In most tissues and organs, the lymphatic circulation is responsible for the removal of interstitial protein and fluid but the parenchyma of the brain and spinal cord is devoid of lymphatic vessels. On the other hand, the literature is filled with qualitative and quantitative evidence supporting a lymphatic function in cerebrospinal fluid (CSF) absorption. The experimental data seems to warrant a re-examination of CSF dynamics and consideration of a new conceptual foundation on which to base our understanding of disorders of the CSF system. The objective of this paper is to review the key studies pertaining to the role of the lymphatic system in CSF absorption. The classical textbook theory assumes that the projections of the arachnoid membrane into the cranial venous sinuses represent the primary sites for CSF clearance and when absorption through these sites is blocked, this leads to disorders of the CSF system. However, this view is increasingly being contested -5.Apart from one study in which Prineas  reportedWe first learn of an affiliation between CSF and lymph through studies conducted over 100 years ago. Schwalbe demonstrated a connection between the subarachnoid space and the cervical lymphatic system in dogs and rabbits with the use of Berlin blue . A similThe cellular parameters associated with the physiological 'coupling' of the CSF and extracranial lymph compartments will no doubt have an influential role in determining how CSF is absorbed and by inference, also create potential pathological targets for obstruction and interference with CSF absorption. The most basic issue is whether CSF convects first into an intervening interstitial compartment  or whether there is some form of direct connection between the CSF and nasal lymphatics. Table Jackson and colleagues have postulated two possible mechanisms for CSF uptake into lymphatics . The firpost-mortem state could have ruptured the lymphatic vessels occasionally. Additionally, it was clear that the longer the period between death and infusion of the contrast agent, the greater the chance of Microfil being observed within the nasal interstitial space due to tissue deterioration.In rats, CSF may move directly from the subarachnoid space into submucosal lymphatics that emerge at the level of the cribriform plate -14. Thispost-mortem. Evans blue dye is an example. However, this does not seem to be true of Microfil. This silastic material was developed to outline vascular networks after injection into a vessel lumen. It is relatively viscous and is unlikely to be taken up readily from an interstitial compartment. In the Microfil studies [post-mortem. This implied that a direct connection had to exist between the CSF and lymph compartments to facilitate uptake into lymphatic vessels.It is of interest to note that some contrast agents can be taken up into lymphatic vessels readily after injection into the interstitial space  studies , the autHistological investigation in the sheep Microfil studies showed that the lymphatic vessels fused to the sheaths of the olfactory nerves within the submucosa proximal to the cribriform plate . Upon clSome studies have reported the existence of a perineural sheath around the olfactory nerves composed of flattened cells . WhetherDirect connections between CSF and lymph would appear to make sense from a theoretical perspective. One might imagine that CSF leaks would be very common if CSF convected routinely into the nasal submucosal interstitium since the fluid would be separated from the air spaces only by a layer of olfactory or respiratory epithelium. The need for effective CSF clearance under a wide variety of intracranial pressures and the requirement to protect the brain from air-borne infection would seem to be best met by a CSF absorption system that limits CSF access to well-defined lymphatic drainage pathways rather than permit random CSF dispersion throughout the extracellular spaces of the olfactory and respiratory submucosa.The most important lymphatic CSF absorption pathway is no doubt the olfactory route leading to cervical lymphatic vessels but there are other nerves that may conduct CSF extracranially. Even though the bulk of evidence favors the olfactory nerves as facilitating CSF-lymph connections, tracers injected into the CSF system appear to exit the cranium along almost all of the cranial nerves including the trigeminal, acoustic , hypogloInjection of tracer into the subarachnoid space resulted in the appearance of tracer in the optic nerve ,23-31. A125I-albumin injected into the subdural space in rabbits was observed to enter plasma [et al, India ink injected into the subarachnoid space of the optic nerve penetrated the arachnoid and entered the interstitial compartment and lymphatics in the dura of the nerve [One possible location for lymphatic CSF absorption that has been ignored generally is the dura itself. In rats, lymphatics exist around the wall of the sagittal sinus, in the areas of the confluence of sinuses in proximity to the mesothelial cells of the subdural spaces and close to the vasculature of the dural tissues . Lymphatr plasma  and it she nerve . There ihe nerve . WithoutLymphatics also appear to play a role in spinal CSF absorption. India ink infused into the ventricles or cisterna magna of rabbits has been found around emerging spinal nerve roots. The tracer passed from the subarachnoid space cul de sac into lymphatic vessels and nodes of the cervical and lumbosacral region . SimilarFrom a quantitative perspective, the drainage of CSF from the spinal cord subarachnoid space plays a role in total volumetric CSF absorption. Studies performed in sheep showed that the relative proportion of CSF absorption by the spinal compartment represents approximately 25% of total CSF clearance .et al [The CNS has a complex extracellular space that connects with the internal (ventricular) and external  CSF through the ependymal layer and pia-mater and the Virchow-Robin spaces . The Viret al -47 suppoet al . Additioet al . TherefoFöldi developed the concept of parenchymal interstitial fluid draining into extracranial lymphatics located in the adventitia of the internal carotid artery . Wang anet al collected lymph from sheep cervical lymphatic vessels overtime after administration of a radioactive tracer into the cisterna magna, and found that there were measurable amounts of tracer in the lymph 1 h after injection  [It is difficult to quantify volumetric CSF absorption by lymphatics due to the complexity of the anatomical pathways involved. Some investigators have simply taken CSF tracer recovery in lymph nodes as a reflection of lymphatic function. For example, Marmarou's group measured a very low recovery in the cervical, retropharyngeal, parotid, and mandibular lymph nodes in cats 8 h after infusion of radioactive albumin into the brain . Howeverjection) .125-albumin. Approximately 32% of CSF was recovered in the cervical lymphatics of sheep [125-albumin was observed in extracranial tissue spaces and lymphatics after continuous infusion into the lateral ventricles [Courtice and Simmonds were among the first to quantify the absorption of a CSF dye into plasma and cervical lymph . They foof sheep . Similarof sheep . When thof sheep . In primntricles .While these studies hinted at an important role of lymphatics in CSF absorption, it was difficult to envision how protein recoveries translated into volumetric data. Additionally, a crucial element in designing an approach to quantify the lymphatic contribution to CSF absorption is the ability to correct the recovery data for errors introduced by filtration of the CSF tracer. In other words, presuming that arachnoid villi and granulations transport CSF into the plasma, the CSF in the plasma will eventually filter into the lymphatic compartment. Without correction, the cannulated lymphatic vessels might receive CSF tracer not only from the CSF compartment directly but also from re-circulated plasma tracer. This would result in an overestimation of the lymphatic contribution to CSF drainage. Similarly, the non-lymphatic contribution to CSF clearance would be underestimated if the loss of CSF tracer due to the normal filtration of proteins from the vasculature were not taken into consideration. Indeed, one study showed that the loss of tracer from sheep plasma was over 5%/h .To correct the tracer recovery data for filtration errors and to permit the estimation of volumetric data from protein tracer approaches, a three-compartment mathematical model was developed and applied to sheep data. The data suggested that 40–48% of all CSF removed from the cranial compartment in adult sheep was cleared by lymphatics . AdditioWhile protein tracer studies have played an important role in focusing attention on lymphatic CSF absorption, perhaps the most striking data have been obtained from studies in which the cribriform plate has been sealed in sheep. In this procedure the nasal mucosa, olfactory nerves and all soft tissue on the extracranial surface of the cribriform plate were scraped away with a curette and the bone surface sealed with either bone wax or tissue glue. Sheep were challenged with constant flow or constant pressure infusions of artificial CSF into the CSF compartment before and after the extracranial side of the cribriform plate was sealed. The rate of CSF absorption was reduced significantly by this blockage and remarkably, the data suggested that the majority (> 80%) of cranial CSF absorption occurred through the cribriform plate at opening CSF pressures in adult  and in n2O [McComb and colleagues noted that an increase in intracranial pressure (ICP) in rabbits and cats resulted in greater levels of a CSF radioactive tracer in the optic nerve, olfactory bulbs, episcleral tissue, and deep cervical lymph nodes ,62. Hasu2O . A tempo2O . Protein2O). At baseline CSF pressures, about 10% of the lymph in sheep cervical lymphatic vessels had its origins as CSF. As ICP was elevated, the proportion increased. At 70 cm H2O ICP, cervical lymph flow rates were 4 fold higher compared to baseline conditions and nearly 80% of the lymph in these ducts was estimated to originate in the CSF compartment [In sheep, cervical lymphatic pressures and flow rates were closely related to ICP . Silver partment .Edema of the brain, elevation of ICP, EEG anomalies and behavioural alterations have been demonstrated after chronic ligation of the cervical lymphatic vessels of dogs ,66. SimiIn recent studies, baseline ICP was elevated after the cribriform plate was obstructed on the nasal side . Mean, dFollowing bolus infusions of saline into the CSF compartment of adult sheep, obstruction of CSF absorption through the cribriform plate increased the peak ICP after infusion and augmented the time required for ICP to return to baseline . MoreoveVery little information is available on this subject but there are some interesting observations that may impact on this issue. Surgical procedures in humans that ablate the olfactory nerves do not seem to be associated regularly with any discernible problems with CSF circulation. It is plausible that CSF might be diverted to the spinal subarachnoid space  to compensate for the obstruction to absorption at the cribriform plate. Nonetheless, it is noteworthy that a study showed that 8% of patients developed hydrocephalus in the immediate postoperative period of cranial base surgery, with half of these patients also exhibiting CSF leaks .Foxc1 mouse mutant demonstrates hydrocephalus and other defects [Foxc2 gene are associated with abnormal development of lymphatic vessels [Lack of development of the olfactory bulbs in humans  and mice defects . The ske defects . Additio defects . While n vessels .The time taken for India ink to move from the CSF into the cervical lymph nodes was increased relative to controls in a model of TGFβ1 induced hydrocephalus in the mouse . This suA lymphatic-CSF relationship would also seem to have immunological implications. For example, a humoral immune response in mice was generated mainly by the deep cervical lymph nodes after injection of sheep red blood cells into various intracerebral sites . In ratsAfter the induction of experimental autoimmune encephalomyelitis in rats, a severe immune response was generated, resulting in cerebral lesions . RemovalThe tenets that form the basis of our understanding of CSF absorption do not appear to have received critical appraisal in recent years. The arachnoid projections into the cranial venous sinuses are believed to represent the primary sites for CSF absorption and current views on the pathophysiology of the CSF system have often focused on impaired CSF clearance through these elements . HoweverThe author(s) declare that they have no competing interests.LK had the primary responsibility of writing and organizing the review. AZ and MJ contributed ideas and helped in the preparation of the manuscript. All authors read and approved the final manuscript.


G-CSF is known to function as a hematopoietic growth factor and it is known to be responsible for leukocytosis. G-CSF-producing tumors associated with leukocytosis include various types of malignancies.We report the case of a 72-year-old man with dedifferentiated chondrosarcoma characterized by dedifferentiated components of malignant fibrous histiocytoma- or osteosarcoma-like features in addition to conventional chondrosarcoma, arising from his pelvic bone. After hemipelvectomy, when local recurrence and metastasis were identified, leukocytosis appeared and an elevated level of serum granulocyte-colony-stimulating factor (G-CSF) was also recognized. The patient died of multiple organ failure 2 months after surgery. Autopsy specimens showed that the histological specimens of the recurrence and metastasis were dedifferentiated components, without any conventional chondrosarcoma components. G-CSF was expressed only in the dedifferentiated components, not in the chondrosarcoma components, immunohistochemically.This is the first report of chondrosarcoma, or any other primary bone tumor, with leukocytosis, probably stimulated by tumor-produced G-CSF from the dedifferentiated components. Granulocyte-colony stimulating factor (G-CSF) enhances differentiation along the neutrophil lineage, and accelerates maturation of metamyelocytes into mature neutrophils. G-CSF also prolongs the survival of neutrophils and their precursors, including stem cells. ConsequDedifferentiated chondrosarcoma accounts for approximately 10% of all chondrosarcomas . DediffeA 72-year-old man, who had been suffering from right hip pain for 7 years when walking, was referred to our institute. Roentogenography of the right pelvic bone showed an expansion of the cortical contour and a soft-tissue mass with punctate calcification figure . MagnetiPreoperative laboratory data were not remarkable. After surgery, the laboratory data also showed leukocytosis predominantly in the neutrophils with an elevated level of C-reactive protein figure . The serIn the current report, we present a case of dedifferentiated chondrosarcoma with leukocytosis and an increased serum G-CSF level. In a previous report, serum G-CSF was not found to be increased in bone tumors, including osteosarcoma, chondrosarcoma, Ewing/PNET and giant cell tumor, compared to the control . TherefoIn the current case, leukocytosis appeared after the surgery, when recurrence and metastasis also appeared. The histology of the recurrent and metastatic lesions was not conventional chondrosarcoma but only dedifferentiated components. Therefore, it would seem that the G-CSF production could have been associated with the dedifferentiated components. Supporting this idea, G-CSF expression was found only in the dedifferentiated components, and not in the conventional chondrosarcoma components, immunohistochemically. As described, the histology of G-CSF-producing soft-tissue tumors was MFH and liposarcoma ,15-18. TThe current case of dedifferentiated chondrosarcoma had a rather long history of 7 years without leukocytosis prior to the initial surgery. The resected specimens had conventional chondrosarcoma components in most part, and dedifferentiated components only in a small part. It is not clear why there was no leukocytosis before the surgery, even though there were dedifferentiated components. One theory is that the volume of dedifferentiated components with G-CSF production was too small to cause leukocytosis. Another theory is that there was no production at the beginning, and dedifferentiated components acquired the function of G-CSF production during the course of tumor development. When leukocytosis appeared, the tumor had already recurred and metastasis had developed, and the patient died 2 months after the surgery. G-CSF has been shown to induce tumor cell proliferation -21. In ap53, Rb and FHIT are reported to be responsible for the "switch" to a high-grade anaplastic sarcoma (dedifferentiated components) from monoclonal origin of demonstrated dedifferentiated chondrosaroma [It is uncertain whether dedifferentiated chondrosarcoma consists primarily of two cell types or whether dedifferentiation develops secondarily. Molecular genetic study of one case has shown a substantial number of genetic alterations, as well as severe aneuploidy and loss of heterozygosity in both the components of dedifferentiated chondrosarcoma . LOH  declare that they have no competing interests.AS drafted the manuscript and carried out the immunohistochemical studies. HY and YO carried out the immunohistochemical studies. KT, SM and KH participated in the design of the study and performed. MT and YI conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.


Granulocyte-colony stimulating factor (G-CSF) is known as a powerful regulator of white blood cell proliferation and differentiation in mammals. We, and others, have shown that G-CSF is effective in treating cerebral ischemia in rodents, both relating to infarct size as well as functional recovery. G-CSF and its receptor are expressed by neurons, and G-CSF regulates apoptosis and neurogenesis, providing a rational basis for its beneficial short- and long-term actions in ischemia. In addition, G-CSF may contribute to re-endothelialisation and arteriogenesis in the vasculature of the ischemic penumbra. In addition to these trophic effects, G-CSF is a potent neuroprotective factor reliably reducing infarct size in different stroke models.Here, we have further delayed treatment and studied effects of G-CSF on infarct volume in the middle cerebral artery occlusion (MCAO) model and functional outcome in the cortical photothrombotic model. In the MCAO model, we applied a single dose of 60 μg/kg bodyweight G-CSF in rats 4 h after onset of ischemia. Infarct volume was determined 24 h after onset of ischemia. In the rat photothrombotic model, we applied 10 μg/kg bodyweight G-CSF daily for a period of 10 days starting either 24 or 72 h after induction of ischemia. G-CSF both decreased acute infarct volume in the MCAO model, and improved recovery in the photothrombotic model at delayed timepoints.These data further strengthen G-CSF's profile as a unique candidate stroke drug, and provide an experimental basis for application of G-CSF in the post-stroke recovery phase. Granulocyte-colony stimulating factor (G-CSF) is a cytokine that stimulates generation of neutrophilic granulocytes, and has been in clinical use for more than 10 years in indications related to counteracting neutropenia, or for bone-marrow transplantations . RecentlCharacterised by this multimodal array, G-CSF appears beneficial for short-term damage parameters such as infarct size, and long-term effects on functional outcome . Based Here we further explore the window of opportunity for G-CSF treatment in firstly, the most accepted model for evaluating neuroprotective effects, rat middle cerebral artery occlusion (MCAO), and secondly, the cortical photothrombotic stroke model for detecting effects on long-term functional outcome.We recently reported an infarct-reducing capacity of G-CSF after middle cerebral artery occlusion (MCAO) when treatment was initiated at 30 min and 2 h after onset of ischemia ,6. Here 3 (n = 24) vs. 223.3 ± 27.3 mm3 (n = 18), p < 0.05; Figure 3 vs. 144.3 ± 22.7 mm3; p < 0.05) as well as the subcortical  part of the infarction.We observed a robust infarct volume reduction by 33 % in the total infarct volume as compared to vehicle-treated rats  did not differ between the groups. In addition, there were no differences in mortality or body weight between all groups (not shown).Sensorimotor deficits in the rotarod test were obvious in vehicle-treated ischemic animals compared to sham-operated rats in the rotarod for up to 6 weeks after the insult Figure . G-CSF tHere we demonstrate a robust infarct-reducing effect of a 4 h treatment delay with G-CSF in a severe hemispheric stroke model (MCAO), viewed as the gold standard for testing neuroprotective effects. The end point for determining infarct volume was 24 h following onset of ischemia, a standard timepoint for neuroprotective studies. We know, however, that the effect of G-CSF is not transient as has been discussed with some experimental drugs, but is also detectable when examining the brain at 72 h post ischemia after a combined CCA/distal MCA occlusion . The achSupport for a delayed window of opportunity for G-CSF in acute stroke models comes from three other independent studies. Treatment effects were investigated in comparatively mild ischemia models producing small hemispheric or cortical infarctions appropriate to assess recovery effects rather than to test the neuroprotective potential of a drug in the acute phase. Six et al. have shown a stunning 55% infarct reducing effect when treatment was delayed for 24 h after transient MCAO in the mouse with a relatively small sample size of n = 6 per group . Shyu et3 to 72.5 mm3 in the vehicle treated group, Figure By comparing the differences of total infarct volumes without and with edema correction, our data indicate an absolute reduction in edema volume in favor for G-CSF  our datIn addition to a rational mode-of-action within stroke pathophysiology, any candidate for further stroke drug development should be explored according to the STAIR criteria . G-CSF fThe effect on functional outcome appears as a separable activity of G-CSF, independent of its neuroprotective potential. This makes G-CSF a highly attractive candidate for recovery enhancement in the subacute or chronic phase of a stroke and increases the likelihood for detecting clinical outcome improvement in acute stroke trials.Aiming at the two principal modes-of-action of G-CSF in the brain, antiapoptosis and regeneration enhancement, we have both studied effects on infarct size with delayed treatment in a severe MCAO model, as well as functional sensorimotor outcome in the cortical photothrombotic model. We find that G-CSF has an acute time window of at least 4 h post-stroke, and an astonishing time window for effects on functional outcome of three days after stroke. The strong effects on functional outcome distuinguish G-CSF from most other candidate stroke drugs in development.Our findings further strengthen confidence in the efficacy of G-CSF both for infarct size reduction, and improvement of functional parameters, and are important guiding lines for future clinical development.2O, 30% O2, and 1% halothane. The right femoral vein was cannulated and used for drug delivery. During the experiment, core body temperature was monitored and maintained at 37°C by a thermostatically controlled heating pad . Middle cerebral artery occlusion (MCAO) was induced with a silicon-coated  4-0 nylon filament (Ethicon) that was introduced into the common carotid artery and advanced into the internal carotid artery as described previously [Male Wistar rats weighing 280 to 320 g received inhalation anesthesia with 70%Neviously . SuccessInfarct volumes were determined 24 h after induction of ischemia by TTC staining as described previously . 2 mm seMale Wistar rats were anesthetised with an intramuscular injection of 100 mg/kg bodyweight ketaminehydrochloride (Ketanest) and 8 mg/kg bodyweight xylazine hydrochloride (Rompun). Anesthesia was maintained with 50 mg/kg bodyweight ketaminehydrochloride and 4 mg/kg bodyweight xylazine hydrochlorideif necessary. A PE-50 polyethylene tube was inserted into the right femoral artery for continuous monitoring of mean arterial blood pressure, and blood gases. The right femoral vein was cannulated by a PE-50 tube for treatment infusion. During the experiment rectal temperature was monitored and maintained at 37°C by a thermostatically controlled heating pad . Photothrombotic ischemia was induced in the rat parietal cortex according to the method of Watson et al. . AnimalsIn all animals Rotarod testing was performed during the light cycle before ischemia after a training period of 3 days, and at 1,2,3,4,5 and 6 weeks after ischemia by an investigator blinded to the experimental groups. Rats were placed on an accelerating Rotarod cylinder (increased speed from 4 to 40 rpm within 5 min). The time the animals remained on the Rotarod was measured, and the trial ended if the animal fell off the rungs or gripped the device and spun around for two consecutive revolutions without attempting to walk on the rungs. An arbitrary time limit of 500 sec was set for the rats on the Rotarod cylinder in training and in the testing procedures. The animals were trained 3 days before ischemia, and the mean duration (sec) on the device was recorded with three measurements.All animal experiments were performed in accordance with national and international regulations, and were approved by the appropriate government authorities. All experiments were performed in a fully randomised and blinded fashion.The values presented in this study are means ± SEM. The two-tailed t-test was used to determine significant difference of infarct volumes. Behavioural measurements were analysed using ANOVA and the Student-Newman-Keuls post hoc test. Area under the curve (AUC) was calculated using the trapezoidal algorithm. Linear regressions were compared using coincidence testing. Statistics were performed with NCSS 2004  or Primer of Biostatistics software. A p value <0.05 was considered statistically significant.RW, CP, CK, RL carried out the animal studies, and participated in the data analysis. AS and WRS carried out data analyses. AS, WRS, SS, AB conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.


Thermus thermophilus and Deinococcus radiodurans belong to a distinct bacterial clade but have remarkably different phenotypes. T. thermophilus is a thermophile, which is relatively sensitive to ionizing radiation and desiccation, whereas D. radiodurans is a mesophile, which is highly radiation- and desiccation-resistant. Here we present an in-depth comparison of the genomes of these two related but differently adapted bacteria.Thermus and Deinococcus after the divergence from their common ancestor, we demonstrate a high level of post-divergence gene flux in both lineages. Various aspects of the adaptation to high temperature in Thermus can be attributed to horizontal gene transfer from archaea and thermophilic bacteria; many of the horizontally transferred genes are located on the single megaplasmid of Thermus. In addition, the Thermus lineage has lost a set of genes that are still present in Deinococcus and many other mesophilic bacteria but are not common among thermophiles. By contrast, Deinococcus seems to have acquired numerous genes related to stress response systems from various bacteria. A comparison of the distribution of orthologous genes among the four partitions of the Deinococcus genome and the two partitions of the Thermus genome reveals homology between the Thermus megaplasmid (pTT27) and Deinococcus megaplasmid (DR177).By reconstructing the evolution of Thermus and Deinococcus lineages have taken divergent paths toward their distinct lifestyles. In addition to extensive gene loss, Thermus seems to have acquired numerous genes from thermophiles, which likely was the decisive contribution to its thermophilic adaptation. By contrast, Deinococcus lost few genes but seems to have acquired many bacterial genes that apparently enhanced its ability to survive different kinds of environmental stresses. Notwithstanding the accumulation of horizontally transferred genes, we also show that the single megaplasmid of Thermus and the DR177 megaplasmid of Deinococcus are homologous and probably were inherited from the common ancestor of these bacteria.After the radiation from their common ancestor, the  Deinococcus spp. and Thermus spp. are believed to belong to a distinct branch of bacteria called the Deinococcus-Thermus group [Thermus genus currently consists of 14 thermophilic species, whereas the Deinococcus genus includes at least eleven, mostly mesophilic species known for their extreme resistance to γ-irradiation and other agents causing DNA damage, particularly, desiccation [D. frigens sp. nov., D. saxicola, have been isolated recently from Antarctic rock and soil samples [D. geothermalis and D. murrayi, are considered to be thermophilic (topt~45–50°C) [us group . The comus group -7. The Ticcation . Interes samples . D. geot45–50°C) .T. thermophilus (TT) and D. radiodurans (DR)  have similar general physiology, both being catalase-positive, red-pigmented, non-sporulating, aerobic chemoorganoheterotrophs [T. thermophilus HB27 and HB8 [D. raduodurans R1 [rotrophs . However and HB8 ,13 becamurans R1 .Despite extensive research, genetic systems underlying both thermophilic adaptations and radiation resistance remain poorly understood. Attempts have been made to detect "thermophilic determinants" in the proteomes of thermophiles using a comparative-genomic approach. This resulted in the delineation of a set of proteins that might be associated with the thermophilic phenotype, although most of these are significantly enriched in thermophiles but not unique to these organisms -17. OtheD. radiodurans might prevent production of reactive oxygen species (ROS) by decreasing the number of reactions involving oxygen [Deinococcus spp. might help scavenge ROS generated during irradiation and post-irradiation recovery [Various hypotheses also have been proposed to explain radiation resistance, some postulating the existence of specialized genetic systems, particularly those for DNA repair and stress response ,26. Receg oxygen ,28, and recovery . Howeverrecovery ,27,28.Several evolutionary processes could potentially contribute to the genome differentiation of TT and DR subsequent to the divergence from the common ancestor: (i) differential gene loss and gain, (ii) acquisition of genes via horizontal gene transfer (HGT) which may be followed by loss of the ancestral orthologous gene (xenologous gene displacement (XGD)), (iii) lineage-specific expansion of paralogous gene families by duplication and/or acquisition of paralogs via HGT; (IV) modification of amino acid composition that could affect protein stability. Here, we experimentally characterize radiation and desiccation resistance of TT in comparison to DR, and, assess the contribution of different evolutionary processes to distinct adaptations of TT and DR, using a variety of comparative-genomic approaches and phylogenetic analysis. We identify the unique feature of the gene repertoires of TT and DR that might contribute to these phenotypic differences, which could be the subject of further experimental work. In addition, we describe the results of a detailed analysis of the proteins predicted to be involved in DNA repair and stress response functions, which are particularly relevant for adaptive evolution of resistance phenotypes.Esherichia coli K12. For DR, the radiation dose yielding 10% colony forming unit (CFU) survival (D10) is ~16 kGy, whereas for TT and E. coli, the D10 dose is ~0.8 kGy and ~0.7 kGy, respectively  accumulation, significantly lowered the cells' IR resistance [2•-), with Mn cycling between the divalent and trivalent states; Mn redox-cycling scavenges both O2•- and hydrogen peroxide [10, ~0.8 kGy) is 0.047 compared to 0.0072 for E. coli , 0.24 for D. radiodurans , and <0.0001 for Pseudomonas putida  [2•- by Mn(II) is highly dependent on the availability of H2O2, which in TT would be expected to become limiting during recovery at 65°C because of thermal decomposition [We recently reported a trend of intracellular Mn/Fe concentration ratios in bacterial IR resistance, where very high and very low Mn/Fe ratios correlated with very high and very low resistances, respectively . We havesistance . These osistance ,29. At hperoxide . In thisposition ; inefficInformation on the presence-absence of orthologous genes in a set of genomes can be used to produce a gene-content tree ,34. The Aquifex aeolicus (AA) and Thermotoga maritima (TM). We found that genes that are present in TT but not DR are significantly more likely to be present in AA and TM than genes present in DR but not TT .As noticed previously, some DR genes that belong to families well-represented in both bacteria and archaea showed clear archaeal affinity . To asseThermus lineage by the flavin-dependent thymidylate synthase, typical of archaea and bacterial thermophiles. In other cases, the substituted analogous enzymes or pathways remain uncharacterized. For instance, the displacement of the folate-dependent thymidylate synthase with the flavin-dependent type in the TT lineage and in other bacteria and archaea correlates with the apparent loss of dihydrofolate reductase , which catalyzes the last step of the tetrahydrofolate biosynthesis pathway. Since tetrahydrofolate is an essential cofactor, a displacement appears most likely. Recently, it has been shown that the halobacterial FolP-FolC fusion protein complemented a Haloferax volcanii folA mutant [Our reconstruction of the evolutionary events that occurred after the divergence of the TT and DR lineages from the common ancestor delineated the sets of genes that likely have been gained and lost by each lineage Figure . We firsA mutant . Thus, ipdxK was likely lost in the TT lineage, whereas DR probably independently acquired pdxH. However, the pathway is likely to be functional in both organisms. Since similar gaps in the pyridoxal phosphate biosynthesis are seen in a variety of prokaryotes [In addition, TT does not encode two enzymes for pyridoxal phosphate biosynthesis , while DR has a complete set of enzymes of this pathway. Our reconstructions suggest that karyotes , it appeSome systems apparently were completely lost in the TT lineage. These include the urease complex, the ramnose metabolism pathway, acyl CoA:acetate/3-ketoacid CoA transferase, fructose transport and utilization, and glycerol metabolism. Notably, most of these systems are also absent in thermophilic bacteria and many thermophilic archaea.In contrast to DR, the genes that appear to have been acquired by TT show a clear connection to the thermophilic lifestyle. In particular, TT seems to have acquired 23 gene families from the set of putative thermophilic determinants , whereasThe Sox-like sulfur oxidation system is among the group of genes that were apparently acquired in the TT lineage. The TT Sox operon is partly similar to the one identified in AA , K+-transporting ATPase (3 genes), and the FeoA/FeoB Fe transport system. This abundance of ion transport systems might be indirectly linked to oxidative stress resistance through regulation of membrane ion gradients and Mn/Fe homeostasis (see above).DR is more dependent than TT on peptide-derived growth substrates  and has Among the genes apparently acquired by DR, two encode multidomain proteins containing distinct periplasmic ligand-binding sensor domains . Another protein, DRA0204, contains the CHASE3 domain  and is lAdditionally, DR has many genes (25 COGs) encoding systems for microbial defense; TT has only 14 COGs in this category, 13 of which are shared with DR. At least 7 genes for restriction-modification system subunits were specifically acquired by the DR lineage, along with several antibiotic-resistance enzymes. This difference might be linked to the reduced metabolic capabilities of DR, which is dependent on nutrient-rich conditions for growth and, perhaps, encounters more microbial species than TT.The previous analysis of the DR genome revealed 15 genes that appear to have been horizontally transferred from unexpected sources, such as eukaryotes and viruses ; only twIt is well-established that HGT has made major contributions to the gene repertoires of most thermophilic bacteria as supported by the presence of numerous genes with unexpectedly high similarity to and/or phylogenetic affinity with genes typical of hyperthermophilic archaea -60. ThesThermoanaerobacter tencongiensis (data not shown).As expected, compared to DR, TT has a notable excess in the fraction of best hits to thermophilic bacteria and archaea for both core and non-core proteins Figure . HoweverAmino acid composition bias is known to affect not only sequence similarity searches but phylogeny reconstruction as well . We testin situ, without disruption of the operon organization [Among the cases of potential XGD supported by phylogenetic analysis, there are two ribosomal proteins, L30 and L15, which are encoded by adjacent genes within a conserved ribosomal operon. The proteins encoded by surrounding genes in these operons showed clear affinity to the corresponding DR orthologs (data not shown). In phylogenetic trees of L30 and L15, the TT proteins reliably clustered with orthologs from bacterial hyperthermophiles and not with the corresponding DR orthologs Figure  and 6B. nization . AdditioThe apparent lineage-specific HGT in DR and TT was not limited to XGD or to acquisition of genes from thermophiles. Additional examples of various types of HGT supported by phylogenetic analysis are given in Table Most bacterial lineages contain unique sets of expanded paralogous gene families . This noSeveral paralogous families are specifically expanded in TT Table . The larNotably, several protein families that are expanded in TT but are absent in DR belong to the set of potential thermophilic determinants   or are ebona fide components of repair systems [Comparative analysis of the well-characterized genetic systems for replication, repair and recombination, and related functions in TT and DR shows that fractions of these genes in the respective genomes are very similar  repair enzymes of TT, the most conspicuous ones are the components of the putative thermophile-specific repair system, which are predominantly encoded on the TT megaplasmid Figure . The funComparison of proteins comprising various (predicted) systems involved in stress response reveals a greater number and diversity of such proteins in DR, which has 26 COGs with relevant functions that are not represented in TT compared to 3 such COGs in TT   and one e stress ,83. Conse stress ,85, wherOxidative stress defense mechanisms also might include control of Mn and Fe partitioning in the cell . ProteinCraterostigma plantagineum [Since desiccation also causes oxidative stress, proteins involved in desiccation resistance belong to the general cellular defense category . Desiccatagineum ; DR alsotagineum .i.e., probably evolved from a distinct genome partition of the common DR-TT ancestor. Apparently, however, the genomes of the megaplasmids have undergone extensive rearrangements since the divergence from the common ancestor because no conservation of gene order could be identified (data not shown).Both TT and DR have multipartite genomes. To examine possible evolutionary relationships between the genome partitions of TT and DR, we analyzed the distribution of symmetrical best hits (putative orthologs) in the single extra-chromosomal element of TT, the pTT27 megaplasmid, and the three smaller genome partitions of DR . Of these, 11 belong to the putative mobile thermophile-specific DNA repair system ,45 Figu. AdditioRecently, the genome of another strain of TT (HB8) has been completely sequenced and became available in public databases . A preliDeinococcus geothermalis (DG) has recently become publicly available [Furthermore, a nearly complete draft genome sequence of vailable . Since Dvailable  and see These observations give rise to two hypotheses: (i) the TT megaplasmid is essential for the survival of the organism at high temperatures. Consistent with this idea, we detected an expansion of a two-component toxin-antitoxin system, which consists of a PIN-like nuclease (toxin) and a MazE family transcriptional regulator (antitoxin) . Such a Specific roles of plasmid-borne genes in recovery from DNA damage have been proposed previously, including class Ib ribonucleotide reductase, periplasmic alkaline phosphatase, and extracellular nuclease, and subsequent analyses revealed at least five other genes implicated in this process  ,76. TherTT and DR share a large core of genes and form a clade in the gene-content tree, which supports the idea that these bacteria form a distinct clade, as indicated previously by phylogenetic analysis of rRNA and various proteins, and that the evolution of their gene complements was, roughly, clock-like. However, major differences between the gene repertoires of TT and DR were observed, indicating that both genomes lost numerous ancestral genes and acquired distinct sets of new genes primarily via HGT. In addition, numerous lineage-specific expansions of paralogous gene families were identified, particularly, in DR.Some of the differences in the gene repertoires of TT and DR can be linked to the distinctive adaptive strategies of these bacteria. For example, TT appears to have acquired many genes from (hyper)thermophilic bacteria and archaea, whereas DR apparently acquired various genes involved in oxidative stress response and other "house-cleaning" functions from diverse bacterial sources.The gene content of the TT megaplasmid (pTT27) and the DR megaplasmid (DR177) are sufficiently similar to conclude that they evolved from a common ancestor. To our knowledge, this is the first evidence of persistence of a megaplasmid beyond the genus level. However, the TT megaplasmid also carries many genes whose functions are implicated in the thermophylic phenotype; in particular, components of the predicted thermophile-specific repair system. These megaplasmids are likely to be essential for the survival of both TT and DR, with their maintenance controlled by toxin-antitoxin systems. Furthermore, the substantial differences between the gene repertoires of the megaplasmids of TT strains HB27 and HB8 indicate that this genome partition has been highly dynamic, with high rates of gene loss and HGT events occurring during evolution.Thermus-Deinococcus clade should allow more definitive comparative-genomic analysis to elucidate the nature of the common ancestor of these bacteria.The evolutionary reconstruction based on the parsimony principle is generally compatible with the idea that the common ancestor of TT and DR was a mesophilic bacterium, whereas the thermophylic phenotype of TT evolved gradually via HGT of genes from thermophiles. Conversely, the radiation-desiccation resistance phenotype of DR might have gradually evolved via HGT of genes from other mesophiles, particularly, with highly developed oxidative stress response systems. However, it should be noted that the TT-DR common gene core includes dozens of genes of apparent archaeal origin or, at least, genes with thermophilic affiliation. Moreover, the DG genome encodes a few additional "thermophilic determinants" that are missing in DR but are unlikely to have been transferred from a thermophilic source independently of TT, as shown by comparative-genomic and phylogenetic analyses described here. Thus, acquisition of a considerable number of archaeal genes might have occurred along the evolutionary branch leading to the common ancestor of TT and DR. Accordingly, at this stage, we cannot rule out the possibility that this ancestor was a moderate thermophile rather than a mesophile. Further sequencing of bacterial genomes of the 600 ~0.9, which corresponds to 107 – 108 colony forming units (CFU)/ml; 1 TT cell/CFU. TT cells grown in TGY were examined for their total Mn and Fe content by ICP-MS (see main text). For radiation resistance assays, cells were irradiated without change of broth on ice with 60Co at 6.8 kGy/hour . At the indicated doses, cultures  were appropriately diluted and plated on solid medium , and CFU counts were determined after 2 days' incubation at 65°C.Irradiations. Three TT colony-isolates were inoculated individually in liquid TGY  and incubated at 70°C. Cells were harvested at OD6–107 cells (25 μl) were transferred to microtiter plates, which were transferred to desiccation chambers containing anhydrous calcium sulfate (drierite) and incubated at room temperature or 65°C. At the indicated times, cells were re-suspended in TGY, and CFU-survival frequencies were determined by dilution-plating on solid medium (65°C).Desiccation. Five separate colony-isolates were pre-grown in TGY as for irradiation trials. Cell samples 10-5. Taxonomic affiliation was determined by best hits in non-redundant database of protein sequences at the National Center of Biotechnology Information  using BLASTP program [The sets of predicted proteins of TT and DR were searched against each other for symmetrical and non-symmetrical hits using PSI-BLAST with expectation (E) value threshold of 10 program  with def program  and CDD- program . Contrad program ,97. The Multiple alignments for phylogenetic analysis were constructed using the MUSCLE program ; columnsThermus and Deinococcus response to irradiation and desiccation and the analysis of Mn/Fe ratio of Thermus. MJD, EVK, and KSM participated in design of experiments and project coordination, and contributed to the writing of the manuscript. All authors read and approved the final manuscript.MVO performed the majority of genomic comparisons, phylogenetic analysis and helped to draft the manuscript. YIW and KSM performed some genomic comparisons and statistical analysis of the data. EKG, VYM, AV and MZ performed the experiments on the comparison of the tables 1S, 2S, 3S, 4S and supplementary figures 1S, 2S, 3S).contains compressed file for all the trees (in PHYLIP format) for 122 proteins from the reconstructed gene set of the DR-TT common ancestor that showed affinity to thermophiles  for proteins from DR-TT core that had best hits to thermophiles.Click here for file


To determine if this increased frequency was due to the nucleotide site having the properties of a mutation hot spot, or some other explanation, we developed a new experimental approach. We examined the spatial distribution of the frequency of the C755G mutation in the germline by dividing four testes from two normal individuals each into several hundred pieces, and, using a highly sensitive PCR assay, we measured the mutation frequency of each piece. We discovered that each testis was characterized by rare foci with mutation frequencies 103 to >104 times higher than the rest of the testis regions. Using a model based on what is known about human germline development forced us to reject (p < 10−6) the idea that the C755G mutation arises more frequently because this nucleotide simply has a higher than average mutation rate (hot spot model). This is true regardless of whether mutation is dependent or independent of cell division. An alternate model was examined where positive selection acts on adult self-renewing Ap spermatogonial cells (SrAp) carrying this mutation such that, instead of only replacing themselves, they occasionally produce two SrAp cells. This model could not be rejected given our observed data. Unlike the disease site, similar analysis of C-to-G mutations at a control nucleotide site in one testis pair failed to find any foci with high mutation frequencies. The rejection of the hot spot model and lack of rejection of a selection model for the C755G mutation, along with other data, provides strong support for the proposal that positive selection in the testis can act to increase the frequency of premeiotic germ cells carrying a mutation deleterious to an offspring, thereby unfavorably altering the mutational load in humans. Studying the anatomical distribution of germline mutations can provide new insights into genetic disease and evolutionary change.The frequency of the most common sporadic Apert syndrome mutation (C755G) in the human fibroblast growth factor receptor 2 gene  Some human disease mutations occur 100–1,000 times more frequently than would be predicted from genome wide studies of mutation in different species. In Apert syndrome, for example, two-thirds of all new causal mutations occur at only one base in the affected gene. This unusually high frequency suggests that something about that DNA base or its local surroundings makes it highly susceptible to mutation. We studied this hypothesis by examining the location of cells containing this mutation in the testes of normal men. We found that mutant cells were not uniformly distributed throughout the testes, as would be expected for random mutations. Instead, we found 95% of the mutants in small clusters containing only a few percent of the total testes cells. A higher-than-average mutation rate could not explain the data. We propose that these mutations arise at the expected rate, but that mutated cells gain a selective advantage that allows them to increase their frequency compared to nonmutant cells. Our results—which argue against the mutational hot spot model in favor of a selection model—suggest how germline selection in animals can alter the mutational load of a species. The frequency of Apert syndrome mutations is 100-1,000 times higher than expected from average mutation rates, and it is due to positive selection in the testis increasing the frequency of germ cells carrying the defect. Each reaction contained 20 mM HEPES (pH 7.5), 30 mM KCl, 50 μM Na4PPi, 2 mM MgCl2, 80 μM of each dNTP, 80–200 nM of each primer, 2 μM Rox, 0.2 X Syber Green I, 0.04 unit/μl TMA31FS DNA polymerase (Roche Molecular Systems), and 25,000 copies of testis or epididymal sperm DNA molecules. Research samples of TMA 31FS DNA polymerase may be obtained from Thomas W. Myers, Director, Program in Core Research, Roche Molecular Systems, 1145 Atlantic Avenue, Alameda, CA 95401, USA (thomas.myers@roche.com). The sequences (see accession numbers) of the C755G-specific PAP primers used in these experiments are: 5'-CCCCACTCCTCCTTTCTTCCCTCTCTCCACCAGAGCGAT(ddG) and 5'-TTTGCCGGCAGTCCGGCTTGGAGGATGGGCCGGTGAGGC(ddC).. The 79-bp PAP product makes detection possible using quantitative PCR . The primers were synthesized by Biosource International (http://www.biosource.com) or Biosearch Technologies (http://www.biosearchtech.com). The PAP primer ending in a ddC was synthesized by conventional methods. The PAP primer ending with ddG required synthesis using 5'-CE phosphoramidites. The ddNTP at the 3' end of each primer is complementary to nucleotide 755 but each to different strands.The assay for C755G mutations resembles an allele-specific PCR (ASPCR) that amplifies mutant but not wild-type molecules using a primer whose 3′ end contains a base that is complementary to the mutant –51. The The PAP cycling conditions included an initial denaturation step of 2 min at 94°C followed by 150 cycles of 6 s at 94 °C and 40 s at 78 °C. Positive reactions could easily be distinguished from negative ones by considering the kinetics of the increase in fluorescence with cycle number and the melting profile of the final PCR product. For sample PAP data, see 4PPi, 2 mM MgCl2, 80 μM of each dNTP, 200 nM of each primer, 2 μM Rox, 0.2 X Syber Green I, 0.04 unit/μl TMA31FS DNA polymerase (Roche Molecular Systems), and 25,000 copies of testis or epididymal sperm DNA molecules/reaction. The sequences of the C755G-specific PAP primers are: 5′-TATTAATATAACTTAGTATCTGTCACCCCAAGGGAACCAA(ddG) and 5′-TATTAATATAGAGTATTGACTCTTATTCTTGGGCTTCGAC(ddC). Note that the ten 5'-most nucleotides of these primers are not complementary to the mutated target sequence. An 81-bp PAP product results. The PAP cycling conditions included an initial denaturation step of 1 min at 94 °C followed by 134 cycles of 6 s at 94 °C and 40 s at 70 °C.For the control, we measured the frequency of a C-to-G transversion at a CpG site on human Chromosome 7. The C nucleotide is known to be polymorphic in the human population (see accession numbers). Testis donor 374 was genotyped and found to be a C/C homozygote and thus his testis and sperm cells could be examined for the presence of C-to-G transversion mutations at this site. To measure the frequency of mutation, 10 μl PAP reactions were carried out in 384-well plates . Each reaction contained 20 mM HEPES (pH 7.5), 30 mM KCl, 50 μM Na6 genomes) were used to estimate the C755G mutation frequency for every piece of testes 374–1 and −2. In those cases where fewer than 25/40 reactions were positive, we took the number (after Poisson correction) as an estimate of the C755G mutation frequency for that piece. If 25 or more reactions were positive, then the experiment was repeated using 10-fold dilutions until a dilution of the DNA from the piece was found to give fewer than 25/40 positives.A total of 40 reactions containing 25,000 genomes each  parts of 854–1 . First, http://www.clontech.com). Over the course of our study, we observed an assay background in human blood DNA of between 6.6 × 10−8 and 2.4 × 10−7, based on experiments using ∼67,000,000 control genomes. This places an upper limit on the possible mutation frequency in human white blood cells, although it would be lower if false positives can also result from artifacts of the assay itself.For all mutation counting, the estimate of the total testis mutation frequency was the average of the frequencies of the pieces weighted by the number of genomes in those pieces. For a positive assay control, ten or 20 Apert C755G mutant molecules from a patient heterozygous for the C755G mutation  were added to 500,000 human genomes and divided into 20 reactions (an average of 0.5 or 1 mutants per reaction). Negative assay controls consisted of 25,000 genomes of nongermline human blood DNA from normal individuals  individual were added to 500,000 human genomes from individuals homozygous for the C allele and divided into 20 reactions . Negative controls consisted of 20 reactions  of blood DNA from individuals homozygous for the C allele.Classical cytological approaches provide our main source of information about human germline development and maturation ,15–28. Ng generations. In this phase, the testis grows exponentially from one self-renewal cell to 2g  self-renewal cells. The total number of divisions in the testis in this phase is (2g  − 1). This corresponds to a total of (2g +1 − 2) daughter cells being produced, and there are this many opportunities for replication-dependent mutations. There is a mutation rate per cell division λ at the disease site. A mutation is shared by all of a cell's descendants, so a mutation early in the growth phase will produce many mutants. To be more specific, a mutation at the kth generation will produce 2g k− mutant descendants. This concept, first noted by Luria and Delbruck ). Since the mutation probability and p are small, the number of self-renewal cells will still stay roughly constant in the adult phase.The second modification we consider is selection. We introduce a new parameter into the model: at each adult phase generation, with probability lication . The occg SrAp cells. We set the units so that 1 is the length of one of the 192 testis pieces. Then a mutation at the kth generation will produce a cubic mutation cluster with length (192 × 2k−)1/3. To consider some examples, a mutation at the first generation means half of the testis is mutated, a mutation at the 8th generation produces a cluster with 91% of the length and 75% of the volume of one of the testis pieces, a mutation at the 21st generation produces a cluster with 5% of the length and 0.01% of the volume of one of the testis pieces. The location of a cluster is random within the testis.Above we discussed the number of mutant cells with regard to our various models, now we will discuss their spatial distribution. For simplicity, we have assumed that the testis is in the shape of a rectangular box, and it is divided into a regular grid of 6 × 8 × 4 = 192 cubic testis pieces . If a mug SrAp cells (mutated or not) in each of the 192 testis pieces. During the adult phase, mutation events occur randomly throughout the testis. If p = 0, then each such mutation event produces only one mutant SrAp cell. If p > 0, then each mutation event produces a cluster of mutant SrAp cells. The number of mutants in a cluster is discussed in the previous paragraphs. For simplicity, we assume that the shape of a cluster is a cube. The size of a cluster is calculated assuming that the cluster has the same density of SrAp cells as the rest of the testis. Depending on the cluster's size, it may be contained within one testis piece or intersect several adjacent testis pieces. Unlike the growth-phase clusters, the SrAp cells in the selection clusters are in addition to the nonmutated SrAp cells in the testis pieces. Due to the exponential growth of these selection clusters, there is a relatively narrow range of realistic parameter values for p. For example, a mutation early in the adult phase will produce a cluster approximately 10−5 the size of the testis if p is 0.01, whereas this same cluster will be larger than the rest of the testis if p is 0.03 or greater. Multiple mutation clusters may intersect the same testis piece and a given cell may only be mutated once (there are no reverse mutations).We assume that at the beginning of the adult phase, there is an equal fraction of the 2http://rd.plos.org/pbio.0050224) to simulate the model and its modifications. We fix the parameters a = 1,127 for donor 374 and a = 943 for donor 854, and consider both the extremes g = 27 and g = 34. First we consider the mutation hot spot model (p = 0). We simulated the model many times to estimate the probability that the simulated mutation frequency is within 5% of the observed mutation frequency as a function of the mutation parameter λ (mutation rate per cell division). The λ that optimizes this quantity is our approximate maximum likelihood estimate  and standard methods can produce approximate 95% confidence sets. For the selection model (p > 0), there are two parameters to consider: p and λ. There are an infinite number of pairings of these parameters' values (a subset of parameter space) for which the model can match the observed mutation frequency. Consequently, we searched for the parameter values which optimized the following two-criteria probability: (a) that the simulated mutation frequency is within √5% of the observed frequency, and (b) that the simulated fraction of testis' genomes in the fewest number of testis' pieces necessary to contain 95% of the mutants is within √5% of this observed quantity. Note that for the mutation hot spot model (p = 0) this two-criteria probability is 0 for all values of λ, since no single value of λ can match both criteria. In general, to estimate the empirical likelihood, we simulated the model 1,000 times for each set of parameter values.We have written a computer program (p = 0) and the selection model (p > 0), we simulated the model under optimal parameters and counted the number of pieces with frequencies in these same categories, and then performed the chi-squared test. For the control site, since the observed frequencies are much lower, we considered the following four categories: [0–4 × 10−6), [4 × 10−6–9 × 10−6), [9 × 10−6–1.4 × 10−5), [1.4 × 10−5+). The notation [x–y) means frequencies f such that x ≤ f < y.To determine whether the distribution of mutations in the 192 pieces is consistent between the model and the data, we used two hypothesis tests. First we counted the number of pieces with mutation frequencies in each of the eight color categories in p > 0), this test is sufficient; however, in order to reject the hot spot model (p = 0), we have also performed a goodness-of-fit test. For each simulation, we computed the following statistic: the minimum fraction of testis pieces which contain at least 95% of the total mutants. We simulated the hot spot model under the optimal parameters and recorded this statistic for those simulations such that the total testis mutation frequency was within 5% of the real data. We repeated this procedure such that 1,000,000 simulations met this criterion . The simulated statistic was never lower than 90%, in contrast with the much lower values for the real data in p-value is <10−6. However, this value is just a function of our patience in running simulations; we contend that for however many simulations are run, this statistic will never be lower than for the data. In order to get a p-value independent of the number of simulations, we fit the distribution of simulated statistics to a t-distribution (admittedly a poor fit) and the statistics for the real data were over 300 standard deviations away, making the p-value less than 10−200. The (anticonservative) chi-squared test rejected the hot spot model with p-value less than 2 × 10−16. Regardless of which p-value is used, there is strong support to reject the hot spot (p = 0) model.Because a mutation cluster can overlap adjacent testis pieces, this dependence makes the chi-squared test anticonservative. In order to not reject the selection model Click here for additional data file.Figure S2Testis 854–1. The testis was divided in half as in (2.1 MB PDF)Click here for additional data file.Figure S3For additional information, see Methods. Only the 3′ portions of the primer sequences are shown.(12 KB PDF)Click here for additional data file.Figure S4Ten PAP reactions were carried out on DNA from a piece of Testis 854–2.(A) Amplification curves (linear plot) show the increase in fluorescence as a function of cycle number. Note that the displacement of the curves from one another does not signal different starting amounts of target DNA, because on average, 90% of the wells should have no more than one target according to the Poisson distribution. Five of the samples gave positive curves and five were negative.(B) Melting profiles of the ten reactions shown in (A). Only the five positive reactions gave the correct melting profile for the expected PAP product. The negative control reactions (unpublished data) indicated no product was formed.(75 KB PDF)Click here for additional data file.Protocol S1(55 KB DOC)Click here for additional data file.Table S1(52 KB XLS)Click here for additional data file.Table S2(40 KB XLS)Click here for additional data file.Text S1(22 KB PDF)Click here for additional data file.www.ncbi.nlm.nih.gov/omim/) accession number for Apert syndrome is 101200. The National Center for Biotechnology Information  Nucleotide accession number for the FGFR2 gene is NM_000141. The Single Nucleotide Polymorphism database (http://www.ncbi.nlm.nih.gov/projects/SNP/) build 127 transversion for the C nucleotide site on human Chromosome 7 is rs3801993.The Online Mendelian Inheritance in Man (


In this study we demonstrate that the hemopoietic growth factor, G-CSF successfully mobilised progenitor cell populations into the peripheral blood in a population of patients despite intensive pretreatment with chemotherapy. Administration of G-CSF increased the numbers of peripheral blood progenitor cells (PBPC) by a median of 76-fold above basal levels. Maximal levels of PBPC were observed on days 5 and 6 after G-CSF treatment. In two patients a second cycle of G-CSF mobilised PBPC to levels comparable with those seen after the first cycle of G-CSF treatment. An earlier hemopoietic cell population (pre-CFC's) was also mobilised with levels increased up to 50-fold above basal levels. Using a standard mononuclear cell leukapheresis technique the PBPC were collected extremely efficiently  and could be further successfully enriched by separation using a Ficoll gradient. For patients who underwent the optimal collection protocol  a total of 32 +/- 6 x 10(4) GM-CFC kg-1 were collected. The ability to mobilise PBPC using G-CSF alone and to successfully and efficiently harvest these cells has important implications for the future of transplantation and high dose chemotherapy procedures.


Midazolam is used routinely to sedate patients in the intensive care unit (ICU). We suspected that midazolam over-sedation was occurring in the ICU of the Guy's and St. Thomas' Trust and that it could be difficult to differentiate this from underlying neurological damage. A sensitive assay for detecting midazolam and 1-hydroxymidazolam glucuronide (1-OHMG) in serum was developed and applied in the clinical setting.In the present study we evaluated a series of cases managed in a mixed medical, surgical and trauma ICU. Serum was collected from 26 patients who received midazolam, were 'slow to wake' and in whom there was suspicion of neurological damage. Patient outcome was followed in terms of mortality, neurological recovery and neurological damage on discharge.P < 0.002), eight of whom died and two were discharged from the ICU with profound neurological damage.Out of 26 patients, 13 had detectable serum levels of midazolam and/or 1-OHMG after a median of 67 hours (range 36–146 hours) from midazolam cessation. Of these 13 patients in whom midazolam/1-OHMG was detectable, 10 made a full neurological recovery. Of the remaining 13 patients with no detectable midazolam/1-OHMG, three made a full neurological recovery; 10 patients were subsequently found to have suffered neurological damage (These findings confirm that prolonged sedation after midazolam therapy should be considered in the differential diagnosis of neurological damage in the ICU. This can be reliably detected by the assay method described. The effects of midazolam/1-OHMG persist days after administration of midazolam has ceased. After prolonged sedation has been excluded in this patient group, it is highly likely that neurological damage has occurred. Midazolam is an intravenous sedative that is commonly used during ventilation in critical illness. It is often regarded as the sedative of choice in the intensive care unit (ICU). According to the findings of our recent electronic survey (93% respondents) , midazolWhen used as a single dose, midazolam's pharmacological characteristics appear favourable, with a rapid onset of action and a short plasma elimination half-life. Midazolam is 94–98% bound to plasma albumin and has a volume of distribution of 1.7 l/kg in healthy individuals . It is eWe suspected that some patients in our ICU, particularly those with renal impairment, were becoming over-sedated with midazolam and the active metabolite 1-OHMG, and that this was complicating the neurological assessment of 'slow to wake' patients. We previously developed a rapid assay for measuring midazolam and its glucuronide metabolite simultaneously : P < 0.002).In contrast, neurological damage was observed in 10 of the remaining 13 patients who had no detectable serum concentrations of midazolam and/or 1-OHMG (midazolam-negative group). Midazolam-positive patients were significantly less likely to have experienced neurological damage . 1-OHMG was detected at a mean of 6800 ± 3432 ng/ml  in the serum of six of the 13 midazolam-positive patients. All six of these patients exhibited a degree of renal impairment  in the face of midazolam levels below the therapeutic range, normally quoted in the critically ill of 100–1000 ng/ml 25 ng/ml  that 1-OOur findings suggest that serum levels of midazolam and/or 1-OHMG in 'slow to wake' patients may be used to aid differentiation between prolonged sedation and neurological damage. Patients found to be midazolam positive using this rapid assay were significantly less likely to have suffered neurological damage. Correct discrimination between neurological damage and prolonged sedation was made for 20 out of 26 patients, indicating a high degree of accuracy. Clearly, the possibility that midazolam-positive patients also have neurological damage remains and must be excluded if these patients do not awaken when serum concentrations of benzodiadepines have fallen to undetectable levels. Additionally, in the midazolam-negative group three patients were discharged with neurological function intact. This of course does not exclude a neurological cause of the coma that had fully resolved on discharge. One patient was receiving the sedating antihistamine chlorphenamine (30 mg/day intravenously) and did not regain full consciousness until it was stopped. In the remaining two patients no other clinical cause of the coma was apparent.The only other agent used routinely in these patients that could have significantly contributed to their reduced level of consciousness was the intravenous opiate fentanyl. Although fentanyl is known to accumulate in critical illness , we coulArguably, the most important finding is that over three-quarters of the 'slow to wake' patients with no detectable serum midazolam/1-OHMG either died or were discharged from the ICU with profound neurological damage, whereas more than three-quarters of those with detectable midazolam/1-OHMG went on to make a full recovery. This observation suggests that prolonged sedation occurs after midazolam therapy and that it can be difficult to differentiate this from neurological damage in the acutely ill patient. The exclusion of midazolam or its metabolite 1-OHMG should be confirmed either by assay detection, as we describe, or by using the short-acting benzodiazepine antagonist flumazenil before a formal diagnosis of neurological damage is made. There are reports ,8 in theWe recommend that use of alternatives to midazolam be considered in this patient group whenever possible, and that if its use is considered essential then steps should be taken to exclude the continuing presence of the drug or its metabolite before an opinion regarding neurological damage is formed.These findings have led to a change in prescribing practice in our ICU. We no longer use midazolam for sedation, and our sedation policy is now based on administering propofol or lorazepam. This view is also supported by the Society of Critical Care Medicine's most recently published guidelines , which rThe results of this investigation confirm that prolonged sedation from midazolam or 1-OHMG should always be considered in the differential diagnosis of neurological damage in critically ill patients who have received midazolam. This can be accurately detected using the assay method described. The sedative effects of midazolam/1-OHMG can persist for days after stopping administration of midazolam. If prolonged sedation can be excluded in these patients, then it is highly likely that neurological damage has occurred.• 	In some patients midazolam is metabolized to its glucuronide, which has sedative properties.• 	Prolonged sedation resulting from this metabolite should be considered when making a differential diagnosis of neurological damage in 'slow to wake' patients.• 	Measurement of midzolam and its metabolite in slow to wake patients will aid the differential diagnosis in these patients.ICU = intensive care unit; 1-OHMG = 1-hydroxymidazolam glucuronide.The author(s) declare that they have no competing interests.All authors participated in the study design, interpretation of results and manuscript preparation. CMK also performed data collection and analyses.


Nine patients with progressive, metastatic disease from primary carcinoma of the colon were entered into a phase I/II study using continuous intravenous infusions of granulocyte-macrophage colony-stimulating factor (GM-CSF) and high dose melphalan (120 mg m-2). GM-CSF was given alone to six patients during the first part of the study to determine a dose that would produce a peripheral leucocyte count (WCC) greater than or equal to 50 X 10(9) 1(-1) and was initially given at 3 micrograms kg-1 day-1 and escalated to 10 micrograms kg-1 day-1 after 10 days. The infusion was discontinued when the WCC exceeded 50 X 10(9) 1(-1) and after a gap of one week, melphalan was given over 30 min. GM-CSF was recommenced 8 h later and was continued until the neutrophil count had exceeded 0.5 X 10(9) 1(-1) for greater than 1 week. One patient achieved a WCC greater than 50 X 10(9) 1(-1) with GM-CSF 3 micrograms kg-1 day-1, but the other five who entered this phase of the study required dose escalation to 10 micrograms kg-1. No toxicity attributed to GM-CSF was seen. After melphalan, the median times to severe neutropenia (less than 0.5 X 10(9) 1(-1] and thrombocytopenia (greater than 20 X 10(9) 1 were seen but these were of short duration (median of 10 weeks). This study demonstrates that GM-CSF given by continuous intravenous infusion produces significant increments of peripheral granulocyte counts at 3 and 10 micrograms kg-1 day-1 and is not associated with any toxicity. The duration of neutropenia and thrombocytopenia induced by high-dose melphalan appears to be reduced by the subsequent administration of GM-CSF to times which are at least as short as have been reported in historical series which have used autologous bone marrow rescue.


This multi-author volume on the blood-cerebrospinal fluid barrier summarizes past and current research in the areas of choroid plexus and cerebrospinal fluid in health and disease. The blood-cerebrospinal fluid barrier functions together with the blood-brain barrier and the meninges, to control the internal environment of the brain. Sited at the choroid plexus epithelium, it secretes the cerebrospinal fluid (CSF), which circulates through the ventricles and around the outside of the brain and spinal cord. Originally assumed to pass an ultra filtrate of plasma into the brain ventricles, the choroid plexus was subsequently shown to secrete a homeostatically controlled electrolyte solution, the CSF . In the choroid plexus development and morphology, section 2 to barrier physiology and molecular biology, section 3 to the barrier in disease states and section 4 to in vivo and in vitro models of the barrier. Many topics are covered in this book and it is possible to select only a few for mention as being of particular interest. The proteins and lipids that make up the intercellular tight junctions are being elucidated at the molecular level, but how permeability is regulated awaits further investigation. The physiological regulation of CSF secretion is another important topic that has received much attention and is yet to be fully elucidated, together with the expression and role of ion transporters and channels. Knowledge of transporters for toxic substances and foreign chemicals and their metabolism by the choroid plexus is expanding rapidly, as are the fundamentals of nucleoside and metal transport and the role of choroid plexus peptides in hormonal signaling in the brain. Choroid plexus function changes with disease and aging and knowledge of the processes involved is important for understanding CNS disease states, such as Alzheimer's, Parkinson's, HIV, and disorders of CSF circulation. The final section provides the reader with a detailed review of the techniques used for investigation of choroid plexus function including in situ perfusion, cell culture and whole animal methods.There has been a requirement for a comprehensive review on the choroid plexus and blood-CSF barrier for a number of years . This muIn conclusion, 'The Blood-Cerebrospinal Fluid Barrier' is a comprehensive book that covers all aspects of the subject and should be on the bookshelf of all researchers involved in brain barriers and brain homeostasis.The author(s) declare that they have no competing interests.Sole author


Serum samples from 82 patients with epithelial ovarian cancer, previously assayed for CA125, were assayed for circulating colony-stimulating factor 1 (CSF-1). An elevated CSF-1 concentration (> 450 U ml-1 or > 5.42 ng ml-1) was significantly associated with a worse survival (P = 0.02). The predictive value of raised CSF-1 levels was retained whether the first available sample for all patients (n = 82) or the first sample at the start of chemotherapy (n = 41) was considered. Mean CSF-1 levels (n = 14) dropped significantly during six courses of platinum-based chemotherapy (P = 0.02). Although an elevated CA125 concentration appeared to be a prognostic indicator in the total population (n = 82), it was not related to prognosis in the group of patients from whom samples had been drawn at the start of chemotherapy. In a Cox proportional hazards model, CSF-1, but not CA125, was significantly associated with outcome following adjustment for stage, grade and degree of surgical clearance.


Supplementing culture medium with granulocyte-macrophage colony-stimulating factor (GM-CSF) partially alleviates this problem by increasing microglial yield. However, GM-CSF has also been reported to transition microglia into a dendritic cell (DC)-like phenotype and consequently, affect their immune properties.It is well appreciated that obtaining sufficient numbers of primary microglia for Staphylococcus aureus (S. aureus) and its cell wall product peptidoglycan (PGN), a Toll-like receptor 2 (TLR2) agonist; the TLR3 ligand polyinosine-polycytidylic acid (polyI:C), a synthetic mimic of viral double-stranded RNA; lipopolysaccharide (LPS) a TLR4 agonist; and the TLR9 ligand CpG oligonucleotide (CpG-ODN), a synthetic form of bacteria/viral DNA.Although the concentration of GM-CSF used in our protocol for mouse microglial expansion (0.5 ng/ml) is at least 10-fold less compared to doses reported to affect microglial maturation and function (≥ 5 ng/ml), in this study we compared the responses of microglia derived from mixed glial cultures propagated in the presence/absence of low dose GM-CSF to establish whether this growth factor significantly altered the immune properties of microglia to diverse bacterial stimuli. These stimuli included the gram-positive pathogen S. aureus and PGN stimulation. Specifically, the induction of tumor necrosis factor-α (TNF-α), macrophage inflammatory protein-2 (MIP-2/CXCL2), and major histocompatibility complex (MHC) class II, CD80, CD86 expression by microglia in response to S. aureus were similar regardless of whether cells had been exposed to GM-CSF during the mixed culture period. In addition, microglial phagocytosis of intact bacteria was unaffected by GM-CSF. In contrast, upon S. aureus stimulation, CD40 expression was induced more prominently in microglia expanded in GM-CSF. Analysis of microglial responses to additional pathogen-associate molecular patterns (PAMPs) revealed that low dose GM-CSF did not significantly alter TNF-α or MIP-2 production in response to the TLR3 and TLR4 agonists polyI:C or LPS, respectively; however, cells expanded in the presence of GM-CSF produced lower levels of both mediators following CpG-ODN stimulation.Interestingly, the relative numbers of microglia recovered from mixed glial cultures following the initial harvest were not influenced by GM-CSF. However, following the second and third collections of the same mixed cultures, the yield of microglia from GM-CSF-supplemented flasks was increased two-fold. Despite the ability of GM-CSF to expand microglial numbers, cells propagated in the presence/absence of GM-CSF demonstrated roughly equivalent responses following We demonstrate that low levels of GM-CSF are sufficient to expand microglial numbers without significantly affecting their immunological responses following activation of TLR2, TLR4 or TLR3 signaling. Therefore, low dose GM-CSF can be considered as a reliable method to achieve higher microglial yields without introducing dramatic activation artifacts. GM-CSeriments -12. Howeeriments -15, whiceriments -16. In aeriments ,18. Overin vitro experiments. Therefore, the primary objective of the present study was to evaluate whether low dose GM-CSF leads to alterations in microglial morphology and/or functional activation in response to a wide variety of PAMPs commonly associated with various CNS infections, namely Staphylococcus aureus (S. aureus) and its cell wall component peptidoglycan (PGN), LPS, polyI:C, and CpG-ODN. Our results demonstrate that low dose GM-CSF led to a significant expansion in microglial numbers without affecting their phagocytic activity or cytokine production profiles in response to the majority of PAMPs examined, with the exception of the TLR9 agonist CpG-ODN. However, we did observe a phenotypic transformation of microglia expanded in the presence of low dose GM-CSF, namely a transition to a DC-like morphology typified by numerous dendrites; however, the functional implication(s) of this change remain to be determined. Therefore, low dose GM-CSF can be successfully utilized as a culture medium supplement to enhance microglial recovery without overtly compromising normal responsiveness to microbial stimuli. Importantly, these findings exclude any potential GM-CSF-induced artifacts in the read-outs of microglial activation routinely used in our studies.During the preparation of our mouse primary mixed glial cultures, we routinely supplement culture medium with low levels of GM-CSF (0.5 ng/ml) to increase microglial yields. Despite the fact that this concentration is approximately ten-fold lower than what has been reported to modulate microglial function and transition into a DC phenotype (i.e. 5–50 ng/ml), we are often questioned regarding the consequences of microglial exposure to GM-CSF during the mixed glial culture period and whether this introduces artifacts in the activation profiles of these cells in subsequent 2 flasks. To minimize variation between microglia expanded in the presence/absence of GM-CSF, mouse pups were procured from litters born on the same day and primary cultures propagated with or without GM-CSF were derived from the same initial mixed glial population  medium while the other half was cultured without GM-CSF).Primary microglia were isolated from inbred neonatal C57BL/6 mice as previously described . BrieflyUpon confluence (7–10 days), flasks were shaken overnight at 200 rpm at 37°C to recover microglia. Microglia from both (+) GM-CSF- and (-) GM-CSF-treated flasks were collected and plated in medium without GM-CSF for all subsequent experiments. The purity of microglial cultures was evaluated by immunohistochemical staining using antibodies against CD11b and GFAP to identify microglia and astrocytes, respectively, and was routinely greater than 95%. Each experiment presented in this paper was initially performed with microglia collected after the first shake and repeated with cells collected after a second shake to confirm that the responses were comparable. Based on our findings that microglial responses were similarly affected in all experiments regardless of when they were harvested, we concluded that microglial responsiveness to microbial stimuli does not significantly differ in cells collected from the first versus subsequent shakes.S. aureus (strain RN6390) was prepared as previously described [S. aureus, poly I:C, and the synthetic CpG oligonucleotide ODN1826 were obtained from InvivoGen . Escherichia coli O11:B1 LPS was purchased from List Biological Laboratories . The doses of stimuli used throughout this report were based on our previous studies that established optimal cytokine responses induced following bacterial stimulation without any evidence of toxicity [Heat-inactivated escribed  and PGN toxicity ,21. All Protein levels of TNF-α, IL-12p40  and macrophage inflammatory protein  were quantified in conditioned medium from PAMP-stimulated microglia using ELISA kits according to the manufacturer's instructions (level of sensitivity = 15.6 pg/ml).To evaluate whether microglia expanded in the presence or absence of GM-CSF demonstrated similar survival profiles following bacterial activation, a standard MTT assay based upon the mitochondrial conversion of  into formazan crystals was performed as previously described .S. aureus isolate that constitutively expresses green fluorescence protein  for 3 h, whereupon Hoechst 33342  was added to visualize nuclei. Cells were washed extensively with PBS and incubated with 0.05% crystal violet in 0.15 M NaCl for 45 seconds to quench any fluorescence emitted by residual extracellular bacteria. Coverslips were viewed under fluorescence microscopy using an excitation wavelength of 460 – 490 nm .Primary microglia expanded with or without GM-CSF were seeded onto 12 mm coverslips in 24-well plates and incubated overnight. The following day, cells were treated with a heat-killed 7 heat-inactivated S. aureus for 24 h, whereupon cells were washed extensively with PBS, fixed in ice-cold methanol, and incubated with PBS/10% donkey serum to prevent non-specific antibody binding  for 30 min at room temperature. Subsequently, microglia were incubated with a MHC class-II antibody  overnight at 4°C. The following day, cells were incubated with a donkey anti-rat biotinylated secondary antibody  and detected using a streptavidin-Alexa Fluor 568 conjugate (Molecular Probes). Subsequently, a directly conjugated CD11b-FITC antibody (BD Pharmingen) was added and cells incubated for 1 h at 37°C, whereupon Hoechst 33342 was used to visualize nuclei. Controls included microglia incubated with secondary antibodies only to assess the extent of non-specific staining. Coverslips were imaged using a Zeiss laser scanning confocal microscope . Hoechst 33342 for nuclear visualization was excited by a 405 nm diode laser, FITC to visualize CD11b immunoreactivity was excited with a 488 nm argon laser, and Alexa Fluor 568 to demonstrate MHC class II expression was excited with a 561 nm DPSS laser, with images collected using the appropriate emissions. The confocal pinhole was set to obtain an optical section thickness of 1.6 μm. To demonstrate co-localization of CD11b, MHC class II, and Hoechst 33342 signals, RGB merges of individual confocal images were performed using the ImageJ software program (NIH Image).Primary microglia expanded in the presence/absence of GM-CSF were seeded onto 12 mm coverslips in 24-well plates and incubated overnight. The following day, microglia were treated with 106 cells/well), and incubated overnight. The following day, cells were treated with 107 heat-inactivated S. aureus for 24 h. At the end of the incubation period, cells were washed twice with PBS, and collected using a cell scraper. A total of 5 × 105 microglia in each group were stained for two-color flow cytometry using CD11b-FITC and CD11c-PE-Cy7 antibodies (both from BD Pharmingen). Fc receptors were blocked with the addition of Fc block™ . In addition, a separate set of microglia were stained with antibodies directed against the co-stimulatory molecules CD40, CD80, and CD86, in addition to MHC class II , and subsequently incubated with a donkey anti-rat IgG FITC-conjugated secondary antibody . Controls included microglia incubated with appropriate isotype control-matched antibodies to assess the extent of non-specific background staining. Cells were analyzed using a FACS Calibur cytometer  with settings based on the staining of microglia with isotype control antibodies alone.Primary microglia expanded in the presence/absence of GM-CSF were seeded into 6-well plates (2 × 106 cells/well), and incubated overnight. The following day, cells were treated with 107 heat-inactivated S. aureus for 24 h or left unstimulated. At the end of the incubation period, cell morphology was visualized by phase-contrast microscopy and images collected using a fixed stage upright epifluorescence microscope  equipped with a 40 × water immersion objective lens and a 12-bit intensified monochrome CCD camera .Primary microglia propagated in the presence/absence of GM-CSF were seeded into 35 mm dishes .Significant differences between experimental groups were determined by the It has been well established that GM-CSF exerts mitogenic effects on primary microglia, effectively expanding cell numbers ,10,12,23To initiate our analysis of low dose GM-CSF effects on microglia, we quantified the relative percentages of microglia recovered from mixed glial cultures continuously propagated in the presence/absence of recombinant mouse GM-CSF (0.5 ng/ml). Upon reaching confluence, mixed glial cultures were collected at three consecutive harvests separated by an interval of 7–10 days. The number of microglia collected after the first harvest was not significantly influenced by GM-CSF; however, after the second and third harvests the number of microglia collected from GM-CSF-negative flasks was significantly lower compared to those supplemented with GM-CSF  was not significantly different between the groups . Importantly, upon recovery of microglia from these cultures, cells are never exposed to exogenous GM-CSF again and are not utilized in experiments for at least 2 days following low dose GM-CSF withdrawal. With this modification, we were able to collect sufficient numbers of microglia for our experiments even after the third harvest, effectively reducing the need for additional microglia cultures. In addition, the main inflammatory responses of microglia following S. aureus stimulation, namely proinflammatory mediator production and phagocytosis, were not affected by propagating cells in the presence of GM-CSF  provides additional support to establish the validity of our findings.The expression of other surface molecules including MHC class II, CD80, and CD86 were not affected by exposure of microglia to low dose GM-CSF. The expression of these molecules has been shown by others to be elevated in neonatal microglia, where cells exhibit a partially activated phenotype in culture ,52,55. I changes ,54. In cell line , we founin vitro culture. Specifically, in our studies, microglia and astrocytes are maintained as mixed cultures during the expansion period. Therefore, it is important to acknowledge that the coordinated effects of endogenous astrocyte-derived factors in combination with exogenous GM-CSF may influence the properties of recovered microglia. As an extension, it is not surprising that different microglial phenotypes are observed when cells are directly purified from the brain parenchyma and cultured in isolation. In addition, compounded with the addition of high levels of exogenous GM-CSF used in other studies, it is not unexpected that microglia assume different biological and functional properties. We propose that the maintenance of primary microglia initially as mixed cultures with astrocytes is more reminiscent of the natural milieu these cells are exposed to in vivo and represents an acceptable model to study the immunological properties of microglia, although we clearly recognize the potential for any in vitro findings to not directly equate to in vivo responses due to the artificial nature of the former. However, the examination of microglia in isolation provides a more simplified approach to investigate the effector functions of these CNS phagocytes in the absence of confounding effects by surrounding cell types.Another important factor to consider is the nature of how primary microglia are propagated during S. aureus. This suggests that our culture paradigm may be successfully used as a method to procure larger quantities of microglia without significantly affecting their downstream responses to microbial stimuli. However, it is important to note that differences in CpG-ODN responsiveness as well as the transition towards a DC-like phenotype were observed in microglia propagated in the presence of low dose GM-CSF, the functional implications of which are currently unknown.Our findings demonstrate that supplementation of neonatal mouse mixed glial cultures with low dose GM-CSF successfully maintained microglial expansion and did not lead to overt alterations in the functional responses of microglia following stimulation with several PAMPs of importance during CNS bacterial and viral infections including PGN, LPS, polyI:C, as well as S. aureus (Staphylococcus aureus); TLR (Toll-like receptor); TNF-α.APC (antigen presenting cell); CD (cluster of Differentiation); CFU (colony-forming units); CNS ; DC (dendritic cell); DMEM (Dulbecco's modified eagle medium); ELISA (enzyme linked immunosorbent assay); FACS (fluorescence activated cell sorting); FBS ; FITC (fluorescein isothiocyanate); GFP (green fluorescence protein); IFN-γ (interferon gamma); IL-12 p40 (interleukin-12 p40); GM-CSF (granulocyte-macrophage colony-stimulating factor); LPS (lipopolysaccharide); M-CSF (macrophage colony-stimulating factor); MHC (major histocompatibility complex); MIP-2/CXCL2 (macrophage inflammatory protein-2); MTT ; ODN (oligonucleotide); OPI ; PAMP (pathogen associated molecular pattern); PGN (peptidoglycan); PBS ; polyI:C (polyinosine-polycytidylic acid);The author(s) declare that they have no competing interests.Both NE and TK directed the work and prepared the manuscript.