Medea, which consists of a maternal toxin and zygotic antidote. The majority of toxin–antidote drive systems require a critical frequency to be exceeded before they spread into a population. Of particular interest, a Z-linked Medea construct with a recessive antidote is expected to induce an all-male population crash for release frequencies above 50%. We suggest molecular tools that may be used to build these systems, and discuss their relevance to the control of a variety of insect pest species, including mosquito vectors of diseases such as malaria and dengue fever.Gene drive systems are genetic elements capable of spreading into a population even if they confer a fitness cost to their host. We consider a class of drive systems consisting of a chromosomally located, linked cluster of genes, the presence of which renders specific classes of offspring arising from specific parental crosses unviable. Under permissive conditions, a number of these elements are capable of distorting the offspring ratio in their favor. We use a population genetic framework to derive conditions under which these elements spread to fixation in a population or induce a population crash. Many of these systems can be engineered using combinations of toxin and antidote genes, analogous to  Gene drive occurs when genetic elements—including genes, gene complexes, entire chromosomes, and endosymbiotic bacteria—increase in frequency in a population even if their presence results in a fitness cost. Gene drive has long been proposed as a mechanism for controlling insect pest populations, beginning with the suggestion by Medea elements  that function by rendering specific classes of offspring arising from specific parental crosses unviable. Under permissive conditions, these systems distort the offspring ratio in favor of the transgenic allele, thus resulting in gene drive. he toxin . The comquencies . Medea rs Semele , Merea , or at one or both copies of the locus per individual (henceforth referred to as gene fixation). The majority of these systems require a significant threshold frequency to be exceeded before they spread into a population, and for these we derive basic properties of the critical frequency that must be exceeded for the construct to spread. We also investigate constructs that induce a population crash—either by causing a deficiency of one gender over the other, or by driving the population toward an unviable genotype. Both autosomal and sex chromosome-linked constructs are considered. While many of the mathematically derived gene drive systems are prohibitively difficult to engineer, a number can be constructed using conceivable toxin–antidote pairs, and are highlighted following the mathematical analysis.Several gene drive systems are outside the scope of this article, and are described by other authors. HEGs and meiotic drive elements, for example, increase the frequency of a favored allele in the gametes of heterozygous parents, and are well described by We use a simple difference equation framework to model the spread of a gene drive system through a randomly mating population. In doing so, we assume discrete generations and infinite population size. Within this framework, we consider both autosomal, X- and Y-linked constructs. These models are easily extended to species for which males are the homogametic sex.T,” and refer to the absence of the construct at the corresponding locus as “t.” Certain offspring of certain parental crosses are rendered unviable by the construct, and in our initial model, viability is independent of offspring gender. The proportion of the kth generation that are individuals of genotypes tt, Tt, and TT are denoted by ku, kv, and kw, respectively. By considering all possible mating pairs, the genotypes of embryos in the next generation are described by the ratio We consider a gene drive construct as a single allele, which we denote by “T allele. Constants c1, …, c14 represent the 14 fundamental ways in which offspring of different parental crosses may be rendered unviable by an autosomal construct . The dynamics of this system differ due to the fact that females carry two copies of the X chromosome, while males carry one. The proportion of the c1, …, c14 represent the 14 fundamental ways in which offspring of different parental crosses may be rendered unviable by an X-linked construct  =  = , these equations can be used to calculate stable and unstable equilibria that summarize the dynamics of these constructs. Analytic expressions for these equilibria are too complex to be useful; however, two equilibrium points are expected to exist in most cases—allele fixation,  = , and allele loss,  = . We are interested in the conditions for which allele fixation is locally stable. Under these conditions, the allele of interest will spread to fixation beginning from a range of population frequencies less than 1.Equations 1–7 describe the population frequency of an autosomal construct from one generation to the next. By setting genotype frequencies equal across generations,  is a requirement for allele fixation.The second eigenvalue is infinite when c6, c11= 0 or hs= 1. This suggests that allele fixation is locally stable when crosses between TT and Tt individuals produce no viable Tt offspring, or when all Tt individuals are unviable . If only one of the crosses between TT and Tt individuals produces unviable Tt offspring , then allele fixation is locally stable provided that s < 1/(2 −h). This condition is satisfied for fitness costs of s < 0.5 regardless of heterozygosity . It is unlikely that gene drive constructs having fitness costs of s≥ 0.5 would be deemed suitable for further development, suggesting that any realistic construct will spread to allele fixation above a certain critical population frequency provided that crosses either between TT males and Tt females or between Tt males and TT females produce no viable Tt offspring.The second eigenvalue is equal to 0 when c1, c6, c11= 1), while the viability of other cross outcomes is unspecified, then allele fixation is unstable for nonzero fitness costs (s > 0) that are greater in homozygotes than heterozygotes (h < 1). However, linear stability analysis is inconclusive in the absence of fitness costs (s= 0) or when fitness costs are dominant (h= 1). In general, a toxin–antidote construct is predicted to spread to allele fixation whenIf all three of the cross outcomes highlighted above are viable  confirm the validity of the above analysis. The simulations also highlight an additional condition for allele fixation—a genetic construct will spread to allele fixation provided that it renders all offspring of parental crosses between TT and tt individuals unviable . This condition holds true under all realistic parameterizations . The requirements outlined in the above four paragraphs encapsulate what we shall refer to as the strong condition for allele fixation—in which allele fixation occurs despite a nondominant fitness cost—and are visualized in c6, c11= 0, h= 0.5, and s= 0.1). It should be noted that constructs outlined in this section are illustrative, and not necessarily straightforward to construct.Numerical iterations of eqs. 1–7 for the 2c1, c6, c11= 1), one or both of the crosses between TT and tt individuals produce viable offspring (c5= 1 and/or c9= 1), and the fitness cost is zero (s= 0) or dominant (h= 1), then simulations are required to determine the stability of allele fixation. Under these conditions, allele fixation is locally stable provided that TT offspring of parental crosses between TT and Tt individuals are viable , and Tt or tt offspring are rendered unviable in at least one parental cross . These requirements lead to what we shall refer to as the weak condition for allele fixation—in which allele fixation only occurs if the fitness cost is zero or dominant—and are visualized in c7, c8, c10, c12, c13= 0 and s= 0). Although allele fixation does not occur for these constructs in the presence of a nondominant fitness cost, the constructs can still spread to a high population frequency. For example, if the construct shown in s= 0.1 (h= 0.5), it is predicted to reach an allele frequency of 94.7% at equilibrium, with 99.7% of individuals having at least one copy of the allele.Numerical iterations also resolve the inconclusive cases mentioned above. If all three of the highlighted cross outcomes are viable . The only exception is if all TT offspring of parental crosses involving Tt individuals are rendered unviable by the construct . In this case, there is an additional requirement for gene fixation to occur—either tt offspring of one or both crosses between Tt and tt parents must be unviable (c12= 0 and/or c13= 0), or the fitness cost due to the construct must be zero (s= 0) or dominant (h= 1). These requirements lead to what we shall refer to as the strong condition for gene fixation—in which gene fixation occurs despite a nondominant fitness cost—and are visualized in c14= 0, h= 0.5, and s= 0.1).An alternative to allele fixation is gene fixation, in which chanisms . NumericTT males and TT females produce viable offspring (c1= 1); however, the dynamics of constructs for which these offspring are unviable (c1= 0) are also of interest, particularly for applied purposes. Allele fixation is not possible in these cases; however, gene fixation is, and some examples of this are provided in Up to this point, we have been considering cases in which crosses between The conditions under which allele fixation is locally stable for an autosomal construct conferring gender-dependent offspring viability are described in the Supporting information. Summarizing these results, an autosomal construct is expected to spread to allele fixation provided thatcm1, represents the proportion of male offspring of crosses between TT males and TT females that are viable; cm6, represents the proportion of male Tt offspring of crosses between Tt males and TT females that are viable; and cm11, represents the proportion of male Tt offspring of crosses between TT males and Tt females that are viable. The corresponding proportions for female offspring are cf1,, cf6,, and cf11,.Here, For an autosomal construct that confers female-specific offspring lethality, the condition for allele fixation simplifies toTT males and TT females must be at least partially viable . This is therefore a requirement for allele fixation. Furthermore, analysis of TT and Tt individuals produces unviable female Tt offspring , then allele fixation is locally stable for modest fitness costs. If all of these highlighted cross outcomes produce viable female offspring , and the fitness cost is zero (s= 0) or dominant (h= 1), then simulations are required to determine the stability of allele fixation. In these cases, allele fixation is locally stable provided that female TT offspring of parental crosses between TT and Tt individuals are viable  and female Tt or tt offspring are rendered unviable in at least one parental cross . Conditions required to induce a population crash are discussed in the Supporting information.In order for allele fixation to be an equilibrium solution of eqs. S1–S13, female offspring of crosses between TT individuals.In all cases for which allele fixation is locally stable, the allele of interest is predicted to spread to fixation beginning from a range of population frequencies less than one. In a few cases, this range of frequencies can be derived analytically; but, in general, numerical simulations are necessary because the release threshold is represented by a family of points in genotype space. Stability analysis reveals just one of these points, and we are most interested in the point having no heterozygotes, because population replacement programs are likely to involve releases of only TT individuals into a population of tt individuals, reveal three circumstances in which the release threshold can be predicted:Numerical iterations of eqs. 1–7 for a variety of initial conditions, assuming a release of equal numbers of male and female If the only unviable offspring are wild type and the construct has no fitness cost, then the release threshold is zero. Otherwise, the release threshold is greater than zero.If the only unviable offspring are heterozygous and the construct has no fitness cost, then the release threshold is 50%.T alleles among the unviable offspring.If only one parental cross gives rise to unviable offspring and the construct has no fitness cost, then the release threshold is equal to the expected proportion of Tt and tt offspring of crosses between Tt males and Tt females unviable; but affects no other parental crosses. Tt offspring have one T allele and one t allele and are expected to be twice as numerous as tt offspring. The proportion of T alleles among expected unviable offspring is therefore 2/6, which corresponds to a release threshold of 33.3% for a construct having no fitness cost. When these rules do not apply, the release threshold can be calculated by numerically iterating eqs. 1–7 for a variety of initial conditions. It should be noted that release thresholds vary with the initial distribution of gender and genotype classes. For some constructs, the release threshold is minimized for a female-biased release . This is a reasonable simplification because many toxins, antidotes, and effector genes are expected to be dominant in action  = .Equations 8–18 describe the population frequency of an X-linked construct across generations. By setting genotype frequencies equal from one generation to the next, these equations can be used to calculate stable and unstable equilibria that summarize the dynamics of X-linked constructs. Two equilibrium points are expected to exist in most cases—allele fixation and allele loss—however, the mathematical description of fixation is surprisingly complicated for an X-linked construct with a fitness cost that is less in Xon e.g., . In thesTo derive the conditions under which allele fixation is locally stable, we calculate the eigenvalues of the Jacobian matrix,If all eigenvalues have modulus less than one, then fixation is locally stable , and theUsing eqs. 8–18 and 24, we find that allele fixation is associated with eigenvalues equal to 0 andc1, c3= 0 or s= 1. This corresponds to the case whereby crosses between XTY males and XTXT females produce no viable offspring, or when all individuals having the construct are unviable. Allele fixation is not an equilibrium solution to eqs. 8–18 under these conditions, and, in fact, both male and female offspring of this cross must be at least partially viable  in order for the construct to be maintained in the population when fixed. This is therefore a requirement for allele fixation.The second and third eigenvalues are infinite when c7= 0 and either or both of c9, c11 are equal to 0. This suggests that allele fixation is locally stable when crosses between XTY males and XTXt females produce no viable XTXt or XtY offspring, or alternatively when crosses between XTY males and XTXt females and crosses between XtY males and XTXT females produce no viable XTXt offspring. Interestingly, if only one of these crosses produces unviable XtY or XTXt offspring , then allele fixation is still locally stable for all realistic fitness costs (s < 0.5). The requirements outlined in the above two paragraphs encapsulate the strong condition for allele fixation of an X-linked construct—in which allele fixation occurs despite a fitness cost—and are visualized in c7, c9, c11= 0, h= 1, and s= 0.1). Once again, constructs outlined in this section are illustrative, and not necessarily straightforward to construct.The second and third eigenvalues are equal to 0 when c1, c3, c7, c9, c11= 1), while the viability of other cross outcomes is unspecified, then allele fixation is unstable for nonzero fitness costs (s > 0). However, linear stability analysis is inconclusive in the absence of a fitness cost (s= 0). In general, an X-linked gene drive construct with a dominant fitness cost is predicted to spread to allele fixation whenIf all five of the cross outcomes highlighted are viable  confirm the validity of the above analysis while resolving the inconclusive cases mentioned above. If all five of the highlighted cross outcomes produce viable offspring , and there is no fitness cost (s= 0), then simulations are required to determine the stability of allele fixation. Under these conditions, allele fixation is locally stable provided that the following rule is satisfied,Numerical iterations of eqs. 8–18 for the 2c1, c3, c7, c9, c11= 1) and if there is no fitness cost (s= 0), then allele fixation will only occur if the number of t alleles among unviable offspring equals or exceeds the number of T alleles among unviable offspring of crosses between XTY males and XTXt females and between XtY males and XTXT females. The one exception, illustrated by the indicator function, tXt females are not counted if XtXt offspring are unviable themselves. This is the weak condition for allele fixation of an X-linked construct—in which allele fixation only occurs in the absence of a fitness cost—and is visualized in c10, c13, c14= 0 and s= 0).Here, we have assumed binary values for all constants. The meaning of this equation is that, if the strong condition for allele fixation is not satisfied ; however, the dynamics of constructs for which this is not the case (c1= 0 and/or c3= 0) are also of interest, particularly for applied purposes. Allele fixation is not possible in these cases; however, gene fixation is. Normally, the conditions that lead to gene fixation—unviable XtXt offspring produced by crosses between XTXt females and XtY males (c14= 0), and unviable XtY offspring produced by crosses between XTXt females and XTY males (c11= 0)—also lead to allele fixation. However, if crosses between XTXT females and XTY males also produce unviable female offspring (c1= 0), then genotypes including the allele of interest fix in the population while the allele itself does not. Some examples of this are provided in Thus far, we have been considering cases in which crosses between XTXT females and XTY males are unviable (c1= 0 and/or c3= 0). Numerical iterations for the 214− 1 possible X-linked constructs reveal a number of possibilities. For an all-male population crash, it is required that all female offspring of crosses between XTY males and XTXT females are unviable (c1= 0); for an all-female population crash, it is required that all male offspring from this cross must are unviable (c3= 0); and to drive the population toward an unviable genotype, both genders are required to be unviable . One configuration that leads to a population crash  is shown in An X-linked construct is also able to induce a population crash if male and/or female offspring of crosses between XTXT and XTY individuals must be released at in order to spread to fixation. Numerical iterations of eqs. 8–18 for a variety of initial conditions, assuming a release of equal numbers of XTXT and XTY individuals into a population of XtXt and XtY individuals, reveal three circumstances, analogous to those for autosomal constructs, in which the release threshold can be predicted:In all cases for which allele fixation is locally stable, the construct is predicted to spread to fixation beginning from a range of population frequencies less than one. For an X-linked construct, this range is described by a release threshold, which is the minimum population frequency that XtXt females and XtY males and the construct has no fitness cost, then the release threshold is zero. Otherwise, the release threshold is greater than zero.If the only unviable offspring are XTXt females and the construct has no fitness cost, then the release threshold is 50%.If the only unviable offspring are XT alleles divided by the expected number of Xt and XT alleles among the unviable offspring.If only one parental cross gives rise to unviable offspring and the construct has no fitness cost, then the release threshold is equal to the expected number of XTXt and XTY offspring of crosses between XTXt females and XtY males unviable. XTXt offspring have one XT allele and one Xt allele and are expected to be equally as numerous as XTY offspring. The expected proportion of XT alleles among unviable offspring is therefore 2/3, which corresponds to a release threshold of 66.7% for a construct having no fitness cost. In other cases, the release threshold can be calculated by numerically iterating eqs. 8–18 for a variety of initial conditions. As for autosomal constructs, these thresholds vary with the initial distribution of genotype classes.To demonstrate the application of the third rule, imagine a construct that renders XMedea element has been engineered by linking a dominant maternal toxin to a dominant zygotic antidote . This construct consists of a dominant maternal toxin linked to a recessive zygotic antidote, represented mathematically as c5, c6, c7, c8, c11, c13, c14= 0  and is released at a frequency above 33.3% in a population  and eqs. 8–18 (for X-linked constructs), we obtain a list of 53 drive elements that can, in principle, be implemented as toxin–antidote systems that spread to fixation or induce a population crash in the presence of modest fitness costs , S1. ForMedea, its male equivalent, and a dominant maternal or zygotic toxin linked to a paternal antidote. These constructs also spread to fixation if offspring lethality is male specific  or female specific . The condition for gene fixation is only satisfied by one X-linked construct—a dominant maternal toxin linked to a heterozygous zygotic antidote.For X-linked constructs , a wide s= 0), this construct causes a population crash for release sizes greater than 50%. The other six constructs all consist of a male-lethal paternal toxin linked to a second toxin and sometimes an antidote. These display different threshold behavior but also induce an all-female population crash.Another distinction between autosomal and X-linked constructs is that simple toxin–antidote combinations inserted at an X-linked locus can induce a population crash. Merea causes a population crash by creating an all-male population (s= 0), this construct causes a population crash for release sizes greater than 50%  and males are homogametic (ZZ). For Z-linked constructs, a paternal toxin or antidote becomes a maternal toxin and antidote, and vice versa. The only other change is that a female-lethal toxin becomes a male-lethal toxin, and vice versa. The most significant result for Z-linked constructs is that pulation . Like itthan 50% . The strWe have considered a variety of single-construct gene drive systems that spread by rendering certain offspring genotypes of certain parental crosses unviable, thus distorting the offspring ratio in their favor under permissive conditions. Literally thousands of constructs having this property are imaginable. We have used simple population genetic models to determine the general properties that single-construct gene drive systems must have in order to drive a gene into a population or induce a population crash. We have also identified a subset of these systems that could, in principle, be engineered using linked pairs of genes encoding a toxin and an antidote.Tt offspring of crosses between TT and Tt individuals are rendered unviable. Alternatively, if all offspring of crosses between TT and tt individuals are unviable, the T allele will also fix. For allele fixation to occur, it is also required that both male and female offspring of crosses between TT individuals are viable. Another possibility is that genotypes including the allele of interest fix in the population while the allele itself does not. This tends to occur if the conditions for allele fixation are not satisfied and tt offspring of crosses between Tt individuals are unviable. Several possibilities are available for inducing a population crash; however, none of these can be engineered with simple combinations of toxins and antidotes.Autosomal constructs are expected to spread to allele fixation in the presence of realistic fitness costs if TY males and XTXT females are viable. X-linked constructs are then expected to spread to allele fixation, even in the presence of modest fitness costs, when XtY or XTXt offspring of crosses between XTY males and XTXt females are rendered unviable, or when XTXt offspring of crosses between XtY males and XTXT females are unviable. Several possibilities are available for inducing an all-female population crash; some of which can be engineered with combinations of toxins and antidotes. For species in which females are the heterogametic sex, analogous constructs lead to an all-male population crash.Analogous conditions exist for driving an X-linked construct into a population. The first requirement is that male and female offspring of crosses between XDrosophila melanogaster with Medea , in which each construct possesses a maternal toxin linked to a zygotic antidote for the maternal toxin on the opposite construct. This is the first synthetic drive system to display clear threshold behavior, providing a proof-of-principle for the development of similar systems in other species.Insect transgenesis is a relatively new field—the first transgenic malaria vector was engineered a little over 10 years ago —and it hth Medea , and morith HEGs . Develop present ; however present  and phenThe gene drive systems listed in Recessive toxins may be generated in several ways, each having its own downfalls. First, it may be possible to discover an endogenous gene which, when disrupted at two homologous loci, leads to lethality—either zygotic lethality, or lethality through a maternal or paternal effect. Such a toxin would be robust to evolutionary pressures because it results from the disruption of an endogenous gene; however, it may be difficult to identify a gene whose only effect when lost is to cause a stage-dependent lethality that can then be rescued through the expression of an antidote at another specific life stage. Second, a toxin gene may be generated that is only active as such when inherited from both parents. However, such a toxin gene would be prone to mutational inactivation because mechanisms for bringing about recessivity without dominant killing are expected to involve multiple components , providing several opportunities for the emergence of antidote-only alleles. Therefore, in general, dominant toxins are preferred.Dominant maternal, paternal, and zygotic toxins are all feasible to engineer. Maternal toxins must be passed to progeny through the female germline, and could consist of mRNAs that encode toxic proteins, or small RNAs that silence the expression of maternally expressed genes whose products are required for embryogenesis . PaternaMaternal, paternal, and zygotic antidotes are all feasible to engineer; but they may not provide protection against all toxin varieties. A maternal antidote could encode for a protein that neutralizes or degrades a toxin, or a small RNA that promotes degradation of a toxin transcript prior to translation. Mothers provide a large amount of material to their eggs, and hence a maternal antidote may be capable of inhibiting a maternal or paternal toxin, or a zygotic toxin expressed early in development when the maternal effect is still active. A zygotic antidote could encode for a protein or small RNA, and could inhibit a zygotic or maternal toxin, provided that the toxin does not have its effect until after the antidote is expressed. A zygotic antidote is unlikely to be capable of inhibiting a paternal toxin, such as a nuclease expressed in sperm, because zygotic transcription is not initiated until some time after fertilization. A paternally provided antidote could inhibit a paternally provided toxin because both act in the same cells . However, given the limited quantity of material sperm provide to the egg, it is unlikely that a paternal antidote could inhibit a maternal or zygotic toxin.A large number of the toxin–antidote constructs listed in Medea, have release thresholds of between 40.0% and 66.7%. Although high, these are much lower than the release ratios achieved for sterile insect programs, which sometimes exceed 90% sterile to wild insects (Merea, which is capable of inducing an all-male population crash for releases proportions greater than 50%.From the above analysis, we identify a number of toxin–antidote systems that are biologically feasible and capable of spreading to fixation or inducing a population crash even in the presence of a fitness cost . All of  insects . The exi insects . FinallyThe gene drive systems discussed here are of great relevance to vector-borne disease control and pest management. Mosquito-borne diseases such as dengue fever, chikungunya, and malaria continue to pose a major health problem through much of the world. Treatments for dengue fever and chikungunya remain elusive, and malaria is proving exceptionally difficult to control in highly endemic areas with insecticide-treated nets, indoor residual spraying, and antimalarial drugs . ConsequAnother area of application is agricultural pest management. Agricultural pests such as butterflies, beetles, flies, and moths have the potential to significantly reduce yields of a variety of crops, and there is a constant effort to control pest populations without excessive use of pesticides . Many in


There have been few reports of severe complications with this treatment. This is the first published case report to our knowledge of cerebrospinal fluid leakage induced by CyberKnife® radiotherapy.CyberKnife® radiation was performed to the residual tumor considering the aggressive feature of the tumor with a molecular immunology Borstel-1 index of 15%. Five months after the treatment, magnetic resonance imaging showed definite tumor shrinkage, and the patient had been living her daily life without any troubles. After another month, the patient was transferred to our clinic because of coma with high fever, and computed tomography demonstrated severe pneumocephalus. Rhinorrhea was definitely identified on admission; therefore, emergency repair of the cerebrospinal fluid leakage was performed using an endoscope. Dural defects at the bottom of the sella turcica were identified under careful endoscopic observation and fat tissue was patched to the dural defects. Follow-up computed tomography proved complete disappearance of air from the cisterns 2 weeks after the surgery, and the patient was discharged from our hospital without any neurological deficits.A skull base tumor was identified on magnetic resonance imaging in a 78-year-old Asian woman with a headache in her forehead. An endoscopic transnasal tumor resection was performed; however, the tumor, invading into the cavernous sinuses and optic canal, was not completely removed. During the subtotal resection of the tumor, no cerebrospinal fluid leakage was observed. Osteosarcoma was histologically diagnosed, and CyberKnife® radiation is one of the effective treatments for skull base tumors; however, the risk of cerebrospinal fluid leakage should be considered when tumor invasion to the dura mater is suspected. Emergency surgical treatment is required when cerebrospinal fluid leakage is induced by the radiotherapy because the leakage is not expected to be healed by palliative treatments.CyberKnife Irradiation is well indicated for unresectable skull base tumors. Fractionated CyberKnifeull base . It is cull base -4. Here ® radiation was applied 1 month after the surgery. The treatment was performed on the basis of the MRI planning  demonstrated an enhanced mass in the sphenoid and ethmoid sinuses with invasion into cavernous sinuses Figure A. The skg Figure A with th® radiation is safer and more effective than conventional radiation or gamma knife treatment [® treatment shrank the osteosarcoma without causing visual deterioration. However, critical CSF leakage occurred. As shown in Figure ® irradiation, and the residual tumor protected the patient from CSF leakage. After the treatment, the irradiated tissue became hypovascular and necrotic change was identified in the residual tumor progressing to the dural defects. As another cause of the CSF leakage, it might be possible that the authors injured dura mater at the sella during the removal of the tumor because the border between the tumor and the dura was not observable at some points. And this pre-existing iatrogenic dural defect caused CSF leakage when the dura shrank in CyberKnife® therapy. To the best of our knowledge this is the first reported case of CSF leakage caused by CyberKnife® therapy, although a few cases of post-radiation CSF leakage were reported in gamma-knife treatment for skull base tumors [® treatment is very effective for skull base tumors; [In skull base tumors, the portions where surgical resection is difficult are also areas where radiation therapy is challenging, such as the cavernous sinus or optic canal, which include cranial nerves and arteries. Cranial nerve palsies and internal carotid artery stenosis caused by external radiation have been reported ,7. To prreatment . In our e tumors -10. Ther tumors; -4 howeve tumors; , should ® radiation should be considered when tumor invasion to the dura mater is recognized. Emergency surgical treatment is required when CSF leakage is identified because the leakage is not healed with conservative treatment.The risk of CSF leakage caused by CyberKnifeWritten informed consent was obtained from the patient for publication of this manuscript and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.CSF: Cerebrospinal fluid; CT: Computed tomography; MRI: Magnetic resonance imaging.The authors declare that they have no competing interests.SMY was a major contributor in writing the manuscript. YI, SMY, and SY performed surgery and analyzed the data. YI and YG accomplished emergency repair of CSF leakage. MM, KH, and AM revised the manuscript with important intellectual content. All authors read and approved the final manuscript.


Trichoderma reesei. Previous reports suggested the presence of three MAPKs in T. reesei: Tmk1, Tmk2, and Tmk3. By exploring the phenotypic features of T. reesei Δtmk3, we first showed elevated NaCl sensitivity and repressed transcription of genes involved in glycerol/trehalose biosynthesis under higher osmolarity, suggesting Tmk3 participates in high osmolarity resistance via derepression of genes involved in osmotic stabilizer biosynthesis. We also showed significant downregulation of genes encoding chitin synthases and a β-1,3-glucan synthase, decreased chitin content, ‘budded’ hyphal appearance typical to cell wall defective strains, and increased sensitivity to calcofluor white/Congo red in the tmk3 deficient strain, suggesting Tmk3 is involved in cell wall integrity maintenance in T. reesei. We further observed the decrease of cellulase transcription and production in T. reesei Δtmk3 during submerged cultivation, as well as the presence of MAPK phosphorylation sites on known transcription factors involved in cellulase regulation, suggesting Tmk3 is also involved in the regulation of cellulase production. Finally, the expression of cell wall integrity related genes, the expression of cellulase coding genes, cellulase production and biomass accumulation were compared between T. reesei Δtmk3 grown in solid state media and submerged media, showing a strong restoration effect in solid state media from defects resulted from tmk3 deletion. These results showed novel physiological processes that fungal Hog1-type MAPKs are involved in, and present the first experimental investigation of MAPK signaling pathways in T. reesei. Our observations on the restoration effect during solid state cultivation suggest that T. reesei is evolved to favor solid state growth, bringing up the proposal that the submerged condition normally used during investigations on fungal physiology might be misleading.The mitogen-activated protein kinase (MAPK) pathways are important signal transduction pathways conserved in essentially all eukaryotes, but haven't been subjected to functional studies in the most important cellulase-producing filamentous fungus  Trichoderma reesei. Physiological investigations of this so-called industrial cellulase-producing ‘workhorse’ are therefore essential in both scientific and technological regards The systematic risk inherent to a fossil fuel based economy has drawn worldwide attention, leading to the proposal of replacing these non-renewable energy sources with renewable energy sources including energy from renewable biomass. One such biomass is lignocellulose, which is abundantly available as agricultural or forestry wastes. Conversion of lignocellulose to energy sources that are compatible to modern industry is a complicated process, in which the most important step is the saccharification of lignocellulose, catalyzed by cellulases produced from filamentous fungi Saccharomyces cerevisiae, in which six MAPK pathways were identified Cells sense their surrounding environments and react to external signals via signal transduction pathways. Among all known signal transduction pathways, the mitogen-activated protein kinase (MAPK) pathway is ubiquitous in almost all eukaryotic species and is one of the most well characterized pathways high-osmolarity glycerol) pathway in S. cerevisiae is involved in combating high osmolarity by activating genes required for viability under hypertonic stress, which include genes in the glycerol synthesis pathway encoding glycerol-3-phosphate dehydrogenase (GPD1) and glycerol–3-phosphatase (GPP2) Aspergillus nidulansMagnaporthe griseaCryphonectria parasiticaNeurospora crassaTrichoderma harzianumAspergillus fumigatusThe HOG (A. fumigatusMagnaporthe oryzaeTrichoderma virensClaviceps purpurea and Fusarium graminearumMycosphaerella graminicola (Mgslt2) and Botrytis cinereahog1 homologues in T. harzianum and A. fumigatus showed unchanged or even increased resistance to CFW and CR, suggesting the hypersensitivity to improved osmolarity is unrelated to weakening of the cell wall Investigations carried out on Slt2p homologues in filamentous fungi suggested the involvement of this MAPK in the maintenance of cell wall integrity in tmkA (a fus3 homologue) deletion strain of T. virenstmk1 (a fus3 homologue) knockout strain of Trichoderma atrovirideN-acetylglucosaminidase- and chitinase-encoding genes increased when tvk1 (a fus3 homologue) was disrupted in T. virensTrichoderma and Aspergillus species, as they are well-known industrial cellulase hyper-producing strains, out of which T. reesei is the most widely studied and used.In filamentous fungi, yeast Fus3p homologues are involved in quite diverse processes, one of which is the production of glycoside hydrolases. Cellulase and chitinase production was upregulated in the T. reesei (syn. Hypocrea jecorina) was first isolated on the Solomon Islands during World War II, and has received considerable improvements over the last seventy years for industrial applications 2+-signaling pathways and factors such as casein kinase II, germinal center kinases and protein kinase C.In silico reconstruction of the MAPK signal transduction cascade in T. reesei identified three distinct pathways, in which three putative MAPKs are involved: the yeast Fus3p homologue Tmk1, Slt2p homologue Tmk2, and the Hog1p homologue Tmk3 T. reesei yet. In this study, by characterizing the properties of the T. reesei Δtmk3 deletion strain, we attempt to identify the role of Tmk3 in T. reesei, particularly in cell wall integrity maintenance and cellulase production that have not been identified for Hog1-type MAPKs in other fungal species. Further comparison between submerged and solid state growth leads to the finding of novel, interesting restoration effects during solid state cultivation. These studies are the first research done on MAPKs in T. reesei, leading up to further in-depth understanding of the regulatory mechanisms of this well-known industrial cellulase hyper-producing workhorse.T. reesei Δku70 strain, derived from the QM9414 uridine auxotrophic pyr4-negative strain TU-6 (ATCC MYA-256), was used as the high transformation efficiency parent strain for the gene deletion experiment, as is previously reported p-nitrophenyl-β-D-glucopyranoside (pNPG), p-nitrophenyl-β-D-cellobioside (pNPC), carboxymethylcellulose (CMC), cytohelicase from Helix pomatia and chitinase from Streptomyces griseus were purchased from Sigma-Aldrich Corporation . p-nitrophenyl-β-D- xylopyranoside (pNPX) was purchased from Tokyo Chemical Industry Co., Ltd. . Congo red was purchased from Tianjin Damao Chemical Reagent Factory . Wheat bran was kindly provided by Longlive Bio-Technology Co., Ltd., Yucheng, Shandong, China. All other chemicals were purchased from Sinopharm Chemical Reagent Co., Ltd. .Phylogenetic tree construction and protein sequence comparison were carried out using the Clustal X2 software T. reesei Δtmk3 was carried out essentially as previously described pyr4 selection marker which restores uridine biosynthesis capabilities in the pyr4-deficient parent strain. Amplification of tmk3 from T. reesei genome was carried out using Kod FX high fidelity enzymes .Construction of T. reesei Δtmk3 was successfully constructed. Genomic DNA extracted from T. reesei Δku70 or Δtmk3 strain was digested using HindIII prior to hybridization. Detection of probe-hybridized DNA fragment was carried out using the DIG High Prime DNA Labeling and Detection Starter Kit I .Southern blotting experiment was used to confirm whether 4SO4, 0.06% MgSO4, 1.5% KH2PO4, 0.08% CaCl2, 0.00005% FeSO4·7H2O, 0.00016% MnSO4·H2O, 0.00014% ZnSO4·7H2O and 0.00002% CoCl2 was first prepared. Spores of T. reesei Δku70 or Δtmk3 strain were harvested after 6 days of growth, and counted using a hemacytometer. For submerged growth, approximately 107 spores were inoculated in submerged media containing 2 g avicel, 2 g wheat bran and 100 ml minimal media solutions. The media for growth of T. reesei Δku70 contain 0.1% uridine. The growth of 100 ml submerged cultures took place in 500 ml flasks at 30°C in a rotary shaker  rotated at 200 rpm. Solid state media contain 6 ml minimal media solution, 2 g avicel and 2 g wheat bran. Approximately 107 spores were inoculated to glass plates containing 10 g solid state media. The plates were subsequently incubated in an incubator  at 30°C without shaking.Minimal media solution containing 0.5% NH6 conidiospores of T. reesei Δku70 or Δtmk3 strain were inoculated on minimal media (MM) plates containing glucose, starch, sucrose, lactose or glycerol, as well as PDA plates. The plates were incubated in an incubator  at 30°C for 4 days. Double-layer avicel plates were prepared by first casting a MM agarose bottom layer containing no carbon sources, and a second MM agarose top layer containing 1% avicel. Approximately 106 conidiospores of T. reesei Δku70 or Δtmk3 strain were inoculated on the plates, which were incubated at 30°C for 4 days prior to examination.Approximately 10T. reesei Δku70 or Δtmk3 hyphae were taken with a bright field microscope  from cultures grown in submerged minimal media containing glucose as the carbon source.Images of 6 spores of T. reesei Δku70 or Δtmk3 strain were inoculated on PDA plates containing various concentrations of NaCl, CFW and CR for sensitivity tests on these chemicals. These plates were subsequently incubated in an incubator  at 30°C for 3 days. The dosages of NaCl used in this assay are respectively 0, 0.3, 0.5, 0.7, 0.9, 1.1 M. The dosages of CFW used in this assay are respectively 0, 10, 20, 30, 40 mg/L. The dosages of CR used in this assay are respectively 0, 75, 125, 150, 175, 200 mg/L. The diameters of colonies on CR- and CFW-containing plates were measured for comparison between T. reesei Δku70 and Δtmk3. Three individual replicates of each experiment were performed.Approximately 10The ATP level was assayed using the Checklite 250 plus ATP kit . The concentration of ATP indicates the average ATP concentration in cultures. Five individual replicates were carried out for ATP level analysis in solid state cultures. Three individual replicates were carried out for ATP level analysis in submerged cultures.T. reesei Δku70 or Δtmk3 culture was also used as a measure of biomass. To measure the DNA content, solid state and submerged cultures were first prepared as is described in this work. For submerged cultures, 1 ml of the culture was drawn from flasks for DNA content determination. For solid state cultures, 25 ml of distilled water was used to suspend the cultures, and 1 ml of suspended culture was drawn for DNA content determination. The cultures were subsequently diluted 5-fold in 15 ml centrifuge tubes, and were subjected to a brief centrifugation to remove supernatant. One milliliter of 10% trichloroacetic acid (TCA) was then added to the tube, thoroughly mixed, and incubated on ice for 3 minutes. The tube was boiled in a water bath  for 30 minutes, followed by centrifugation at 10,000 rpm for 10 minutes. Absorbance at 260 nm in the supernatant was subsequently measured using a UV-visible spectrophotometer . The total DNA content in the cultures was calculated from A260. Three individual replicates were carried out for each assay.The DNA content of a pNPC, CMC, pNPG and pNPX following published protocols pNPCase, CMCase, pNPGase and pNPXase activities in solid state cultures. Three replicates were performed to determine extracellular protein concentrations, pNPCase, CMCase, pNPGase and pNPXase activities in submerged cultures.Protein concentration was determined using the Lowry method T. reesei Δku70 and T. reesei Δtmk3 were assayed similarly to previous reports 6 spores of T. reesei Δku70 or Δtmk3 strain were inoculated in submerged media containing 2% glucose. The mycelia were harvested after 3 days of growth, and subsequently dried by heating at 105°C for 4 hours in an oven  for the determination of dry cell weight (DCW). Sixty mg of mycelia (14 mg DCW) of both T. reesei Δku70 or Δtmk3 strain were heated at 80°C for 90 minutes in 1 ml of 6% KOH using a water bath , followed by addition of 0.1 ml glacial acetic acid. The treated mycelia were further centrifuged in a microcentrifuge  at 13,000 rpm for 10 minutes. The pellet was suspended in 0.5 ml phosphate buffer (pH 6.3) and digested using 0.1 U chitinase by incubation at 37°C for 1 hour. The reaction system was centrifuged again at 13,000 rpm for 10 minutes. Zero point two five mg of cytohelicase was subsequently added to 500 μl supernant, followed by incubation at 37°C for 1 hour. The amount of released N-acetylglucosamine was assayed according to previous reported procedures Chitin contents of T. reesei Δku70 or Δtmk3 strain growing in solid state media and submerged media containing 2% avicel and 2% wheat bran for the examination of expression of cellulase-, hemicellulase-, chitin synthase- and β-1,3-glucan synthase-coding genes, as well as from T. reesei Δku70 or Δtmk3 strain growing in submerged media containing 2% glucose and 0 or 0.15 M NaCl for the examination of expression of glycerol-3-phosphate dehydrogenase- and α,α -trehalose-6-phosphate synthase-coding genes. cDNA was synthesized using PrimeScript RT reagent kit with gDNA erase  from Takara Bio Inc. .Total RNA was extracted from TM II  as the dye. Three individual biological replicates and three individual technical replicates for each biological sample  were carried out. The relative abundance of genes was calculated using the 2−ΔΔCt method as previously described Real-time PCR reactions were carried out on a LightCycler 480II Real-Time PCR system  using SYBR Premix EX TaqT. reesei and other previously characterized MAPKs showed Tmk3 apparently cluster with Hog1-type MAPKs from other species  The growth of T. reesei Δtmk3 on each minimal media plate containing a tested carbon source  is significantly worse than the parent strain; 2) Only slightly slower growth was observed when T. reesei Δtmk3 was grown on complete media (PDA plates); 3) a significantly larger transparent zone was observed around the colony of T. reesei Δtmk3 in comparison to the parent strain, when the two strains were grown on double layer plates containing avicel  cellobiohydrolase is the most abundant enzyme in secreted T. reesei cellulases T. reesei Δtmk3 can be interpreted primarily as the increase of cellobiohydrolase secretion.The larger transparent zone around the colony on avicel-containing double layer plate is an indication of improved avicelase production in T. reesei parent and Δtmk3 strains was also compared during submerged cultivation. The levels of ATP production and DNA content were used as measures of biomass accumulation. As is shown in tmk3-deficient strain, suggesting the important role Tmk3 plays in vegetative growth.Growth of T. reesei parent and Δtmk3 strains to elevated osmotic pressure by growing them on plates containing various concentrations of NaCl. It appears that T. reesei Δtmk3 colonies ceased to develop at the NaCl level of 0.5 M, while the parent strain can still grow in the presence of 0.9 M NaCl  and five α,α-trehalose-6-phosphate synthase-coding genes  functioning in trehalose biosynthesis. Unlike in S. cerevisiae, the upregulation of these genes in response to improved osmolarity is not apparent in T. reesei parent strain  are present in the genome of T. reesei. The transcriptional abundance of these genes in T. reesei parent and Δtmk3 strains was investigated with real-time PCR. The transcription of all these genes except for Trire2_67600 was detected, and all the detected genes except for Trire2_55341 are significantly (2∼8 folds) downregulated in T. reesei Δtmk3 (T. reesei parent strain (4.19±0.07 percent DCW) than in T. reesei Δtmk3 strain (3.48±0.07 percent DCW).The fungal cell wall is primarily composed of proteins and polysaccharides including chitin, β-1,3-glucan and β-1,6-glucan ei Δtmk3 , suggestT. reesei. Interestingly, instead of Hog1-type MAPKs, Slt2-type MAPKs were reportedly involved in cell wall integrity maintenance (as is summarized in the introduction section). The unaltered or even increased resistance to CR and CFW in hog1 deletion strains of T. harzianum and A. fumigatus further suggested Hog1 is not involved in cell wall integrity pathways in these two filamentous fungi T. reesei Δtmk3 hyphae in minimal media is similar to that of A. fumigatus ΔmpkA (MpkA is a Slt2 homologue in A. fumigatus) hog1 deletion strains of M. grisea and A. nidulans when exposed in high salt media due to their incapability to cope with high osmotic pressure T. reesei Δtmk3 and other filamentous fungi in which hog1 homologues were inactivated T. reesei Δtmk3 even under low osmolarity.Our results reported in this study clearly suggest the involvement of Tmk3 in the maintenance of cell wall integrity in T. reesei parent and Δtmk3 strains under submerged cultivation conditions showed significantly decreased production of FPA, extracellular protein, pNPCase activity that measures cellobiohydrolase activity, CMCase activity that measures endoglucanase activity, pNPGase activity that measures β-glucosidase activity, and pNPXase activity that measures β-xylosidase activity in T. reesei Δtmk3 , endoglucanase I (EGI), endoglucanase II (EGII), β-glucosidase I (BGI) and β-xylosidase I (BXL1) (The comparison of cellulase production from ei Δtmk3 . TranscrI (BXL1) .T. reesei Δtmk3 cannot be solely explained by the reduced biomass accumulation, because the transcriptional levels of cellulase- and hemicellulase-coding genes are clearly downregulated. These observations lead to the suggestion that Tmk3 is involved in cellulase production and/or induction. In T. reesei, several transcription factors were shown to regulate cellulase production, including the activator Xyr1 T. reesei, and the phosphorylation specificity of the transcription factors has not been identified.The decrease in cellulase and hemicellulase production in T. reesei physiology is that the way T. reesei is cultured in these investigations does not resemble their natural habitats. In nature, T. reesei grows on solid lignocellulosic particles, instead of in submerged media which are normally used for T. reesei cultivation during physiological studies. There are considerable differences between these two environments: the level of encountered moisture varies significantly; the exposure to oxygen is different; and in the natural environment, secreted proteins do not diffuse like during submerged cultivation. We therefore compared the performance of T. reesei Δtmk3 in submerged media and solid state media, in order to further identify the role of Tmk3 in T. reesei.One common problem on the studies of T. reesei Δtmk3 was grown on solid state media , as well as extracellular and cytoplasmic factors T. reesei Δtmk3 grown during solid state cultivation showed a clear restorative effect in comparison to submerged cultivation. The molecular mechanisms underlying these effects vary on different aspects of cellular physiology, but the unified final restorative effect suggests T. reesei physiology is evolved to favor solid state cultivation. Although secreted proteins or metabolites can be produced more efficiently from filamentous fungi during submerged cultivation, therefore benefiting industrial application, the submerged condition might actually alter fungal growth and metabolism, and care needs to be taken when assumptions are made that growth under certain submerged growth conditions is ‘optimum’ or ‘physiological’, particularly during mechanistic studies on fungal physiology.Our studies of T. reesei. Although sequence comparison suggests Tmk3 is homologous to Hog1p from S. cerevisiae, their functions appear to vary significantly. While Hog1p from S. cerevisiae and Tmk3 from T. reesei are both involved in the resistance to high osmolarity, the mechanism of this function differs: induction of osmotic stabilizer resistance genes in S. cerevisiae and derepression of these genes in T. reesei. Further investigations showed the function of Tmk3 in more novel aspects. Our phenotypic analysis, chemical sensitivity studies and transcriptional profiling all suggested Tmk3 functions in the cell wall integrity signaling pathway similarly to Slt2-type MAPKs, a role never identified for Hog1-type MAPK in filamentous fungi before. We further observed that deletion of tmk3 leads to an apparent decrease in cellulase transcription, and suggested the involvement of Tmk3 in cellulase production regulation by phosphorylating transcription factors.Results reported herein lead to the suggestion of the physiological roles in high osmolarity resistance, cell wall integrity maintenance and cellulase production of a Hog1-type MAPK Tmk3 in T. reesei were significantly reduced upon tmk3 deletion. The degree of reduction was much smaller after transferring to solid-state media. The mechanism of the restoration of growth likely differs from the mechanism of the restoration of cellulase production, but the similarity between the effects suggests T. reesei is evolved to favor solid state cultivation.When grown in submerged media, biomass accumulation and cellulase production of In conclusion, our results show the participation of Tmk3 in high osmolarity resistance and two novel aspects: cell wall integrity and cellulase production regulation. The restorative effect identified during solid cultivation, particularly regulation of cellulase production, is worthy of further investigations for the identification of other regulatory pathways involved in cellulase production.Figure S1Trichoderma harzianum;Trtmk3, Tmk3 from Trichoderma reesei; Ncos2, Os-2 from Neurospora crassa; Cpmk1, Mk1 from Cryphonectria parasitica; AfsakA, SakA from Aspergillus fumitagus; AnhogA, HogA from Aspergillus nidulans; TvtmkB, TmkB from Trichoderma virens; Trtmk2, Tmk2 from T. reesei; Fgmgv1, Mgv1 from Fusarium graminearum; Bcbmp3, Bmp3 from Botrytis cinerea; Cpmk2, MK2 from Claviceps purpurea; AfmpkA, MpkA from A. fumigatus; TvtmkA, TmkA from T. virens; Tvtvk1, Tvk1 from T. virens; Trtmk1, Tmk1 from T. reesei; Tatmk1, Tmk1 from Trichodera atroviride.Phylogenetic analysis of MAPKs from filamentous fungi. The phylogenetic tree was constructed by the neighbour-joining method. Bootstrap values are shown at each note, and are calculated from 1000 trees. Bar, evolutionary distance of 0.1. Thhog1, Hog1 from (TIF)Click here for additional data file.Figure S2T. reesei and Hog1 from Saccharomyces cerevisiae. Trtmk3, Tmk3 from T. reesei; Schog1, Hog1 from S. cerevisiae.Sequence alignment of Tmk3 from (TIF)Click here for additional data file.Figure S3T. reesei parent and Δtmk3 strains. M, DNA molecular size marker; Δtmk3, T. reesei Δtmk3; parent, T. reesei parent strain. Indicated by arrows are the predicted sizes of DNA fragments hybridized with the probe.Southern blotting analysis of (TIF)Click here for additional data file.


CD14To study the effect of GM-CSF, IL-4 and endothelin -1 (ET-1) alone or in combination on monocyte differentiation into myofibroblasts.+ cells were isolated from peripheral blood from 14 SSc patients and healthy controls by positive selection and incubated with different combinations of GM-CSF, IL-4 and ET-1 for 14 days. Type-1 collagen and α-SMA were detected by Western blot, qPCR and confocal microscopy. HLA-DR, CD11c and CD14 expression was analysed by flow cytometry. A collagen gel contraction assay was performed for functional myofibroblast assessment.CD14GM-CSF both induced collagen and α-SMA expression after 14 days. ET-1 further increased GM-CSF-induced collagen expression in a dose dependent manner up to 30-fold. IL-4/GM-CSF combination leads to a more DC-like phenotype of monocytes associated with reduced collagen and α-SMA expression compared to GM-CSF alone. Collagen and α-SMA expression was higher in monocytes from SSc patients and monocytes were more prone to obtain a spindle form. In contrast to controls, ET-1 and IL-4 alone were sufficient to induce α-SMA expression in monocytes from SSc patients. Despite the induction of α-SMA expression, monocyte-derived myofibroblasts only had a moderate capability of contraction in functional analyses.SSc monocytes display increased maturation towards myofibroblasts demonstrated by their phenotype and α-SMA expression when compared to monocytes from healthy controls, however only with minor functional contraction properties. Apart from their well known capacity to differentiate into dendritic cells (DC) and macrophages, they also have the potential to differentiate into collagen-producing fibrocytes + monocyte numbers are increased, with an activated status demonstrated by their expression of CD68, CD204 and Siglec-1 +CD34+ fibrocytes have been found in higher numbers in lungs of patients with SSc-associated interstitial lung disease Systemic sclerosis (SSc) is an autoimmune multisystem connective disease where low grade inflammation and vasculopathy lead to increased extracellular matrix (ECM) deposition in affected tissues, notably the skin, lungs and gastrointestinal tract Myofibroblasts are key effector cells in fibrosis by their capacity to synthesize collagen and contractile α-smooth muscle actin (SMA). Myofibroblasts differentiate either directly from fibroblasts e.g. under the influence of TGF-beta or Endothelin-1 (ET-1) in vitro monocyte survival, proliferation and as trigger of macrophage differentiation, whereas the combination with IL-4 drives differentiation of monocytes into DC In vitro culture of monocytes in the presence of GM-CSF for a prolonged time stimulates the formation of CD14low cells with a DC morphology and high viability, expressing HLA-DR, CD86 and CD11a but low CD1a Granulocyte-macrophage colony-stimulating factor (GM-CSF) is the main differentiation factor for monocytes ET-1 plays an important pathogenic role in SSc. ET-1, which is highly expressed in vessels, acts as vasoconstrictor but is also known for its function as differentiation factor We hypothesised that activated monocytes from SSc patients have a dysregulated differentiation potential upon stimulation with GM-CSF, IL-4 and also ET-1 into ECM producing, myofibroblast-like cells, thereby participating in fibrosis.Peripheral blood was collected from 14 patients with limited (n = 9) or diffuse (n = 5 ) SSc . The mean disease duration was 8.6 years (range 2–20 years). Six patients were on treatment with immunosuppressive medication. Patient characteristics are summarized in 6 cells per ml and treated for 14 days with 50 ng/ml GM-CSF, 50 ng/ml IL-4  or 0–500 ng/ml ET-1 . The culture medium was replenished every 3 days. Light microscopy was used to observe morphological changes. For quantification, spindle shaped cells per 5×5 cm field were counted and normalized to the cell number.PBMCs were isolated from peripheral blood from SSc patients or anonymised healthy controls (n = 8) using Lymphoprep™ . Monocytes were isolated from the PBMC fraction using CD14 microbeads . Monocytes were seeded into tissue culture plates in a density of 1×10Total cell extracts were prepared in 20 mM Tris (pH 7.5), 1 mM EDTA, 100 mM NaCl, 1% Trition X-100, 0,5% Desoxycholate, 0,1% SDS and Protease inhibtor cocktail . Protein extracts were subsequently separated by SDS-PAGE on 4–20% gradient gels  and blotted on PVDF membranes. To detect proteins, Western blot analysis was performed using anti-type 1 collagen, anti-alpha SMA , anti-vimentin (DAKO) and anti-GAPDH antibodies . Western Blots were quantified by using ImageJ software.5 monocytes were seeded in 8-well Culture slides  and cultured for 14 days. After fixation with 4% Formaldehyde and permeabilisation with 0.02% Triton X-100 cells were blocked in 10% goat serum. Specimens were stained with anti-α-SMA  and 4′,6-Diamidine-2′-phenylindole dihydrochloride (DAPI)  and mounted in Mowiol 4–88 . For imaging of the slides a Leica SP2 AOBS UV confocal microscope with a 63× oil immersion objective was used.For immunfluorescence staining 1.5×105′-CAA GAG GAA GGC CAA GTC GAG G -3′, reverse 5′-CGT TGT CGC AGA CGC AGA T -3′). To quantify the relative expression levels of these genes, the expression of 18S mRNA was also determined in the same cDNA samples using a gene-specific primer pair (forward 5′ CGA ATG GCT CAT TAA ATC AGT TAT GG-3′ reverse 5′-TAT TAG CTC TAG AAT TAC CAC AGT TAT CC-3′).Quantitative assessment of mRNA expression levels was achieved by 2-step real-time PCR (qRT-PCR). Total RNA was extracted using the RNeasy Mini Kit . Reverse transcription of mRNA into cDNA from 0.5–1 µg of the extracted total RNA was conducted using the QuantiTect RT Kit (Qiagen). Subsequently, the cDNA was subjected to qRT-PCR in triplicate using the ABi HT 7900 system  and the nonspecific DNA binding dye SYBR green I for the detection of PCR products . Procollagen was amplified with gene-specific oligonucleotide primer pairs  and IL-4 by flow cytometry . IL-4 wi5 CD14+ monocytes were isolated from patients with SSc and healthy controls and incubated for 14 days according previous experiments in medium supplemented with 10% FCS and different combinations of ET-1, GM-CSF and IL-4. GM-CSF-induced α-SMA expression appeared higher in samples from SSc patients  . The addFor a morphological analysis, we stained healthy or SSc monocytes incubated with GM-CSF for 14 days with α-SMA antiserum for confocal microscopy. We found that monocytes from SSc patients expressed more α-SMA and obtained a more spindled shape than monocytes from healthy controls . After n5 CD14+ monocytes on collagen gels under before mentioned conditions with GM-CSF, IL-4 or ET-1 . In our experiments, GM-CSF, IL-4 or ET-1 stimulation led to a different expression pattern between type-1 collagen and α-SMA. Further studies are needed to determine whether this reflects outgrowth of different cellular subsets. To confirm the functional relevance of monocyte differentiation we performed contractility assays which showed that monocyte-derived myofibroblasts result in a maximum of 30% contraction of collagen gelx, compared to 5% by the untreated monocytes. In comparison with a 95% contraction by fibroblasts, the contractile property of monocyte-derived myofibroblast is modest. We also could not detect a clear difference between monocyte derived myofibroblasts from SSc patients and healthy individuals, respectively. Possible explanations for this partial effect could be the lower size of the myofibroblasts compared to fibroblasts with an impaired fiber architecture and the necessity of a further contractile stimulus after the differentiation process. However, this finding also shows that the differentiation process from monocytes into myofibroblast under the tested conditions is not complete and that it might require additional stimulation or further differentiation in vivo.Phenotype analysis by flow cytometry revealed that monocytes cultured with IL-4 express higher of levels HLA-DR, and slightly higher levels of CD11c. CD14Nevertheless, our findings do show a potential of monocytes to differentiate into spindle formed myofibroblasts with a higher expression of matrix proteins which suggest that albeit reduced functional properties, these cells are involved in SSc associated fibrosis, vasculopathy or skin contractions and that notably GM-CSF is a key player in this differentiation process. In future studies, the differentiation potential of monocytes in SSc has to be characterized by functional analyses and compared with the histological analysis and serum levels of GM-CSF, ET-1 and IL-4 in the same individuals.Figure S1GM-CSF upregulates Vimentin in monocytes from SSc patients and healthy controls. Monocytes from healthy controls or SSc patients were cultured for 14 days with GM-CSF, IL-4 or ET-1. Vimentin levels were detected by Western blot analysis. GAPDH was used as loading control.(TIF)Click here for additional data file.


With the establishment of such an in vitro model of urothelial cells with fully developed urothelial plaques and functional properties equivalent to normal bladder urothelium, new perspectives have emerged which challenge prevailing concepts of apical plasma membrane biogenesis and blood-urine barrier development. This may hopefully provide a timely impulse for many ongoing studies and open up new questions for future research.The primary function of the urothelium is to provide the tightest and most impermeable barrier in the body, i.e. the blood-urine barrier. Urothelial plaques are formed and inserted into the apical plasma membrane during advanced stages of urothelial cell differentiation. Currently, it is supposed that differentiation with the final formation of urothelial plaques is hindered in cultured urothelial cells. With the aid of the high-resolution imaging technique of freeze-fracture replica immunolabelling, we here provide evidence that urothelial cells  Urothelial plaques are ultrastructurally distinctive, highly ordered structures made of crystalline arrays of 16-nm hexagon shaped protein particles consisting of four integral membrane proteins called uroplakins i.e. UPIa, UPIb, UPII and UPIIIa Although uroplakins were previously found in cultured UCs In view of these conflicting data, we have now sought detailed evidence for or against the presence of urothelial plaques positive for uroplakins in cultured UCs by using the high-resolution imaging technique of freeze-fracture EM combined with immunogold labelling, i.e. freeze-fracture replica immunolabelling (FRIL). Furthermore, the ultrastructural differentiation of cultured UCs was determined by thin section and scanning EM and the barrier function was assessed by measurement of transepithelial electrical resistance (TER).in vivo. Our findings illustrate the recent impact of the FRIL technique in demonstrating the localization of uroplakins and the structure and formation of urothelial plaques in UCs both in vivo and in vitro.Using these different experimental approaches, we show that in long-term cultures the UCs form an urothelium, in which the superficial UCs express uroplakins and form uroplakin-positive urothelial plaques indistinguishable from those of superficial UCs 2+ concentration was 2 mM. Normal UCs, gently scraped with a scalpel blade from the bladder of an adult male mouse, were used as a control, i.e. UCs in vivo.The experiments were approved by the Veterinary Administration of the Slovenian Ministry of Agriculture and Forestry (permit no. 34401-1/2010/6) in compliance with the Animal Health Protection Act and the Instructions for Granting Permits for Animal Experimentation for Scientific Purposes. Urinary bladders were obtained from adult male mice; strain ICR CD1. Bladders were handled aseptically and immediately immersed in medium for urothelial cells, i.e. UroM consisting of equal parts of MCDB 153 medium  and Advanced-DMEM medium  and supplemented with adenine (15 µg/ml), insulin (5 µg/ml), hydrocortisone (0.5 µg/ml), phosphoetanolamine (0.1 M), glutamax (4 mM), streptomycine (100 µg/ml) and penicillin (100 µg/ml). The final Ca2, 10–12 explants for each mouse bladder), which were transferred onto 0.4 µm porous membranes . Each explant was orientated and spread out, so that the urothelium was on the upper side. Culture medium was introduced into the well containing the porous membrane, so that the urothelium was positioned at the air-liquid interface. Medium was changed daily with the exception of weekends. After 2 months, the molecular and ultrastructural status of UCs growing onto the porous membrane was analyzed.Primary mouse urothelial cultures were prepared as described previously th week, when the UCs reached confluence till the 17th week). Cultured urothelia were grown in medium UroM for 14 weeks. Then one-third of cultured urothelia was propagated in a medium supplemented with 2.5% fetal bovine serum  and two-thirds were propagated in the same medium for an additional 3 weeks. For measuring TER an epithelial voltohmmeter  and STX2 electrodes were used. The measured TER was corrected by subtracting the mean resistance of blank porous membranes (150 Ωcm2) and the results were expressed as Ωcm2 ± SE. Urothelial TER values were averaged and compared by a two-tailed Student's t-test; p values less than 0.05 were considered statistically significant.TER across the urothelia grown on porous membranes  was measured for 4 months  with 30 mM sucrose, pH 8.3, overnight at room temperature. The SDS removed the bulk of the biological material from the replica so that structure was visible by EM, leaving a single lipid monolayer and associated integral and surface proteins adherent to the replica  between the maximum calliper diameter of urothelial plaques in UCs in vivo  and in vitro . Variations in the number of uroplakin particles in the urothelial plaques were found in both UCs in vivo and in vitro. The immunogold label was confined to the E face of the apical PM  in the cytoplasm . DFVs arin vitro retain the capability for synthesis of the uroplakin particles and their arrangement into urothelial plaques.Urothelial plaques are unique and specific assemblies of highly differentiated superficial UCs composed of uroplakins in vitro established urothelia . TER measurements over the course of the 4 months revealed that urothelia grown in such culture conditions form “tight” epithelia. Urothelia grown in a medium UroM without FBS had a mean TER from 534 ± 20 Ωcm2 to 1165 ± 85 Ωcm2.In order to examine whether our rothelia  are “tigin vitro established long-term normoplastic urothelial cultures, the high TER is maintained by the well-developed cell junctions, and not with the uroplakin-positive urothelial plaques. This interpretation is consistent with the findings of Hu and co-workers Thin section and scanning EM of the long-term urothelial cultures clearly demonstrated well-developed tight junctions between superficial UCs . Our preth week. TER measurements revealed that addition of 2.5 % FBS induces the significant increase in TER (th to 17th week the urothelia grown with FBS had enhanced the barrier function. TER measurements over the course of the 3 weeks revealed a mean TER from 816 ± 22 Ωcm2 to 1721 ± 45 Ωcm2.To evaluate the viability and functional responsiveness of the long-term urothelial culture, one-third of cultured urothelia was transferred into a medium supplemented with 2.5% FBS in the 14e in TER . From thin vitro model. Moreover, the findings support the idea that UCs in long-term urothelial culture retain their viability and still respond to extracellular signals, e.g. the added serum.These findings show the significant effect of serum on urothelial TER, which is in agreement with our recent study on the hyperplastic urothelial models in vitro has been achieved. Apart from positive labelling of uroplakins in cytoplasmic DFVs and apical PM, FRIL has also provided new insights into the dynamics of urothelial plaque formation. Among the key questions to be addressed in future studies are whether additional types of protein other than uroplakins are involved in urothelial plaque formation and how the final size of an urothelial plaque is determined and regulated.Our results have a number of applications. The established urothelial culture model can be used as a research tool for investigating the cellular-molecular mechanisms of apical PM biogenesis, which are crucial for development of the blood-urine barrier. The prevailing hypothesis proposes that differentiation with the final formation of urothelial plaques is hindered in cultured UCs. By revisiting the localization of uroplakins in cultured UCs with the FRIL technique, the first unequivocal evidence for uroplakin-positive urothelial plaques in UCs in vivo and in vitro. The information this approach provides is unique and we expect that further exploitation of the FRIL technique in future studies may advance our understanding of the biology of the blood-urine barrier.To sum up, the findings discussed illustrate the recent impact of the FRIL technique in demonstrating the localization of uroplakins and the structure and formation of urothelial plaques in UCs both


The cerebrospinal fluid (CSF) tap test (TT) has been regarded as an important test for the prediction of shunt effectiveness in patients with suspected idiopathic normal pressure hydrocephalus (iNPH). Although its specificity and sensitivity are reportedly high, there remains some disagreement over this point. Herein, the TT as a test for predicting shunt effectiveness was investigated in our multicenter prospective study named SINPHONI and strategies to increase its predictability were examined.One hundred suspected iNPH patients with the following entry criteria were enrolled in the study: (1) 60 to 85 years old, (2) one or more of the NPH triad signs, (3) ventriculomegaly (Evans index > 0.3), (4) high convexity tightness in coronal-section MRI, and (5) no antecedent disorders. Changes in NPH triad symptoms were assessed using the iNPH grading scale and other measures before and after removal of 30 ml lumbar CSF. A positive response to TT was pre-defined by specific improvements on the grading and other scales. A ventriculoperitoneal shunt was performed with a programmable valve. The sensitivity and specificity of the TT was calculated with a contingency table. A decision tree analysis was performed to increase the predictability of the TT.Among 100 patients, 80 were shunt responders. A statistically-significant variable between shunt responders and non-responders was CSF pressure. The changes in single variables in the iNPH grading scale after TT showed high specificity with low sensitivity. In contrast, change of the total score in the iNPH grading scale showed a relatively high sensitivity of 71.3% with specificity of 65%. A decision tree analysis revealed that using the iNPH grading scale total score and pre-shunt CSF pressure ≥ 15 cmH20, sensitivity increased to 82.5%, without a decrease in specificity.The sensitivity and specificity of the TT for predicting shunt responsiveness were optimum when improvement on any iNPH grading scale was combined with CSF pressure ≥ 15 cmH20. To increase the sensitivity of the TT, further effort is necessary.NCT00221091.This study is registered with ClinicalTrials.gov, with the number  Hakim and Adams first reported improvement of NPH symptoms by removal of 15 ml CSF using a lumbar tap [patients . Since tpatients -10. Becapatients ,12. The patients -6. Contipatients -17, but patients . SpecialThis study is registered with ClinicalTrials.gov, with the number NCT00221091.In 2004, a multicenter, prospective study of idiopathic normal pressure hydrocephalus (SINPHONI) was conducted in Japan . Briefly. The score of each domain ranges from 0 to 4. Grade 0 indicates normal and grade 1 indicates subjective symptoms but no objective disturbance. Grade 2, 3 and 4 indicate mild, moderate and severe disturbances, respectively. The change of gait was evaluated 1 or 2 days after the tap, while change of cognition and urination was evaluated at one week. Assessment was done by neurosurgeons in most cases. Response to the TT was pre-defined by three major scales: iNPHGS, TUG and MMSE. An improvement in one point or more on the iNPH grading scale , more than 10% improvement in time on TUG, or more than 3 points improvement in the MMSE was regarded as TT-positive. Improvement in any of the total scores of iNPHGS, TUG or MMSE was defined as positive with an additional variable of Tap-any. The sensitivity and specificity of these pre-defined variables as predictors of a response to shunt surgery were calculated. Furthermore, to increase predictability in the responders during clinical practice, a decision tree analysis was applied.A lumbar tap with removal of 30 ml of CSF was performed in all patients. CSF pressure (CSFP) was measured at the site of puncture. Before and after the tap, all patients were evaluated using the iNPH grading scale (GS) , the MinA ventriculo-peritoneal shunt with a Codman-Hakim programmable valve™ , with the initial pressure setting determined from a quick reference table  was instp < 0.05.Statistical analysis was performed using JMP statistical software version 9 . Statistical comparison was made between shunt responders and non-responders on baseline data, and pre-tap state of iNPHGS, TUG and MMSE Table . BaselinAmong the complete patient group, gait disturbance was noted in 91%, cognition disturbance in 80% and urination disturbance in 60%. The number of males vs. females was 58 vs. 42, and median age was 75  years. Median Evans index was 34.6 (range 32.7-38) and median CSFP at lumbar tap was 12 (range 9-14) cm H2O.p < 0.05 (Table p < 0.005). Among the non-responders, pneumonia was noted in three and surgery-related complications in two , while vascular events including cerebral and cardiac infarction in three and femoral fracture in two, occurred among the responders  is considered to be useful for predicting a favourable response to shunt surgery, particularly in iNPH patients. In previous studies, the volume of CSF removed has varied from 30 ml ,8, 40 ml24 hours . Improve2O was the best threshold for differentiation. This increased the sensitivity to 82.5%, while the specificity remained at 65%. This suggested that patients with higher CSF pressure would be shunt responders even if their symptoms did not improve by one point or more in the iNPHGS after TT.The sensitivity and specificity of the TT have been reported previously as ranging from 72% to 100% for the former and from 33% to 100% for the latter ,7,8. TheIn contrast with TT, continuous CSF drainage has been reported to provide higher sensitivity and specificity, ranging from 50% to 100% and from 60% to 100%, respectively ,14-16. Aet al. reported there were asymptomatic people with MRI features of iNPH in their population-based study [The SINPHONI study revealed high achievement in the treatment of iNPH patients without support of the TT . The SINed study . They ma20 increased the sensitivity up to 82.5% without a decrease in specificity. Thus, the TT is valid as an initial invasive test to predict the response to shunt for elderly patients having disturbances of gait, cognition and/or urination.The value of the TT for predicting shunt effectiveness was investigated in iNPH patients using the SINPHONI data. The sensitivity and specificity changed with different variables and improvement in any iNPH grading scale showed a sensitivity of 71.3% and specificity of 65%. A decision tree analysis revealed that any improvement on iNPHGS followed by inclusion of patients with CSFP higher than 15 cm HCogn: cognition; FN: false negative; FP: false positive; GS: iNPH grading; scale; iNPH: idiopathic normal pressure hydrocephalus; IQR: interquartile range; MMSE: Mini-Mental State Examination; SINPHONI: Study of idiopathic normal pressure hydrocephalus on neurological improvement; TN: true negative; TP: true positive; TT: tap test; TUG: timed up and go test; Urin: urination.This investigator-initiated study was supported in part by Johnson & Johnson K.K., Nihon Medi-Physics Co. Ltd, and Daiichi Pharmaceuticals Co. The funding sources for the study had no role in the design and conduct of the study, in the collection, analysis, and interpretation of the data, or in the preparation, review, or, approval of the manuscript. Drs. Ishikawa, Hashimoto, and Mori have received honoraria from companies that manufactured the devices discussed in this article, including Johnson & Johnson K.K. (Japan) and Nihon Medi-Physics Co. Ltd. Drs. Kuwana and Kazui have received honoraria from Johnson & Johnson K.K. (Japan).All authors had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. MI, MH and EM: drafting of the manuscript. NK and HK: critical revision of the manuscript for important intellectual content. All authors have read and approved the final version of the manuscript.


Human African trypanosomiasis progresses from an early (hemolymphatic) stage, through CNS invasion to the late  stage. In experimental infections disease progression is associated with neuroinflammatory responses and neurological symptoms, but this concept requires evaluation in African trypanosomiasis patients, where correct diagnosis of the disease stage is of critical therapeutic importance.T.b.rhodesiense HAT patients recruited in Eastern Uganda. Paired plasma and CSF samples allowed the measurement of peripheral and CNS immunoglobulin and of CSF cytokine synthesis. Cytokine and immunoglobulin expression were evaluated in relation to disease duration, stage progression and neurological symptoms. Neurological symptoms were not related to stage progression (with the exception of moderate coma). Increases in CNS immunoglobulin, IL-10 and TNF-α synthesis were associated with stage progression and were mirrored by a reduction in TGF-β levels in the CSF. There were no significant associations between CNS immunoglobulin and cytokine production and neurological signs of disease with the exception of moderate coma cases. Within the study group we identified diagnostically early stage cases with no CSF pleocytosis but intrathecal immunoglobulin synthesis and diagnostically late stage cases with marginal CSF pleocytosis and no detectable trypanosomes in the CSF.This was a retrospective study on a cohort of 115 rhodesiense HAT. Neurological signs are observed in both early and late stages, and while intrathecal immunoglobulin synthesis is associated with neurological signs, these are also observed in cases lacking a CNS inflammatory response. While there is an increase in inflammatory cytokine production with stage progression, this is paralleled by increases in CSF IL-10. As stage diagnostics, the CSF immunoglobulins and cytokines studied do not have sufficient sensitivity to be of clinical value.Our results demonstrate that there is not a direct linkage between stage progression, neurological signs of infection and neuroinflammatory responses in  Trypanosoma brucei rhodesiense and T.b.gambiense, is clinically defined by two diagnostic stages, an early stage where the parasites appear to be localised to the blood and lymphatic systems, and a late stage where the parasites are also localised in the central nervous system (CNS) and cause a meningoencephalitis, which is fatal if untreated. We studied the progression between these stages of infection in T.b.rhodesiense infections in Uganda. Progression from early to late stage was associated with an increase in inflammatory responses in the CNS as measured by analysis of the cerebrospinal fluid. However, contrary to predictions based on experimental model studies, neither disease stage progression nor CNS inflammatory responses were directly associated with development of neurological symptoms. Our results suggest that biological basis of the boundary between the two diagnostic stages in this infection may not be clear cut, with implications for therapeutic decision making.Human African trypanosomiasis, caused by the parasites  Trypanosoma brucei ssp. After inoculation of the parasite by the tsetse fly vector, the disease progresses through two stages. In the hemolymphatic, or early stage of disease, parasites proliferate in the blood and lymphatic system. In the meningoencephalitic, or late stage, parasites penetrate the blood brain barrier (BBB) and persist and proliferate in the CNS, causing an encephalitic reaction that leads to death if untreated or inadequately treated T.b.gambiense is endemic to West and Central Africa, with a chronic course of infection in which late stage may not commence for months or years after infection, and for which there is recent evidence for asymptomatic infection T.b.rhodesiense is endemic in East and Southern Africa, is distinguished by the SRA (serum resistance associated gene) and exhibits a more acute pattern of progression than T.b.gambiense, although there is considerable diversity in progression rate that may be related to parasite virulence variation and host immunogenetics Human African trypanosomiasis (HAT), also known as Sleeping Sickness, is caused by the protozoan hemoflagellate T.brucei infection demonstrate that dysregulated inflammatory responses are a major contributor to the pathophysiology of infection, both systemically Animal model studies of post-mortem material In humans, direct measurements of immune cell activation in the brain are not possible for obvious ethical reasons. While gross inflammatory pathology analogous to that observed in rodent models has been described in T.b.rhodesiense are currently treated with arsenical drugs that induce a severe and sometimes fatal reaction known as the post-treatment reactive encephalopathy (PTRE) in about 10% of treated patients, half of whom die as a result giving an overall drug mortality of 5% T.b.gambiense infection there is some evidence that this value is too low and that effective early stage treatment may still be administered in patients with up to 20 WBC/µl in the CSF. It has also been proposed that HAT cases with between 5 and 20 CSF WBC/µl fall into an intermediate stage category, regardless of whether trypanosomes are detected in the CSF T.b.gambiense infections Staging is critical to therapeutic decision making as late stage infections of T.b.rhodesiense HAT T.b.rhodesiense infection.As part of a study of the clinical evolution of This study was conducted according to the principles expressed in the Declaration of Helsinki. All patients recruited received written and verbal information explaining the purpose of this study and gave informed written consent. All protocols were approved by ethics committees in Uganda  and UK (Grampian Joint Ethics Committee). Ethical consent forms were designed in English and also translated into local languages. Consent was given as a signature or a thumb-print after verbal explanation. For those under 16 consent was given by their legal guardian, and for those whose clinical condition prohibited full understanding of the recruitment process, consent was gained from a spouse or other family member.115 HAT patients were recruited at Serere Health Centre, Serere District, Eastern Uganda, between August 2002 and July 2003. This cohort of cases was drawn from of a larger multi-centre study, for which study sites, recruitment protocols, treatment regimens, disease progression characteristics and clinical examination methods have been published elsewhere Staging was carried out in accordance with WHO criteria Plasma and lumbar CSF were collected from all patients as part of routine diagnostic and stage determination procedures. Paired plasma and CSF samples were frozen in liquid nitrogen within 1 h of collection, and maintained in liquid nitrogen until required for analysis.Control plasma and CSF samples for cytokine analysis were obtained from 17 HAT suspects at Serere Health Centre and 18 HAT suspects at the LIRI Health Centre, Tororo, Uganda respectively who were all later diagnosed as non-infected. In some assays, limitations on the volume of plasma and CSF available meant that a subset of patient samples that was effectively randomly selected was analysed. Cytokines IFN-γ, IL-6 and IL-10 concentrations were measured in CSF using a solid phase analyte capture sandwich ELISA  as previously described in QALB). The cut off for dysfunction was calculated using the formulaLOC) was evaluated using the method of Reiber QLIM defines the upper limit of the immunoglobulin quotient (Q(Ig)) in the absence of intrathecal Ig synthesis. Intrathecal Ig synthesis results in a Q(Ig) lying above QLIM. The level of intrathecal synthesis of each isotype (IgLOC) is derived from the formulaBlood-CSF barrier function was evaluated using the albumin quotient (s). Diagnostic outcomes as dependent variables were tested on cytokine concentrations using logistic regression, and diagnostic power was assessed using receiver operating characteristic analysis. Diagnostic panel candidates were selected using mixed stepwise logistic regression and optimal diagnostic cut offs were determined using the prediction profiler in JMP6.0 .None of the continuous variable parameters examined could be transformed to normality. Therefore differences between groups were tested using the Mann-Whitney U-test, or the Kruskal Wallis test followed by Dunn's post-hoc test. Bivariate correlations were evaluated using Spearmann's rank correlation coefficient . Also, as would be expected given that CSF trypanosome and WBC are diagnostic criteria, both were significantly higher in late stage cases compared to early stage cases.Bloodstream parasitemias were scored on thick blood films. Late stage median parasitemia was lower than in early stage, but this difference was not significant . HoweverALB), blood brain barrier (BBB) dysfunction was indicated in 6% of early stage cases and 42% of late stage cases.Total CSF protein was significantly higher in late stage cases compared to early stage cases and also increased in relation to duration of disease . Plasma Lim) Plasma IgG and IgA levels  did not s = 0.41 p<0.001). There was no significant difference between early and late stage cases for IFN-γ (s = 0.46 p<0.01).The CSF IL-10  concentror IFN-γ . TNF-α lor IFN-γ  in all cor IFN-γ  were sigor IFN-γ  concentrs = 0.42 p<0.01); IL-6 and IL-10 (rs- = 0.38 p<0.05); IL-6 and IFN-γ (rs = 0.37 p<0.05) and a negative relationship between TGF-β and IL10 (rs = −0.4 p<0.001).When the relationships between the expression of each of the CSF cytokines were evaluated, significant positive relationships were identified between TNF-α and IL-10 (rIL-10 and TGF-β concentrations were significantly predictive of diagnosis stage in univariate logistic regression (likelihood ratio test p<0.0001 for both cytokines). Each log unit increase in CSF IL-10 and TGF-β concentrations were associated with odds ratios (OR [95% CI]) for late stage diagnosis of 4.0 (2.5–7.4) and 0.04 (0.01–0.0.17) respectively. To determine if either would be of utility as a late stage diagnostic, receiver operating characteristic (ROC) curves were analysed. For IL-10 the area under the ROC curve (AUROCC) was 0.85 but in order to achieve 100% specificity a cut off value of 275 pg/ml only offered 14% sensitivity. For TGF-β, the AUROCC was 0.86, and similarly the cut off for 100% specificity (50 pg/ml) only offered 5% sensitivity. An optimal combined panel of CSF IL-10, TGF-β, and IgM concentration was identified using stepwise logistic regression analysis and with discriminatory cut off levels for late stage determined on the patient data of IL-10>66.4 pg/ml; TGF-β<159.5 pg/ml and CSF IgM>89.2 mg/l. This panel offered an AUROCC of 0.97, with 70% sensitivity for 100% late stage specificity.Neurological signs including altered gait, tremors, incontinence, cranial nerve neuropathy , somnolence and reduced Glasgow coma score (GCS) were observed in both early and late stage patients, indicating early onset of neurological involvement, with only moderate coma (GCS<12) being unrepresented in any early stage cases . Of the loc) or cytokine concentrations  in relation to the presence or absence of gait ataxia, tremors, urinary incontinence, although cases with facial nerve palsies exhibited a slight increase in intrathecal IgA concentration (median (IQR) 0.0 (0.0–4.6) mg/l versus 0.0 (0.0–0.8) mg/l p<0.05). However, cases with moderate coma (GCS<13) exhibited substantially and significantly higher levels of intrathecal synthesis of all Ig isotypes as well as IL-10 and IL-6 . The increase in CSF protein is accounted for at least in part by the large and significant increases in CSF IgM, IgG and IgA concentration. Similar increases have been described previously for T.b.gambiense infection ALB levels with both stage progression and disease duration, and indicates a reduction of either CSF production in the choroid plexus or outflow of CSF into venous circulation. In order to measure intrathecal synthesis of immunoglobulin we used the hyperbolic discrimination curve QLIM as cut off for non-CNS derived Ig LOC) was detected in few (12%) early stage cases but commonly (70%) in late stage cases. In contrast to the findings from similar studies in T.b.gambiense infection T.b.rhodesiense HAT was predominantly a single class (IgM) response and 2 or 3 class responses were considerably less common. Overall, while intrathecal Ig synthesis in late stage infection provides clear evidence of the activation of humoral immune responses by trypanosomes in the CNS, it does not offer the sensitivity or specificity required to be effective as a stage diagnostic. None of the early stage cases in this study where intrathecal Ig synthesis was detected relapsed (over a 1 year follow up period) after suramin treatment, indicating that the diagnostic decision to classify these cases as early stage was correct. In T.b.gambiense infection a similar frequency of early stage cases exhibiting Ig synthesis has been described In the CSF, total protein increased with stage progression and reported disease duration, consistent with previous observations for both T.b.rhodesienseT.b.gambiense infection in vitro models of murine brain macrophage activation CSF cytokine concentrations were measured as indicators of cellular immune activation in the CNS. Increases in IL-10 and IL-6 concentration with stage progression are consistent with previous studies in both Cytokines in the CSF have previously been proposed as potential stage diagnostic markers T.b.rhodesiense HAT, it is possible to test the hypothesis that neurological dysfunctions observed in HAT are manifestations of inflammatory neuropathology. Neurological signs  were equally probable to be observed in both early and late stage cases, and the only significant difference in incidence of neurological symptoms was for moderate coma which was never observed in early stage cases. This result is consistent with observations in a larger multi-centre study in Uganda Through analysis of immunoglobulin and cytokine responses in the CNS in relation to stage progression in T.b.gambiense infection, there may be an “intermediate” or “early-second stage” of infection T.b.rhodesiense HAT. However such an interpretation would require the study of considerably more cases of this type, and while all three cases showed normal BBB function, 2 of the cases presented an intense intrathecal IgM synthesis  of cases of HAT with CSF WBC concentration 6 and 20/µl. These cases were classified as late stage cases, however it was noted that these were the only 3 cases in the study where trypanosomes were not detected in the CSF even after double centrifugation. It is intriguing to speculate that these cases might present the first indication that, as is the case in ynthesis  despite T.b.rhodesiense HAT, increasing levels of intrathecal immunoglobulin synthesis and CNS pro-inflammatory cytokine expression were associated with disease progression from early to late stage, although these were of limited diagnostic value. While intrathecal immunoglobulin synthesis was associated with the development of coma, it was not associated with any of the other typical neurological sequelae of HAT, which were also not related to CSF inflammatory or counter-inflammatory cytokine levels. Neuroinflammatory responses in correctly diagnosed early stage cases and cases with a similarity to the intermediate stage of T.b.gambiense HAT suggest there may be an early CNS involvement prior to the detectable invasion of the brain by the parasite and that effects on the CNS may be mediated indirectly by the parasite while it is still localised in the haemolymphatic system.In conclusion, in Figure S1Quotient diagrams for CSF IgG, IgA and IgM in HAT. (a) early stage and (b) late stage HAT patients. Values above the upper discrimination line  indicate intrathecal synthesis, with the intrathecal fraction being indicated with reference to the dashed lines representing 20%, 40%, 60% and 80% of total CSF Ig. In panel (b) the cases marked with solid triangles represent the 3 individuals with CSF WBC between 6 and 20 cells/µl.(TIF)Click here for additional data file.Checklist S1STROBE checklist.(DOC)Click here for additional data file.