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  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  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% 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  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 , according to the manufacturers instructions. The main steps were as follows: Mouse monoclonal IgG antibodies to MLH1  MSH2 , MSH6  and PMS2  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  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  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 , 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  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.


Myasthenic crisis is the most serious life-threatening event in generalized myasthenia gravis (MG) patients. The objective of this study was to assess the long-term impact of thymectomy on rate and severity of these attacks in Iranian patients.We reviewed the clinical records from 272 myasthenic patients diagnosed and treated in our neurology clinic during 1985 to 2002. Fifty-three patients were excluded because of unconfirmed diagnosis, ocular form of MG, contraindication to surgery, concomitant diseases and loss to follow-up. The Osserman classification was used to assess the initial severity of the disease. Frequency and severity of the attacks were compared between two groups with appropriate statistical tests according to the nature of variables. Multivariate logistic regression analysis was used to assess the predictors of myasthenic crisis in the group of patients without thymoma.110 patients were in thymectomy group and the other 109 patients were on medical therapy. These two groups had no significant differences with respect to age at onset, gender, Osserman score in baseline and follow up period. 62 patients  had reported 89 attacks of myasthenic crisis. 20 patients of 62 (32%) were in thymectomy group and 42 (68%) were in the other group. There was significant difference between the two groups in number of patients with crisis . In addition, these attacks were more severe in group of non-thymectomized patients as the duration of ICU admission was longer and they needed more ventilatory support during their attacks. Regression model showed thymectomy and lower age at onset as two predictors of decrement in myasthenic crisis rate in non-thymomatous MG patients.It is suggested that frequency and severity of myasthenic attacks as important endpoints in evaluation of MG patients. Thymectomy seems to have a preventive role on rate and severity of these attacks. Myasthenia Gravis (MG) is the mostly known autoimmune disease in human, mediated by autoantibodies against the nicotinic receptor of acetylcholine in neuromuscular junctions . Two carMyasthenic crisis refers to a rapid deterioration in neuromuscular function with respiratory compromise due to ventilatory muscle insufficiency or weakness of upper airway musculature or both . These aIn general, four methods of treatment are currently in use : antichoAlthough there is no convincing evidence regarding the use of thymectomy in non-thymomatous MG patients ,13, mostThymectomy effects on occurrence and intensity of respiratory crisis have not been studied yet. Concerning value of this life-threatening event in MG patients, we studied the role of thymectomy on long-term frequency and severity of these attacks.We reviewed the clinical records from 272 myasthenic patients diagnosed and treated in the neurology clinic of Shariati Hospital, Tehran, during 1985 to 2002. All included patients had positive history and physical examination, confirmed with positive Tensilon test and electromyography (with repetitive nerve stimulation test). Searching for thymomas, all patients had a chest computed tomography (CT) scan.The following patients were excluded from the study: 3 patients with doubtful test results and unconfirmed diagnosis; 25 patients with ocular form of MG (as these patients have no presentation of crisis); 2 patients with thymoma (and two episodes of crisis in the previous year), but contraindicated for surgery because of coronary artery disease; 6 patients with thyroid function abnormalities (because of their known effects on induction and reduction of myasthenic crises); 2 patients with concomitant rheumatoid arthritis (and consumption of high doses of corticosteroids and immunosuppressives); and 15 patients lost from follow-up with incomplete data. 219 MG patients enrolled in the study.Myasthenic crisis was defined as an attack that compels physicians to hospitalize the patient and carefully supervise patient's respiratory capacity . In mostAll included patients were eligible for surgery. Patients with CT confirmed thymoma underwent elective thymectomy as soon as possible. In the first visit after diagnosis confirmation in non-thymomatous patients, the advantages and drawbacks of operation (including risks of anesthesia in MG patients) had been explained to them and deciding between the two choices of thymectomy or conservative therapy had been left to the agreement between physicians and patients. No particular protocol had been established for these patients and physicians had only a consultant role for patients with no force on them.For clinical assessment of the initial severity of the disease, the classification of Osserman  had beent test and analysis of variances (ANOVA) as parametric tests, Mann-Whitney U test as non-parametric test, and Pearson's chi-square test for qualitative differences . P values less than 0.05 were taken to indicate statistical significance. Continuous variables are shown as the mean ± standard deviation (SD) and non-parametric variables with their median and range. Odds ratios and relative risks with 95% confidence intervals (95% CI) were calculated to assess the proportional risk of crisis between two groups.The participants' baseline characteristics and follow-up data were analyzed by Student's In the subgroup of patients without thymoma, multivariate logistic regression analysis was used to assess the relationship between frequency of myasthenic crisis and several risk factors such as sex, start age of the disease, Osserman score and thymectomy. According to its percentile, start age was divided into three categories as bottom one-third (under percentile 33), middle one-third (between percentiles 34 and 66), and top one-third (over percentile 67).110 patients underwent thymectomy  and 109 patients were on conservative therapy. Baseline characteristics and follow-up periods are shown in Table Totally, 62 patients in both groups experienced 89 attacks of respiratory failure during their follow-up, which accounts for 28.3% of the study population. As it is shown in Table To assess the severity of each attack, we considered duration of ICU admission (days), need to respiratory support and need to plasmapheresis or IVIG during crisis as indicating variables. As it is shown in Table U test revealed significant difference in this regard (P < 0.001). Severity indexes did not differ significantly between thymomatous and non-thymomatous patients Table Pathology reports in thymectomized patients revealed 16 cases of thymoma. Fifty percent of these patients (8 of 16) had experienced crisis, which accounts for 40% (8 of 20) of thymectomized patients with history of crisis. Mann-Whitney Totally, there were 203 patients without thymoma. Regression analysis in this subgroup of patients revealed that group of non-thymectomized patients as well as two third of patients with higher ages at the beginning of the disease were more prone to respiratory attacks Table option to increase the probability of remission or improvement in these patients [Persuasive evidence for thymectomy in non-thymomatous generalized myasthenic patients is not on hand. In an attempt to establish standards in this regard, American Academy of Neurology advised thymectomy as only an patients . In thiset al. in 1981, 15 of 28 thymectomized patients experienced 21 crises during follow-up period [Surprisingly, none of these studies has considered myasthenic crisis frequency as their study endpoint. For instance, in the study of Cohen p period . In anotp period . This stIn our experience, persistence on thymectomy for all myasthenic patients would not be so wisely. Response to thymectomy is highly variable among these patients. Method of gaining agreement between physicians and patients would bring some benefits. As it is shown in Table When myasthenic crisis occurrence is considered as the main endpoint of treatment in myasthenic patients, thymectomy has a great impact. Patients on conservative therapy had risk of about 3-fold of thymectomized patients to encounter respiratory attacks. However, the overall frequency of myasthenic crisis among our study population could be considered more than similar reports ,4, perhaSome previous studies have shown the benefit of thymectomy in the control of symptoms of MG. It has been revealed that delay in surgery could worsen the prognosis of MG  and the During myasthenic crisis, Weakness of the respiratory muscles may be out of proportion to that of other skeletal muscles. In rare cases, ventilatory failure is the only clinically apparent manifestation of the disease ,25. In aGenerally, infection is the most common trigger for myasthenic crisis . Higher The main limitation of this study was its retrospective design and the lack of randomization. Apparently, within a period of 17 years, many changes have occurred to diagnostic and therapeutic facilities as well as knowledge and attitude of physicians. These changes could have significant consequences on the outcome of myasthenic patients. Although various operative approaches seem not to differ drastically , variousIn conclusion, we suggest evaluation of various aspects of myasthenic crisis as an important endpoint in long-term assessment of generalized MG patients. Moreover, we showed that thymectomy has a preventive role on rate and severity of myasthenic attacks in age and disease severity matched groups of patients. However, this effect needs further evaluation in upcoming prospective studies.None declared.In advance, suggestion of the design of the study was from our professor AkS. Data extraction and initial analysis were done by AlS and HT. SA participated in the design and implementation of the study. AM performed additional analyses and wrote the first draft of the paper. MS and SA both had helpful and valuable comments in revising the paper. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


C. elegans and S. cerevisiae genomes were 25%–50% more constrained prior to duplication than the genes that failed to leave duplicates. We further show that conserved genes have been consistently prolific in generating duplicates for hundreds of millions of years in these two species. These findings reveal one way in which gene duplication shapes the content of eukaryotic genomes. Our finding that the set of duplicate genes is biased has important implications for genome-scale studies.A central goal in genome biology is to understand the origin and maintenance of genic diversity. Over evolutionary time, each gene's contribution to the genic content of an organism depends not only on its probability of long-term survival, but also on its propensity to generate duplicates that are themselves capable of long-term survival. In this study we investigate which types of genes are likely to generate functional and persistent duplicates. We demonstrate that genes that have generated duplicates in the  Gene duplication is a key process in genome evolution. These authors show that highly conserved genes duplicate more often and are therefore likely to contribute more to the content of eukaryotic genomes Gene duplication is the most important source of new genes and consequently a vital source of genetic novelty . RecentlSaccharomyces cerevisiae  mutational generation, (2) fixation in a population, and (3) preservation through a period when they may be functionally redundant, may favor some genes over others. For example, gene duplicates that lead to an advantageous increase in gene dosage will be preferentially fixed by positive selection, as has been observed in bacteria and revisiae . For othrevisiae , and whicis-regulatory regions, expressed in many tissues . This measure of protein evolution has been shown to be related to several important properties of genes, including dispensability, level of expression, and the number of protein–protein interactions (S. cerevisiae and Caenorhabditis elegans with those that have not.For these reasons we chose to investigate a bias in the molecular attributes of the genes that duplicate. One very informative gene attribute is the rate of protein evolution defined as the number of nonsynonymous substitutions per nonsynonymous site in a given time (ractions . We chosS. cerevisiae and C. elegans (the “study genes”), but instead measured evolutionary rates in two distantly related outgroup species, Drosophila melanogaster and Anopheles gambiae (such a pair of orthologs is referred to as a “representative pair”). Because evolutionary rates for a particular gene are highly correlated in diverse lineages  (S. cerevisiae evolve on average more than 50% slower than the representative pairs of singletons (0.192 versus 0.302), while in C. elegans the difference exceeds 25% (0.230 versus 0.296).The number of duplicate pairs and singleton genes identified in the genomes of or both) . The repA. gambiae and D. melanogaster. Results from this analysis echoed those of the KA comparisons: representative pairs of duplicate genes in both S. cerevisiae and C. elegans are much less likely to have accumulated insertions or deletions than representative pairs of singletons   .KA and the indel rate. First, some of the orthologs identified in D. melanogaster and A. gambiae have undergone duplication in these lineages. This could both affect their rates of evolution, as discussed above, and also lead to the identification of the slowest evolving paralog in D. melanogaster and A. gambiae for the representative pairs of study genes. The latter effect can lead to an artificially low estimate of the evolutionary rates. To test for this possibility, we repeated our analysis using only representative pairs that have not duplicated in either D. melanogaster or A. gambiae. Although this analysis included substantially fewer genes (see p < 0.005 for both organisms). Second, we wanted to make sure that the bias is not due to the peculiarly slow evolution of duplicate genes in multigene families. A reanalysis for only those duplicated genes (in the study organisms) with no other paralogs in the genome revealed very similar results (data not shown). Third, it is possible that our conservative definition of “singleton” may have artificially biased the set of singletons towards rapidly evolving genes. This could be true if slowly evolving singleton genes tend to possess anciently conserved, widely shared protein domains. By generating homology to other genes, these domains may make these singletons fall below the conservative E-value cutoff that we used. To test this possibility, we relaxed our criteria for singleton genes to include all those genes with no E-value less than 10–10. The average rate of evolution for this group of singleton genes was no different than for the former set (data not shown).To further validate these conclusions, we wanted to test several potential sources of error in our analysis of both enes see , the resKS) for the representative pairs of the two classes of genes. This measure is customarily used as a proxy for mutation rate because substitutions at synonymous sites are generally thought to be selectively neutral. However, in many genes, especially those expressed at high levels, synonymous sites appear to be under selection, as evidenced by codon bias. For such genes, the rate of synonymous evolution will underestimate the rate of mutation , gene class (whether the representative pair was for a duplicate or singleton), and KS (between representative pairs). Our results, presented in S. cerevisiae duplicate genes have a lower KS than representative pairs of singleton genes (Spearman Correlation column), but that this correlation disappears when we control for codon bias . Thus, in the case of S. cerevisiae, the higher codon bias of the slowly evolving representative pairs completely accounts for the differences in KS between the two groups. For C. elegans, the KS of the representative pairs for duplicate genes is in fact marginally higher than that for singleton genes, and this slight trend remains when codon bias is taken into account. Thus, mutational differences cannot account for the differences in the rate of protein evolution in either S. cerevisiae or C. elegans.To overcome this complication, we computed the partial correlation coefficients between each of the three factors: codon bias (measured by the codon adaptation index [CAI]  in D. meKS above. D. melanogaster), and KA (of the representative pairs). This analysis revealed some important differences in how the duplication bias is generated in S. cerevisiae and C. elegans.We can also use the level of codon bias to gain additional insight into the potential reasons for the generation and maintenance of duplicate copies of conserved genes. Codon bias is a proxy for the level of expression , while tS. cerevisiae show that the CAI of the representative pairs of duplicates is greater than that of the representative pairs of singleton genes. This indicates that the genes leading to preserved duplicates in S. cerevisiae tend to be unusually highly expressed (p < 0.001). In contrast, for C. elegans, duplicate genes do not appear to be biased towards highly expressed genes (p > 0.1). This difference may reflect a disparity in the mutational generation, fixation, or preservation of duplicates in these two organisms. This analysis also reveals that when codon bias is held constant, the relationship between KA and gene class persists in both organisms. In the case of C. elegans, the correlation coefficient between gene class and KA remains nearly identical when CAI is held constant. For S. cerevisiae, the partial correlation coefficient between KA and gene class does decrease when CAI is held constant (but remains highly significant), implying that the slower evolution of representative pairs of the duplicated genes in S. cerevisiae is partly mediated by preferential duplication of highly expressed genes. To validate these conclusions, we repeated the same analysis using CAI values in the study organisms rather than in D. melanogaster. This analysis revealed very similar results (data not shown).First, both direct and partial correlations for S. cerevisiae and C. elegans lineages, we plotted the evolutionary rate of representative pairs and the average CAI (both in D. melanogaster and in the study organisms) for duplicate pairs of different age classes (where age is measured by KS between the duplicate study genes) , this analysis captures the uniformity of the bias in these lineages. For both organisms, slowly evolving genes appear to have led to the duplicate genes in all age classes (covering hundreds of millions of years). For C. elegans, both the evolutionary rates of the representative pairs and their CAI values remain virtually constant for duplicated genes of all ages. In addition, the CAI values for the duplicate pairs of different ages in C. elegans are very similar to the CAI values for singletons—the only exception is a slight elevation in the CAI for duplicate pairs in the KS range from 1 to 1.5. By contrast, the plot for S. cerevisiae reveals that young duplicate genes (KS < 2.0) tend to have representative pairs with a lower KA than those of older pairs, and this trend is paralleled by the elevated CAI of these young duplicate pairs.To determine whether conserved genes have been preferentially duplicated throughout the history of the y genes) . While lS. cerevisiae is that duplicate pairs with a high codon bias are expected to have a depressed value of KS, as discussed above, and thus will appear younger than they really are. To overcome this problem, we corrected KS estimates for S. cerevisiae genes based on their CAI using a simple approach recently developed for this species  , KA estimates for representative pairs of duplicate genes in S. cerevisiae are much lower than for duplicate genes in C. elegans, whereas for older duplicate genes (KS > 2), the KA estimates are roughly equivalent in both S. cerevisiae and C. elegans.A problem for interpreting this trend in cies see   . If the S. cerevisiae  many of the genes that are duplicated in a single individual are deleterious and thus are quickly removed from the population or (2) many of the duplicate genes that reach fixation in a population do so because of positive selection for the duplicate copy, rather than reaching fixation neutrally by genetic drift.For the first mechanism to work, increases in the dosage of slowly evolving genes must be less likely to have deleterious consequences to organismal fitness than increases in the dosage of more rapidly evolving genes. Recent empirical work, however, has shown that the opposite might be true. In particular, data from yeast have shown that less dispensable, slowly evolving genes are more likely to be haploinsufficient than dispensable genes . This imS. cerevisiae and bacteria  many of the duplicate gene copies that arise in a population are lost quickly to nonfunctionalizing mutations  and (2)  tissues , or of g tissues . Because tissues , these mS. cerevisiae or C. elegans  an increased rate of evolution for the duplicated genes immediately following duplication e.g.,  and (2) S. cerevisiae, C. elegans, D. melanogaster, and A. gambiae were downloaded from GenBank  at http://www.ncbi.nlm.nih.gov/Ftp/index.html. To identify duplicate and singleton genes, a reciprocal protein BLAST  and eliminated all representative pairs with paralogs with an E-value of less than 0.1 . Results from the reanalysis revealed significant trends similar to those found when using all representative pairs.To test whether including duplicate pairs and singleton genes with representative pairs possessing paralogs in the D. melanogaster, S. cerevisiae, and C. elegans genomes using the program CodonW . The table used to calculate CAI for S. cerevisiae is the standard table included in the package. We obtained the appropriate codon usage tables for C. elegans and D. melanogaster from studies by We obtained CAI values for genes in the S. cerevisiae, we used CAI values of each pair to help obtain a better relative estimate of their ages. This was necessary because duplicate pairs with a high codon bias effectively have fewer neutral synonymous sites, resulting in the gross underestimation of their age based on KS alone (S. cerevisiae is given by KS = rt(1 – c), where r is the rate of synonymous substitution in genes with no codon bias, t is time, and c is codon bias as measured by CAI . Rearranging this equation yields the formula KS′ = rt = KS/(1 – c), which we used to obtain corrected estimates of the age of duplicate pairs in S. cerevisiae. No such correction was made for C. elegans genes because they were not shown to have a significantly higher codon bias than singleton genes and because no simple means of correction is presently known.For duplicate genes in S alone ; Li 1997


The importance of low high-density lipoprotein cholesterol (HDL-C), elevated non HDL-C , and an isolated low HDL-C is rapidly emerging. The antiatherosclerotic roles of reverse cholesterol transport and the pleiotropic antioxidant – anti-inflammatory mechanistic effects of HDL-C are undergoing rapid exponential growth.In 1997 a 53-year-old Caucasian male presented with a lipoprotein profile of many years duration with an isolated low HDL-C and uric acid levels in the upper quintile of normal. He developed an acute myocardial infarction involving the right coronary artery and had percutaneous transluminal coronary angioplasty with stenting of this lesion. He also demonstrated a non-critical non-flow limiting lesion of the proximal left anterior descending coronary artery at the time of this evaluation.Following a program of global risk reduction this patient has done well over the past 7 years and remains free of any clinical signs and symptoms of atherosclerosis. His HDL-C and uric acid levels are currently in the normal physiological range.Low HDL-C and isolated low HDL-C constitute an important risk factor for atherosclerosis. Therapies that lead to a return to normal physiologic range of HDL-C may result in the delay of atherosclerotic progression. MRH, a 53-year-old Caucasian male (physician) developed an acute inferior myocardial infarction (MI) associated with bradycardia and occasional PVCs. Emergency medication included aspirin, nitroglycerin and a bolus of TPA.The cardiology team preformed PCTA at the site of near complete blockage of the right coronary artery with successful stent placement. At this time a non-critical 40% lesion located in the proximal left anterior descending coronary artery was noted, which was not manipulated. The patient was discharged following 24 hours of stable monitoring.Herpes Simplex encephalitis 1989 with full recovery.Relapsing fever 1971 full recovery, spontaneous left pneumothorax times two (1982–83), lumbar fusion back surgery 1985, and Mother with CVA (cerebellar) age 58 full recovery. Died of Hodgkin's lymphoma 64. Brother with type 1 diabetes mellitus with onset at age 29  died in sleep age 51.Father with CVA (vertebrobasilar) age 75 with full recovery, COPD, died in sleep while recovering from TIA and pneumonia age 84.Grandparents lived to their 80s and died of old age.High stress family physician who seldom drank alcohol and smoked a pipe occasionally. Blood pressure at times of high stress would elevate to 140/85–88 and return to 120–130s/ 70–75 at times of non-stress in the office. He was physically active with no dedicated exercise programFive months prior to MI and reflective of numerous metabolic profiles over the preceding decades.Total cholesterol 198 mg/dLTriglycerides 154 mg/dLHDL-C 34 mg/dL. HDL-C (1970–1973 32 mg/dL and 34 mg/dL)LDL-C calculated 120 mg/dLNon HDL-C = (198-34) = 164Total Chol/HDL ratio = 6.2 > than 5 and is highUric acid 6.5 mg/dLBlood sugar non-fasting 102 mg/dLHomocysteine first week post MI fasting: 28 mcmol/LLFTs, electrolytes, calcium and phosphorus, serum iron, renal function, and CBC were all in normal range.Patient started a program reflecting the global risk reduction approach described in the RAAS acronym table  and is cCurrent Laboratory Values 2004:Total cholesterol: 138 mg/dLTriglycerides: 94 mg/dLHDL-C: 45 mg/dLLDL-C calculated: 74 mg/dLNon HDL-C: (138-45) = 93Total Chol/HDL ratio = 3.0Uric acid: 6.5 mg/dLBlood sugar: Fasting 80 mg/dL, 2 hour post prandial 118 mg/dLHomocysteine: 7.2 mcmol/LLp(a): 4.2 mg/dL in normal range immediate post MI and again at this time: 4.3 mg/dL.hs-CRP: 0.7 mg/L.LFTs, electrolytes, calcium and phosphorus, serum iron, renal function, and CBC are all in normal range.This patient has done well over the past seven years and remains free of any clinical signs and symptoms of cardiovascular disease. While this patient will always remain a CHD risk, his current laboratory values remain in a normal physiological range. As noted above his HDL-C and uric acid levels are currently in the normal physiological range and his hs-CRP remains in the second quartile.According to Framingham risk scores associated with the National Cholesterol Education Program Adult Treatment Panel III (NCEP ATP III) guidelines , few wouIf we score this patient according to the estimate of 10-year risk for men he gets 6 points for age 53, 2 points for total cholesterol 160–199 age 53, 3 points for being a pipe smoker, 2 points for HDL being < 40 mg/dL, and 1 point for systolic blood pressure 140–159 untreated. This totals 14 points and results in an estimated 10-year risk for men of 16%, which is less than the 20% recommended for more aggressive therapy.Even if we apply the NCEP ATP III guidelines of having two plus risk factors: Male sex, hypertension, smoking, and low HDL-C with a 10 risk < or = to 20% we obtain the following recommendations: LDL-C goal < 130 mg/dL, initiation of TLC if LDL-C is = or > 130 mg/dL, consideration of drug therapy if LDL-C is > or = to 130 mg/dL after three months of TLC. It is important to note that our patient had a LDL-C of 120 mg/dL prior to his event. Even if we look at the non HDL-C levels, which are allowed to be 30 mg/dL higher than LDL-C goals we have a patient with a non HDL-C of only 164. MRH became a CHD risk patient within a short period of time of 5 months.The importance of low HDL-C and cardiovascular disease associated with the lipid triad  found in the metabolic syndrome (metS) and overt type 2 diabetes mellitus (T2DM) and a contributing factor to the elevated non HDL-C discussed in the current NCEP ATP III guidelines or the patients with isolated low HDL-C is rapidly evolving.The accelerated atherosclerosis (atheroscleropathy) associated with the metS and T2DM has been previously reviewed and is definitely a serious problem associated with the current epidemic of obesity – diabesity and T2DM .Both isolated low HDL-C and elevated non HDL-C  levels are difficult to get to known NCEP ATP III recommendations and this task usually requires combination therapy. These therapies consist of therapeutic life style changes and pharmacotherapy including statins, fibrates, selective cholesterol inhibitors such as ezetimibe, and niacin in addition to a global risk reduction of all non HDL-C existing risk factors table  5]..5].In this case report a focus on isolated low HDL-C is appropriate. This case report demonstrates a marked improvement of all lipid parameters including his low HDL-C. However, this marked improvement is not always as simple as this case and therefore, both the patient and the clinician need to be very patient, as well as, creative in order to achieve global risk reduction .In 1977 the Tromso Heart Study demonstrated that CAD patients have HDL-C levels 35% lower than controls and those patients with low HDL-C are three times more likely to develop CAD than those with elevated LDL-C . These eet al. were able to show an inverse association of high HDL-C and low coronary risk was as statically as strong as the direct association of high LDL-C and high coronary risk in a cohort of men and women age 40–82 followed for 12 years who were free from CAD at study entry. At any level of cholesterol low HDL-C increases the rate of CHD [The inverse relation of HDL-C to CHD events has been widely discussed since the original publication of data from the Framingham study 1986) ,8. Caste6 ,8. CasThe NCEP ATP III guidelines clearly defines a level < 40 mg/dL as an independent risk factor for CHD . RaisingMichael Miller has stated: "Low HDL-C is the most common lipoprotein abnormality in patients with CHD and is predictive of CHD events, even when total cholesterol levels are normal" .et al., found that the prevalence of isolated low HDL-C as a risk factor for CHD mortality to be present in one out of six or 16.6 % while studying a 21-year follow up of 8000 men [Goldbourt U 8000 men . FurtherThere are at least eight secondary causes for low HDL-C table  and at lHDL-C is synthesized in the intestine and liver and is extremely important in reverse cholesterol transport from the tissues to the liver for disposal. It works in conjunction with the ABCA1 cholesterol transporter within the intimal macrophages  associated with overt T2DM and multiple risk factors associated with accelerated atherosclerosis may result in a damaging effect to HDL-C and interfere with the ABCA1 transporter in reverse cholesterol transport via a mechanism of oxidation and nitration of tyrosine residues on the apo A-1 lipoprotein outer shell of HDL-C lipoprotein . This biLifestyle changes that are important in raising low HDL-C consist of smoking cessation, weight loss, exercise, and the use of alcohol in moderation.HDL-C has numerous positive effects on the endothelium and arterial vessel wall, which decrease non-diabetic atherosclerosis and the accelerated atherosclerosis – atheroscleropathy associated with metS and overt T2DM table .NCEP ATP III allows the clinician to factor in the additional risks associated with novel, emerging risk markers such as our patients elevated homocysteine. Other risk markers would be the lipid markers: Elevated triglyceride, remnant lipoproteins, lipoprotein (a), an abnormal TC/HDL-C ratio, small dense LDL particles, HDL subspecies, and apolipoprotein A and B. The non lipid markers would include: An elevated glucose, inflammatory markers , coagulation markers , and fibrinogen), the emerging roles of matrix metalloproteinases (MMPs) and of course the established risk marker of an elevated homocysteine. It is interesting to note that Qujeq D et al., noted a negative correlation between total homocysteine and HDL-C levels  in a study evaluating 126 patients  with unequivocal changes of acute myocardial infarction in the electrocardiogram as compared to 135 normal healthy controls, while noting a positive correlation between total homocysteine and LDL-C levels  [The reader may note that the patients' uric acid level was quite elevated prior to his acute coronary event and that this level returned to a very normal level following global risk reduction and aggressive therapy for his multiple risk factors in addition to his isolated low HDL-C. Although not a considered a risk factor or even an emerging, novel risk marker, uric acid may be a quite sensitive marker of underlying redox and oxidative stress. Uric acid levels greater than 4 mg/dL may be considered a red flag in those patients, such as our case report, with high risk for CHD .When viewing the sources for atherosclerosis it is important to note that there are two routes for accumulation of atherogenic lipoproteins (input and outflow) within the arterial intima and subsequent remodeling of the arterial vessel wall.L-A avw = L-in - L-out.The atherosclerosis equation: Lipoprotein Accumulation (retention) in the arterial vessel wall = Lipoprotein in - lipoprotein out. non HDL-C = total Cholesterol – HDL-C .L-in, would equal the net lipoproteins derived from the GI tract (absorption) plus that synthesized by the liver. Lipoprotein out is strictly via reverse cholesterol transport to the liver and secretion via bile into the gut. L-in is primarily the beta lipoproteins or apolipoprotein B containing lipoprotein particles, whereas L-out depends primarily on the alpha lipoproteins, apolipoprotein A or HDL-C. The beta lipoproteins are atherogenic and the alpha lipoproteins are antiatherogenic. From this analogy one can see can see why non HDL-C was so important in the recent NCEP ATPIII guidelines: This is also why the recent global (52 countries) INTERHART study found the ApoB/ApoA-1 ratio (the ratio of atherogenic lipoproteins to non atherogenic lipoproteins) to be the best predictor of CHD (odds ratio of 3.25 for top verses lowest quintile) as compared to the other eight other risk factors  delivering the atherogenic apoB lipoproteins. While the kitchen sink would represent the accumulation of atherogenic lipoproteins within the arterial vessel wall or L-Aavw.In a like manner, the DRAIN would represent L-out or HDL-C or apoA-1 lipoproteins. From this analogy it can easily be seen that if there is inadequate HDL-C or apoA-1 the atherogenic kitchen sink will overflow and result in acute coronary syndromes as happened in our case report figure .While the treatment of isolated HDL-C may seem overwhelming at times, it will be rewarding for both the clinician and the patient as demonstrated by the our case study. This patient has done well for seven years and it is anticipated he will continue to do well with his laboratory values now in a sustained, normal physiologic range.Additional tests by nuclear magnetic resonance spectroscopy (NMR LipoProfile) would assist us in knowing the LDL particle number (LDL-P) and would assist us in even more aggressive therapy. In addition to his current goals he has met, he should have an LDL-P under 1000 micromol/L and small LDL-P under 700 micromol/L.Apo A-1Milano and Apo A-1Paris have resulted in a marked increase in research interest for the HDL-C lipoprotein particle and its future manipulation [Even though we have discussed LDL-C from a quantity perspective, due to an isolated low HDL-C, we should additionally be aware that there exists and equally important role for the quality of HDL-C . Recentlpulation . In the pulation . Recentlpulation . Additiopulation .Recently John Snow, M.D. (1813–1858), a legendary figure in the field of epidemiology, of London, England was honored . He hypoCould low HDL-C be the "pump handle" of atherosclerosis and CHD?ABCA-1: ATP binding cassette transporter A-1BMI: body mass indexCHD: coronary heart diseaseCAD: coronary artery diseasehs-CRP: highly sensitive C reactive proteinT2DM: type 2 diabetes mellitusmetS: metabolic syndromeHDL-C: high density lipoprotein cholesterolLDL-C: low density lipoprotein cholesterolLFTs: liver function testsPCTA: percutaneous transluminal coronary angioplastyVLDL-C: very low density lipoprotein cholesterolTC: total cholesterolTLC: therapeutic lifestyle changesTPA: tissue plasminogen activatorThe author(s) declare that they have no competing interests.MRH conceived the idea to write this manuscript. MRH and SCT wrote, and edited this manuscript together.


A critical issue is to what extent such plasticity involves persistent changes of glutamate receptor subtypes and many prior studies have suggested a main role for alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors in mediating the effect. Our previous work in hippocampal slices revealed that, under pharmacological unblocking of NMDA receptors, both AMPA and NMDA receptor mediated responses undergo a slowly developing depression. In the present study we have further adressed this phenomenon, focusing on the contribution via NMDA receptors. Pharmacologically isolated NMDA receptor mediated excitatory postsynaptic potentials (EPSPs) were recorded for two independent synaptic pathways in CA1 area using perfusion with low MgFollowing unblocking of NMDA receptors, there was a gradual decline of NMDA receptor mediated EPSPs for 2–3 hours towards a stable level of ca. 60–70 % of the maximal size. If such an experimental session was repeated twice in the same pathway with a period of NMDA receptor blockade in between, the depression attained in the first session was still evident in the second one and no further decay occurred. The persistency of the depression was also validated by comparison between pathways. It was found that the responses of a control pathway, unstimulated in the first session of receptor unblocking, behaved as novel responses when tested in association with the depressed pathway under the second session. In similar experiments, but with AP5 present during the first session, there was no subsequent difference between NMDA EPSPs.Our findings show that merely evoking NMDA receptor mediated responses results in a depression which is input specific, induced via NMDA receptor activation, and is maintained for several hours through periods of receptor blockade. The similarity to key features of long-term depression and long-term potentiation suggests a possible relation to these phenomena. Additionally, a short term potentiation and decay (<5 min) were observed during sudden start of NMDA receptor activation supporting the idea that NMDA receptor mediated responses are highly plastic. In the present study, we will further develop the concept of gradually decaying responses.Hippocampal synapses display a variety of activity dependent changes that may represent basic elements of memory. Of foremost interest are long-term potentiation (LTP) and depression (LTD), especially forms that depend on N-methyl-D-aspartate (NMDA) receptor activation and therefore can attain "associative" properties -3. The schannels . Dependieceptors . Severaleceptors . We have of time . FollowiOne critical issue regarding LTP, LTD as well as other forms of glutamatergic synaptic plasticity, is the relative contribution of different glutamate receptor subtypes in creating the synaptic modification. Knowledge about this matter may be helpful in elucidating the underlying modification. While a selective change of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors has been cherished -10, espe2+ solution in the presence of NMDA receptor antagonist AP5 to allow for pathway equalization (see Methods) without evoking NMDA EPSPs. Synaptic transmission was then entirely blocked by adding AMPA receptor antagonist CNQX, followed by unblocking of NMDA receptors by wash out of AP5. During this time, only one pathway was stimulated, keeping the other one silent for later use. As illustrated in Fig. 2+ remained stable for several hours .AMPA EPSPs were initially recorded in low MgTo exclude the possibility that the observed decay of NMDA responses were due to deterioration of slices, implying a general decrease of essential physiological processes, the experiment was repeated for the other pathway, i.e. the one that had not previously expressed NMDA responses (continuation in the same set of 8 slices). As seen in Fig. The same experiment was used to address the question of persistency as well as input specificity of the slowly developing depression of NMDA EPSPs. As can be seen in Fig. The above results suggest that the progressive decay observed in a single pathway as an instant event actually represents a long-term, pathway-specific change that can be assessed long later by comparison across pathways. To further examine the relation between the instantaneously recorded depression and the one measured about 90 min later, the relation between the two was plotted as illustrated in Fig. To pinpoint the induction mechanism, in terms of a pre- versus postsynaptic location, experiments were carried out in a similar way as above except for keeping AP5 in the solution during the first session see Fig. . Hence, Depending on the type of synaptic modification, EPSP waveforms may change in different ways, and previous work in our lab has demonstrated that NMDA EPSPs are more prone than AMPA EPSPs to show these changes . Taking While our data show that the time window matters for measuring NMDA EPSPs, all the above results were qualitatively similar regardless of which window was used. It can be noted, however, that the depression of NMDA EPSPs by the end of a recording session was less pronounced using early measurements than late ones .In 12 out of the 20 experiments, the fiber volley was well separated from the stimulus artifact allowing it to be properly measured. No significant change was detected, the end-of session value amounting 103 ± 2 % of the value at the EPSP peak .In the above, NMDA receptor activation occurred gradually while the antagonist AP5 was washed out. This is in line with the experimental protocol used in our previous work on composite EPSPs containing both AMPA and NMDA components . HoweverIn order to determine whether the transient potentiation had any obvious relation to the slow depression of NMDA EPSPs, the relation between the two was examined. Thus, the degree of initial potentiation was calculated by comparing the pathways just after stimulation was started and the depression was determined, as before, by comparing the end-of-session value with the peak value  activation of afferents. The decline was found to be a form of long-term synaptic depression with an induction linked to NMDA receptor activation and with an expression that was maintained through periods without such activation. Several of its basic characteristics were similar to those of conventional LTP and LTD, suggesting a possible relation to these phenomena.in vitro due to declining viability of biological tissue or other experimental imperfections. Such unspecific "run down" can not account for the present findings since the gradual depression of responses could be repeated in the same slice, using a previously undepressed pathway. On the other hand, if the experiment was repeated twice in the same pathway, the second occasion revealed a diminished NMDA EPSP that showed little further decay. Together, these results show that the depression is input specific and long lasting and that it can saturate. Moreover, the lack of associated changes of the fiber volley speaks against a failure of axon conductance [Decaying responses is a potential side effect in long-term, electric recording ductance , favorinWhile both pre- and postsynaptic expression mechanisms appear feasible, certain mechanisms of induction can be excluded. For instance, a decrease in probability of glutamate release due to a direct depletion of the vesicle pool is unlikely since AMPA EPSPs could be evoked for several hours without significant decay . EveThe input specificity and NMDA dependent induction of the current depression conform with basic properties of conventional LTP and LTD . The depGradually decaying, NMDA receptor mediated responses were observed previously in our lab during recording of composite AMPA-NMDA EPSPs for several hours ,23. AtteWhile we observed that isolated NMDA EPSPs decay "spontaneously", most prior studies employing such EPSPs did not report a decay. It might be that limitations of recording time concealed the effect and cell dialysis during whole cell recording could also be a limiting factor. Actually, a recent study, recording "novel" responses under whole cell conditions, reported on decaying AMPA EPSPs but constant NMDA EPSPs . The posStandard LTP/LTD experiments compare relatively stable periods of recording before and after induction of the synaptic modification. This was not possible in the present case, since merely test stimulation evoked the decay. Therefore, comparisons were generally made between synaptic pathways subjected to different stimulus paradigms. The induction of depression in a single pathway during an initial 2 h period caused a subsequent difference between NMDA responses of the two pathways throughout a subsequent test period. The degree of initial decay was closely related to the later difference between pathways, suggesting that once depression occurred it could be maintained through periods of receptor blockade until testing was performed. Our data suggest a duration of the depression of more than 3 h after the initial induction period. This is in the range commonly referred to as "late", which is believed to involve special biochemistry such as gene expression and protein synthesis ,26. WhetThe gradual depression of NMDA EPSPs was found to saturate after 2–3 h as evidenced by both single and double session experiments. This is in line with several other forms of NMDA-dependent plasticity, including LTP, LTD and chemically induced variants, which are shown to be saturable -29. WhetPrevious work on conventionally induced LTD revealed an essential role for protein phosphatases in mediating the synaptic modification ,31. ConsNMDA receptors also have a number of other regulatory sites, allowing for modulation by glycine, polyamines, calcium, and redox agents  and theyAlthough a postsynaptic modification appears to be the primary choice, a presynaptic one that is initiated postsynaptically is also conceivable. In previous attempts to distinguish between pre- and postsynaptic mechanisms, LTD was compared with depression caused by various pharmacological agents with respect to the ability to influence the waveform of EPSPs . While LWhile the main line of experiments employed a smooth start of NMDA receptor activation following the gradual washout of AP5, another set of experiments made use of sudden activation by awaiting full washout until stimulation was started. Compared to smooth activation, there was an additional, transient potentiation that largely decayed within 20–30 stimuli. This is in accord with a previous study in hippocampal slices showing that stopping stimulation of composite AMPA-NMDA EPSPs for 10–60 min (and one case of isolated NMDA EPSP for 10 min) resulted in a transient potentiation when stimulation was resumed .2+ via the NMDA channels. Similar mechanisms of receptor desensitization/inactivation might be responsible in the present case in forming the transient phase after starting stimulation. Some details remain unexplained by this simple model, such as the biphasic character of the transient phase in terms of initial growth and subsequent decay. One can speculate that a minor LTP, or short-term potentiation, might be induced by the sudden activation of NMDA receptors and so would contribute to the initial growth, although the underlying cause is not addressed in this kind of explanation.Several other studies describe decaying NMDA responses in relation to inactivation or desensitization of receptors ,45. Acco2+ solution was used in our case to unblock NMDA receptors, similar unblocking may occur naturally in response to depolarization. Several important issues are still not settled. Is the saturation of the NMDA EPSP depression an absolute matter or can it be overcome, leading to further down regulation and possibly silencing of synapses? Conversely, is it possible to reverse, i.e. de-depress, the change by LTP or similar processes, allowing for bidirectional control? Further research is needed to resolve these questions.The above results emphasize that NMDA receptor mediated responses are highly plastic and that mere test stimulation can induce a short-term potentiation as well as a slowly developing depression that persists for several hours. The depression was input specific and saturable, and its induction required NMDA but not AMPA receptor activation in conformity with conventionally induced LTP and LTD, suggesting a relation to these phenomena. While a low Mg2 2, MgCl2 2, NaHCO3 26, NaH2PO4 1 and glucose 10, oxygenated by 95% O2, 5% CO2. The hippocampus was dissected out and transverse 400 μm thick slices were prepared by a vibratome or tissue chopper. The slices were initially kept in the same solution at room temperature for at least 60 min. As required, slices were then transferred to one or several "submerged type" recording chambers. During the experiment, slices were perfused at 30°C by a solution similar to that above except that the concentration of Mg2+ was 0.1 mM. The usage of low Mg2+ allowed for expression of NMDA receptor mediated responses.Experiments were performed on 12 to 18 day old Sprague-Dawley rats. The animals were decapitated after isoflurane (Forene) anesthesia in accordance with the guidelines of the Swedish Council for Laboratory Animals. All animal procedures were approved by the Local Ethics Committee at Göteborg University. The brain was removed and placed in an ice-cold artificial cerebrospinal fluid solution containing (in mM) NaCl 119, KCl 2.5, CaClStimulation was delivered as 0.1 ms negative constant current pulses via monopolar tungsten electrodes. For each slice, two stimulating electrodes were placed in the apical dendritic layer of CA1 pyramidal cells on either side of the recording electrode to provide for stimulation of two separate sets of afferents. Field EPSPs were recorded by using a glass micropipette filled with 3 M NaCl (4–10 MΩ resistance). The basal test stimulus frequency was 0.1 Hz with stimuli delivered alternately to the two electrodes, successive stimuli being separated by 5 s. To test the effect of stimulus interruption, one of the two electrodes was given no stimulation during a certain time, the other one remaining stimulated at 0.1 Hz.Recording commenced by monitoring isolated AMPA EPSPs in the presence of AP5 (50 μM) to block NMDA responses. A low concentration of CNQX (1 μM) was used to partially suppress the AMPA responses. In this way, somewhat larger stimulus strengths could be applied, suitable for evoking isolated NMDA EPSPs in the later part of the experiment. During the time of AMPA EPSP recording, the stimulus strengths were adjusted for each slice to equalize the synaptic inputs of the two pathways. This was essential for later comparison of NMDA EPSP across pathways. After obtaining a baseline of equal AMPA responses, the concentration of CNQX was raised to 10 μM which entirely blocked synaptic responces. The remaining non-synaptic response, consisting of stimulus artifact and presynaptic volley, was used to define "true zero".To study NMDA receptor mediated responses, CNQX (10 μM) was maintained in the solution while AP5 was washed out for one or several 2 h periods, referred to as sessions in the following. In between the sessions as well as afterwards, synaptic transmission was again blocked by applying AP5 (50 μM), framing in the sessions by periods of recording non-synaptic responses. Under the sessions, various tests were made depending on the purpose of investigation. Usually one input remained silent during the first session and stimulation was not resumed until after synaptic transmission was reblocked. In another kind of experiment, the initially silent pathway was reactivated in the early part of the first session after NMDA receptors were unblocked, providing a means for sudden start of NMDA receptor activation.Signals were amplified, filtered and transferred to a PC clone computer for on-line and off-line analysis by specially designed electronic equipment (based on an Eagle Instruments multifunction board) and own developed computer software. AMPA EPSPs were measured using an early time window (first 1.5 ms after the fiber volley) while NMDA EPSPs were measured using both an early (first 5 ms after volley) and a late (35–45 ms after artifact) time window. The late measurement was used in presenting most of the results, allowing easy comparison with previous work in our lab that estimated the NMDA component of composite EPSPs via a late measurement . SimilarMeasurements were calculated by integrating the curve along the specified time window after substraction of the prestimulus baseline. All values were corrected by substracting the corresponding measurements of the non-synaptic potential obtained after total blockage of the EPSPs (except when measuring the fiber volley). The final data were quantified as relative values compared to a reference level defining 100 %. While the initial baseline formed a natural reference for AMPA responses, the choice was less obvious for NMDA responses, leading us to use the highest level of responses for one of the pathways in one of the experimental sessions (selected to make sense). Results are expressed as mean ± S.E.M. Statistical comparisons were made using Student's t-test.Drugs were obtained from Tocris Cookson, UK; prefabricated stimulating electrodes were obtained from World Precision Instruments, FL USA, type TM33B.AMPA, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid;AP5, D(-)-2-amino-5-phosphonopentanoic acid;CA, cornu ammonis;CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione;EPSP, excitatory postsynaptic potential;LTD, long-term depression;LTP, long-term potentiation;NMDA, N-methyl-D-aspartate;MD planned and carried out most of the experiments including data analysis, and compiled the manuscript. RL carried out experiments, participated in the planning process and helped in shaping the final manuscript. HPX carried out the initial experiments establishing the effect of NMDA EPSP depression. BJ was responsible for logistics planning and participated in experiments. HW conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.


As an alternative to direct DNA sequencing of PCR products, random PCR-RFLP is an efficient technique to discriminate between species. The PCR-RFLP-method is an inexpensive tool in forensic science, even if the template is degraded or contains only traces of DNA from various species.Interspecies-specific DNA sequence polymorphisms in the mitochondrial cytochrome b gene were analyzed using PCR-RFLP technology to determine the source  of blood traces obtained from a leaf.Bos taurus), sheep (Ovis aries), goat (Capra hircus), roe buck (Capreolus capreolus) and red deer (Cervus elaphus).The method presented can be used for the discrimination of cattle ( Determination of the species from which traces of source material, such as blood stains on a leaf, originate can sometimes be a difficult task in forensic DNA analysis. For instance, insurance claims that involve car accidents with animals require authentication. Species identification is also essential in food quality control-procedures or for the detection and identification of animal material in food samples. Numerous analytical methods that rely on protein analysis have been developed for species identification, such as electrophoresis techniques ,2, immunHowever, before applicable for routine analysis, species-specific diagnostic polymorphisms or mutations must be determined.If these mutations affect restriction enzyme sites, a simple PCR-RFLP can be used as a "sceening tool" for detection.One area in which the PCR-RFLP screening tool would be useful is in training hunting hounds. Before hounds are accepted and approved for hunting, they have to be evaluated by different tests. These tests include an examination of the dog's ability for winding and trailing. To examine these abilities normally a track is prepared consisting of minute amounts of blood from game animals, e. g., wild boar, spotted on a few leaves that are distributed in the forest or field. A trained hound should be capable of winding and trailing without any problems. However, in a case that came to our attention, hounds were unable to wind and it was assumed by the owners that the examiner had used blood from domestic animals. Hence, the question as to the source of the track  was open.Bos taurus), sheep (Ovis aries), goat (Capra hircus), roe buck (Capreolus capreolus) and red deer (Cervus elaphus).To solve this problem we established genetic test (PCR-RFLP) that focused on the use of diagnostic polymorphisms in the mitochondrial cytochrome b gene: Our method can be used for the discrimination between the following mammal species: cattle  and the pattern of bands from the blood sample of one of the dry leaves. As controls, DNA-samples from known species were used. Specific RFLP patterns that distinguish between 5 species are analyzed here. To estimate the exact size of fragments, DNA sequence information was used. C. hircus shows a single fragment of 182 bp, whereas with C. capreolus a fragment of 162 bp was obtained. The RFLP pattern of C. elaphus consisted of 2 fragments of 108 bp and 54 bp respectively. The blood sample on the leaf shows four fragments: 114 bp and 68 bp-fragments, which are characteristic for B. taurus, and a 105 bp and 75 bp fragment which can be assigned to O. aries. This result indicates that the blood sample on the leaf represents a mixture from two species (B. taurus and O. aries). The results were obtained from two independent DNA extractions and confirmed several times by independent PCRs, as well as by DNA sequencing. Amplifications were repeated and species identification was verified by diagnostic Dloop sequencing [B. taurus and three nucleotide differences were observed.For the analysis, DNA was prepared from blood remains on different leaves. The DNA was amplified as described in Methods. Figure quencing . The DloB. taurus and O. aries is still possible for a ratio of 59:1 can lead to false results, but with the designed primer pair this problem was circumvented. For this reason we designed maximally discriminatory primers and the mismatches in each primer are sufficient to exclude human DNA under stringent PCR conditions. The primers were tested with different amounts of added DNA. This method can be used for analysis of mixed samples, since up to three species in different proportions can be determined. The RFLP test using other tissues, e.g., muscle, hair with roots, bones, and saliva, yielded reproducible results. Faeces have not been tested so far.However, when minimizing target DNA, the bands tend to fade away on the agarose gel. The presence of a specific PCR-RFLP for the species analysed, a fragment length of less than 200 bp and the exclusion of contaminating sequences improved methods for existing species determination. The principle of RFLP is often used in food analysis . HoweverThe speed and the efficiency of current nucleotide technology, such as automatic sequencing, will permit the identification of additional taxa. However, the establishment of a PCR-RFLP test would likely need further extensive experimentation.B. taurus, O. aries, C. hircus, C. capreolus and C. elaphus. However, after further development, the tested mitochondrial nucleotide sequences may allow the forensic identification of other animal species.In summary, we were able to show that a simple PCR-RFLP is efficient in differentiating between ® Tissue Kit  according to the manufacturers' handbook. Isolated DNA was diluted in 50 μL HPLC-H2O and used for further analyses.DNA was extracted from blood and tissue samples using QIAampBone samples were roughly ground with a pestle and mortar, then finely powdered in a Retsch mill. Bone powder (0.3 g) was incubated in 1.5 mL of 0.5 M EDTA (pH 8.3) for 20 h while rotating. The suspension was centrifuged for 4 min at 4000 rpm. The supernatant was transferred to a fresh tube or to an automated nucleic acids extraction system  and 1.6 mL sterile distilled water  was added. As the extraction procedure was automated the volumes of reagents dispensed may have varied between runs. Five hundred microliters of Proteinase K was added and the mixture incubated for 1 h at 58°C with shaking. Three milliliters of phenol/chloroform/isoamyl alcohol  were added and the mixture was further incubated at room temperature for 6 min while shaking. The phases were allowed to separate by incubating at room temperature for 8 min without shaking and the organic phase and interphase, if present, were discarded. Chloroform  was added to the aqueous phase and the mixture incubated for 6 min at room temperature while shaking. The phases were again allowed to separate by incubating at room temperature for 8 min without shaking and the organic phase and interphase, if present, were discarded. Ninety microliters of sodium acetate (pH 4.5) and 3.2 mL of 100% isopropanol were added followed by incubation for 2 min with shaking. Five microliters of Glasmilk (Dianova) were added and the suspension was shaken for another 10 min. To obtain a pellet, the solution was filtered through Precipitette filters (Applied Biosystems) or centrifuged for 3 min at 5000 rpm. The pellet was washed with 80% ethanol and eluted into 50 μL sterile distilled water . Five to ten microliters of extract were used for PCR amplification or the extract was stored at -20°C. Glasmilk was not removed prior to amplification.For RFLP analysis, a 195 bp long PCR fragment was amplified from mitochondrial cytochrome b region. The following primer pair was used for amplification, CB7u (5'-GCGTACGCAATCTTACGATCAA-3') and CB7l (5'-CTGGCCTCCAATTCATGTGAG-3').2; 150 μM dNTPs; 0,18 μM of each Primer and 2 U AmpliTaq Gold (PE Applied Biosystems). Cycling conditions included a denaturation step at 95°C for 3 minutes, followed by 32 cycles of 95°C for 1 minute, primer annealing at 54°C for 1 minute and elongation at 72°C for 1 minute in a thermocycler (Hybaid).The PCR was carried out in a total volume of 50 μL consisting of 10 ng DNA, 60 mM KCl; 12 mM Tris-HCl; 2.5 mM MgClThe 195 bp fragment was digested with TSP509 (New England Biolabs) for two hours at 65°C. The resulting fragments were separated by gelelectrophoresis in a 2.5 % agarose gel.A single fragment of the mitochondrial DNA Dloop region was amplified using primers Dloopu (5'-AAATGTAAAACGACGACGGCCAGTAATCCCAATAACTCAACAC-3') and Dloopll (5'-AAACAGGAAACAGCTATGACCACTCATCTAGGCATTTTC-3').2, pH 8.3) and Q-solution, 100 μM for each dNTP, with 1 M Taq Polymerase and 10 pmol of each primer. Four microlitres of the DNA-extract were added to the PCR mix. The amplification was carried out with initial denaturation at 95°C for 10 min, followed by 35 cycles of one denaturation step at 94°C for 40 sec, primer annealing at 52°C for 40 sec and primer extension at 72°C for 45 sec in a Hybaid thermocycler. PCR-products were purified using the QIAEX II Gel Extraction Kit  according to the manufacturers' instructions. Sequencing was performed using ABI-Prism™ Dye Kit V3 (Applied Biosystems) in a 10 μL volume containing 2 μL purified PCR-product and 5 pmol of primer. Sequencing reactions underwent 27 cycles of 30 sec at 94°C, 30 sec at 50°C and 3 min at 60°C in a Techne thermocycler. The dye terminators were removed by sephadex-G45 column purification (Millipore). Sequencing reactions were electrophoresed for 2 h on an ABI Prism® 3100 genetic analyzer (Applied Biosystems) according to the manufacturers' instructions.Amplifications were performed in a final volume of 20 μL in 10 × PCR buffer  and observed approximately 100% sequence identity compared with Ovis aries. Furthermore, we investigated different cattle breeds and we could not find any sequence differences within the tested cytochrome b DNA-fragment.Regarding closely related species, we analyzed mouflon DNA (IP performed the DNA extractions, PCR-RFLP analysis and mtDNA Dloop DNA sequencing.JB developed and provided the PCR-RFLP protocol for species identification.BB was responsible for funding, supervision of the research project, manuscript writing and editing as well as scientific correspondence.