Escherichia coli. There are over 60 genes that are influenced by H-NS many of which are involved in metabolism. To determine the significance of H-NS-regulated genes in metabolism and stress tolerance, an hns mutant of E. coli O157:H7 was generated  and its growth, metabolism, and gastrointestinal passage compared to the parent strain (43895) and strain FRIK47001P harboring pSC0061 which contains a functional hns and 90-bp upstream of the open-reading frame.H-NS is a DNA-binding protein with central roles in gene regulation and nucleoid structuring in hns mutant grew slower and was non-motile in comparison to the parent strain. Carbon and nitrogen metabolism was significantly altered in the hns mutant, which was incapable of utilizing 42 carbon, and 19 nitrogen sources that the parent strain metabolized. Among the non-metabolized substrates were several amino acids, organic acids, and key metabolic intermediates  that limit carbon acquisition and energy generation. Growth studies determined that the parent strain grew in LB containing 14 to 15% bile or bile salts, while the hns mutant grew in 6.5 and 9% of these compounds, respectively. Conversely, log-phase cells of the hns mutant were significantly (p < 0.05) more acid tolerant than the parent strain and hns mutant complemented with pSC0061. In mouse passage studies, the parent strain was recovered at a higher frequency (p < 0.01) than the hns mutant regardless of whether log- or stationary-phase phase cells were orally administered.The E. coli O157:H7 hns mutant decreased its ability to survive passage through mice. Collectively, these results expand the influence of H-NS on carbon and nitrogen metabolism and highlight its role in the ability of O157:H7 strains to respond to changing nutrients and conditions encountered in the environment and its hosts.These results demonstrate that H-NS is a powerful regulator of carbon and nitrogen metabolism as well as tolerance to bile salts. It is likely that the metabolic impairments and/or the reduced bile tolerance of the  Escherichia coli O157:H7 is a human pathogen that causes hemorrhagic colitis in humans and in some cases may incite hemolytic uremic syndrome [E. coli O157:H7 persistence has not been fully elucidated although Naylor et al. [E. coli O157:H7 in the bovine intestinal tract and the environment require that it closely control metabolism and stress protection.syndrome ,3. Adultsyndrome . The comE. coli nucleoid [hns transcription is subject to auto repression [E. coli are either transcriptionally or translationally controlled by H-NS [Shigella and Salmonella [H-NS is a cytoplasmic protein that is abundant in the nucleoid . It playnucleoid ,8. The lpression ,10. The  by H-NS ,11. Seve by H-NS ,11-15. Tlmonella ,13,16-19E. coli O157:H7, a detailed understanding of the consequences of modulators that are responsive to environmental conditions is particularly important. To this end, an hns mutant was constructed to define the growth and metabolic changes and any subsequent impact on gastrointestinal passage in this important human pathogen.Although there has been considerable work on gene regulation by H-NS, there has been comparatively little work on its impact on bacterial metabolism and stress tolerance. Considering the varied and changing environments encountered by hns structural gene and upstream regulatory region was amplified by PCR from genomic DNA of E. coli O157:H7 ATCC 43895. The nucleotide sequence of the EcoRI-BamHI fragment was determined and was identical to the sequenced gene in E. coli O157:H7 EDL933 [E. coli SM10 λpir to E. coli O157:H7 strain ATCC 43895 were kanamycin resistant, ampicillin sensitive, sucrose positive, and O157 positive. Confirmation of a double crossover in which wild-type hns was replaced with hns::nptI was confirmed by PCR (data not shown). PCR analysis of genomic DNA from the parental strain (ATCC 43895) with primers choi9917 and choi9918 generated a 375-bp fragment; whereas, genomic DNA from the hns::nptI mutant (FRIK47001P) resulted in a DNA fragment approximately 1.6-kb in length. The 1.6-kb fragment is in agreement with the projected size of the DNA fragment containing wild-type hns (375 bp) and the nptI gene (1.2 kb).A 695-bp fragment that includes the 7 EDL933 . Transcohns mutant (FRIK47001P) in LB at 37°C was slower and reached a lower maximum optical density than the parent strain or FRIK47001P containing pSC0061 (data not shown). The doubling times of the parent, complemented, and mutant strains were 27, 34, and 36 minutes, respectively. The maximum OD600 nm achieved by the parent and complemented strains was 1.3 to 1.4 after 18 h of incubation while the hns mutant reached a maximum OD600 nm = 1.0. Similar to the results in LB, the growth of the hns mutant in LB supplemented with mucin or in fecal slurries was slower and reached a lower final density than the parent strain. In addition, the colony size of the hns mutant on agar media was smaller than the parent and complemented strains. The presence of kanamycin in the growth media did not influence the growth of the mutant. These results are in agreement with results with hns mutants of non-pathogenic E. coli which had greater doubling times than the parent strains [s level in the hns mutant, which is partially suppressed in hns rpoS double mutants [hns mutant tested negative for the H7 antigen and was non-motile in motility medium (data not shown); however, the O157 antigen was still detected. The absence of H7 is consistent with previous data showing that H-NS is involved in the control of flagella gene expression and that hns mutants are non-motile [The growth of the  strains ,21. One  mutants . The hnsn-motile ,23.hns mutant was more susceptible to inhibition by low pH, NaCl, bile (Table hns mutants [hns mutant at the different temperatures was 4.5 to 5.0 which was 0.5 to 1.0 pH unit higher than the parent and complemented strains with the greatest difference observed at the lowest incubation temperature (16.5°C). Both the parent and complemented strains grew at pH 4.0 at each incubation temperature. Similar to the results with pH, the hns mutant grew in LB supplemented with a maximum of 4–6% NaCl compared to 6–7% for the parent and complemented strains. The test strains tolerated the highest concentration of NaCl when incubated at 25°C. There was one exception in which the hns mutant complemented with pSC0061 grew in a maximum of 4% NaCl at 16.5°C which was equivalent to the maximum level tolerated by the hns mutant. It is unclear why the complemented strain differed from the parent strain in this case where in all other growth conditions the results were the same. An unexpected finding was the ability of the E. coli O157:H7 to grow in LB containing bile compounds at 14–15%. The hns mutant was significantly more sensitive to bile salts and bovine bile preparations and grew in the presence of a maximum of 6.5% and 9.0% of these compounds, respectively. The growth of the strains in the presence of bile was further studied in LB containing 5% bile salts. Although the hns mutant grew, it had a greatly extended lag period (approximately 40 h) in comparison to the parent and complemented strains (<12 h)  than the parent strain when challenged in synthetic gastric fluid (pH 2.0) -regulated genes in the absence of H-NS. Stationary phase increased the acid tolerance compared to log phase in all three strains and the numbers decreased by less than 1 log10 CFU/ml after 3 h of acid challenge  than the hns mutant.The acid tolerance of the parent, ) Figure . These fe Figure . Howeverhns mutant strains challenged in LB acidified with acetic or lactic acids (data not shown).In contrast to the findings with acid challenges conducted in synthetic gastric fluid that is acidified with hydrochloric acid, there was no difference in the survival of the parent and hns mutant, glutamate decarboxylase (GAD) activity was measured (ΔpH/h/108 CFU). The hns mutant had significantly less (p < 0.01) GAD activity than the parent strain and strain FRIK47001P complemented with pSC0061 (p < 0.001) when examining stationary-phase cells (data not shown). These findings provide one possible explanation for the reduced acid tolerance in comparison to the parent strain of the hns mutant in stationary phase.In an attempt to discern the reduced acid tolerance in stationary-phase cells of the hns mutant was incapable of utilizing 42 carbon and 19 nitrogen sources that the parent strain metabolized  and trehalose, dulcitol, D-ribose, and dextrin. In addition, eight organic acids, including pyruvate, were not used as a sole carbon sources by the hns mutant . The findings from this metabolic characterization greatly expand the role of H-NS as a regulator of carbon and nitrogen metabolism in E. coli.Metabolic characterization using Phenotype Microarrays showed that the d Tables . The abint Table . H-NS ises Table . NitriteE. coli O157:H7 negative and that few bacteria grew on the selective agar that were similar to the inoculation strain . The administration of the parent strain, hns mutant (FRIK47001P), and hns mutant complemented with pSC0061 did not result in signs of illness in ICR mice. Although mice were tested for 3 days post inoculation, all mice that shed the inoculated E. coli O157:H7 strain tested positive one day after inoculation and some mice continued to shed the strain at day 2. No fecal samples tested positive at day 3. The results from three trials are shown in Table hns mutant tested positive at a dose of 103 CFU. The results between the parent and hns mutant were statistically different (p < 0.01) at a dose of 104. Similar results were obtained with stationary-phase cells of the parent strain, which was detected in a total of 17 of 27 mice inoculated. The passage of the hns mutant was not improved when stationary-phase cells were administered, and the feces from only 1 mouse (dose of 104 CFU) of 27 inoculated tested positive. At doses of 103 and 104 CFU, the results with stationary-phase cells of the parent strain were statistically different  from those obtained with the hns mutant. The passage of the hns mutant complemented with a functional hns in pSC0061 was similar to that of the parent strain (data not shown). The diminished passage of the hns mutant was further demonstrated when the percent of the dose surviving passage through the mouse was calculated. Approximately, 7.9% of the parent strain and 4.2% of the hns mutant complemented with pSC0061 were recovered in feces compared to only 0.2% of the hns mutant that was determined from the few fecal samples that harbored the mutant. These results demonstrate that H-NS significantly impacts gastrointestinal passage in mice.Testing of fecal samples prior to inoculation determined that mice were tant FRIK7001P, anE. coli O157:H7 must respond to changing environmental conditions whether it is present in the gastrointestinal tract or the environment [E. coli associated with the metabolism of specific substrates as well as stress protection [s (rpoS), and two-component systems  [hns mutants occurring as a result of indirect effects on gene expression [ironment . Metabolironment ,27. H-NSotection ,9,15; sund YedW) . Thus, Hpression ,27.hns mutant of E. coli O157:H7, the growth was slower than the parent strain. Although it is not completely understood, the increased doubling time in hns mutants is partially related to the increased level of σs and the possibility of secondary site mutations that affect growth [s at the posttranscriptional level [s increases in log-phase cells of hns mutants [s in hns mutants is the increase in the heat-shock chaperone DnaK which enhances σs stability [s results in the production of σs-regulated proteins some of which are involved in stress protection [hns mutant is a result of the combined effects of the increased level of σs [In our studies of an t growth . H-NS neal level ,28,29, a mutants ,30. Anotel of σs  and alteel of σs ,40.hns mutant was also more susceptible to inhibition by NaCl and bile salts than the parent strain , while the growth of the hns mutant was limited to a maximum of 9.0 and 6.5%, respectively. Bile salts are produced by the liver to breakdown fat and are deleterious to biological membranes. E. coli must tolerate bile salts that can reach concentrations of 20 mM in the duodenum and even higher in the small intestine [hns mutant. H-NS is known to influence expression of at least 5% of the genes in E. coli, many that encode for membrane proteins [E. coli O157:H7 strains.The growth of the in Table . These dntestine . The redproteins . The posE. coli O157:H7 shedding in cattle [hns mutant in comparison to the parent strain in log-phase cells is consistent with the expression of σs-regulated proteins [hns mutant, and hns complemented strains) in comparison to log-phase cells. However, the hns mutant was less acid tolerant than the parent strain in stationary phase. The gad system comprised of GadA and GadB, two isoforms of glutamate decarboxylase, and gadC that encodes for a glutamate/γ-aminobutyrate antiporter [hns mutant did not utilize glutamate as a sole carbon source; therefore, we compared glutamate decarboxylase activity in stationary-phase cells of the mutant and parent strains. The specific activity of glutamate decarboxylase was reduced in the hns mutant (p < 0.01) compared to the parent strain. The decreased specific acitivity of glutamate decarboxylase may explain the difference in acid tolerance between the hns mutant and parent strain in stationary-phase cells.Acid tolerance is one characteristic that influences the persistence of n cattle  and may n cattle . The incproteins . In stattiporter -38 is onhns mutant was significantly less than the parent strain . Gene expression profiles of mucus-grown E. coli have identified genes involved in the catabolism of gluconate, N-acetylglucosamine, sialic acid, glucuronate, mannose, fucose, and ribose are induced and considered preferred carbon sources [hns mutant while the E. coli O157:H7 parent strain used all seven carbon sources . Another possibility is that bile salts may have affected passage of the hns mutant through mice. In addition to being inhibitory, bile in association with H-NS is known to repress virulence genes [hns mutant. Considering that the intestinal tract contains at least 500 bacterial species [hns mutant. Studies with gnotoxenic mice would be particularly valuable in addressing the contributions of competing flora on the survival of the hns mutant. Based upon the results from this study, the most plausible explanation for the reduced passage of the hns mutant through mice is the regulatory impairments that influence growth and stress protection.There are a number of possible antimicrobial factors in the gastrointestinal tract that may be responsible for the diminished capacity of the al tract ,42. Althce genes . However species , it is aE. coli O157:H7 to respond to changing conditions it encounters in its host and the environment. This is the first report of the high tolerance of E. coli O157:H7 strains to bile and bile salts that may be of significance in localization and persistence within its bovine host and possibly during human infection. Studies are in progress to identify, which proteins that are influenced by H-NS contribute to bile tolerance. The results from this study also establish H-NS as a potential target for control of this important human pathogen.Results from this study significantly expand the importance of H-NS in carbon and nitrogen metabolism Figure  as well E. coli were grown in Luria-Bertani (LB) broth at 37°C. Antibiotics  were added to media when appropriate: ampicillin, 100 μg/ml; kanamycin, 50 μg/ml; and tetracycline, 10 μg/ml. For log-phase and stationary-phase cultures, 5 μl of an overnight culture was used to inoculate 5 ml of LB and incubated with shaking (150 rpm) at 37°C to an optical density at 600 nm = 0.5  and optical density at 600 nm = 1.1 , respectively.The bacterial strains and plasmids used in this study are listed in Table hns structural gene and flanking regions was amplified from genomic DNA of E. coli O157:H7 ATCC 43895 by PCR using oligonucleotide primers  containing an EcoRI (choi0005) and BamHI (choi0006) site on the respective 5'-ends. After PCR amplification, the PCR fragment was digested with EcoRI and BamH1 and ligated into pBR322, previously digested with the same enzymes, to produce pSC0061. The nucleotide sequence of the cloned fragment was determined (University of Wisconsin-Biotechnology Center). The suicide vector, pCVD442, was used to generate hns mutant in E. coli O157:H7 ATCC 43895 by homologous recombination. The hns gene in pSC0061 was inactivated by insertion of nptI encoding for aminoglycoside 3'-phosphotransferase (confers resistance to kanamycin) digested from pUC4K [PstI and gap filled with Klenow fragment. nptI was ligated with a partial PstI digest of pSC0061 carrying hns which has a PstI site present within its open reading frame (ORF). The construct (pSC0062) was digested with EcoR1 and BamH1 to liberate the 1.9-kb hns::nptI fragment. The fragment was blunt-ended with Klenow and ligated with SmaI-digested pCVD442 [E. coli SM10 λ pir [tra gene to recipient strain E. coli O157:H7 43895. Conjugation and mutant selection were conducted as described previously [hns ORF and confirm the presence of nptI in hns (1.6-kb fragment) (data not shown). FRIK 47001 (hns::nptI) was complemented by transforming FRIK 47001 with pSC0061.A DNA fragment containing the  pCVD442  to produ10 λ pir  carryingeviously . TranscoE. coli O157:H7 strain ATCC 43895 and hns::nptI mutant (FRIK47001) were evaluated for their ability to use carbon and nitrogen sources using PM1, PM2, and a PM3 plates, respectively. Strains were grown on R2A agar (Biolog) overnight at 37°C and sufficient cells were transferred to inoculating fluid to equal the 85%T turbidity standard (Biolog). FRIK47001 was resuspended in inoculating fluid containing glucose rather than succinic acid when inoculating the PM3 plate because it was unable to use succinic acid as a carbon source. Each well was inoculated with 100 μl of the standardized cell suspension, incubated at 37°C for 48 h, and the results recorded.The analysis of sole carbon and nitrogen source utilization was conducted with Phenotype MicroArrays™ . This method is used to identify the function and global interactions of a gene on cellular metabolism ,49. The hns mutant, and hns mutant complemented with pSC0061. Cells were used to inoculate the adjusted LB medium at a final concentration of 105 CFU/ml and incubated at 16.5, 25, and/or 37°C without shaking. The minimum pH for growth was determined in the pH range of 3.5 to 7.0 at 0.5 increments. The following were added to LB at the following ranges: NaCl, 0 to 10% (0.5% increments); bile salts no.3 (Difco), 0 to 16%; bovine bile (Sigma), 0 to 16%; porcine stomach mucin (Sigma), 2.5 mg/ml; bovine submaxillary gland mucin (Sigma), 2.5 mg/ml; and cefixime (Lederle), 0.2 to 0.8 μg/ml. Inoculated tubes were incubated at the designated temperature and observed daily for turbidity for up to 21 days. The growth of the strains in the presence of bile salts and mucin were studied in more detail. Growth in LB supplemented with 5% bile salts or 2.5 mg mucin/ml was monitored spectrophotometrically at 600 nm using a Bioscreen Analyzer at 37°C for 48 h . Plates were shaken for 30s prior to each optical density reading. The growth of strains was also monitored in fecal slurries (mouse feces diluted 1:10 in sterile deionized water). The fecal slurries were inoculated at a final concentration of approximately 105 CFU/ml from overnight cultures grown in LB, and then incubated at 37°C. Samples were removed and the number of CFU/ml determined by plating on MacConkey sorbitol agar  supplemented with 2.5 μg potassium tellurite/ml (Fisher) and 0.05 μg cefixime/ml  [Growth studies were conducted in LB adjusted to the appropriate pH or supplemented with NaCl, bile, bile salts, or cefixime using the parent, ver, NY)  and enum600 = 0.5) and stationary (OD600 = 1.1) phase cells were used to inoculate flasks containing 50 ml of synthetic gastric fluid or acidified LB to achieve a final concentration of ca. 105 CFU/ml. Flasks were incubated at 37°C with shaking (150 rpm). Samples were removed periodically and then plated on tryptic soy agar  using a Model D Spiral Plater . The number of colony forming units (CFU) was determined after 24 h of incubation at 37°C. The survival curves shown are the mean values from at least three trials.Acid tolerance was assessed in synthetic gastric fluid adjusted to pH 1.5 and LB acidified with acetate or lactate (pH 4.4) as previously described . Log . Three trials were conducted, and in each trial three mice were inoculated with each concentration of the test strain. Fecal samples were collected each day for 3 days after inoculation and tested for the presence or absence of E. coli O157:H7. Fecal samples were diluted (1:10) in modified EC broth supplemented with novobiocin (20 μg/ml) and incubated at 37°C with shaking (150 rpm) for 18–24 h. Following enrichment, 20 μl of Dynabeads® anti-E. coli O157  was added to 0.5 ml of the enriched fecal culture and wash buffer (0.5 ml), incubated, and washed as described by the manufacturer. The resuspended beads were streaked on MacConkey sorbitol agar supplemented with potassium tellurite/ml and cefixime/ml [E. coli O157:H7 colonies. Suspect colonies were confirmed as O157 by agglutination . The minimum detection level of this procedure was approximately 100 CFU/g.The passage studies were conducted as previously described . In brieshaking 1 rpm for The data presented are the mean values from at least three trials. The acid tolerance of strains was compared using the inverse of slopes from linear regression plots of survivor curves. The data from acid challenges were analyzed for statistical differences by one-way analysis of variance. A z-test for was used to evaluate significant differences in the proportions of mice testing positive between inoculation strains . All calhns mutant and pSC0061. CWK designed and coordinated the project and wrote the manuscript.IE, KCJ, DJB, and BV conducted laboratory experiments, analyzed data, and contributed to the writing of the manuscript. SHC contributed to the initial concept of this project and constructed and confirmed the


Nitrosoureas are the drugs most effective in the treatment of patients with intracerebral malignant glioma. Their limiting toxicity is delayed myelosuppression. A prospective, randomised crossover study of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) was performed in patients receiving BCNU for relapsed glioblastoma, to investigate whether the resulting haematological toxicity profile could be modified by rhGM-CSF. Adequate data for analysis were obtained in 13 patients. Following BCNU, the nadir neutrophil count was higher in 12 out of 13 patients during the rhGM-CSF-protected cycles compared with the unprotected cycles. The median nadir was also significantly higher . Five episodes of neutropenia (< 2 x 10(9) l-1) occurred during the unprotected cycles compared with none in the rhGM-CSF-protected cycles (P = 0.076). There was no evidence of any effect on platelets. This result shows that the haematological toxicity profile following therapeutic doses of BCNU can be modified. It suggests that rhGM-CSF and other growth factors should be investigated for clinical efficacy in chemotherapy using nitrosoureas.


The testis is the third common site of relapse after primary treatment of childhood acute lymphoblastic leukaemia, but in adults relatively few testicular relapses of acute lymphoblastic leukaemia have been reported. In the present investigation the differences in the behaviour of leukaemia in immature and mature rat testis and the interactions of testicular and leukaemic cells were studied. Intraperitoneal injection of rat T-leukaemic cells to sexually immature animals induced testicular infiltrations in 100% of animals in 17 days. The infiltrations were small and located perivascularly in the interstitial tissue. Intraperitoneal injection of T-leukaemic cells to sexually mature animals induced testicular infiltrates in 42% of the animals. Leukaemic cells injected directly to the lymph sinusoids of sexually immature and mature testis proliferated rapidly causing testicular enlargement. The M(r) > 5 K fraction of extracts of 50 days old normal rat testes inhibited 3H-TdR incorporation of both normal and leukaemic ConA-stimulated rat lymphoblasts significantly. The same fraction of extracts of testes of 25 days old rats did not have any effect on 3H-TdR incorporation. The normally occurring pubertal increase in the lymphocyte inhibitory effect of the M(r) > 5 K fraction of testis extracts on 3H-TdR incorporation of PBL was prevented following either intraperitoneal or intratesticular injection of rat leukaemic lymphoblasts administered at the age of 25 days. The present observations suggest that physiological pubertal changes in the permeability of vascular endothelium and immunosuppressive effect of the testis may be important explanatory factors for the smaller number of testicular relapses in men compared to boys seen after treatment of ALL.


Measurement of soluble HLA in body fluids has a potential role in assessing disease activity in autoimmune disorders.We applied a solid phase, enzyme-linked immunoassay to measure soluble HLA class I (sHLA-I) and class II (sHLA-II) molecules in the saliva and cerebrospinal fluid (CSF) in 13 untreated patients with relapsing-remitting form of multiple sclerosis (MS). For comparison purposes, we also studied saliva from 53 healthy subjects.Saliva from normal controls had detectable sHLA-I levels in 41 of 53 individuals studied, with values ranging from 9–100 ng/ml (mean = 41 ± 2.8 ng/ml). sHLA-I was undetectable in the saliva in 11 of 13 MS patients, and in none of the CSF specimens. In contrast, mean sHLA-II concentration in the saliva of MS patients was significantly increased compared to controls . The mean CSF sHLA-II level (369 ± 16 unit/ml) was equivalent to the mean sHLA-II concentration measured in saliva (mean = 386 ± 52 unit/ml) (P = 0.7). In patients with brain magnetic resonance imaging (MRI) enhancing lesions (n = 5), reflective of more active disease, CSF sHLA-II averaged 356 ± 26 unit/ml compared to 380 ± 51 in saliva. Similarly, in patients with non-enhancing lesions (n = 8), CSF sHLA-II averaged 377 ± 18 unit/ml compared to 390 ± 77 unit/ml in saliva. Thus, the mean sHLA-II concentration in saliva and CSF was essentially equivalent for MS patients with or without enhancing plaques.Our data suggest that the measurement of soluble HLA in saliva, specifically sHLA-II, correlates with the level found in the CSF. Therefore, if sHLA correlates with disease activity in MS, as has been proposed, saliva measurements provide a noninvasive correlate of CSF measurement. The human major histocompatibility antigens, HLA, are generally cell bound, but trace amounts exist in soluble form -3. TheseIn the neurological realm, there is a possible alteration of sHLA-I and/or sHLA-II levels as a reflection of disease activity in multiple sclerosis (MS). Clinical and brain magnetic resonance imaging (MRI) disease activity in MS is associated with fluctuations in sHLA-I and sHLA-II levels in the serum and cerebrospinal fluid (CSF) of patients with MS -14. HoweTheoretically, we would expect that measurement of sHLA in CSF would be most likely to reflect central nervous system (CNS) disease activity if indeed such measurement could serve as a monitor of a disorder such as MS. However, CSF exams are invasive and not without potential complications. Therefore, we sought to determine whether more readily accessible body fluid, specifically saliva, might provide correlative sHLA measurements in an autoimmune-mediated CNS disease such as MS.We analyzed CSF and saliva from thirteen consecutive Caucasian patients with relapsing-remitting form of MS (RRMS) defined by the McDonald criteria . None ofSaliva samples were collected through expectoration preceded by rinsing of the mouth with sterile water. The resultant salivary samples were collected into test tubes and stored at -20°C until subsequent assay. CSF was collected by standard sterile lumbar puncture technique after the informed consent was reviewed with the patient and signed.Brain MRI was performed using a 1.5 T machine with a standard quadrature head coil. The imaging protocol included sagittal T1-, axial T1-, T2-weighted, and fluid attenuated inversion recovery (FLAIR) images. All MRI scans were performed before and after (Gd-DTPA) infusion. Axial T2-weighted and pre- and post-contrast T1-weighted images were used for assessment of MS plaques. The images were independently interpreted using inspection and computer-assisted techniques by a neuroradiologist. Detection of lesions, compatible with MS, was made by visual inspection as was determination of the absence or the presence of contrast enhancing lesion. Computer based software allowed comparison of the lesions among different groups. Comparisons were made between all 13 MS patients who were subgrouped into either those with and those without enhancing plaques in their brain MRI scans.2-microglobulin), Ab2.06, L203 and IVA-12 (anti-HLA-DR) were obtained from the American Type Culture® collection . These lines were expanded and the mAbs were produced in BALB/c mice as described previously . However, whether sHLA from other body fluids follows a similar pattern remains to be determined as evidenced by the reciprocal relationship between CSF and serum sHLA-I levels in MS reported by these same investigators .It appears from this preliminary study that sHLA-II is the predominant class of sHLA molecules present in the CSF and saliva of MS patients. This is in contrast to CSF and saliva sHLA-I, which we have found to be in non-detectable quantities. The reduced sHLA-I and augmented sHLA-II observed in these body fluids may reflect the active stage of RRMS, triggered by the stimulation of immune system in the absence of immunosuppressive therapy. Our results indicate that measurement of saliva sHLA-II may be a potential noninvasive biological marker of disease activity in a primary CNS disease such as MS.The author(s) declare that they have no competing interests.Dr. Adamashvili has contributed to this manuscript by providing expertise for laboratory measurement of soluble HLA-I and soluble HAL-II.Drs. Minagar and Kelley have contributed to this manuscript by recruiting and examining multiple sclerosis patients, interpretation of data and preparing the manuscript.Dr. Gonzalez-Toledo has contributed to this manuscript by interpreting the neuro-radiology studies and generating neuro-radiology data.Dr. Featherston has contributed to this manuscript by doing statistical analysis and generating the figures.


Myocardial infarction can be difficult to diagnose after lung surgery. As recent diagnostic criteria emphasize serum cardiac markers (in particular serum troponin) we set out to evaluate its clinical utility and to establish the long term prognostic impact of detected abnormal postoperative troponin levels after lung resection.We studied a historic cohort of patients with primary lung cancer who underwent intended surgical resection. Patients were grouped according to known postoperative troponin status and survival calculated by Kaplan Meier method and compared using log rank. Parametric survival analysis was used to ascertain independent predictors of mortality.From 2001 to 2004, a total of 207 patients underwent lung resection for primary lung cancer of which 14 (7%) were identified with elevated serum troponin levels within 30 days of surgery, with 9 (64%) having classical features of myocardial infarction.The median time to follow up (interquartile range) was 22 (1 to 52) months, and the one and five year survival probabilities (95% CI) for patients without and with postoperative troponin elevation were 92% (85 to 96) versus 60% (31 to 80) and 61% (51 to 71) versus 18% (3 to 43) respectively (p < 0.001).T stage and postoperative troponin elevation remained independent predictors of mortality in the final multivariable model. The acceleration factor for death of elevated serum troponin after adjusting for tumour stage was 9.19 (95% CI 3.75 to 22.54).Patients with detected serum troponin elevation are at high risk of early mortality with or without symptoms of myocardial infarction after lung resection. Postoperative myocardial infarction is not a well studied topic after lung surgery. From the few reports in the literature, the frequency to range between 0.7% to 2% ,2, but iRecently, the diagnostic criteria for myocardial infarction has evolved from a history of chest pain, EKG changes and a rise in serum cardiac markers, to an emphasis on serum cardiac markers (in particular serum troponin) as the primary diagnostic modality . The norTo evaluate the clinical utility of troponin assay after lung surgery, we set out to assess the frequency of classical supporting features of myocardial infarction and to establish the long term prognostic impact of detected abnormal postoperative troponin levels in patients who underwent lung resection for primary lung cancer.We studied a historic cohort of patients from 2001 to 2004 (the start date reflects the availability of troponin assay at our institution) with known or suspected primary lung cancer who underwent intended surgical resection. As cervical mediastinoscopy and biopsies were performed prior to thoracotomy for all patients with clinically suspected N2 disease, additional lymph node sampling was only undertaken in selected patients with unexpected or suspicious findings during thoracotomy. Staging was performed according to Mountain 1997 . We exclPatients were identified from our hospital National Health Service (NHS) database of Office of Population, Censuses and Surveys – Classification of Surgical Operations and Procedures codes (OCPS), and data obtained from case notes and histopathology reports. Survival was documented as the date of last follow up in a hospital outpatient clinic, and mortality documented as the date of death from patient records or the NHS strategic tracing service .® AccuTnI™ troponin I assay  with 0.04 μg/l as the upper reference limit of normal. The pathology database was screened for each patient individually to determine if a troponin sample was requested. Troponin levels of 0.04 μg/l or more within 30 days of surgery were classified as elevated in this study. Patients were grouped according to the presence of known post operative troponin elevation, and patients with normal or no known postoperative troponin levels were classified as "no troponin elevation".Troponin I was analysed using the AccessCategorical data are presented as frequency (%) and continuous data as mean with standard deviation (SD) or median with interquartile range (IQR). Comparisons of baseline categorical data between the two groups were made using Fisher's exact test and continuous data compared using two tailed t-test.Parametric and non-parametric survival analyses were applied. Actuarial survival was estimated using the Kaplan-Meier method and compared using the Log-rank test. to Parametric survival analysis (log-logistic accelerated failure time model) was used to model the hazard function, ascertain the individual contribution of factors associated with survival and to compare the risk adjusted survival between the two groups. The survival analysis was repeated with non-parsimonious propensity score multivariable adjustment, and one-to-one (nearest neighbor) matching on age, sex, tumour stage, nodal status and operation extent. Statistical analyses were performed using R version 2.0.0 .st January 2001 to 1st January 2004, a total of 231 patients underwent lung resection for the presumed diagnosis of primary lung cancer. We excluded 26 patients who did not prove to have primary lung cancer. Of the 205 patients remaining, 41 had a troponin sample requested, 19 were elevated, of which 14 (7%) were elevated within 30 days of surgery. The remaining 5 patients in which the troponin levels were detected as elevated more than 30 days from surgery were analysed in the no troponin elevation group. The mean age (SD) of the cohort was 66 (11) years, and 133 (65%) were men. The baseline characteristics of the two groups are summarised in table From 1Of the 14 patients with elevated postoperative troponin levels, the mean peak level was 0.936 μg/l, and the majority (10/14) were detected within the first 5 postoperative days  was 22 (1 to 52) months, with the one and five year survival probabilities (95% CI) for patients without and with postoperative troponin elevation as 92% (85 to 96) versus 60% (31 to 80) and 61% (51 to 71) versus 18% (3 to.43) respectively , corresponding to 9.19 (95% CI 3.75 to 22.54).The univariable prognostic predictors are summarised in table For patients with known troponin elevation, the instantaneous risk of death  with a non-significant p-value for the coefficient for the propensity score (p = 0.11). The results remained robust even with one-to-one matching of the 14 patients with known elevated troponin levels to controls .Normally, troponin is not detectable in the serum after lung surgery ,7. The rThe relationship to myocardial infarction is more difficult to delineate, as 36% (5/14) of patients with elevated serum troponin levels did not have chest pain or EKG evidence of ischemia. After thoracic surgery, we routinely use thoracic epidural analgesia supplemented with non-opiate oral analgesics. We believe that this practice is common and potentially attenuates symptoms of ischaemic chest pain. In addition, it might be more difficult for patients to distinguish between new onset chest pains against the background of thoracotomy wound pain. Classical ST-elevation on EKG was present in only 5 (36%) patients. Therefore, it can be difficult to establish a diagnosis of postoperative myocardial infarction in the setting of thoracic surgery.Serum troponin levels may be useful in this regard, apart from greater cardio-specificity (compared to CK-MB), minute elevations were able to convey long term prognostic information. The upper reference limit 0.04 μg/l) in our study was five times lower than the reference limit of 0.20 μg/l required by the assay used in Vikenes et al. [ μg/l in On one end of the spectrum of severity of myocardial infarction, myocardial necrosis detected on isolated serum troponin elevation may be a specific method to identify patients at a high risk of early death (within six months of surgery). Established postoperative myocardial infarction lies on the other extreme of the spectrum, and raises the 30-day mortality by an odds ratio of 32 [We highlight a potential use for serum troponin as a screening investigation to identify patients with a poor prognosis, regardless of further symptoms of myocardial infarction. This is consistent with observation that isolated troponin elevation is an independent predictor of mortality across the spectrum of the chest pain syndromes . In totaAn unfortunate corollary of the retrospective nature of our study is the inability to confidently identify the patient risk factors that are associated with troponin elevation. Although patients with postoperative troponin elevation were significantly older, age alone is not a powerful discriminating feature. At a rate of 7% (95% CI 4 to 11) and estimated 9 fold acceleration in the risk of death, the clinical utility of serum troponin estimation after lung resection appears to be an important topic for further study. Importantly, only 5 of the 14 patients had a preoperative history of ischaemic heart disease, of which all had symptoms of stable angina and good performance status, not meeting the accepted criteria for further cardiovascular screening [To date, there are no guidelines that address the management of myocardial infarction specifically after thoracic surgery. General guidelines for non-cardiac surgery stress the need for medical management  and risk factor modification for secondary prevention . Despite1 of 2.2 l in patients in the troponin elevation arm was not different from our usual preoperative patient population.The main limiting factors are the retrospective study design on a relatively small group rendering our study susceptible to bias. Unfortunately, sample size was limited by the relatively recent introduction of troponin assay at our institution. We have assumed that atrial fibrillation was associated with myocardial necrosis as we have found no evidence to suggest that atrial fibrillation alone elevates serum troponin independently of myocardial necrosis. However, our findings are not altered in respect of this relationship. Although we were not able to adjust for preoperative lung function in our analyses, the mean preoperative FEVIt is clear that serum troponin is rapidly becoming the cardiac marker of choice, and prospective studies on unselected patients will be able to more accurately establish its utility as a prognostic marker. Further work would be able to determine if elevated troponin levels result from procedures that involve the pericardium , and ascertain if it carries similar prognostic implication.After lung surgery, approximately a third of patients with troponin elevation do not have classical symptoms of myocardial infarction. Atrial fibrillation was a common presenting feature leading to the identification of elevated serum troponin. Patients with detected serum troponin elevation are at high risk of early mortality after lung resection for primary lung cancer.


Bacterial meningitis is often associated with cerebral compromise which may be responsible for neurological sequelae in nearly half of the survivors. Little is known about the mechanisms of CNS involvement in bacterial meningitis. Several studies have provided substantial evidence for the key role of nitric oxide (NO) and reactive oxygen species in the complex pathophysiology of bacterial meningitis.In the present study, serum and CSF levels of NO, lipid peroxide (LPO) (mediators for oxidative stress and lipid peroxidation); total thiol, superoxide dismutase (SOD) (antioxidant mediators) and S-100B protein (mediator of astrocytes activation and injury), were investigated in children with bacterial meningitis (n = 40). Albumin ratio (CSF/serum) is a marker of blood-CSF barriers integrity, while mediator index  is indicative of intrathecal synthesis.Compared to normal children (n = 20), patients had lower serum albumin but higher NO, LPO, total thiol, SOD and S-100B. The ratios and indices of NO and LPO indicate blood-CSF barriers dysfunction, while the ratio of S-100B indicates intrathecal synthesis. Changes were marked among patients with positive culture and those with neurological complications. Positive correlation was found between NO index with CSF WBCs ; CSF-LPO with CSF-protein ; total thiol with LPO indices ; S-100B and Pediatric Glasow Coma Scores ; CSF-LPO with CSF-S-100B ; serum-total thiol with serum S-100B .This study suggests that loss of integrity of brain-CSF barriers, oxidative stress and S-100B may contribute to the severity and neurological complications of bacterial meningitis. Bacterial meningitis is the most severe and frequent infection of the central nervous system (CNS). It remains an important public health problem worldwide. Even wi2.), hydrogen peroxide and hydroxyl radical [The mechanisms of CNS damage during meningitis have not been conclusively identified. During bacterial infections, neutrophils and macrophages gather at the site of infection to combat the microorganisms. Blood-derived and brain-resident immune cells employ reactive oxygen species (ROS) as part of their host defense mechanisms against invading bacteria. In this process, these cells consume molecular oxygen, which is converted into toxic superoxide anion  (markers of oxidative stress and lipid peroxidation); total Thiol and superoxide dismutase (SOD) (markers of antioxidant activity). S-100B protein is a marker of astrocytic reaction or glial damage. We inve3, CSF: blood glucose ratio <60% or CSF glucose < 40 mg/dl and protein > 40 mg/dl in absence of CSF hemorrhage and positive CSF culture[3) with predominance of mononuclear cells, normal CSF glucose and chloride concentrations and normal or increased protein. We did not do CSF culture, serology or PCR for neurotropic viruses, 3) fetal and neonatal malformations; chromosomal abnormalities or developmental delay, 4) perinatal asphyxia, 5) immunodeficiency, 6) endocrine diseases as diabetes mellitus and obesity, 7) purpura, 8) impairment of hearing prior to diagnosis of sepsis, 9) neuromuscular diseases, and 10) recent neurosurgery or ventriculoperitoneal shunt.All consecutive children , aged <15 years (mean: 71.8 ± 8.23 months), blood pressure (99 ± 19 mmHg), admitted to the Pediatric Department, Hospital of Infectious Diseases , Assiut, Egypt, carried the diagnosis of bacterial meningitis, were included in this study. Septic meningitis was diagnosed by the presence of the following: CSF polymorphonuclear cell count > 5 mmF culture. The diaClinical data were collected as follow: 1) demographics , coexisting medical conditions, antibiotic pretreatment, vaccination status, temperature and duration of fever at the time of presentation and occurrence and timing of seizures, 2) physical examination findings, 3) routine tests for biochemistry and hematology were done on the date of lumbar tap including complete blood count, kidney and liver function tests, 4) chest x-ray, urine and stool analyses were done as indicated per patient, 5) CSF analysis included white and differential cell count, protein and glucose concentrations, gram-stained CSF smear examination and culture for bacteria, 6) the severity of brain involvement was assessed using the Pediatric Glasgow Coma Scale or scoring system (PGCS). This isUpon hospital discharge (survivors or non-survivors), summary data including total hospital stay and complications were collected for all patients.Serum and CSF samples were collected from patients upon admission. Only blood samples were obtained from the control children as our ethical committee did not permit CSF intake from normal children. CSF was collected under strict non-infected conditions. Standard hematological and biochemical analysis of peripheral blood were also documented. All samples were tested for white blood cell count with differential and bacterial cultures. Levels of CSF glucose, protein were determined by routine laboratory procedures. Methods for CSF processing and bacterial culture used in this study have been described previously. BrieflyNitric oxide (NO) was determined by a classic griess reaction with sulfanilic acid and alpha naphthylamine in diluted sulfuric acid medium as described by Ding et al. LPO was3/albumin serum concentration) was calculated as an indicator of blood-CSF barrier dysfunction in patients with meningitis[3/mediator serum concentration) with the albumin ratio , it is possible to infer whether the mediators of similar molecular weight to albumin are passively transferred across the disrupted blood-CSF barriers or intrathecally synthesized. The latter is indicated by calculation of the blood-derived mediator indices as follow: mediator index is equal to the mediator ratio (CSF/serum) divided by albumin ratio (CSF/serum). The higher the mediator ratio than albumin ratio indicates local intrathecal substance production. Estimation of marker indices seems to have a significance for blood-deriver proteins or substances but it is not applicable for brain-derived proteins or substances as S-100B which has an intrathecal fraction of ~99% [Albumin is an exclusively blood-derived CSF protein. It is transferred to the CSF via passive diffusion through the intact blood-CSF barriers. The CSF-to-serum albumin concentration was found to be 1:205. Increased albumin concentrations in CSF must always be due to blood CSF barrier dysfunction. In this study, albumin ratio . The serThe analysis was done using SPSS version 11.5. Data were expressed as mean ± 2SD or as a percentage when appropriate. One-way ANOVA or Mann-Whitney U tests were used for comparison between different groups. The Pearson's and Spearman correlation coefficient tests were used to evaluate associations between measured parameters (continuous and non-continuous variables). For all tests, p < 0.05 was considered significant.3, neutrophils: 92 ± 13% and lymphocytes: 8 ± 13%) as compared to our normal lab reference values . CSF-culture was positive in 37.5% (n = 15) of patients. Organisms identified were streptococcus pneumonia , staphylococcus aureus , haemophilus influenza  and neisseria meningitides .This is a cross-sectional study. CSF biochemistries of the samples examined confirmed the diagnosis of bacterial meningitis   and rigidity of the 4 limbs . Headache and drowsiness without other manifestations were observed in 30% (n = 12) which may be non-specific manifestations due to medications. Three patients with rigidity had streptococcus pneumonia and the 2 patients with 6th cranial nerve palsy had haemophilus influenzae and Neisseria meningitidis (one patient each). Mortality rate was 5% (n = 2). Organisms identified were streptococcus pneumonia , staphylococcus aureus , haemophilus influenza  and neisseria meningitides . The high rate of culture negative disease  was caused by multiple factors including the use of antibiotics before admission, a common problem in Egypt and many other developing countries [The frequent presenting clinical manifestations include: fever , vomiting , headache , seizures , nuchal rigidity , drowsiness , diarrhea , cough  and bleeding tendency . According to PGCS, moderate  to severe  scores were identified in 40% of children. Fundus examination was normal. Neurological evaluation at the time of hospital discharge  revealed persistence of specific neurological manifestations in 12.5% (n = 5) of patients in the form of squint ; CSF-LPO with CSF-protein ; NO and LPO indices ; total thiol with LPO indices ; S-100B and PGCS ; CSF-LPO with CSF-S-100B ; serum-total thiol with serum S-100B . In healthy controls, a positive correlation was found between serum S-100B with serum LPO .The results of this study may suggest the following: 1) Dysfunction of blood-CSF barriers occurs with bacterial meningitis, 2) Oxidative stress has an important role in the pathogenesis of bacterial meningitis, 2) Brain damage does occur in bacterial meningitis as evidence by increased intrathecal synthesis of S-100B, NO and LPO biomarkers, 3) the finding of association between the levels of mediators of oxidative stress, antioxidants and S-100B may suggest their role in disease severity and the occurrence of neurological complications.In the present study, the increased albumin ratio (CSF/serum) is an indicator of morphological and/or biophysical dysfunction of the blood CSF barriers caused by inflammation. Albumin is the main fraction (80%) of CSF proteins. Increased albumin concentrations in CSF must always be due to blood CSF barriers dysfunction (100% blood-derived protein). Furthermore, it has been suggested that the mediators' CSF-to-serum ratio and indices  are better for diagnostic purposes rather than relying on CSF concentrations only and to infer whether the blood-derived mediators of similar molecular weight to albumin are passively transferred across the disrupted blood-CSF barriers or intrathecally synthesized. This has been attributed to the fact the difference in the CSF levels of these mediators throughout the day caused by CSF turnover and the to-and-fro motions of the CSF by cardiac cycles or the circadian rhythms. According to the laws of diffusion, it is important to know that all blood proteins traverse capillary walls of blood-CSF barriers by passive diffusion (molecular flux) into brain, extracellular fluid and CSF according to their molecular weight, e.g. smaller molecules like IgG (150 kDa) (serum/CSF ratio: 500:1), albumin (67 kDa) (serum/CSF ratio: 205:1) are fast in exchange than large molecules e.g. IgM (900 kDa) which are slower in exchange and subsequently form a steeper blood-to-CSF concentration gradient (IgM: serum/CSF ratio: 3400:1).In this study, serum and CSF levels of NO were elevated in children with meningitis. NO CSF/serum ratio and index are suggestive of local production in the CNS as well as passage through the disturbed blood brain barrier. Some authors suggested that the increased level of CSF-NO in patients with bacterial meningitis is due to blood-CSF barriers dysfunction or disturbed permeability as a result of the inflammatory process,23. BactA positive correlation was found between NO-index and CSF-WBCs. Another interesting result in this study is that the serum-NO level was higher in patients with gram positive CSF culture compared to negative culture indicating that the NO production is related to the presence of the organisms. Previous reports indicated that higher levels of NO are associated with severe disease . CSF pleIn this study, serum LPO levels were elevated in patients with meningitis which points to the presence of a general status of oxidative stress affecting cellular membranes-7,9,26.In this study, the high levels of NO, regardless of its source, have been associated with membrane lipid peroxidation. There is a substantial body of work implicating ROS, inflammation and oxidative stress in the development of cerebral edema, vascular damage, CSF pleocytosis and intracranial complications in bacterial meningitis and meningoencepahlitis,7. It isIn the present study, S-100B concentrations were increased. The results of S-100B CSF/serum ratio suggest increased intrathecal synthesis. Its levels were higher in patients with positive than with negative cultures and in presence of neurological complication than in their absence. A positive correlation was identified between CSF-S-100B and CSF-LPO and between S-100B ratio and the severity of meningitis as indicated by PGCS. The positive correlation was found between serum-S-100B with serum-total thiol lead to the suggestion that the damaged astrocytes may increase the formation of S-100B according to severity of oxidative stress.S-100B is a low molecular weight mainly brain-derived protein . In healthy subjects, natural autoantibodies of IgG class to S-100B are present in serum. However, the exact source of S-100B in the blood is unknown. At normal concentrations, it is able to protect hippocampal neurons against glutamate toxicity. It was The exact mechanisms that regulate the increased intrathecal levels of S-100B protein in bacterial meningitis are unknown. Inflammation ., glutamTo summarize, the present investigation provides a new perspective for the clinical study of S-100B, with special reference to neurologic sequelae after bacterial infections. We believe that this study has potential clinical importance in the field of pediatrics and biological processes although the results of this work in some of its parts are confirmatory to the previously established data and our suggestions of their role in the pathogenesis of bacterial meningitis are speculative in nature. This is the main limitation of this study. Further experimental studies are needed to confirm the direct or indirect causal relationship between bacteria, inflammation, oxidative stress and S-100B, markers in the pathogenesis of bacterial meningitis. Although CSF remains the biological fluid of choice, further investigations are needed in other biological fluids.The results of this study may have clinical and research implications. It may contribute to further understanding of factors involved in the pathogenesis of brain compromise in bacterial meningitis. It seems to be multifactorial and not due to direct infection of CSF or to the presence of toxins. It involves disruption of the blood-CSF barriers caused by inflammatory process, oxidative stress and impaired astrocyte function. If the results of this study are supported by further research, this may lead to the use of distinct profiles of mediators as possible biomarkers for the prediction of prognosis, monitoring response to treatment and improvement of management strategies of patients with bacterial meningitis.CNS: central nervous system; CSF: cerebrospinal fluid; BBB: blood-brain barrier; LPO: lipid peroxide; NO: nitric oxide; SOD: superoxide dismutaseThe authors declare that they have no competing interests.SAH carried out the clinical evaluation of the patients, collection of serum and CSF samples from patients, participated in the design of the study, statistical analysis and drafted the manuscript. EAH carried out the laboratory analysis of albumin and S100-B protein from serum and CSF and participated in the design of the study and its statistical analysis. MMZ carried out the laboratory analysis of NO, LPO, total thiol and SOD from serum and CSF and participated in the design of the study. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Cerebrospinal fluid (CSF) contacts many brain regions and may mediate humoral signaling distinct from synaptic neurotransmission. However, synthesis and transport mechanisms for such signaling are not defined. The purpose of this study was to investigate whether human CSF contains discrete structures that may enable the regulation of humoral transmission.Lumbar CSF was collected prospectively from 17 participants: with no neurological or psychiatric disease, with Alzheimer's disease, multiple sclerosis, or migraine; and ventricular CSF from two cognitively healthy participants with long-standing shunts for congenital hydrocephalus. Cell-free CSF was subjected to ultracentrifugation to yield supernatants and pellets that were examined by transmission electron microscopy, shotgun protein sequencing, electrophoresis, western blotting, lipid analysis, enzymatic activity assay, and immuno-electron microscopy.Over 3,600 CSF proteins were identified from repeated shotgun sequencing of cell-free CSF from two individuals with Alzheimer's disease: 25% of these proteins are normally present in membranes. Abundant nanometer-scaled structures were observed in ultracentrifuged pellets of CSF from all 16 participants examined. The most common structures included synaptic vesicle and exosome components in 30-200 nm spheres and irregular blobs. Much less abundant nanostructures were present that derived from cellular debris. Nanostructure fractions had a unique composition compared to CSF supernatant, richer in omega-3 and phosphoinositide lipids, active prostanoid enzymes, and fibronectin.via volume transmission within the nervous system that are for slower, more diffuse, and of longer duration than synaptic transmission.Unique morphology and biochemistry features of abundant and discrete membrane-bound CSF nanostructures are described. Prostaglandin H synthase activity, essential for prostanoid production and previously unknown in CSF, is localized to nanospheres. Considering CSF bulk flow and its circulatory dynamics, we propose that these nanostructures provide signaling mechanisms  Third, functional effects can be invoked via CSF: intracerebroventricular (icv) and intracisternal infusion of sodium or neuropeptides effect appetite [Physiological signaling  regions ,2 and ma regions , suggest regions , unlike appetite , drinkinappetite , sleep [appetite , and paiappetite ; and icvappetite .2 [In spite of accumulating evidence for non-synaptic transmission, it is not known how the biosynthesis and transport of signals are regulated within the circulating CSF. For instance, neurotransmitters in solution would be rapidly inactivated, such as hydrolysis of acetylcholine by acetylcholinesterase in the CSF. Moreover, integral membrane proteins may not function optimally in aqueous CSF, such as the prostaglandin H synthase (PGHS) required to synthesize the sleep-inducing prostaglandin D2 . The scoHere, we demonstrate that CSF consistently has a matrix of membrane- and protein-rich nano-scaled structures with many signal transduction components bounded by lipid membranes. These structures include features of vesicles containing acetylcholine, large dense-core vesicles (LDCVs), exosomes, and spherical structures with functional prostaglandin H synthases (PGHS) -1 & -2. People in health and disease states have these structures that contribute to CSF heterogeneity, and provide unique transport and signaling capabilities throughout CSF-contacting brain surfaces.2 (PLA2) from Naja Mossambica ; cyclooxygenase activity assay and phosphatidylinositide ; electrophoresis chemicals and criterion gels ; synthetic lipid standards ; uranyl acetate, Whatman #2 filters, EPON, and 200 mesh nickel formvar/carbon coated grids ; and paraformaldehyde powder The following were purchased: Quant-iT protein assay kit ; ultrafree-MC filters and immobilon-P polyvinylidine chloride (PVDF) membrane ; 1-Step™ NBT/BCIP ; centrifuge tubes 326814 and 344057 were used in 50 ti and 50.1 sw rotors on an L8-70 ultracentrifuge ; ammonia, ultrapure HPLC grade water, parafilm, methanol, and chloroform ; luna silica normal phase HPLC column , glutaraldehyde, phosphate buffered saline (PBS), sucrose, triethanolamine, phenylmethanesulphonylfluoride (PMSF), leupeptin, bovine serum albumin (BSA), glycine, Triton X-100, bovine brain phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS), and phospholipase AADP-ribosylation factor-like protein 2 (ARL2) was a generous gift from RA Kahn . 3-oxo-5-alpha-steroid 4-dehydrogenase 2 (S5A2), secretogranin-3 (SCG3), prostaglandin G/H synthase 2 (PGHS-2) were rabbit polyclonal antibodies made by MGH & John Rush  from synthetic peptides; S5A2 from CAGAGHHRFYLKMFEDYPKSRKALIPFIF; SCG3 from CAGAGKEAKEKETLITIMKTLIDFV; PTGDS from APEAQVSVQPNFQQD . The folTwenty lumbar and two ventricular CSF samples were collected prospectively for compositional studies after obtaining informed consent from 19 different participants, three of whom were sampled on two separate occasions . Twenty one mL of lumbar CSF was collected from the L2/3 or 3/4 inter-spaces, between 1 and 5 pm, with opening pressure measured in cm CSF in the recumbent position. Two ventricular CSF samples (each of approximately 250 mL) were collected by conventional external drainage  at room temperature over 24 h, with pressure measured continually in cm CSF, referenced to the external auditory meatus.Selection for lumbar samples was based on participants that represent health, migraine, inflammatory, or degenerative brain disorders. Selection for ventricular samples was based on participants that had received long-term shunts for congenital hydrocephalus and had normal cognitive function. Normal (n = 3): no classifiable neurological or psychiatric disorder after detailed structured interview and clinical assessment; Multiple sclerosis (n = 2): diagnosis of clinically definite multiple sclerosis based on national criteria ; AlzheimAll CSF was prepared, aliquoted, and analyzed immediately or stored frozen within 1 h of collection. Table Concentrations of total protein (in triplicate) were determined using a microplate-based Quant-iT protein assay kit with BSA, 0-500 μg/mL as standard, as recommended by the manufacturer. For all protein shotgun sequencing (below), CSF fractions were denatured (6 M urea), reduced, amidocarboxymethylated, washed with 100 mM ammonium bicarbonate (pH 8), filtered on Viva Spin 500 , digested with trypsin  overnight at 37°C, and quenched with formic acid.Liquid chromatography and mass spectrometry (LCMS) was applied to the S1 of two different samples  for separation of the peptide mixture on the 10 cm PicoFrit separation column. Peptides that eluted from the reverse phase column were analyzed by a Finnigan LTQ™ linear ion trap mass spectrometer that was equipped with a nano-electrospray ion source (both Thermo Fisher Scientific). Data-dependent mass spectral acquisition (MS/MS) was enabled and allowed for the MS/MS analysis of the most intense ion in the range of 450-1600 m/z (full scan) with the following dynamic exclusion settings: repeat count, 1; repeat duration, 0.5 min; exclusion duration, 3.0 min.This was performed using the Proteome X Linear Ion-Trap System (Thermo Fisher Scientific). The system was fitted with a strong cation exchange column, SCX 320 μm ID × 100 mm (Thermo Fisher Scientific) and two C18 reversed-phase nanotrap columns: IntegraFrit Trap, 75 μm ID × 25 mm, Biobasic™ C18 followed by a PicoFritTM nanobore HPLC column with a 15 μm i.d. pulled tip, 75 μm ID × 10 cm Biobasic™ . 100 μg of S1 protein digest was injected onto the SCX column and peptides were eluted onto a nanotrap column by successively injecting 20 μl of NHA modified version of the Pepfinder Kit with a Surveyor HPLC, autosampler, and nanoflow solvent delivery (Thermo Fisher Scientific) was used to present the CSF sample to an LTQ ion trap mass spectrometer equipped with a nano-electrospray ion source (Thermo Fisher Scientific) and 30 μm PicoTip emitter . The Pepfinder kit was modified from its original form to contain a 5 × 0.3 mm Zorbax™ C-18 peptide trap  or a 0.075 × 25 mm Biobasic C18 IntegraFrit Trap combined with a 100 μm ID × 25 cm BioBasic 18 nanobore C-18 separation column (both from New Objective). 1 - 4 μg of CSF S1 protein digest was injected onto the trap, washed, and then eluted onto and through the C-18 column with a pseudo-exponential gradient profile, from 0-80% B in ~4 hr in following gradient increments; 0.1%/min in 50 min, 0.2%/min for 50 min, 0.25%/min for 40 min, 0.33%/min for 60 min, 0.44%/min for 45 min and 4%/min for 5 min . The mass spectrometer was operated in a data-dependent MS/MS mode and dynamic exclusion was enabled. Gas-phase fractionation with three distinct scan ranges  was used to maximize the number of peptides identified as described . The num® algorithm [-5 were accepted for protein analysis. Post-translational modifications were assessed using a fully automated, de novo sequencing software program, DeNovoX (Thermo Fisher Scientific). A comprehensive list of non-redundant proteins was generated from all analysis runs (n = 15), using Excel sorting and Pivot Table functions. Results were compared to existing identifications [MS/MS spectra obtained from these LCMS analysis of the S1 protein digests of sample #s 1 & 2  with Bioworks 3.2 in full tryptic and semi-tryptic search mode using the Sequest algorithm implementation of SORCERER . For unit resolution data, a precursor tolerance of 2Da was used, while 100 ppm tolerance was applied for high mass accuracy data. Positive identifications were established using the probability calculations in Bioworks 3.2, with a statistical expectation of > 10E-5. Results from tryptic and semi-tryptic searches were combined and filtered after export into Excel. A comprehensive list of non-redundant proteins was generated from all analysis runs (n = 6) using Excel sort and Pivot Table functions. Results were compared to existing identifications .S1 samples (1.5 mL) from #s 1, 2, 5, & 6  then eluted through a silica column with a chloroform/methanol/water/ammonium hydroxide gradient [Lipids were extracted from S3 fluids and P3 pellets using the method of Bligh Dyer . After rgradient . The eluPrecursor ion scans (PIS) and neutral ion loss (NIL) for different lipids were obtained using a full scan MS infusion experiment on a triple quadrupole mass spectrometer, TSQ Quantum (Thermo Fisher Scientific) operated at a spray voltage of 4500 V, sheath gas pressure of 40 units, auxiliary gas pressure of 0, capillary temperature of 225°C and collision pressure of 1.5 units. Negative ions were acquired in the profile mode with 13 different scan events after collision induced dissociation (20-24 V) of deprotonated precursor ions or the neutral loss of specific groups from lipids. Negative PIS of 196.3 (mass range 650-950), 171.14 (mass range 600-900), 240.96 (mass range 750-1200), 168.17 (mass range 600-900) were used to monitor PE, PG/PA, PI and SPM, respectively. Negative NIL of 86.99 (mass range 650-900) and 50.13 (mass range 400-1000) were used to monitor PS and PC/LPC/PAF, respectively. Lipids containing eicosanpentanoeate , arachidonate  and docosahexaenoate  were detected using PIS of these ions in a mass range from 600-1200. Peak intensities were integrated, processed, and mole quantities determined using ICIS and Xcalibur software (Thermo Fisher Scientific). Mole quantities were determined from standard curves obtained using known amounts of lipid standards (0-400 ng).2 at 37°C overnight in reaction buffer. The same incubation was carried out with heat-denatured enzyme, controls without enzyme. Reaction products were stored at 4°C until assayed either by LCMS or TEM with negative staining as described above.P3 samples were incubated with PLAmax kinetic microplate reader at 595 nm . PGHS activity (nmol/min/mg protein) was calculated as described by the kit manufacturer .All assay components were pre-equilibrated to the room temperature except for the PGHS-2 Standard that was kept on ice. Detection was based on measuring the peroxidase activity of cyclooxygenase by colorimetrically monitoring the appearance of oxidized N, N, N', N'-tetramethyl-p-phenylenediamine at 595 nm in 96 well plates. Either Dup-697 (PGHS-2 inhibitor) or SC-560 (PGHS-1 inhibitor) was added to inhibitor wells and both inhibitors were added into background wells. Standard, samples, and backgrounds were analyzed in duplicates. The plate was carefully shaken and after 5 min of incubation at room temperature, absorbance was read on the VPressure was normal at all lumbar (<150 cm) and ventricular (< 15 cm) CSF collections. All fluids were clear, with < 5 white blood cells per mL, and no red blood cells were seen. All samples had total protein content within the normal range of 0.1 - 0.5 g/L.Using sample #s 1 & 2 , based on specific staining at their predicted molecular weight: FINC, SNAP 23, PGHS-2, RALA, S5A2, ARL2, and SCG3. We also found that semaphorin 4D, chromogranin A & B, and the known SNARE complex proteins synaptotagmin, syntaxin, and synaptobrevin were enriched in P3, but with insufficient samples for statistical analysis.To explore the sources of these structures, we considered that the abundant spherical structures would come from the choroid plexuses and the ependymal lining of the CSF, and might be derived from similar-sized synaptic vesicles , LDCVs , or exosSince nanostructures are purified in P3 fractions, we performed iTEM to find whether the proteins identified on western blots Figure  localize2 (Naja mossambica). This depleted the major phospholipid peaks as measured by mass spectrometry, and removed > 90% of the spherical structures as measured by TEM (data not shown).The morphology of the abundant spheres visible by TEM Figure  suggestsp < 0.005).Liquid chromatography of S3 and P3 lipids partially resolved PI into two peaks consisting of polyunsaturated fatty acids (PUFA), (PI-peak-1), and saturated fatty acids, (PI-peak-2), Figure  &9D. Thep = 0.0005).The presence of eicosanoid metabolic enzymes  in the initiation of normal sleep, when it is applied via the CSF selectively to the ventral rostral brainstem [2  is an unstable intermediate in PGD2synthesis, and its local synthesis by the integral membrane protein PGHS is required for production of downstream prostaglandins. We therefore assayed PGHS activity in the CSF fractions.To test whether CSF nanostructures have functional enzymes, we selected the prostanoid pathway because our protein and lipid studies showed both their enzymes and substrates present in CSF. Furthermore, Hayaishi's group demonstrated a physiological effect for prostaglandin Drainstem , and manrainstem -37. HoweWe wanted to test PGHS activity from a large quantity of CSF because PGHS-2 staining structures were infrequent on iTEM Figure  and P3 wvia the CSF. However, there is little information about the mechanisms by which such communications are regulated. A lack of structures to transport, protect, localize, and eventually transduce signals at an appropriate receptor poses major restrictions on transmission within the 150 mL of fluid. We now report details of the morphology and biochemistry of unique structures in CSF that have the potential to overcome many, if not all, of these limitations.A variety of experiments, as outlined in the background, support volume transmission between brain regions CSF proteomic studies have recently revealed many proteins that are substantially different from those found in plasma ,38-41. OWhen classes of CSF protein components from published data were compared based on GO component categories, around 40% are membrane proteins . ZougmanOur TEM data demonstrates small particles on filtered CSF. The presence of these nano-sized particles from filtration provides evidence that these structures are not formed during the ultracentrifugation procedure. The presence of identical nanostructures in CSF from two participants that were collected and prepared freshly as compared to stored CSF, demonstrates that the nanostructures are not a product of sample storage.Past reports of CSF sub-cellular structures have been interpreted as resulting from blebbing, apocrine secretion, apoptosis events, or cellular debris ,42-45 an2 caused dissolution of the TEM nanostructures, and depleted major P3 phospholipids as determined by LCMS, demonstrate that lipid membranes enclose these spherical structures. These membranes provide an appropriate environment for some of the abundant transmembrane proteins found in our shotgun sequencing experiments of CSF , persons with intermittent disability (migraine), and persons with serious brain pathology of either an inflammatory (multiple sclerosis) or degenerative disease (Alzheimer's disease) suggests strongly that these structures are ubiquitous in CSF. Though the unknown functions of these nanostructures require much further study, our data hints at multiple roles.The different electrophoretic and western blots of P3s in this nanostructure-rich fraction are evidence that the P3 fraction from CSF contains biochemically distinct components. The FINC immunostain is greatly enriched in P3 fractions that mainly contain the varied blobs and strands. The known functions of FINC as an adhesive extracellular molecule involved in neurite development  support Our data Figure  also sho2, are present  for synthesis of the obligatory prostanoid intermediate, PGH Figures  &8A and  Figures  in CSF.  Figures . These r Figures  and lipa & -2 forvia CSF. For example, topical acetylcholine has been consistently shown to dilate cerebral vessels [Membrane-bound nanospheres with acetylcholine provide potential protection from acetylcholinesterase in CSF, thus enabling a humoral mode for vesicle neurotransmission  vessels , raising9 and 1012 nanospheres are produced per day, comprised of 5 μg total protein. These relatively small amounts of CSF nanostructures may have physiological roles since the readily releasable synaptic vesicle pool size ranges from as low as 5 to as many as 5,000 per single synapse, as reviewed by Südhof [For both healthy as well as those with brain disorders we estimate, assuming production and turn over at the same rate as the fluid, between 10y Südhof . While trd ventricle of about 5 mm/sec [rd ventricle to move to receptors in the medial hypothalamic wall of the same ventricle. For a simple circulation of pre-synthesized signal within this small space, we estimate transmission speed will be in the order of a second. At the other extreme, such as nanostructures originating in a ventricle, migrating to a more distant subarachnoid location, and requiring signal synthesis, we estimate speeds ranging from minutes to several hours, since total CSF exchanges 3-5 times a day and is stagnant in some regions [Brain extracellular fluid is known to diffuse ions, dopamine, proteins, and 35 nm particles ,56 in sp5 mm/sec ,58. Figu regions ,58. Bulk regions -7,53,59.Human lumbar and ventricular CSF samples demonstrate abundant membranous CSF structures, 30-200 nm in size from both healthy and sick participants. Compared to the supernatant, these structures have unique protein and lipid compositions, contain acetylcholine, and have complete prostanoid pathways from membrane phospholipids to specific receptors. Variation in CSF nanostructures may be informative in both health and disease studies, but they require enrichment since they are diluted by more abundant fluid components. We anticipate that further study of these biochemically and morphologically unique CSF nanostructures will identify their roles in modulating brain functions and dysfunctions.2: prostaglandin D2; PGH2: prostaglandin H2; PGHS: prostaglandin H synthase; PI: phosphatidylinositide; PIS: precursor ion scan; PLA2: phospholipase A2; PMSF: phenylmethanesulphonylfluoride; PS: phosphatidylserine; PTGDS: prostaglandin D synthase; PUFA: polyunsaturated fatty acid: PVDF: polyvinylidine difluoride; RALA: Ras-related protein ral-A; S1: CSF 3,000 g supernatant; S2: CSF 17,000 g supernatant; S3: CSF 200,000 g supernatant; S5A2: 3-oxo-5-alpha-steroid 4-dehydrogenase 2; SCG3: secretogranin 3; SE-1: statistical expectation value; SNAP-23: synaptosomal-associated protein 23; SNARE: soluble N-ethylmaleimide-sensitive fusion attachment protein receptor; SPM: sphingomyelin; TEM: transmission electron microscopy (iTEM: immuno-TEM).AA: arachidonic acid; ARL2: ADP-ribosylation factor-like protein 2; BSA: bovine serum albumin; CM: ceramide; CS: cerebroside sulfate: CSF: cerebrospinal fluid; DHA: docosahexaenoate; EPA: eicosanpentanoeate; FINC: fibronectin; GO: gene ontology; LCMS: liquid chromatography mass spectrometry; LDCV: large dense core vesicle; LPC: lysophosphatidylcholine; NIL neutral ion loss; P1: CSF 3,000 g pellet; P2: CSF 17,000 g pellet; P3: CSF 200,000 g pellet; PA: phosphatidic acid; PAF: platelet-activating factor; PBS phosphate buffered saline; PC: phosphatidylcholine; PE: phosphatidylethanolamine; PG: phosphatidylglycerol; PGDThe authors declare that they have no competing interests.MGH conceived and designed both the human subjects and laboratory aspects of the project, recruited and diagnosed study participants, collected some of the CSF, carried out some of the shotgun protein sequencing experiments, performed the ultracentrifugation and sample preparations for biochemical and electron microscopy studies (TEM and iTEM), analyzed all data, and drafted the manuscript. ANF participated in design of all experiments, carried out the lipid analyses, and participated in manuscript revisions. EO carried out electrophoresis, western blots, developed and carried out the enzyme activity assays. PL carried out the early ultrafiltration of nanostructures and participated in the electron microscopy and analysis of these filters. RPC and GM recruited and diagnosed study participants, collected CSF, and participated in manuscript revisions. JNC helped design and supervise all electron microscopy experiments. RGB and AFH helped design and carry out most of the protein shotgun sequencing experiments, and participated in manuscript revisions. All authors read and approved the final manuscript.CSF proteins from repeated analyses of two independent samples. CSF proteins identified by Uniprot number, name, and GO component category, from 15 shotgun sequencing analyses of sample #s 1 & 2 in Table Click here for fileCSF proteins from the P3-enriched pellet and the S3 supernatant. CSF proteins identified from a single, high resolution shotgun sequencing run of two sets of P3 and S3 samples from one participant collected on two independent occasions (sample #s 3 & 3' in Table Click here for file


We have previously demonstrated a 33% response rate in patients with primitive neurectodermal tumours after the direct injection of 131I-monoclonal antibodies (MAbs) into the cerebrospinal fluid (CSF). Dose-limiting toxicity is myelosuppression due to the passage of the radioimmunoconjugate from the CSF to the blood compartment. This occurs at doses of 2220 MBq of 131I-MAb and above, although this is not seen in all patients studied and appears to be related to the degree of prior therapy received. Rather than attempting to improve the efficacy of this approach to the treatment of disseminated disease within the CSF compartment by dose escalation and haemopoietic rescue, we have explored the possibility of repeatedly administering the radioimmunoconjugate. Eight patients were recruited to the study, two of whom received two and six of whom received three injections of 131I-MAb. After repeated administration of 131I-MAb pharmacokinetic data revealed that, with one exception, the radioimmunoconjugate cleared from the CSF compartment with similar kinetics, while its residence time in the blood decreased with each injection. This was due to the development of an anti-mouse Ig response in the blood. Clearance of 131I-MAb from the ventricular CSF appears to be independent of the presence of an anti-mouse Ig response in this compartment. The differential clearance of the radioimmunoconjugate from the ventricular CSF and from the blood results in a marked increase in the therapeutic index that can be achieved. Up to 5920 MBq of 131I-MAb was administered as the third injection of radioimmunoconjugate and combined doses of up to 12,500 MBq were given without either haematological or neurological toxicity. These data illustrate that dose escalation and thus an increase in the dose rate delivered to tumour cells within the CSF is possible if ways are found to reduce the residence time of the radioimmunoconjugate in the blood compartment. Suggestions as to how this can best be achieved are reviewed in detail.


Proteins controlling mitochondrial dynamics are often targeted to and anchored into the mitochondrial outer membrane (MOM) by their carboxyl-terminal tail-anchor domain (TA). However, it is not known whether the TA modulates protein function. GDAP1 is a mitochondrial fission factor with two neighboring hydrophobic domains each flanked by basic amino acids (aa). Here we define GDAP1 as TA MOM protein. GDAP1 carries a single transmembrane domain (TMD) that is, together with the adjacent basic aa, critical for MOM targeting. The flanking N-terminal region containing the other hydrophobic domain is located in the cytoplasm. TMD sequence, length, and high hydrophobicity do not influence GDAP1 fission function if MOM targeting is maintained. The basic aa bordering the TMD in the cytoplasm, however, are required for both targeting of GDAP1 as part of the TA and GDAP1-mediated fission. Thus, this GDAP1 region contains critical overlapping motifs defining intracellular targeting by the TA concomitant with functional aspects. Mitochondria are dynamic organelles that constantly fuse and fragment. Critical components that regulate and coordinate fusion and fission of the mitochondrial inner and outer membrane (MOM) have been identified Many proteins involved in the regulation of mitochondrial dynamics are located at the MOM and contain a C-terminal membrane tail anchor (TA). For example, mitofusins span the MOM twice with the N- and the C-terminus facing the cytosol Notable examples of classical TA-proteins of the MOM include small import receptors of the preprotein translocase of the outer mitochondrial membrane (TOM complex) Although TA-proteins lack a N-terminal targeting signal, they specifically integrate into a limited number of organelles such as the endoplasmic reticulum (ER), mitochondria, peroxisomes, and chloroplasts GDAP1 lead to the peripheral neuropathy Charcot-Marie-Tooth disease (CMT), affecting both Schwann cells, the myelinating glia of the peripheral nervous system, and neurons S-transferases (GSTs) The nervous system-enriched GDAP1 (ganglioside induced differentiation associated protein 1) is a mitochondrial fission factor located at the MOM This study was aimed at determining the precise topology of GDAP1, the motifs involved in MOM targeting and integration, and the relationship between TA structure and the fission function of the protein.To determine the topology of GDAP1, we required an appropriate epitope-tagged version of the protein. Large C-terminal tags severely impair the posttranslational translocation of ER-targeted TA proteins in vitro translated GDAP1, Fis1, Mfn2 or luciferase to the post-nuclear supernatant of Hela cells  already led to significant loss of mitochondrial localization  Mitochondrial targeting of GDAP1 is not critically dependent on individual basic aa within the TA; (2) C-terminally TMD-flanking clusters of basic aa play a key role in targeting; (3) A cluster of basic aa N-terminally bordering the TMD is also required for mitochondrial localization; strikingly, even individual aa of this cluster are concurrently crucial for GDAP1 function; (4) The residues immediately adjoining HD1 N- or C-terminally  are not required for targeting, but R310 is required for fission. Interestingly the mutation R310Q was found in patients with CMT Next, we examined the role of the aa sequences of the TMD and HD1 on mitochondrial targeting and fission activity. To this end, we constructed GDAP1 mutants with scrambled TMD and HD1  and a mutant lacking HD1  and dispMOM targeting of TA proteins has been suggested to depend on short TMDs We next asked whether the particular TMD and the flanking GDAP1 C-terminus are critical for fission-inducing activity. Thus, we constructed a chimeric protein in which the TMD and the C-terminus of GDAP1 are replaced by that of VAMP1B, a tail-anchored MOM protein with no known function in mitochondrial fission et alWattenberg GDAP1 is a mitochondrial fission factor of the MOM and mutations affecting this protein cause a subtype of the inherited neuropathy CMT in vitro comparable to the integration of the classical TA protein Fis1. We found that GDAP1 integrates into the membrane exclusively dependent on the TA-domain and not on other cytosolic domains like an N-terminal targeting sequence, the GST-domains, or HD1. In contrast, the two TMD-containing MOM protein Mfn2 did not integrate in our assay system. A similar integration of in vitro translated TA-proteins into the MOM has previously been demonstrated in digitonin-permeabilized HeLa cells Concerning mitochondrial targeting, our protease-protection assays revealed that GDAP1 spans the MOM once with its C-terminal TMD. The N-terminal part with the GST domains is located in the cytosol. The short C-terminal tail is located in the IMS. These findings are in agreement with the definition of classical TA-proteins that span the membrane with a single transmembrane domain close to the C-terminus The classification of GDAP1 as a TA protein of mitochondria is further supported by the following results: First, MOM TA protein targeting is regulated by basic aa flanking the TMD In contrast to the clusters of basic residues surrounding the TMD of GDAP1, the basic residues K291 and R310, adjoining the more N-terminally located HD1, are not required for MOM targeting. They are, however, essential for GDAP1-mediated fission activity. Similarly, HD1 deletion or scrambling of its aa sequence severely impair fission activity of the mutant GDAP1 proteins but without affecting mitochondrial localization. These experiments indicate a key role for HD1 in GDAP1 function without major involvement in mitochondrial targeting and GDAP1 insertion into the MOM. Indeed the R310Q mutation, which has lost the ability to induce mitochondrial fission, was found in CMT patients Potential GST activity of GDAP1 might modify the lipids of the MOM to allow fission since the GDAP1 sequence harbors both a GST-N and a GST-C domain The generation of GDAP1-, EGFP-GDAP1- and GDAP1-EGFP- expression constructs was described previously Polyclonal rabbit anti-GDAP1 antibodies have been previously described t-test. For quantification of the mitochondrial morphology, 450 to 600 transfected cells were counted and categorized into five distinct mitochondrial morphologies: Aggregated, tubular, mixed, vesicular and fragmented as previously described COS-7 cells and HeLa cells were cultured in DMEM (Invitrogen) containing 10% FCS (Brunschwig) until 80% confluent. COS-7 cells were transfected using Fugene 6 (Roche), HeLa cells using Lipofectamine2000 (Invitrogen) according to the manufacturers' protocols. Cells were fixed at the indicated time points. Immunofluorescence procedures were performed as described previously HeLa cells were transfected with either GDAP1 or GDAP1-FLAG expression constructs. After 24 hours, fractions were isolated and mitochondria were enriched as described previously in vitro from the respective cDNAs in the pcDNA3.1 vector using the TNT Quick Coupled Transcription/Translation System (1 µg plasmid / reaction). The protocols for membrane integration and differential centrifugation were adapted from Niemann, et al. in vitro-synthesized proteins for 60 minutes at 4°C. Both subsequent centrifugation steps were performed for 20 minutes at 10000 g. Equal volumes of supernatants and pellets were analyzed by autoradiography using ENHANCE (Perkin Elmar) according to the manufacture's protocol. To confirm the integration of in vitro translated GDAP1-FLAG this radiolabeled protein was digested with 50 mg/ml proteinase K for 30 min at 4°C without (pure in vitro translat) or after membrane integration (60 min incubation with post nuclear supernatant on ice). The digest was stopped with 5 mM PMSF for 10 min. Immunoprecipitation was done with the anti-FLAG antibody (s.a.) in IP-buffer ). The samples were incubated with the antibody at 4°C for 2 h. Protein A-sepharose  was added for an additional hour. Samples were washed three times in IP-buffer, boiled at 90°C for 10 min in SDS-buffer  and analyzed by autoradiography.Proteins were synthesized Figure S1GDAP1-EGFP is peripherally attached to the MOM. (A) COS-7 cells were transiently transfected with either mtEGFP (a–c), wt GDAP1 (d–f), or the C-terminal EGFP-tagged construct GDAP1-EGFP (g–i). Fifteen hours after start of transfection, cells were co-stained with MitoTracker to analyze mitochondrial localization. GDAP1-EGFP shows only partial mitochondrial localization. In addition, the expression of GDAP1-EGFP causes mitochondrial aggregation. (B) Upper panel: Cartoon of a cell before and after permeabilization of the cell membrane with low concentrations of digitonin showing the release of cytosolic proteins . Subsequent treatment with trypsin digests cytosol-exposed membrane-bound protein parts . The used digitonin and trypsin concentrations do not affect the mitochondrial membrane integrity . Lower panel (b–g): GDAP1-EGFP and mtDsRED expressing COS-7 cells were permeabilized with 50 µM digitonin and treated in parallel with 250 µM trypsin for the indicated time points. Images were taken after permeabilization and trypsin digest. The cytosolic GDAP1-EGFP signal is washed out early due to permeabilization of the cell (c). Only the GDAP1-EGFP associated with mitochondria remains detectable but is lost over time of the protease digest (b–d). The mitochondrial targeted marker mtDsRED is not washed out or degraded by the protease (e–g). These results indicate that GDAP1-EGFP partly associates with mitochondria but the long-extended C-terminus fails to translocate across the MOM. Bars, 10 µm.(10.01 MB TIF)Click here for additional data file.Figure S2In-vitro translated GDAP1-EGFP peripherally attaches to membranes. The post-nuclear supernatant of HeLa cells was incubated with the in vitro-translated GDAP1-GFP and the mitochondrial pellet was resuspended in buffer (control), in 1 M NaCl, 0.1 M carbonate (pH 11), or in buffer with 0.1% TritonX-100, and centrifuged to separate the soluble protein supernatants (S) from membranous pellets (P). Upon treatment with sodium chloride or carbonate GDAP1-GFP was extracted as was the intermembrane space protein Cytochrome C (1.47 MB TIF)Click here for additional data file.Figure S3Effect of TMD length on mitochondrial targeting and fission activity. (A) Schema of GDAP1 TMD aa sequence and constructs with altered TMD length. TMD hydrophobicities are given in brackets. (B) Confocal immunofluorescence analysis of transfected COS-7 cells reveals mitochondrial targeting for all recombinant proteins (a–l), albeit for TMD+5 with reduced efficiency (g–i). (C) Quantification of mitochondrial localization. Significant mislocalization was detected for TMD+5. (D) Analysis of fragmentation-inducing activity of mutants revealed no significant difference compared to wt GDAP1 (GDAP1). Bars, 10 µm.(9.88 MB TIF)Click here for additional data file.Figure S4Quantitative analysis of mitochondrial targeting. Quantification of mitochondrial localization of recombinant proteins used in this study in transiently transfected COS-7 cells. Abbreviations are explained in the text.(2.66 MB TIF)Click here for additional data file.Figure S5Quantitative analysis of expression levels. (A) Expression levels of tested GDAP1 point mutants, (B) mutants with varying TMD length, and (C) tagged and chimeric variants of GDAP1 were comparable to wt GDAP1 (GDAP1) protein in transfected COS-7 cells. The abbreviations are explained in the text. Quantification was performed by calculating the ratio of anti-GDAP1/anti-beta actin signals on Western blots of cell lysates from sister plates of those used for morphological analysis (n = 3).(9.89 MB TIF)Click here for additional data file.Materials and Methods S1Supplemental Materials and Methods(0.05 MB DOC)Click here for additional data file.


The Kearns-Sayre syndrome is a mitochondrial disorder  that causes ophthalmoplegia, retinopathy, ataxia and brain abnormalities such as leukoencephalopathy. In this syndrome, the choroid plexus epithelial cells, unlike brain cells, are greatly enlarged and granular, consistent with their inability to adequately transport folate from blood into cerebrospinal fluid (CSF), and homovanillic acid (a dopamine metabolite) from CSF into blood. This inability to transport folates from blood into CSF (and brain) adequately, causes cerebral folate deficiency that can be partially reversed by very high doses of reduced folates. The Kearns-Sayre syndrome is a disease that interferes with key choroid plexus functions and is a cause of generalized choroid plexus failure. Besides secreting most of the cerebrospinal fluid (CSF), the choroid plexus (CP) epithelial cells, a locus of the blood-CSF barrier, like renal tubule cells, have myriad other functions. These include the active transport of essential micronutrients into CSF from blood, e.g., methyltetrahydrofolic acid (MeTHF) and ascorbic acid (AA) . From CSIn recent years, the mechanisms responsible for some of these functions of the CP have been worked out on a molecular basis. For example, the vectorial transport of MeTHF  from blood into CSF via the CP is due to a three-part active transport system in series: first, the folate receptor alpha (FRα) transfers MeTHF from blood into CP endosomes; then MeTHF is released from the endosomes into the CP cytoplasm by the proton-coupled folate transporter (PCFT); and finally MeTHF is released from the cytoplasm into CSF by facilitated diffusion via the reduced folate carrier (RFC) . AnotherThe principal objective of this paper is to answer a question we asked about thirty years ago: Can the CP fail as an organ in a generalized way in disease states ? If so, In humans, there are now examples of specific CP functions that fail as a result of inherited and acquired conditions. For example, there are two different genetic causes of inability to pump MeTHF from blood into CSF via CP. These can be termed human knockouts (KOs). This can be due to KO of either FRα or PCFT ,3. TheseThe above examples are extremely revealing but do not answer the question about generalized CP failure - akin to renal failure - secondary to diffuse damage to the CP epithelial cells . HoweverIn vivo, in humans with KSS one known functional consequence is the inability to transport adequate amounts of MeTHF into CSF with consequent CFD [In KSS, there are prominent anatomical changes in human CP epithelial cells that are greatly enlarged and full of eosinophilic granules . In vivouent CFD -7,9. Mosuent CFD -7,9. In Unlike MeTHF, the HVA concentrations in CSF are elevated in KSS  almost certainly due to decreased clearance of HVA from CSF, presumably by the CP epithelial OAT-3 . There iAssociated with the low CSF MeTHF and the raised HVA and CSF protein in KSS, is a devastating progressive neurological disorder that begins before the age of twenty. KSS manifests itself clinically with ophthalmoplegia, retinopathy, ataxia and other abnormalities -7. We poThus, KSS can be considered a cause of diffuse CP failure. Presumably, beside MeTHF and HVA transport, many other important functions are abnormal. For example, we would predict AA concentrations in CSF will be low. Hence, if in fact the concentration of AA is low in CSF, KSS patients might benefit from high doses of intravenous dehydroascorbic acid (DHA). DHA enters brain via the glucose transporter at the blood- brain barrier (via the transporter glut 1) and is converted to AA in situ in brain [KSS is an example of a systemic mitochondrial disorder that affects the CP anatomically and physiologically. It is clear that the CP, like several other tissues  in KSS, suffers from excess mitochondrial DNA heteroplasmy. This presumably leads to insufficient ATP concentrations to drive cellular processes  in CP with consequent low CSF and CNS MeTHF concentrations, as well as elevated CSF HVA, and other so-far unmeasured CSF abnormalities. Our increased understanding of KSS has led to amelioration of the syndrome (with high doses of reduced folates), but there is clearly more work to be done. KSS is an example of the importance of basic research in unraveling complex clinical disorders.AA: ascorbic acid (vitamin C); ATP: adenosine triphosphate; CFD: cerebral folate deficiency; CP: choroid plexus; CSF: cerebrospinal fluid; DHA: dehydroascorbic acid; FRα: Folate receptor alpha; HVA: homovanillic acid; KSS: Kearns-Sayre Syndrome; MeTHF: methyltetrahydrofolic acid; OAT-3: organic acid transporter 3The authors declare that they have no competing interests.RS and CEJ both analyzed the literature, wrote the manuscript and worked on the revision. Both authors have read and approved the final version of the manuscript.