microfil gains entry into the dural venous sinuses is unknown but is currently under investigation. However, the concept of direct CSF transport into the superior sagittal sinus continues to be problematic. We used several experimental approaches to assess this issue in previous studies in sheep. The most informative technique was to employ the classical approach of Mann to look for enrichment of a radioactive CSF tracer in the superior sagittal sinus compared to a peripheral vein . The Manstically ,41,42.2O  no enrichment was observed. When the gradient favouring absorption was greater than 20 cm H2O, a modest enrichment of tracer was noted [In our investigations in adult sheep, at a CSF-superior sagittal sinus pressure gradient of around 10 cm Has noted .A similar result was obtained with tracer enrichment experiments in newborn lambs. The latter result was surprising. Adult sheep have abundant arachnoid projections in the superior sagittal sinus which are visible when stained with Evans blue dye but, in the neonatal lamb, arachnoid projections are few or absent . TherefoIn our studies the maximum enrichment of CSF tracer in the superior sagittal sinus was observed only within the first few minutes after increasing intracranial pressure . Within 2O , no enrichment was observed. At pressure gradients greater than this level, the concentration of the tracer was significantly higher than in the peripheral circulation. Therefore, the lack of enrichment of the CSF tracer in the superior sagittal sinus at normal physiological intracranial pressures does not appear to be due to downstream lymphatic transport of the CSF tracer. Contrary to the 'classical' view, direct transport of CSF into cranial veins  may represent an auxiliary system that is recruited when intracranial pressures are high or when other transport pathways are compromised.To determine if the lack of CSF tracer enrichment in the superior sagittal sinus was due to lymphatic transport into peripheral veins, we created what we thought would be the conditions most favourable to measure clearance via the arachnoid granulations. We sealed the cribriform plate and prevented CSF transport into the spinal subarachnoid compartment thus negating much of the lymphatic transport capacity. Nonetheless, even under these conditions when the CSF-superior sagittal sinus pressure gradient was raised to 20 cm HIn summary, we have never been able to obtain persuasive evidence that direct CSF transport into the venous system is a physiological reality. In contrast, the qualitative and quantitative support for nasal lymphatic role in CSF absorption is gaining momentum.In this report, we present the first direct evidence for CSF-lymph connections in primates including monkeys and one human. The fact that the Microfil distribution patterns were very similar in the seven species tested suggested that the lymphatic transport of CSF represented an important pathway by which CSF is cleared from the subarachnoid space in mammalian species. A workable hypothesis can be developed around the concept that defects in lymphatic CSF transport may contribute to syndromes characterized by elevated intracranial pressure and/or hydrocephalus.The author(s) declare that they have no competing interests.MJ: conceived of the study, and participated in its design and coordination.AZ: developed the application of the Microfil method to the analysis of CSF transport pathways, participated in all experiments, created an image catalogue and helped in the preparation of the manuscript.CP: aided in the experiments in human, sheep, rats and mice and helped in the preparation of the manuscriptGS: performed the studies in rats and miceDA: in conjunction with AZ performed the studies in humans and monkeys and assisted in all experiments.All authors read and approved the final manuscript.