HIV infection and progression to AIDS is characterized by the depletion of T cells, which could be due, in part, to apoptosis mediated by the extra-cellular HIV-encoded Tat protein as a consequence of Tat binding to tubulin. Microtubules are tubulin polymers that are essential for cell structure and division. Molecules that target microtubules induce apoptosis and are potent anti-cancer drugs. We studied the effect on tubulin polymerization of three Tat variants: Tat HxB2 and Tat Eli from patients who are rapid progressors (RP) and Tat Oyi from highly exposed but persistently seronegative (HEPS) patients. We compared the effect on tubulin polymerization of these Tat variants and peptides corresponding to different parts of the Tat sequence, with paclitaxel, an anti-cancer drug that targets microtubules.We show that Tat, and specifically, residues 38–72, directly enhance tubulin polymerization. We demonstrate that Tat could also directly trigger the mitochondrial pathway to induce T cell apoptosis, as shown in vitro by the release of cytochrome c from isolated mitochondria.These results show that Tat directly acts on microtubule polymerization and provide insights into the mechanism of T cell apoptosis mediated by extra-cellular Tat. AIDS is due to human immunodeficiency virus HIV-1) infection of CD4 T cells and is characterized by the cell death of HIV-infected lymphocytes and uninfected bystander cells  infectio,6. Tat wVinca.alkaloids. The MDAs inhibit microtubule dynamics in living cells and lead to apoptosis after cell cycle disturbance [c, leads to the caspase cascade activation and thus to apoptosis [in vitro by the release of cytochrome c from isolated mitochondria [Another mechanism was proposed that involves microtubules to induce Tat-mediated apoptosis . We showturbance -24. Theyturbance ,26. The poptosis . Moreovechondria .The aim of this study was to evaluate the consequence of Tat binding to microtubules and the correlation with the mitochondria-mediated T cell apoptosis. We used three Tat variants with sequences from 99 to 101 that have the size of the main Tat variants found in the field . The HIVThe minimal concentration of tubulin (Cr) necessary to obtain tubulin polymerization without drug was 7 μM in our buffer conditions . We measWhen the temperature of the samples was decreased to 10°C, we observed a complete depolymerization for tubulin alone (line 1) and tubulin with 4 μM and 8 μM Tat (lines 2 and 4 respectively). Even at the highest concentrations of Tat (16 μM and 20 μM), at 10°C the turbidity decreased to the original values (data not shown). By contrast, in the same conditions, we did not observe any depolymerization in the presence of paclitaxel (line 3). However, full depolymerization in the presence of paclitaxel was obtained with a longer incubation at 4°C (data not shown). The reversibility of following an incubation at 10°C or 4°C strongly suggests that turbidity enhancement is not related to precipitation or aggregation but to microtubule formation.We also observed that when Tat concentration is increased to stoichiometric quantities with tubulin, the turbidity values increased dramatically to an absorbance greater than 2.These values are superior to the absorbance obtained in the same conditions using the stoichiometric quantity (15 μM) of paclitaxel, which induces effective tubulin polymerization. Turbidity is known to be a function of the total weight concentration of scattering particles only when the particles have small diameters compared with the wavelengths of the incident light ,35. ThusWe evaluated the ability of different Tat variants to modify tubulin polymerization. In our conditions, when 15 μM of tubulin are incubated at 37°C with 8 μM Tat HxB2, Eli or Oyi, the turbidity plateau values at 350 nm are 1.01, 1.61 and 0.80 respectively (Table To identify the sequence(s) region(s) implicated in tubulin, peptides overlapping the sequence of Tat HxB2 and Tat Oyi were synthesized. The sequences of peptides 38–72 and 73–99 derived from both Eli and Oyi are indicated in figure p = 0.037) using one-sided ANOVA. The percentages of apoptotic cells in the presence of 1 μM Tat Oyi were similar to the control cells (0.1 %)  staining. This method allows the quantification of apoptotic cells by the observation of the characteristic nuclear fragmentation. After 20 hours treatment, the percentage of apoptotic cells in the presence of 10 μM Tat HxB2 (11 %) and 10 μM Tat Eli (12.5 %) were higher than those obtained with 10 μM Tat Oyi 3.5 %) Fig. . These d.5 % Fig. %) Fig. . Thus, aPaclitaxel stabilizes the microtubule network by inducing the formation of pseudoasters in mitotic cells and the formation of bundles, structures that correspond to strongly associated microtubules, in interphasic cells . Considei.e. 48 h) in the presence of paclitaxel leads to 90 % of cell death (data not shown). After 20 hours treatment, the percentage of the G2/M cells in the presence of 1 μM Tat HxB2 or 10 μM Tat Oyi were 28 % and 32 % respectively and similar to the 33 % observed in the untreated cells  whereas the level of cytochrome c with 0.2 μM of Tat Eli (lane T0.2) was comparable to the untreated mitochondria (lane T-). In parallel, cytochrome c levels decreased slightly in mitochondria treated with Tat, as revealed by Western Blot of the pellets  with FASTMoc chemistry according to the method of Barany and Merrifield  on 4-hyd275 nm = 1.07 L·g-1·cm-1 in 0.5% SDS in neutral aqueous buffer or ε275 nm = 1.09 L·g-1·cm-1 in 6 M guanidine hydrochloride.Lamb brain tubulin was purified by ammonium sulfate fractionation and ion-exchange chromatography, stored in liquid nitrogen and prepared for use as described ,46. DuriIn vitro studies have been derived from experiments that show that the variation of temperature permits the disassembly/reassembly cycles of tubulin in the presence of GTP and Mg2+ [2, and 3.4 M glycerol, pH 6.5. The various concentrations of Tat or paclitaxel were mixed with tubulin at 4°C. The reaction was started by warming the samples to 37°C in a 0.2 × 1 cm cells, and the mass of polymer formed was monitored by turbidimetry at 350 nm using a Beckman DU7400 spectrophotometer thermostated. Depolymerization occurs when the temperature is lowered to 10°C. The data showed in fig 350 nm values correspond to values without the buffer. The microtubule mass was determined by centrifugation assay. Samples were taken at the turbidity plateau at 37°C. They were ultra-centrifuged at 50,000 rpm for 15 min at 37°C using fixed angle rotor TLA 100.2 in a TL100 Beckman apparatus. In these conditions, microtubules precipitate in the pellet and non- polymerised tubulin remains in the supernatant. Pellet and supernatant were loaded separately on 10 % acrylamide SDS-PAGE, colored with Coomassie blue after migration  containing 10 % fetal bovine serum (BioWhittaker), 2 mM glutamine , 1 % penicillin and streptomycin . Exponentially growing cells (105 cells/ml) were seeded 3 days before paclitaxel or Tat treatment.The lymhoblastoid Jurkat cell line was cultured in RPMI 1640 supplemented with 2 mM ultraglutamine , penicillin (100 U/ml), streptomycin (100 μg/ml), and 10 % heat-inactivated fetal calf serum . Human neuroblastoma SK-N-SH cells were routinely maintained at 37°C and 5 % COc antibody  was used at 1/1000. Secondary antibody was goat antimouse monoclonal antibody conjugated with peroxidase . The antibodies used for immunofluorescence microscopy were anti-α-tubulin  and FITC mouse anti-goat IgG .Stock solution of paclitaxel (Sigma) was prepared in DMSO. The final concentration of DMSO used in cell culture was less than 0.05 %. For Western blotting, anti-VDAC antibody  was used at 1/500 and anti- cytochrome 5 cells/ml were incubated in the absence or presence of Tat or paclitaxel at different concentrations . The proportion of hypodiploid cells was used as an estimate of apoptosis. Cell death was further analysed using fluorescence microscopy of cells stained with DAPI. 5 × 105. Jurkat cells/ml were incubated at 37°C for 20 hours with various Tat or paclitaxel in Lab-Tek chamber slides. . After centrifugation for 10 min at 3500 rpm, spin cells were fixed with 3.7 % formaldehyde, permeabilized with 0.1 % saponin and treated with PBS containing 10 μg/ml 4'6-diamidino-2-phenylindole (DAPI) (Sigma). The morphology of the cell nuclei was observed with a fluorescence microscope using an excitation wavelength of 350 nm. Nuclei were considered to have the normal phenotype when glowing bright and homogenously. Specific well drawn line up chromosome were seen in equatorial plates for cells in metaphase. Apoptotic nuclei were identified by the condensed chromatin gathering at the periphery of the nuclear membrane or a total fragmented morphology of nuclear bodies. Cells were counted and the percentage of normal., mitotic and apoptotic nuclei determined. Data indicated in the figure Jurkat cells at a concentration of 5 × 10see Fig. . After 26 Jurkat cells/ml were treated for 6 hours with 10 μM Tat HxB2 or 1 μM paclitaxel and were processed for tubulin indirect immunofluorescence staining as previously described [10escribed . After i7 cells were suspended in a sucrose buffer  at 4°C, homogenized with 50 strokes in a glass homogenizer , and centrifuged twice at 800 g for 10 min. The supernatants were then centrifuged at 15,000 g for 10 min at 4°C. Mitochondrial pellets were immediately washed three times in the sucrose buffer. Isolated mitochondria were aliquoted and incubated for 2 hr at 37°C with various Tat concentrations. After centrifugation , pellets and supernatants were carefully separated. Mitochondrial pellets were lysed in lysis buffer  and loaded in parallel with the supernatants on a reducing 15 % SDS-PAGE gel. The gel was processed for immunoblotting and revealed using antibodies against VDAC and cytochrome c. Anti-VDAC antibody  was used to ensure that equal amounts of mitochondrial proteins were loaded on the gels and that no mitochondria or lyzed mitochondria were present in the supernatant. Secondary antibodies were added and visualization was carried out using an enhanced chemiluminescence detection kit . The results presented are representative of four independent experiments.Mitochondria were isolated by cell fractionation from the neuroblastoma SK-N-SH cell line as previously described . 9 × 107HIV, human immunodeficiency virusMDA, microtubule damaging agentVDAC, voltage-dependent anion selective channel.The author(s) declare that they have no competing interests.JdM carried out most of the experimental work, performed the experimental design and drafted the manuscript. MC performed part of the cell, mitochondrial and immunofluorescence study directed by DB. PB performed tubulin purification, part of tubulin experiments on polymerization, quantification of microtubules and electron microscopy directed by VP. GRC, SL, SO, DE and JW synthezised the peptides and characterized them biochemically and biophysically. CP performed FACS studies. EPL participated in the design of the study, the redaction of the manuscript and funded the studies.


Reports exist of transmission of culture in nonhuman primates. We examine this in a troop of savanna baboons studied since 1978. During the mid-1980s, half of the males died from tuberculosis; because of circumstances of the outbreak, it was more aggressive males who died, leaving a cohort of atypically unaggressive survivors. A decade later, these behavioral patterns persisted. Males leave their natal troops at adolescence; by the mid-1990s, no males remained who had resided in the troop a decade before. Thus, critically, the troop's unique culture was being adopted by new males joining the troop. We describe (a) features of this culture in the behavior of males, including high rates of grooming and affiliation with females and a “relaxed” dominance hierarchy; (b) physiological measures suggesting less stress among low-ranking males; (c) models explaining transmission of this culture; and (d) data testing these models, centered around treatment of transfer males by resident females. A unique less-aggressive suite of behaviors that affects the overall structure and social atmosphere of a wild baboon troop potentially represents an intergenerational transfer of social culture A goal of primatology is to understand the enormous variability in primate social behavior. Early investigators examined interspecies differences, e.g., that pair-bonding is more common among arboreal than terrestrial primates . Attenti(Pan troglodytes) across Africa demonstrate variability in 39 behaviors related to tool use, grooming, and courtship  related to the overall structure and social atmosphere of the troop. We also document physiological correlates of this troop atmosphere, the transmission of relevant behaviors past their originators, and possible mechanisms of transmission.Nearly all such cases of nonhuman culture involve either technology , food acquisition, or communication. In this paper, we document the emergence of a unique culture in a troop of olive baboons In the early 1980s, Forest Troop slept in trees 1 km from a tourist lodge. During that period, an open garbage pit was greatly expanded at the lodge. This attracted an adjacent baboon troop, Garbage Dump Troop, which slept near the pit and foraged almost exclusively there.By 1982, many Forest Troop males went to the garbage pit at dawn for food. While such refuse eaters did not differ in age distribution (data not shown) or average dominance rank from non–refuse eaters, they were more aggressive ; such agIn 1983, an outbreak of bovine tuberculosis occurred, originating from infected meat in the dump. From 1983 to 1986, most Garbage Dump animals died, as did all refuse-eating Forest Troop males ; no other Forest Troop animals died .These deaths greatly altered Forest Troop composition, such that there were fewer adult males and more adult females; this more than doubled the female:male ratio . By 1986Because of these events, observations of the troop were stopped, and only censusing was done until 1993. Research was begun on Talek Troop, approximately 50 km away.In 1993, informal observation of Forest Troop indicated that the behavioral features seen by 1986 had persisted. Critically, by 1993, no adult males remained from 1983–1986; all current adult males had joined the troop following 1986. Thus, the distinctive behaviors that emerged during the mid-1980s because of the selective deaths were being carried out by the next cohort of adult males that had transferred into the troop. Focal sampling on Forest Troop recommenced in 1993, in order to document this phenomenon. Data from Forest Troop 1993–1996  were compared with two other data sets that served as controls: observations from 1993–1998 on the Talek Troop (henceforth T93–98), and observations of Forest Troop itself prior to the deaths . These two control data sets did not differ significantly from each other and were combined, henceforth T93–98/F79–82.Males of F93–96 and T93–98/F79–82 had similar rates of approach–avoidance dominance interactions (data not shown). Moreover, dominance stability did not differ, as measured by the percentage of approach–avoidance interactions which represented a reversal of the direction of dominance within a dyad of males of adjacent rank . There was also no difference in the average tenure length of the highest-ranking male (approximately a year).Despite those similarities, dominance behavior in F93–96 differed from the two control cases in ways that, arguably, made for less stress for low-ranking males. A first example concerns approach–avoidance dominance interactions between males more than two ranks apart in the hierarchy. The overwhelming majority of such interactions were won by the higher-ranking individual. Because a male is rarely seriously threatened by an individual more than two ranks lower in the hierarchy, interactions between individuals that far apart typically represent harassment of or displacement of the subordinate by the higher-ranking male, rather than true competition. In T93–98 and F79–82, approximately 80% of approach–avoidance interactions were between males more than two ranks apart in the hierarchy. In contrast, a significantly smaller percentage of approach–avoidance interactions were soin F93–96 A. InsteaPatterns of aggression also differed between F93–96 and T93–98/F79–82 in a way that suggested a less stressful environment for subordinates in F93–96. The troops had similar overall rates of aggressive interactions . HoweverWe examined the data for reconciliative behavior  in F93–Quantitative data on affiliative behaviors were not available for F79–82. However, F93–96 males socially groomed more often than did control T93–98 males A this di2R of correlation between rank and reproductive success: 0.25 ± 0.25 and 0.54 ± 0.10, respectively, n.s.).Sexual behavior did not differ between F93–96 and T93–98/F79–82. The percentages of nonpregnant, nonlactating females in estrus per day did not differ . Moreover, the relationship between male rank and reproductive success did not differ , hypertension, and decreased levels of high density lipoprotein cholesterol, growth factors, and circulating lymphocytes . We testThis was the case A. In F79Subordinate F93–96 males were spared another stress-related physiological marker. Experimental anxiety was induced by darting males, intramuscularly, with β-carboline-3-carboxylic acid, a benzodiazepine receptor antagonist which induces behavioral and physiological indices of anxiety in primates . Males wβ-carboline-3-carboxylic acid had no effect on behavior in high-ranking males in T93–98 or F93–96 B.The druThus, in the more typical F79–82 and T93–98 troops, subordination had distinctive stress-related physiological correlates. In contrast, F93–96 males lacked these rank-related differences.A decade after the deaths of the more aggressive males in the troop, Forest Troop preserved a distinct social milieu accompanied by distinct physiological correlates. Critically, as noted, no adult males in F93–96 had been troop members at the end of the tuberculosis outbreak. Instead, these males had subsequently transferred in as adolescents, adopting the local social style. A number of investigators have emphasized how a tolerant and gregarious social setting facilitates social transmission e.g., , exactly(Macaca mulatta) housed with stumptail macaques (M. artoides) assume the latter's more conciliatory style ((Papio anubis) and hamadryas baboons (P. hamadryas) differ in social structure, and females of either species experimentally transferred into a group of the other adopt the novel social structure within hours  or aversive ones  contingent upon their assimilating the troop tradition. In such models, a key question is who “instructs.” Much as with the term “culture” being used with respect to animal behavior, the use of the term “instruction” has also generated some controversy, with some preferring the concept of “active behavioral modification” by others bringing about the change. As a striking example of that, when young male cowbirds learn to produce their local song, they initially produce an undifferentiated repertoire of songs, and females react to the production of appropriate dialect with copulation solicitation displays, thus providing positive reinforcement and shaping those behaviors .In “observational models” applied to chimpanzee tool use, young learn nut cracking by observing and copying adults. As applied to F93–96, transfer males would model behavior upon that of resident males.In “facilitation models” of the chimpanzee example, proximity to adults and their hammers increases the likelihood of the young experimenting with hammers and deriving the skill themselves. As applied to the baboons, male F93–96 behaviors would be an implicit default state where, in the absence of the more typical rates of male aggression , females broadly tend to become more affiliative, and in the context of more affiliative female behavior, transfer males broadly tend to become less aggressive. As perhaps a way of stating the same, the default state may emerge because of the atmosphere of a troop with a high female:male ratio .Finally, a “self-selection model” may apply to the baboons, in which particular kinds of males were more prone to transfer into such a troop .We assessed these models by analyzing cases where adolescent males transferred on known dates and were observed for at least 2–6 mo afterward. Thus, we searched for behavioral patterns involving new transfer males that might differ between F93–96 (five such transfers) and T93–98/F79–82 (12 transfers).Many interactions involving new transfer males did not differ . TransfeWe then examined affiliative interactions between females and new transfer males, and found striking differences between F93–96 and T93–98/F79–82, in that F93–96 females treated new transfer males in the same affiliative manner that they treated resident males. F93–96 transfer males had a shorter latency until first being groomed by or presented to by a female than did T93–98/F79–82 transfer males A. The dB. While These data allow some insight as to the mechanisms of social transmission in F93–96 . The lack of contingency in the treatment of transfer males by residents argues against instruction; commensurate with this, there is relatively little evidence for “instruction” in nonhuman primate cultural transmission . The simThe data instead support either observational or facilitative/default models. Insofar as resident males in all troops interacted with transfer males similarly, transmission in F93–96 could have involved observation only if such observations were of how resident males interacted with females or each other. Some, but not all, studies support observational models of social transmission in other primates ; there aThis analysis raises the possibility that there is no social transmission, but that the F93–96 pattern is merely the emergent outcome of the 2:1 female:male ratio. To test this, we analyzed the five available studies of baboon troops with adult female:male ratios of 2 or more which contained quantitative data comparable to the present data . The keyThe unconditional  nature of the default model is puzzling, in that it requires that females be relatively affiliative to recent transfer males who, nonetheless, are initially aggressive to them. This seems counter to the long-standing emphasis in primatology on individual relations . Precedent for this unexpected implication comes from the social epidemiology literature concerning “social capital,” in which health and life expectancy increase in a community as a function of communitywide attributes that transcend the level of the individual or individual social networks .In summary, we have observed circumstances that produced a distinctive set of behaviors and physiological correlates in a troop of wild baboons. Moreover, these behaviors were taken on by new troop members; while obviously not conclusive, the data suggest that this most likely occurs through observational or facilitative/default models. Finally, somewhat uniquely in nonhuman primate studies, these findings concern the intergenerational transfer of social, rather than material culture.These findings raise some issues. There appear to be adverse health consequences of the stress-related physiological profile of subordination in typical baboon troops . The disA converse issue concerns circumstances that might destroy the F93–96 culture. The culture might be destroyed if numerous males transfer into the troop simultaneously, or if a male transfers in who, rather than assuming the F93–96 culture, instead takes advantage of it. Game theory suggests that F93–96 would be vulnerable to such “cheating.” Another issue concerns the fate of natal males from F93–96 when they transfer elsewhere. Reciprocal altruism models  suggest Finally, these findings raise the issue of their applicability to understanding human social behavior and its transmission. Human history is filled with examples of the selective loss of demographic subsets of societies . The present data suggest that demographic skews may have long-term, even multigenerational consequences, including significant changes in the quality of life in a social group.(Papio anubis) living in the Masai Mara Reserve of Kenya. Olive baboons live in multimale troops of 30–150 animals, with polygamy and considerable male–male aggression. Males change troops at puberty and, as adults, achieve ranks in somewhat fluid dominance hierarchies. In contrast, females remain in their natal troop, inheriting a rank one below that of their mother.Subjects were a troop, Forest Troop, of olive baboons Subjects were observed each summer from 1978–1986, and continuously since 1993. An additional troop, Talek Troop, was observed continuously since 1984. Behavioral data were collected as 20-min focal samples . During Reproductive success was indirectly estimated from frequencies of matings and consortships . The value of any given consortship or mating was adjusted by the probability of a fertile mating occurring that day .Endocrine data were collected under circumstances allowing for measures of basal steroid hormone levels . Subject


Journal of Experimental & Clinical Assisted Reproduction is an Open Access, online, electronic journal published by BioMed Central with full contents available to the scientific and medical community free of charge to all readers. Authors maintain the copyright to their own work, a policy facilitating dissemination of data to the widest possible audience without requiring permission from the publisher. This Open Access publishing model is subsidized by authors (or their institutions/funding agencies) in the form of a single £330 article processing charge (APC), due at the time of manuscript acceptance for publication. Payment of the APC is not a condition for formal peer review and does not apply to articles rejected after review. Additionally, this fee is waived for authors whose institutions are BioMed Central members or where genuine financial hardship exists. 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A fascinating case, with much to learn about diagnosis and treatment of patients with abnormal CSF results. After learning from the case, take our online quiz A 17-year-old woman, who was born in Bangladesh, presented to an accident and emergency department in the United Kingdom with a history of being unwell for 24 hours. She had a headache and fever, and was vomiting. On questioning, there was no photophobia or neck stiffness. She lives in the United Kingdom and had been for a holiday to Bangladesh 4 months previously. The family members were all well, and none had similar symptoms. The woman had no previous medical history and was on no medication.9 per millilitre , with 84% neutrophils. Her erythrocyte sedimentation rate was 15 mm in the first hour , and her C-reactive protein was less than 5 mg/l . A malaria screen was negative, and renal and liver function tests were normal.On examination, she looked unwell, with a temperature of 38.5 °C, pulse 96 beats per minute, blood pressure 112/52 mm Hg, and a respiratory rate of 16 breaths per minute. She had a Glasgow Coma Score of 14, there was no neck stiffness, and her ocular fundi were said to be normal. There were no other significant findings on examination. Immediate investigations showed a normal blood count apart from a raised white blood cell (WBC) count of 11.1 × 10Haemophilus antigens, and bacterial culture was subsequently negative. The patient was given 2 g of ceftriaxone intravenously immediately, and ceftriaxone treatment was continued following the CSF results. Aciclovir IV, at a dose of 10 mg/kg every 8 hours, was added to cover the possibility of herpes encephalitis.A clinical diagnosis of probable viral meningitis was made pending the results of further investigations. Chest X ray was normal, as was an unenhanced CT brain scan. Lumbar puncture showed a normal cerebrospinal fluid (CSF) opening pressure, and the CSF was not blood stained. Laboratory analysis showed 1,606 WBC/ml , of which 60% were lymphocytes with protein of 1.08 g/l  and glucose of 2.9 mmol/l , with a corresponding plasma glucose of 5.7 mmol/l . No organisms were seen on Gram stain. The CSF was negative for pneumococcal, meningococcal, and recurrent episodes of apparent aseptic meningitis.The differential diagnosis was now viral, bacterial, tuberculous (TB), fungal, or malignant meningitis, or sarcoidosis. Additional investigations were requested, including polymerase chain reaction (PCR) on CSF for viruses and tuberculosis. There were no risk factors for HIV infection (HIV seroconversion can present with meningitis). Mollaret’s meningitis (recurrent aseptic meningitis associated with herpes simplex virus) was a possibility , though The following morning the patient’s temperature returned to normal at 37 °C, and she felt better. Referral was made to the Infectious Disease Service. Twenty-four hours after admission, she was afebrile but clinically worse, with marked headache and vomiting. A day later she spiked a fever of 39 °C. No organisms were cultured from the CSF, urine, or blood. CSF bacterial antigens, cryptococcal antigen, and CSF auramine stain (for mycobacteria) were also all negative.The following day the patient complained of further headache, nausea, blurred vision, and photophobia. In addition, she was noted to have bilateral large pupils, which did not react to light, and very pink optic nerves. Papilloedema was thought likely, and no other neurological signs were detected. The original CSF sample was negative on PCR for tuberculosis and herpes simplex virus, C-reactive protein remained at less than 5 mg/l, and a further head CT scan with contrast was normal.The diagnosis was revised to meningoencephalitis. Other agents—such as listeria, tuberculosis , and viThe fixed, dilated pupils were of major concern, and urgent ophthalmic review was requested. Examination by the ophthalmologist showed reduced vision at 6/60 right eye and 6/36 left eye. On testing with Ishihara charts, the patient had severely reduced colour vision. Her pupils were large and non-reactive to light or accommodation in both eyes. The eyes were inflamed, with cells present in the aqueous and vitreous humours. The optic nerves were swollen and very pink , but theThe combination of the clinical symptoms, signs, and ocular features was characteristic of Vogt-Koyanagi-Harada (VKH) syndrome . All antThis young patient presented acutely with a fever and some signs suggestive of meningitis. She was initially treated as having viral meningitis, but the CSF findings indicated that other aetiologies needed to be considered. In particular, in a woman who previously lived in and recently visited Bangladesh, with a lymphocytic meningitis and borderline CSF glucose, tuberculosis had to be considered. Initially the ocular symptoms and signs were not a prominent feature, but the signs were likely to have been present when the patient was first seen.The typical CSF changes associated with meningitis of different aetiologies are shown in There is a wide range of infectious causes of meningitis worldwide. The likely infecting organism will be determined by the age and immune status of the patient plus the situation in which the infection was contracted. Thus, in an immunocompetent adult in the UK, enteroviruses are the commonest cause of viral meningitis, with meningococcus and pneumococcus the commonest bacterial agents. Tuberculosis is more common in people who have lived in a highly endemic area. In other parts of the world, the differential diagnosis may include viral infections such as West Nile virus in the continental United States and Japanese B encephalitis in Asia, or other pathogens such as rickettsiae, borrelia (Lyme disease), and protozoa. In the immunocompromised host, listeria must be considered, and there is an increased risk of fungal infection and tuberculosis. Finally, it is important to consider sexual exposure, as both secondary syphilis and HIV seroconversion may present with meningitis.It is important, therefore, in the evaluation and management of patients presenting with a meningoencephalitis that the differential diagnosis be continually reviewed if the patient is not responding to therapy . When apVKH syndrome ,5,6 is aVKH syndrome is usually bilateral, but occasionally the eyes can be affected asymmetrically so that one is very mildly involved. The syndrome is accompanied by marked intraocular inflammation, and there are choroidal infiltrates  associatThe disorder is caused by an immune response to melanin and affects parts of the body where melanin is found. The initiating stimulus for this response is unknown, but T-cells sensitised to melanin-associated antigens are found in the peripheral blood. In the ear, the melanocytes of the inner ear are the target, and the inflammatory response here results in hearing loss and balance problems. In longstanding untreated cases, depigmentation may occur in other sites such as skin .Consider meningitis in the differential diagnosis of a patient presenting with fever and headache.CSF analysis is essential to confirm meningitis and as part of establishing the cause.Consider non-infectious causes when a patient does not respond rapidly to therapy.Blurring of vision must be investigated and may help in determining the underlying diagnosis or the presence of papilloedema.SutlasPNUnalAFortaHSenolSKirbasD2003Tuberculous meningitis in adults: Review of 61 casesInfection31387391Early suspicion and appropriate long-term anti-tuberculosis therapy together with corticosteroids may reduce mortality and morbidity in patients with TB meningitis14735380RedingtonJJTylerKL2002Viral infections of the nervous systemArch Neurol59712718This review is an update on diagnosis and treatment12020250ThomsonRBJrBertramH2001Laboratory diagnosis of central nervous system infectionsInfect Dis Clin North Am1510471071This paper discusses conventional tests, such as culture, and others such as antigen testing and PCR11780267RotbartHA2000Viral meningitisSemin Neurol20277292The virology, pathogenesis, epidemiology, clinical manifestations, diagnostic studies, and established and potential antiviral therapies for viral meningitis are discussed. A differential diagnosis of the aseptic meningitis syndrome is provided11051293NegriniBKelleherKJWaldER2000Cerebrospinal fluid findings in aseptic versus bacterial meningitisPediatrics105316319Polymorphonuclear cell predominance in the CSF does not discriminate between aseptic and bacterial meningitis10654948KamondiASzegediAPappASeresASzirmaiI2000Vogt-Koyanagi-Harada disease presenting initially as aseptic meningoencephalitisEur J Neurol7719722This paper describes the neurological and eye signs in VKH syndrome11136362SeehusenDAReevesMMFominDA2003Cerebrospinal fluid analysisAm Fam Physician6811031108Lumbar puncture is frequently performed in primary care, and this review outlines the interpretation of the clinical and laboratory findings14524396ShahKHEdlowJA2002Distinguishing traumatic lumbar puncture from true subarachnoid hemorrhageJ Emerg Med236774The purpose of this article is to assist emergency physicians in distinguishing traumatic lumbar punctures from subarachnoid hemorrhage12217474


Atrial septal defect closure is now routinely performed using a percutaneous approach under echocardiographic guidance. Centrally located, secundum defects are ideal for device closure but there is considerable morphological variation in size and location of the defects. A small proportion of atrial septal defects may have multiple fenestrations and these are often considered unsuitable for device closure. We report three cases of multiple atrial septal defects successfully closed with two Amplatzer septal occluders. Atrial septal defect (ASD) closure is now commonly performed using a transcatheter, percutaneous approach and with the Amplatzer septal occluder, large defects can be safely closed ,2. DevicThe majority of patients require a single device for closure of the ASD but a small proportion of patients may have more than one defect in the atrial septum. This can be difficult to diagnose using transthoracic echocardiography (TTE) as abnormal colour flow obscures the origins of the shunt, particularly if the second defect is situated inferiorly. We report three cases of patients referred for ASD closures that were found to have multiple ASDs and the techniques used to close these defects.2) was 400. Repeat TTE continued to demonstrate left to right shunting and the patient was readmitted for a further device closure six months later. At cardiac catheterisation, the second defect was identified low in the secundum septum between the fossa ovalis and the mouth of the coronary sinus. The defect was successfully closed with a 9 mm Amplatzer septal occluder with no evidence of obstructed flow in either the coronary sinus or the inferior vena cava. Equivalent chest x-ray radiation dose for the second procedure was 187. The patient remained well with no evidence of residual shunt six months following the procedure.A 34-year old woman was referred for consideration of percutaneous ASD closure. The ASD had been diagnosed when the patient was 12 years old and TTE had suggested that the right ventricle was dilating. At cardiac catheterisation there were mildly elevated right ventricular systolic pressures and a pulmonary to systemic flow ratio of over two. The secundum ASD was estimated to be 15 mm wide using TTE with aneurysmal formation of the interatrial septum. The patient was admitted for percutaneous ASD closure and underwent uncomplicated placement of a 17 mm Amplatzer septal occluder. Transesophageal echocardiography (TEE) during the procedure revealed the presence of a second ASD near the inferior vena cava and a small post-procedure shunt. The septal occluder did not completely cover both defects. Equivalent chest x-ray radiation dose  but it likely that simultaneous deployment will increase the procedure risk ,6. When There is considerable variation in atrial septal defect anatomy. A small proportion of patients with an ASD have more than one defect and these can be closed using conventional septal occluders under transoesophageal echocardiography guidance. The use of intracardiac echocardiography should allow more accurate device positioning, particularly defects located low in the atrial septum.


The success of coronary artery bypass grafting (CABG) is limited by poor long-term graft patency. Saphenous vein is used in the vast majority of CABG operations, although 15% are occluded at one year with as many as 50% occluded at 10 years due to progressive graft atherosclerosis. Intravascular ultrasound (IVUS) has greatly increased our understanding of this process. IVUS studies have shown that early wall thickening and adaptive remodeling of vein grafts occurs within the first few weeks post implantation, with these changes stabilising in angiographically normal vein grafts after six months. Early changes predispose to later atherosclerosis with occlusive plaque detectable in vein grafts within the first year. Both expansive and constrictive remodelling is present in diseased vein grafts, where the latter contributes significantly to occlusive disease. These findings correlate closely with experimental and clinicopathological studies and help define the windows for prevention, intervention or plaque stabilisation strategies. IVUS is also the natural tool for evaluating the effectiveness of pharmacological and other treatments that may prevent or slow the progression of vein graft disease in clinical trials. The success of coronary artery bypass grafting (CABG), although the gold standard for the treatment of multivessel coronary artery disease, is limited by poor long-term vein graft patency . Early vAlthough the cellular and molecular mechanisms underlying vein graft disease have been systematically investigated, the time course and development of this process in patients after coronary bypass has only recently been defined as a consequence of the increasing use of intravascular ultrasound (IVUS). Quantitative coronary angiography, traditionally the predominant imaging modality used to assess the severity of vein graft disease underestimates the severity of vein graft remodeling and athermanous plaque development by measuring the vessel lumen in only two dimensions ,14. In cGlagovian remodeling was first described as a radial enlargement of the entire cross sectional area of a vessel in response to intramural atheroma . First iIVUS changes correlate closely with the findings of experimental and clinical studies. In porcine saphenous vein bypass grafts, in the first week after grafting, adventitial medial and neointimal thickening occurs as a consequence of increased shear stress, surgical preparative injury and the subsequent activation of multiple growth factor and cytokine cascades. This is associated with the infiltration of inflammatory cells, medial smooth muscle cell proliferation and migration to form a neointima . AdventiThe early changes seen in the vessel wall of vein grafts are similar to those seen during vessel remodeling in atherosclerotic coronary artery segments . In normEarly vein graft changes can be viewed as adaptive, however they also predispose the graft to later accelerated graft atherosclerosis . SeveralRisk factors for, and the microscopic appearance of vein graft atherosclerosis are largely similar to those in coronary arteries and it is reasonable to suggest that similar pathological mechanisms are at work, however these occur over a much more rapid time course in vein grafts ,4. AtherIVUS studies have clearly shown that early 'adaptive' or pathological changes occur within weeks of grafting and that occlusive atheroma, in susceptible individuals occurs within 1 year. IVUS studies have therefore defined the window in which strategies to inhibit vein graft disease might be effective. Furthermore, in addition to its advantages over coronary angiography, IVUS in vein grafts has been shown to be both accurate ,16 and rex vivo prior to implantation using pharmacological or other methods that may inhibit subsequent disease is a feature unique to vein graft disease. There are many examples of this being successfully achieved in experimental models. Pre-treatment with rapamycin [The ability to manipulate vein grafts apamycin , paclitaapamycin , have beapamycin  and endoapamycin  as has tapamycin . Targeteapamycin . StabiliIVUS has significantly contributed to our understanding of vein graft failure. It also serves as the natural tool for the development of clinical strategies that may lead to significant improvements in vein graft patency and more importantly for better long-term quality of life and longevity for patients with coronary artery disease. The introduction of clinical trials to address this Achille's heel of coronary bypass surgery are long overdue.None declared.G Murphy performed the literature review and prepared the manuscript. G Angelini conceived the idea for the manuscript and prepared the manuscript.


During granulopoiesis in the bone marrow, the nucleus differentiates from ovoid to lobulated shape. Addition of retinoic acid (RA) to leukemic HL-60 cells induces development of lobulated nuclei, furnishing a convenient model system for nuclear differentiation during granulopoiesis. Previous studies from our laboratory have implicated nuclear envelope composition as playing important roles in nuclear shape changes. Specifically noted were: 1) a paucity of lamins A/C and B1 in the undifferentiated and RA treated cell forms; 2) an elevation of lamin B receptor (LBR) during induced granulopoiesis.The present study demonstrates that perturbation of cytoskeletal elements influences nuclear differentiation of HL-60 cells. Because of cytotoxicity from prolonged exposure to cytoskeleton-modifying drugs, most studies were performed with a Bcl-2 overexpressing HL-60 subline. We have found that: 1) nocodazole prevents RA induction of lobulation; 2) taxol induces lobulation and micronuclear formation, even in the absence of RA; 3) cytochalasin D does not inhibit RA induced nuclear lobulation, and prolonged exposure induces nuclear shape changes in the absence of RA.The present results, in the context of earlier data and models, suggest a mechanism for granulocytic nuclear lobulation. Our current hypothesis is that the nuclear shape change involves factors that increase the flexibility of the nuclear envelope (reduced lamin content), augment connections to the underlying heterochromatin (increased levels of LBR) and promote distortions imposed by the cytoskeleton (microtubule motors creating tension in the nuclear envelope). Granulopoiesis, the differentiation of peripheral blood granulocytes, involves dramatic nuclear and cytoplasmic structural changes . Committenvelope-limited chromatin sheets" or ELCS. The suspected role of LBR was confirmed in subsequent studies [Several established tissue culture cell lines have been investigated as model systems for understanding the events and mechanisms of granulopoiesis . Previou studies ,9, which studies  and the  studies . The mecbcl-2 cells [in vitro granulopoiesis. Prolonged exposure of HL-60-bcl-2 cells to CD or TX does lead to perturbations of nuclear shape, independently of RA induced nuclear differentiation.The position of the interphase nucleus within the cell appears to be regulated by microtubules and associated dynein  and acti-2 cells , we demoHL-60/S4 cells were cultivated for up to four days in medium with (or without) 1 μM RA and with (or without) 1 μM CD. Daily samples of these four cultivation conditions were cytospun on microscope slides and Wright-Giemsa stained for examination. The image data from day 3 are presented Figure . Controlbcl-2 cells, which are more refractory to undergoing rapid apoptosis.The previous publication  also exabcl-2 cells for up to three weeks following addition of 1 μM RA. For the sake of analysis, we distinguished four nuclear morphology categories: ovoid  1 μM RA for up to 7 days, cytospun onto microscope slides, methanol-fixed, air-dried and stained with Wright-Giemsa. The results were generally similar to those described above for HL-60/S4 cells. Several examples of Wright-Giemsa stained HL-60-bcl-2 cells treated with CD or with RA plus CD are shown in Figure -bcl-2 cells.HL-60-bcl-2 cells were exposed to 0, 0.1 or 1.0 μM NC, with 1 μM RA for up to 10 days. Samples were harvested for Wright-Giemsa staining at 1, 2, 3, 4, 7 and 10 days; for immunostaining at day 8. Figure bcl-2 cells at day 2 or day 4 after the addition of RA and nuclear morphology was observed on day 8. NC had a clear inhibitory effect upon RA induced nuclear lobulation, even when added 4 days after RA. Thus the inhibitory effect of NC is not an early event in granulocytic differentiation. As noted earlier, we have observed essentially 100% cell death of HL-60/S4 cells exposed to 0.1 μM NC by day 2. Viability of HL-60-bcl-2 cells was much better; but protection was not complete. By day 8 of 0.1 μM NC, viability had dropped to ~23%. This could also be observed as increased levels of cellular debris in the Wright-Giemsa stained slides. The cells shown in Figures -bcl-2 cells.HL-60myc [myc amplification [bcl-2 cells indicated that as early as day 2, addition of 0.1 or 1.0 μM TX resulted in the appearance of indented and lobulated nuclei in the absence of RA. Consequently, Wright-Giemsa staining analysis of TX treated cells (in the absence of RA) was performed for up to 10 days  of RA. Apparently the cytotoxicity of TX is potentiated in cells with elevated levels of c-myc . HL-60 cfication ,25. Prels Figure . There ws Figure . Clearlybcl-2 cells contrasts with earlier observations on RA treated "polarized" HL-60/S4 cells [bcl-2 cells, than previously observed for HL-60/S4 cells (data not shown). It is possible that the HL-60/S4 cells are more readily "activated", like normal neutrophils, than are HL-60-bcl-2 cells. They also differentiate to granulocytic form faster (~3 to 4 days), compared to HL-60-bcl-2 cells (~7 days).The position of the centrosomal region was visualized with monoclonal antibodies against γ-tubulin Figure . In all bcl-2 cells, compared to HL-60/S4 cells, allowing a window of time for determining the effects of these MT modifying chemicals on nuclear shape and nuclear differentiation.Prolonged exposure of HL-60 cells to various cytoskeleton-modifying chemicals  are very harmful to cell viability, inducing rapid apoptosis. The present study was conducted primarily on a Bcl-2 overexpressing subline of HL-60 cells, which is more refractory to apoptosis and exhibits the chemically induced differentiation properties of the parent cell line . Lethal bcl-2 and HL-60 cells observed in this study. The first difference: ~10% of the undifferentiated HL-60-bcl-2 cells exhibit micronuclei, compared to ~0.8% of HL-60 cells [-myc) and appear to form dynamically during S phase [bcl-2 cells exhibited immunostaining of centromeres, heterochromatin and nucleolar antigens  with multilobed nuclei, which we almost never observed with HL-60/S4 cells. There may be some clinical significance to this latter observation. A small percent of granulocyte nuclei with 5 or more nuclear lobes in human blood smears is considered diagnostic for megaloblastic anemia (vitamin B12 or folic acid deficiency) [bcl-2 cells suggests that delayed or dysfunctional apoptosis might play a role in these human diseases. It is of interest that neutrophil nuclear multilobulation (hypersegmentation) has been described in two circumstances that delay apoptosis: 1) Glucocorticoid administration to patients induces hypersegmentation [in vitro glucocorticoid treatment of neutrophils prolongs their survival [in vitro treatment of neutrophils with G-CSF prolongs their survival [The second difference: RA treated HL-60-on up to % with muentation , and in survival -39. 2) Gsurvival , and in survival ,41.bcl-2 cells by simultaneous exposure to NC  to 0.01–0.1 μM TX for up 20 hours, followed by incubation in drug-free media for up to 72 hours, led to nuclear envelope "unraveling" and clustering of nuclear pores . The autr Figure . The bunAlthough far from a complete understanding of all the molecular forces involved in shaping the granulocytic nucleus, a working model that incorporates existing data and emerging concepts provides a useful perspective for future experimentation Figure . The probcl-2 demonstrates that nuclear lobulation correlates with low levels of lamins A/C and B1 coupled with a rise in LBR levels  has been correlated with nuclear envelope folds or invaginations . We haveThe proposed model for granulocytic nuclear lobulation yields several testable predictions: 1) Expression of lamins A/C and B1 in HL-60 cells should strengthen the nuclear envelope, minimizing nuclear lobulation following exposure of the cells to RA; 2) "Knock-down" or expression of a "dominant negative" form of LBR in HL-60 cells would be expected to suppress granulocytic lobulation; 3) Overexpression of dynamitin in HL-60 cells should inhibit dynein activity , preventEmploying Bcl-2 overexpressing HL-60 cells, which are more refractory to induced apoptosis than the parent cell line, we demonstrated that disruption of the MTs by nocodazole prevented nuclear lobulation in response to RA treatment. These results implicate the necessity of an intact MT system for granulocytic nuclear shape. Cytochalasin D, on the other hand, did not suppress RA induced nuclear lobulation. Combined with the decreasing levels of intermediate filaments (vimentin) during differentiating granulocytic forms of HL-60 cells, the role of the MT system appears to be quite central to the nuclear shape change. Recent models on the role of a MT bound motor (dynein) in facilitating mitotic nuclear envelope breakdown suggest that similar tension forces on the differentiating granulocyte nucleus, combined with continued influx of nuclear envelope components, could explain nuclear invaginations and lobulation.bcl-2 [Two HL-60 cell sublines were employed in this study: HL-60/S4 , which abcl-2 , which abcl-2 ,4.all-trans retinoic acid (RA), nocodazole (NC), taxol (TX), cytochalasin D (CD). Stock solutions of these chemicals were stored at -20°C as described earlier [The following chemicals were purchased from Sigma-Aldrich :  earlier ,4.Guinea pig antisera were the generous gifts of two Ph. D. students in the laboratory of H. Herrmann : anti-LBR and anti-emerin, from C. Dreger; anti-lamin A and anti-lamin B1, from J. Schumacher. Goat anti-lamin B was obtained from Santa Cruz Biotechnology Inc. . Rabbit anti-4 × dimethyl H3K9 (lysine 9 on histone 3) was generously provided by T. Jenuwein . Human auto-antisera anti-centromere (CREST) and anti-nucleolus (antigens unknown) were purchased from Antibodies Inc. . Rabbit anti-calreticulin was from Calbiochem . Mouse monoclonal antibodies against α-tubulin, γ-tubulin and Golgi p58 were all purchased from Sigma-Aldrich. Mouse monoclonal anti-vimentin (3B4) was a gift of H. Herrmann and has been described before . FITC-, For Wright-Giemsa staining, cells were cytospun onto ethanol-cleaned microscope slides, fixed in room temperature methanol for 15 min, air-dried and stained as described earlier . For anaIn the majority of immunostaining experiments the procedure followed that described previously , with sobcl-2 cells over a three-week period (simultaneously with preparation of slides for Wright-Giemsa staining), were analyzed by immunoblotting as previously described [SDS total cell extracts, obtained from undifferentiated and RA treated HL-60-escribed . No atteRA, retinoic acid; MT, microtubules; NC, nocodazole; TX, taxol; CD, cytochalasin D; ELCS, nuclear envelope-limited chromatin sheetsALO performed the microscopy and prepared the figures. DEO performed the tissue culture, immunostaining and immunoblotting. Both authors were involved in the conception of the study and have read and approved the final manuscript.


Immunoglobulin rearrangement involves random and imprecise processes that act to both create and constrain diversity. Two such processes are the loss of nucleotides through the action of unknown exonuclease(s) and the addition of P nucleotides. The study of such processes has been compromised by difficulties in reliably aligning immunoglobulin genes and in the partitioning of nucleotides between segment ends, and between N and P nucleotides.A dataset of 294 human IgM sequences was created and partitioned with the aid of a probabilistic model. Non-random removal of nucleotides is seen between the three IGH gene types with the IGHV gene averaging removals of 1.2 nucleotides compared to 4.7 for the other gene ends (p < 0.001). Individual IGHV, IGHD and IGHJ gene subgroups also display statistical differences in the level of nucleotide loss. For example, within the IGHJ group, IGHJ3 has average removals of 1.3 nucleotides compared to 6.4 nucleotides for IGHJ6 genes (p < 0.002). Analysis of putative P nucleotides within the IgM and pooled datasets revealed only a single putative P nucleotide motif (GTT at the 3' D-REGION end) to occur at a frequency significantly higher then would be expected from random N nucleotide addition.The loss of nucleotides due to the action of exonucleases is not random, but is influenced by the nucleotide composition of the genes. P nucleotides do not make a significant contribution to diversity of immunoglobulin sequences. Although palindromic sequences are present in 10% of immunologlobulin rearrangements, most of the 'palindromic' nucleotides are likely to have been inserted into the junction during the process of N nucleotide addition. P nucleotides can only be stated with confidence to contribute to diversity of less than 1% of sequences. Any attempt to identify P nucleotides in immunoglobulins is therefore likely to introduce errors into the partitioning of such sequences. The variable domain of the immunoglobulin heavy chain (IGH) is encoded by the IGHV (variable), the IGHD (diversity) and the IGHJ (joining) genes. In developing B cells these genes are brought together via a process of recombination involving the selection of one of each gene type from sets of genes present within the genome . The briThe addition of N nucleotides is performed by the enzyme terminal dideoxynucleotidyl transferase (TdT), and in the IGH locus this addition can occur at both the D to J and the V to D-J joins . The regP nucleotides are derived from the asymmetric opening of hairpin loops that form at gene ends as part of the rearrangement process . The opeThe mechanism of immunoglobulin gene rearrangement was first proposed by Tonegawa in the late 1970's [The very few reports of exonuclease removal in the literature mainly describe analysis of murine sequences -14. ThesR, also offers standardised nomenclature [Recent advances in data standardisation in immunogenetics has allowed for improved statistical analysis. The standardisation emanates from IMGT-ONTOLOGY ,17 upon nclature  and stannclature . Despitenclature ,22.The development of a statistically based algorithm for the partitioning of immunoglobulin sequences  as an alThe collection of human IgM sequences from public databases resulted in a dataset of approximately 1500 sequences. The exclusion of fetal, moderately and highly mutated (>5 mutations) and disease associated sequences reduced the dataset to 306 sequences. Further exclusions were made of those sequences that showed signs of IGHV gene replacement or the utilization of multiple D genes. Five sequences utilized two D gene segments, as identified using strict criteria as previously described  . Average removals from IGHV region ends were significantly lower than removals from IGHD and IGHJ region ends . On average, only 1.2 nucleotides were lost from IGHV region ends while average removals of 4.7 nucleotides were evident from each of the IGHD region ends as well as from the IGHJ region ends.The average number of nucleotides removed from each gene subgroup within the three genes was calculated to identify differences in processing of sequences at the gene subgroup level Figure . It was Removals from D gene subgroups were examined at each of the region ends. The removals from the 5' D end did not reveal significant differences, but significant differences were seen between the subgroups at the 3' end  Figure . More exComparison of average nucleotide loss for each of the six IGHJ subgroups revealed significant differences between the average removals . The low level of removals from IGHJ3 was notable. On average just 1.3 nucleotides were removed from IGHJ3 sequences, while an average of 6.4 nucleotides were removed from IGHJ6 sequences  Figure .A more detailed examination of exonuclease removals from the IGHJ genes was undertaken, to investigate the influence of W and S motifs. The presence of these motifs in the first 15 5' nucleotides of the IGHJ ends was considered. IGHJ ends containing 5' S motifs showed significantly lower average removals than those lacking a 5' S motif . The IGHJ genes whose sequences did not possess an S motif within the first 15 5' nucleotides had, on average, three more nucleotides removed Figure .Putative P nucleotides were identified among those gene ends that remained untrimmed by exonuclease activity during the process of IGH V-D-J rearrangement. Examples of nucleotides that satisfied the P nucleotide criteria were observed at 3' V, 5' D, 3' D and 5' J gene ends. The identified nucleotides ranged in length from 1 to 4 nucleotides. The observed P nucleotides fell into twenty-three sets based upon unique sequences, and the gene end at which they were observed (Table A single case of significance was observed among the putative P nucleotide sequences. This was for a sequence of 3 nucleotides (GTT) which was associated with the 3' end of the IGHD region. The occurrence of 3 'GTT' sequences in the dataset remains the only significant putative P nucleotides even if the alpha value is increased to 0.01. Using a 0.05 significance level, 6 sets out of 23 appear significant, however this conclusion carries a 69% chance of being incorrect and that the results occurred by chance.Although putative P nucleotide sequences are present in 10% of sequences most of these are likely to have arisen as the result of N nucleotide addition. P nucleotides can only be confidently attributed to less than 1% of sequences with three sequences from the IgM dataset contained statistically significant P nucleotides out of the 245 IGH V-D-J rearrangements examined. The overall contribution of P nucleotides to junctional nucleotides was 9 nucleotides out of 2899 junctional nucleotides, or 0.3% of junctional nucleotides, within the IgM dataset. The probability of 'GTT' occurring within an N region is 0.007875, therefore, the sequence could be expected to occur twice at the observed position in the 245 junctions examined, by chance alone. Of the three identified P nucleotides it is therefore possible that only one is a true P nucleotide. The contribution of P nucleotides to junctional nucleotides could therefore be as low as 0.1%, with P nucleotide inclusions occurring in less than 0.5% of sequences.H gene replacement [Investigation of the role played by nucleotide loss and addition in the generation of immunoglobulin diversity has been limited by the ability of researchers to accurately determine gene ends. The development of a statistically based partitioning method has allowed this study to gain insights into the nature of nucleotide loss and addition in the expressed human immunoglobulin repertoire. Analysis of 294 human IgM sequences revealed significant differences between average nucleotide losses from different heavy chain genes segments. IGHV genes suffer less removal in comparison to other genes, suggesting that a process or processes may act to prevent the removal of critical components or to select against sequences in which such removals have occurred. Critical components may include the conserved TGT that defines the start of the CDR3  and the lacement -25.The extension of the analysis to the gene subgroup level showed significant differences among removals from the IGHV gene subgroups, among the IGHD subgroups at the 3' end of the D gene and between the six IGHJ gene subgroups. The most striking contrast was observed for the IGHJ gene subgroups, specifically for IGHJ3 and IGHJ6. Removals from IGHJ3 averaged only a single nucleotide, while IGHJ6 on average lost in excess of 6 nucleotides. The differences observed as part of this study suggest that the loss of nucleotides during the creation of human heavy chain immunoglobulin sequences is not random.Unique 'patterns' of exonuclease removal between gene subgroups have previously been reported in murine immunoglobulins ,28, howeThe nucleotide composition of gene has previously been stated to influence exonuclease processing in the murine system . The preConsideration of the W and S motif composition of the 3' D gene segments showed the IGHD2 and IGHD3 gene subgroups to be rich in W motifs and to lack S motifs (data not shown). These two subgroups showed higher average removals compared to other IGHD subgroups. The relationship between nucleotide composition and exonuclease activity could therefore explain the significant differences observed at the 3' end of the IGHD gene subgroups.IGHD2 and IGHD3 are both long D genes. A relationship between D gene length and exonuclease activity may have therefore been acting to influence exonuclease processing. Examination of exonuclease activity of D genes grouped by length did reveal significant differences (data not shown), however, these differences were only evident at the 3' end of the D genes and as the analysis was confined to IGHD2 and IGHD3 sequences, it is difficult to conclude whether sequence length has a role.The influence of nucleotide composition on exonuclease removals from heavy chain gene is easily examined in the IGHJ genes, due to the small number of alleles and the clear division of sequences based on the presence or absence of S and W motifs. Significant differences in the V genes were not further examined as the larger number of alleles made sample groups too small to allow for meaningful statistical analysis. The absence of significant differences at the 5' end of the IGHD genes may result from the lack of distinct differences in the nucleotide composition of these sequences. This would make any variations in exonuclease processing more subtle, and thus a larger sample size would be necessary to observe any differences.P nucleotide addition has been reported to contribute to diversity in between 10% ,8,29 andIt should be noted that even those P nucleotides accepted on the basis of statistical analysis carry a degree of uncertainty. Accounting for the possible misidentification of TdT additions among P nucleotides suggests that the contribution of P nucleotides to junctional nucleotides may be even lower than 0.3%. The identification of P nucleotides during partitioning of immunoglobulin sequences introduces a greater margin of error than would result from their exclusion from partitioning processes. For example, the rare nature of P nucleotide inclusion in rearranged immunoglobulins means that in a dataset of 1000 sequences less than 10 sequences may possess P nucleotides. Arbitrary identification of putative P nucleotides in the same dataset could however lead to 100 of the sequences being identified as having P addition. The error is therefore smaller if P nucleotides are not allocated as part of the partitioning process.The statistical demonstration of the phenomenon of P nucleotides by Meier and Lewis utilized altered recombination substrates, where the IGHV, IGHD and IGHJ gene regions were replaced by restriction sites on a plasmid vector . The recin vitro evidence in support of the formation of hairpin loops as part of the immunoglobulin rearrangement mechanism [Substantial echanism ,30,31 anechanism ,33. The R, [Human IgM sequences were obtained from public nucleotide databases; IMGT/LIGM-DB available through the IMGT, The IMmunoGeneTics Information SystemR, , the EntR,  and the R, . The seqThe determination of genes and N regions of the sequences within the dataset was performed with the aid of a statistical analysis of point mutations . This meThe focus of the study upon exonuclease removal required careful definition of rules for the identification of nucleotide losses. Preliminary alignment of sequences were performed using IMGT/V-QUEST . Where iD gene determination was performed using previously described criteria , where tDifficulties with immunoglobulin partitioning are often experienced, especially in the determination of gene ends. In this study, runs of consecutive nucleotide differences between a IGH V-(D)-J rearrangement and a germline sequence at a gene end were always attributed to exonuclease removal, rather than mutation of the gene end. The portion of a IGH V-(D)-J rearrangement that contained such differences, with respect to the germline sequence, was allocated to the N region, and the gene was considered to have undergone exonuclease processing at the gene end.Situations where IGHV or IGHJ ends revealed a series of differences and similarities to the germline sequence required the consideration of each possible combination of exonuclease removal and point mutation that could have led to the creation of the observed region end. Probabilities were calculated for each 'path' to the observed segment end. Mutability scores for the region end were used to determine the likelihood of point mutations contributing to the region end. TdT addition probabilities were used to calculate the likelihood of N additions generating particular nucleotide sequences. The TdT probabilities used were p(G) = 0.35, p(C) = 0.35, p(A) = 0.15, p(T) = 0.15 [Sequences that lacked exonuclease removals from gene region end(s) were examined for the presence of P nucleotides . Self-coThe contribution of P nucleotides to immunoglobulin diversity was investigated through data generated by the investigations reported here. Unique putative P nucleotides were identified and their appearance was tallied within the dataset. The number of junctions that displayed a lack of exonuclease activity at one or more gene ends was also calculated. The cumulative binomial probability of observing a given number of P nucleotides or greater was then calculated for each putative P nucleotide sequence in an attempt to estimate the contribution of P nucleotides to immunoglobulin diversity.The effect of W and S motifs upon exonuclease activity was investigated within the IGHJ genes. The IGHJ genes were grouped based upon the relative location of such motifs. W motifs were defined as sequences of 3 or more consecutive A or T nucleotides. S motifs were defined as sequences of 2 or more consecutive G or C nucleotides. The presence of these motifs was considered within the first 15 5' nucleotides of the IGHJ genes. Three sets were established, based upon the observed configurations of the motifs in the IGHJ subgroups; S motif followed by W motif, S motif only and W motif only  with an alpha value of 0.05.An analysis of P nucleotides was performed by calculating the probability that putative P nucleotides may actually have resulted from N nucleotide addition by TdT. Probabilities were calculated as described by Meier and Lewis . The pron is the observed number of P nucleotides and N is the total number of sequences containing junctional inserts equal to or greater than the length of the P nucleotide(s) being examined, and p is the expected frequency of the observed P nucleotide sequence, which was calculated using reported TdT frequencies [where, quencies . ProbabiKJ carried out the dataset creation and partitioning, performed the statistical analysis and drafted the paper. BG aided in the design of the study and the development of the statistical analysis. WS advised on study design and development. AC devised the study, aided in study design and co-ordination and revised the manuscript. All authors read and approved the final manuscript.294 Partitioned human IgM Sequences 294 human IgM sequences were partitioned using a statistically based model and used in the examination of exonuclease activity and P nucleotide addition in the expressed human repertoire. The file is in Microsoft Excel format.Click here for file


The cause-and-effect relationship between innate immune activation and neurodegeneration has been difficult to prove in complex animal models and patients. Here we review findings from a model of direct innate immune activation via CD14 stimulation using intracerebroventricular injection of lipopolysaccharide. These data show that CD14-dependent innate immune activation in cerebrum leads to the closely linked outcomes of neuronal membrane oxidative damage and dendritic degeneration. Both forms of neuronal damage could be blocked by ibuprofen and alpha-tocopherol, but not naproxen or gamma-tocopherol, at pharmacologically relevant concentrations. This model provides a convenient method to determine effective agents and their appropriate dose ranges for protecting neurons from CD14-activated innate immunity-mediated damage, and can guide drug development for diseases, such as Alzheimer disease, that are thought to derive in part from CD14-activated innate immune response. Activated innate immunity is associated with several degenerative and destructive brain diseases including Alzheimer disease (AD), HIV-associated dementia (HAD), ischemia, head trauma, stroke, cerebral palsy, and axonal degeneration in multiple sclerosis . In thisLPS specifically activates innate immunity in peripheral organs through a well-described Toll-like receptor (TLR)-dependent signaling pathway ,3. There2, respectively . In. In14]. in vitro, important limitations arise in living systems where extensive, highly active enzymatic pathways have evolved to metabolize many of the commonly measured products, such as 4-hydroxynonenal [in vivo is measuring F2-isoprostanes (F2-IsoPs) [in situ  , product-NeuroPs . Since D in vivo .4-NeuroP accumulation in cerebrum of wt mice exposed to ICV LPS and observed a delayed, transient elevation that peaks at approximately 24 hr after exposure and then returns to baseline by 72 hr post exposure [We first determined the time course of Fexposure . It is i4-NeuroPs (Table 2 receptor subtype 2 (EP2), one of four prostaglandin E2 (PGE2) receptors expressed in brain and one of the two PGE2 receptors expressed by microglia, have no neuronal oxidative damage in response to ICV LPS [in vivo [in vivo is restricted to microglia among parencymal cells in brain [in vivo is dependent on CD14, MyD88, p50 of NF-κB, iNOS, and EP2.We next used a series of mice, all on the C57Bl/6 genetic background, lacking specific genes to establish the determinants of neuronal oxidative damage in this model. Our results showed that genetic ablation of one co-receptor CD14), the required adaptor (MyD88), or one arm of the initial signal cascade (the p50 subunit of NF-κB) each completely blocks an LPS-induced increase in cerebral F4, the re ICV LPS . There a ICV LPS ,11,22-24[in vivo ,26. Seco[in vivo -32, it din brain ,26. Thusin vivo to the extent that is observed in AD brain without any neurodegeneration?These data left us with an apparent conflict. We have clearly demonstrated neuronal oxidative damage to mouse cerebrum following ICV LPS that is of a magnitude comparable to diseased regions of AD brain . HoweverTo address this question, we decided to examine directly the dendritic compartment of neurons, which is largely transparent to the standard histological techniques used so far to investigate ICV LPS-induced damage. Using Golgi impregnation and Neurolucida-assisted morphometry of hippocampal CA1 pyramidal neurons , we firsWe next pursued the molecular determinants of ICV LPS-induced dendritic degeneration using the same genetically altered mice that we used above Table . We obsein vivo neuroprotective doses of nonsteroidal anti-inflammatory drugs and anti-oxidants that are being evaluated as potenital protectants from AD. Indeed, a major criticism leveled against nonsteroidal anti-inflammatory drugs (NSAIDs) is that the concentrations that appear to be neuroprotective in epidemiologic studies are lower than those that classically considered anti-inflammatory doses. Moreover, there is some data suggesting that some NSAIDs, such as ibuprofen and naproxen, that may differ in their effectiveness as AD protectants despite being equivalent anti-inflammatory agents in peripheral assays of inflammation suggesting alternative mechanisms of action in AD [4-NeuroPs. For ibuprofen, the EC50 for suppressing ICV LPS-induced F4-NeuroPs is between 0.1 and 0.5 μg/ml and the maximal effect is reached by 1.4 μg/ml, considerably lower than the classic anti-inflammatory dose. In contrast, naproxen is without effect up to 1.4 μg/ml and thus an EC50 cannot be calculated from these data. As with F4-NeuroPs, ibuprofen completely protects both dendrite length and spine density . AT partially suppresses ICV LPS-induced F4-NeuroPs at 10 mg/kg and completely suppresses F4-NeuroP formation and both reduction in dendrite length and reduction in spine density at 100 mg/kg (Figure in vitro and lacks a specific transporter in vivo, does not, as expected, protect from neuronal oxidative damage or dendritic degeneration at the same dose.Next, we extended our studies to tocopherols, natural antioxidant products with a number of proposed actions  including Figure . GT, an Our data show that CD14-dependent activation of cerebral innate immunity leads to an acute, transient increase in oxidative damage to neuronal membranes that coincides with reversible dendritic degeneration. Although we did not directly test TLR-4 deficient mice in our studies, given what is know about LPS receptor activation and the fact that TLR-2-/- mice were not protected from neuronal damage caused by ICV LPS, these data argue strongly for CD14/TLR-4-dependent neuronal damage in our model. Moreover, using a wide array of genetically altered mice, we observed complete concordance between dendritic degeneration and neuronal membrane oxidative damage. In combination, these data suggest that these two events are mechanistically related, perhaps with neuronal membrane oxidative damage being a proximate contributor to dendritic degeneration in the context of innate immune activation.One obvious, commonly voiced criticism of the model described here is that it produces an acute stress that does not correspond to chronic neurodegenerative diseases. However, it has yet to be shown whether the stress to individual neurons in these protracted diseases truly is chronic or instead the integration of innumerable microscopic acute stresses over many years. Finally, to the extent that CD14-dependent innate immunity activation contributes to neurodegenerative diseases, such as AD and HAD, the model described here provides a convenient means to screen experimental therapeutics and rapidly optimize dosing and timing parameters before moving to more complex animal models or clinical trials.2 receptor subtype 2; F2-IsoPs: F2-isoprostanes; F4-NeuroPs: F4-neuroprostanes; GT: γ-tocopherol; HAD: HIV-associated dementia; ICV: intracerbroventricular; iNOS: inducible nitric oxide synthase; LPS: lipopolysaccharide; MPO: myeloperoxidase; NSAIDs: nonsteroidal anti-inflammatory drugs; PG: prostaglandin; PGE2: prostaglandin E2; TLR: Toll-like receptor; wt: wild type.AA: arachidonic acid; AD: Alzheimer disease; AT: α-tocopherol; Aβ: amyloid beta; COX-2: cyclooxygenase 2; DHA: docosohexaenoic acid; EP2: prostaglandin EThe authors declare that they have no competing interests.


Protein expression vectors that utilize the bacteriophage T7 polymerase/promoter system are capable of very high levels of protein production. Frequently, however, expression from these vectors does not reliably achieve optimal levels of protein production. Strategies have been proposed previously that successfully maintain high expression levels, however we sought to determine the cause of induction failure.We demonstrated that decreases in protein overproduction levels are not due to significant plasmid loss nor to mutations arising on the plasmid, but instead largely are attributable to chromosomal mutations that diminish the level of functional T7 RNA polymerase, resulting in decreased expression from the plasmid. Isolation of plasmid DNA from non-expressing strains and reintroduction of the plasmid into a T7 RNA polymerase-producing strain such as BL21(λDE3) reproducibly restored high level protein production.Our results suggest that a major contributing factor to decreased expression levels in T7 based systems is chromosomal mutation resulting in loss of functional T7 RNA polymerase. Consistent with this hypothesis, we found that optimal protein overproduction was obtained reproducibly from T7 promoters using freshly transformed cells that had not been subjected to outgrowth during which mutations could accumulate. Escherichia coli using a gene expression system that allows selective overexpression of a cloned gene , Sec (pJW25) , and Tus (pJW25)  were genrmations . Plasmid600 of 0.4 by addition of IPTG to a final concentration of 1 mM. Growth was allowed to continue for 2 hours after addition of IPTG. Uninduced controls were grown the same except no IPTG was added. Cells were lysed by boiling in SDS [All strains were grown in LB medium . When plg in SDS , and prog in SDS .600 of 0.4 for induction. Thus, the bacteria were subcultured only once. For continuous subculturing experiments, samples were removed before addition of IPTG and used to inoculate fresh LB plus ampicillin media at a dilution of 1:1000.For experiments using newly transformed BL21(λDE3), colonies were picked directly from the transformation plate and inoculated into 5 ml LB containing ampicillin for overnight growth. The overnight culture was diluted 1:1000 into fresh LB with ampicillin and grown to an ABacteriophage T7 mutant Δ4107 , which wGenomic DNA was isolated using the Qiagen DNeasy kit. Oligonucleotides used as primers (t7pol1 – gattaacatcgctaagaacg and t7pol2 – gattcatgtcgatgtcttcc) were obtained from Midland Certified Reagents, Midland, TX. PCR was performed using the FailSafe PCR kit  following manufacturer's recommendations. PCR products were visualized by agarose gel electrophoresis.IPTG – isopropyl-β-D-galactosidePCR – polymerase chain reactionLB – Luria-BertaniSDS – sodium dodecyl sulfateJGGV was responsible for the original observations of inconsistent expression levels and the data for Figure