Human T-lymphotropic virus type 1 (HTLV-1) -associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a rare chronic neuroinflammatory disease. Since the disease course of HAM/TSP varies among patients, there is a dire need for biomarkers capable of predicting the rate of disease progression. However, there have been no studies to date that have compared the prognostic values of multiple potential biomarkers for HAM/TSP.Peripheral blood and cerebrospinal fluid (CSF) samples from HAM/TSP patients and HTLV-1-infected control subjects were obtained and tested retrospectively for several potential biomarkers, including chemokines and other cytokines, and nine optimal candidates were selected based on receiver operating characteristic (ROC) analysis. Next, we evaluated the relationship between these candidates and the rate of disease progression in HAM/TSP patients, beginning with a first cohort of 30 patients (Training Set) and proceeding to a second cohort of 23 patients (Test Set). We defined “deteriorating HAM/TSP” as distinctly worsening function (≥3 grades on Osame's Motor Disability Score (OMDS)) over four years and “stable HAM/TSP” as unchanged or only slightly worsened function (1 grade on OMDS) over four years, and we compared the levels of the candidate biomarkers in patients divided into these two groups. The CSF levels of chemokine (C-X-C motif) ligand 10 (CXCL10), CXCL9, and neopterin were well-correlated with disease progression, better even than HTLV-1 proviral load in PBMCs. Importantly, these results were validated using the Test Set.As the CSF levels of CXCL10, CXCL9, and neopterin were the most strongly correlated with rate of disease progression, they represent the most viable candidates for HAM/TSP prognostic biomarkers. The identification of effective prognostic biomarkers could lead to earlier detection of high-risk patients, more patient-specific treatment options, and more productive clinical trials. HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a rare neurodegenerative disease caused by infection with human T-lymphotropic virus type 1 (HTLV-1). HTLV-1 infects 10–20 million people worldwide, and, depending on the region, 0.25–3.8% of infected individuals develop HAM/TSP. As the disease progresses, chronic inflammation damages the spinal cord and lower limb and bladder function gradually decline. In the worst cases, even middle-aged patients can become perpetually bedridden. Today, there are treatments that may alleviate the symptoms to a certain degree, but there is no cure that can halt disease progression, and there are no known biomarkers to indicate the level and speed of disease progression. In this study, we successfully identified three promising candidate biomarkers. We believe that the use of these biomarkers could lead to more accurate prognoses and more prudent, patient-specific treatment plans. We not only hope that these biomarkers are sensitive enough to use as selection criteria for clinical trials, but also that measurements of these biomarkers can be used to accurately evaluate drug effectiveness. In short, the biomarkers we identified have the potential to help more effectively treat current HAM/TSP patients and to pave the way for new drugs to potentially cure future HAM/TSP patients. Human T-lymphotropic virus type 1 (HTLV-1) is a human retrovirus associated with persistent infection of T-cells HAM/TSP is characterized by unremitting myelopathic symptoms such as spastic paraparesis, lower limb sensory disturbance, and bladder/bowel dysfunction Several candidate prognostic biomarkers with elevated levels in HAM/TSP patients have already been identified in the peripheral blood and cerebrospinal fluid (CSF). In the peripheral blood, such candidates include the HTLV-1 proviral load in peripheral blood mononuclear cells (PBMCs) and serum levels of the soluble IL-2 receptor (sIL-2R) We hypothesized the existence of biomarkers capable of differentiating stable and deteriorating HAM/TSP patients. In this retrospective study, a preliminary experiment was first conducted to select the most promising candidate biomarkers by comparing blood and CSF levels in HAM/TSP patients and control subjects . Four caThe study was designed and conducted in accordance with the tenets of the Declaration of Helsinki. The protocol in this study was approved by the Ethics Review Committee of St. Marianna University School of Medicine (No. 1646). Prior to the collection of blood or CSF samples, all subjects gave written informed consent permitting the analysis of their samples for research purposes as part of their clinical care.Between April 2007 and February 2013, we enrolled 53 HAM/TSP patients according to the inclusion and exclusion criteria shown in Blood and/or CSF samples were obtained within a one-hour window for each subject. Peripheral blood samples were collected in heparin-containing blood collection tubes and serum-separating tubes. Plasma and PBMCs were obtained from the former tubes and serum was obtained from the latter. PBMCs were isolated with standard procedures using Pancoll® density gradient centrifugation . Plasma and serum samples were stored at −80°C until use. CSF was collected in polypropylene tubes. A small amount of CSF was used for routine laboratory tests, which included total protein, cell count, and IgG level. The remaining CSF was aliquoted into cryotubes and stored at −80°C until undergoing further analysis. All tests in this study were performed on samples from these frozen stocks.The serum concentration of sIL-2R was determined using an ELISA . HTLV-1 proviral load was measured using real-time PCR, following DNA extraction from PBMCs, as previously described CSF cell count was determined using the Fuchs–Rosenthal chamber . Total protein and IgG levels in the CSF were measured using a pyrogallol red assay and a turbidimetric immunoassay, respectively. The anti-HTLV-1 antibody titer was determined using the gelatin particle agglutination test . CSF concentration of sIL-2R was determined using an ELISA . CSF neopterin level was measured using high-performance liquid chromatography. IFN-γ and six chemokines  were measured using a CBA (BD Biosciences). The CSF concentrations of three chemokines  and IL-17A were measured using commercially available ELISA kits . All assays were conducted according to the respective manufacturers' instructions.The 53 total HAM/TSP patients without any history of HAM/TSP-targeting treatments were interviewed using a questionnaire  to deterGraphPad Prism 5  was used to plot graphs and perform statistical analyses. Differences between the two subject groups were tested using the Mann-Whitney U-test. Receiver operating characteristic (ROC) analysis was performed to examine the sensitivity and specificity of individual biomarkers. For the ROC analyses, an area under the ROC curve (AUC) of 1.0 was used to represent a perfect test with 100% sensitivity and 100% specificity, whereas an area of 0.5 was used to represent random discrimination. Spearman's rank correlation test was employed to investigate the correlation between the four CSF markers  and the proviral load in PBMCs. To compare the four CSF markers between three groups , we used the Kruskal–Wallis test followed by Dunn's post-hoc tests. P-values<0.05 were considered statistically significant.p≤0.0001, In order to identify candidate blood markers for HAM/TSP, the concentrations of IL-1β, TNF-α, and IFN-γ were measured in plasma samples from four ACs and four HAM/TSP patients. Plasma levels of IL-1β and TNFα were below the detection limits  except in one patient with HAM/TSP. Plasma IFN-γ levels showed no significant differences between ACs and HAM/TSP patients . Therefore, these quantities were not measured in additional samples . The prop<0.0001 overall, p = 0.0019 and p = 0.0119, respectively; In order to identify candidate CSF markers for HAM/TSP, elevated levels of various potential markers were screened for in CSF samples from HAM/TSP patients. CSF IL-17A was detectable (>3.0 pg/mL) in only one of eight HAM/TSP patients screened (including six deteriorating-type patients), and the level in this one patient (deteriorating-type) was negligible (4.0 pg/mL). CSF IFN-γ was detectable (>1.8 pg/mL) in only 3 of 10 HAM/TSP patients screened (six deteriorating patients), and the levels in all three were negligible (range 3.3–4.2 pg/mL). Therefore, these cytokines were not measured in additional patients. Total protein, cell count, IgG, neopterin, sIL-2R, and nine chemokines  were also measured in the CSF of 30 untreated HAM/TSP patients and in eight HTLV-1-infected control subjects (seven ACs and one patient with smoldering ATL). The results indicated that CSF levels of CXCL10, neopterin, and CXCL9 were remarkably higher in HAM/TSP patients compared to control subjects . The results revealed that all five CSF markers were significantly higher in the deteriorating group compared to the stable group , but thaTo validate the results obtained using the Training Set, the same nine markers were compared between deteriorating and stable patients using the Test Set (a second cohort of 23 HAM/TSP patients that had not undergone HAM/TSP-targeting treatment). As shown in The demographics of the HAM/TSP patients versus the control subjects for both the blood tests and CSF analyses were compared and evaluated for statistical significance . There wSimilarly, the demographic and clinical characteristics of stable versus deteriorating HAM/TSP subjects in both the Training and Test sets are shown in To investigate the potential influence of disease duration as a secondary variable, a new test group was created containing only those patients for whom the disease onset date was 7–13 years prior to the sample collection day. Patients fitting this criterion were selected from the 53 total available from both the Training and Test sets: eight stable patients and ten deteriorating patients; we confirmed that there was no significant difference in disease duration between these two groups. The results remained consistent with our previous findings: CSF CXCL10, CXCL9, and neopterin were all elevated in deteriorating patients with respect to stable patients p<0.01, .Four stable HAM/TSP patients were left completely untreated and followed for a period of three to five years. Within this time, one patient rose one grade on the OMDS scale, and the other three experienced no change in OMDS grade at all. The levels of CSF CXCL10 and neopterin remained consistently low over time .To date, there have been few well-designed studies that have evaluated the relationship between biomarkers and HAM/TSP disease progression. In a previous retrospective study with 100 untreated HAM/TSP patients, a significant association was demonstrated to exist between higher HTLV-1 proviral load in PBMCs and poor long-term prognosis; however, the predictive value of high proviral load appeared to be too low to qualify it as a marker for disease progression in clinical practice In this study, elevated CSF cell count, neopterin concentration, and CSF levels of CXCL9 and CXCL10 were well-correlated with disease progression over the four year period under study, better even than HTLV-1 proviral load in PBMCs  and 4. AThe HTLV-1 proviral load in the CSF as well as the ratio of the proviral load in the CSF to that in PBMCs have been reported to be effective for discriminating HAM/TSP patients from ACs or multiple sclerosis patients infected with HTLV-1 From our results, we concluded that of the potential biomarkers under study, CXCL10, CXCL9, and neopterin are the most fit for determining the level of spinal cord inflammation, and thus the most fit for predicting disease progression in HAM/TSP patients. Although the CSF cell count is an easily measurable inflammatory marker, it is not sensitive enough to reliably detect the level of spinal cord inflammation. Numerous patients with CSF cell counts within the normal range exhibited high levels of other inflammatory markers, such as neopterin and CXCL10 . In factWe also explored the possibility of combining multiple biomarkers via multiple logistic regression to form a combination more sensitive and specific than individual markers, but the results indicated that there is not much to be gained from combinations (data not shown).While there were no significant demographic differences between subject groups, the clinical characteristics of stable versus deteriorating HAM/TSP patients of course differed widely , and S2.The main limitation of our retrospective study is that our samples were collected from patients at the end of the four year period during which the extent of progression was analyzed as opposed to the beginning of the four year period, which would have been optimal for directly measuring their prognostic powers. Of course, the patients with severe HAM/TSP symptoms began undergoing treatment soon after sample collection, rendering any observations on disease course after sample collection un-useable for analysis in this study. While this situation is non-ideal, we hypothesize that biomarker levels in a given patient do not substantially change over a few years' time. We were actually able to monitor the biomarker levels of four untreated HAM/TSP patients over 3–5 years, and the levels remained relatively stable in all four subjects over time , support+ Th cells differentiate from naïve T-cells to members of the Th subset , and each one expresses its own characteristic chemokine receptors The results of this study indicate that CXCL9 and/or CXCL10 may play a key role in the pathogenesis of HAM/TSP by recruiting more inflammatory cells to the spinal cord lesions. In this study, we measured the levels of the chemokines in the CSF that might play a part in inducing the migration of T-helper (Th) cells. CD4In conclusion, in this retrospective study, we have demonstrated that CSF levels of CXCL10, CXCL9, and neopterin are promising candidate prognostic biomarkers for HAM/TSP. These biomarkers may provide a means for the early identification of patients at increased risk of debilitating disease progression, those that may need anti-inflammatory therapies to limit or prevent this, and for evaluating the efficacy of such therapies. This initial identification of prognostic biomarkers for HAM/TSP should be followed by a future multicenter prospective clinical study.Figure S1Diagram illustrating the biomarker selection process. A total of 26 biomarker candidates including 9 in the blood and 17 in the CSF underwent the following selection processes: 1) pre-screening of the cytokines for presence in HAM/TSP patients, 2) selection for markers elevated in HAM/TSP patients with respect to controls (AUC>0.8), 3) selection for markers elevated in deteriorating HAM/TSP patients with respect to stable patients (AUC>0.8) in a cohort termed the Training Set, 4) validation of the selected markers by evaluating again (AUC>0.8) in a second cohort termed the Test Set. The darkening of an arrow's color represents that marker's failure to meet the selection criteria, and the termination of an arrow indicates that no further testing was conducted for that marker. CYT = cytokine, HTLV-1 PVL = HTLV-1 proviral load, Ab Titer = anti-HTLV-1 antibody titer, AUC = area under the ROC curve.(TIF)Click here for additional data file.Figure S2Questionnaire on the development of motor disability over time as measured using Osame's Motor Disability Score (OMDS). The first and second columns indicate the OMDS numerical value and description, respectively. Doctors interviewed the patients and filled in the table according to the following instructions: in the bottom row, write the ages at which symptoms listed to the left first appeared, and above the age check the box in the row corresponding to the symptom.(TIF)Click here for additional data file.Figure S3Rate of disease progression in HAM/TSP patients without any history of HAM/TSP-targeting treatment. Each line illustrates the change in OMDS over time for an individual patient after disease onset for (A) all patients in the Training Set (n = 30) and  only deteriorating patients (n = 11) including three particularly rapidly progressive patients (shown as solid black circles) and  only stable patients (n = 14).(TIF)Click here for additional data file.Figure S4Comparison of CSF levels of nine chemokines in control subjects and HAM/TSP patients. The CSF levels of nine chemokines  were compared between control subjects  and HAM/TSP patients . Horizontal bars indicate median values. The Mann-Whitney U-test was used for statistical analysis.(TIF)Click here for additional data file.Figure S5Low sensitivity of CSF cell count for detection of HAM/TSP. (A) Sensitivities of four potential CSF markers for detection of HAM/TSP. For CSF CXCL10, CXCL9, and neopterin, dotted lines indicate reference values, defined as mean for control subjects +3 standard deviations. For CSF cell count, the dotted line represents the pre-established reference value of 15/3 mm3. The sensitivity of CSF cell count was much lower than those of the other CSF markers. (B) Direct comparison of the sensitivities of CSF cell count and the other three CSF markers. The horizontal dotted lines all represent the reference value for CSF cell count (≤15/3 mm3), and each vertical dotted line indicates the reference value for each of the other CSF markers. With these lines drawn, one can see in the shaded area the numerous patients with CSF cell counts within the normal range but abnormally high levels of each of the other inflammatory markers, thus directly illustrating the comparatively low sensitivity of CSF cell count.(TIF)Click here for additional data file.Figure S6Changes in levels of CSF markers and OMDS over time in four untreated HAM/TSP patients. The three graphs illustrate the changes over time in CSF CXCL10 (top), neopterin (middle), and OMDS (bottom) for four untreated stable HAM/TSP patients. The patients were observed for 60 months (No. 1), 56 months (No. 2), 49 months (No. 3), and 39 months (No. 4).(TIF)Click here for additional data file.Figure S7Significant positive correlation between the proviral load in PBMCs and four CSF markers. HTLV-1 proviral load in PBMCs was compared with the levels of each of four CSF markers  in HAM/TSP patients (n = 53). Data analysis was performed using the Spearman's rank correlation test.(TIF)Click here for additional data file.Figure S8Significant higher CSF levels of CXCL10, CXCL9, and neopterin even in stable HAM/TSP compared to controls. The levels of four CSF markers  were compared among three groups  assembling patients from both Training and Test Sets combined. The horizontal bar indicates the median value for each group. Statistical analysis was performed using the Kruskal–Wallis test followed by Dunn's post-hoc tests. ns: not significant, * P<0.05, *** P<0.001.(TIF)Click here for additional data file.Table S1Demographics of HAM/TSP patients and control subjects. There were no significant differences in the demographics of HAM/TSP patients versus control subjects.(DOCX)Click here for additional data file.Table S2Demographics and clinical characteristics of HAM/TSP patients (Training set + Test Set). Among the HAM/TSP patients from the Training and Test Sets pooled together, deteriorating patients experienced disease onset significantly later in life and had lived with the disease for shorter periods.(DOCX)Click here for additional data file.


Surfactant proteins (SP) have been studied intensively in the respiratory system. Surfactant protein A and surfactant protein D are proteins belonging to the family of collectins each playing a major role in the innate immune system. The ability of surfactant protein A and surfactant protein D to bind various pathogens and facilitate their elimination has been described in a vast number of studies. Surfactant proteins are very important in modulating the host's inflammatory response and participate in the clearance of apoptotic cells. Surfactant protein B and surfactant protein C are proteins responsible for lowering the surface tension in the lungs. The aim of this study was an investigation of expression of surfactant proteins in the central nervous system to assess their specific distribution patterns. The second aim was to quantify surfactant proteins in cerebrospinal fluid of healthy subjects compared to patients suffering from different neuropathologies. The expression of mRNA for the surfactant proteins was analyzed with RT-PCR done with samples from different parts of the human brain. The production of the surfactant proteins in the brain was verified using immunohistochemistry and Western blot. The concentrations of the surfactant proteins in cerebrospinal fluid from healthy subjects and patients suffering from neuropathologic conditions were quantified using ELISA. Our results revealed that surfactant proteins are present in the central nervous system and that the concentrations of one or more surfactant proteins in healthy subjects differed significantly from those of patients affected by central autoimmune processes, CNS infections or cerebral infarction. Based on the localization of the surfactant proteins in the brain, their different levels in normal versus pathologic samples of cerebrospinal fluid and their well-known functions in the lungs, it appears that the surfactant proteins may play roles in host defense of the brain, facilitation of cerebrospinal fluid secretion and maintenance of the latter's rheological properties. The healthy central nervous system has conventionally been considered to be almost completely independent of the immune system In general, the blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (BCSFB) limit the access of cells and proteins circulating in the bloodstream to the brain Surfactant proteins are part of the pulmonary surfactant, a thin layer covering the alveolar surface, decreasing the surface tension between the air and the alveoli of the lungs to prevent the collapse of the small airways at the end of expiration. The pulmonary surfactant consists of lipids, mainly phosphatidylcholine and phosphatidylglycerol, and surfactant proteins A, B, C and D. The proteins are essential for the proper function of the surfactant and can be divided into two major structural and as a consequence functional groups.Mycobacterium tuberculosis by alveolar macrophages and modulate the intracellular mechanisms of killing the invading pathogen Pseudomonas aeruginosa, Haemophilus influenzae and Klebsiella pneumoniaeSurfactant protein A (SP-A) and surfactant protein D (SP-D) are molecular factors of the innate immune system; they facilitate the elimination of numerous pathogens in the lungs. For example, they promote phagocytosis of Staphylococcus aureus, Klebsiella pneumoniae) and bacteriostatic (Pseudomonas aeruginosa) effects Surfactant protein B (SP-B) and surfactant protein C (SP-C) are small hydrophobic proteins with the main function of decreasing surface tension at the air-liquid interface in distal airways SP-C binds bacterial lipopolysaccharide (LPS) and modifies pro-inflammatory cytokine-mRNA in monocytes after LPS stimulation Extra-pulmonary production of SPs and their importance for host defense and maintenance of physiological conditions has been demonstrated in a number of tissues and organs amongst them the salivary system Considering the specific localization of SPs at anatomical–physiological barriers  and their importance as innate defense molecules, we hypothesized that the four known surfactant proteins are molecular components of the innate immune system of the CNS. We particularly assumed their role as pathogen-binding proteins at the barriers between blood and CNS, protecting the highly vulnerable tissue from bacteria, viruses and fungi circulating in the bloodstream.In addition, regarding the known effects of SPs on biological fluids, we proposed the presence of SPs in cerebrospinal fluid, acting as regulators of its rheological properties.The study was conducted in compliance with Institutional Review Board regulations, informed consent regulations, and the provisions of the Declaration of Helsinki.Because of the origin of tissue samples and the background of acquisition of csf samples no ethics committee approval was necessary.The brain samples were obtained from body donors  at the Institute for Anatomy and Cell Biology Halle-Wittenberg, Germany. Prior to death every body donor dedicated his body to the Institute for Anatomy and Cell Biology for scientific and academic purposes, stating this in his last will. Tissue samples were anonymized. All tissues used were dissected from the cadavers within 9–24 hours post mortem. Prior to dissection, the medical history of each body donor was studied and donors with neurological syndromes were excluded. The brain of each cadaver was sagitally split in half. One half was fixed in 4% paraformaldehyde, the other half was further dissected and the regions of interests  were immediately snap-frozen in liquid nitrogen and stored at −80°C for further biomolecular investigation.Samples of cerebrospinal fluid  were generously donated by the Institute of Medical Microbiology, Martin-Luther-University, Halle/Wittenberg. The samples were collected for routine medical diagnostics to confirm or disprove CNS-infections and the remnants were stored over years at −80°C for possible further testing. Informed consent was written for the lumbar puncture and the acquisition of cerebrospinal fluid. After the death of the patient the related CSF sample was approved for scientific purposes and released to the Institute for Anatomy and Cell Biology together with the information about gender and diagnoses of the patients. The CSF samples were anonymized. Samples of cerebrospinal fluid from patients with stroke (ischemic or hemorrhagic infarction of the brain), autoimmune inflammation of the CNS  and infections of the CNS were included in the study. In order to sustain comprehensibility of the underlying cause for the alteration of SP concentrations in the CSF, the following specimens were excluded: CSF samples with blood contamination, CSF samples from patients with stroke/cerebral infarction plus evidence of bacterial growth, CSF samples from patients with hemorrhage into the subarachnoid space and CSF samples from patients without a diagnosed CNS illness but with a laboratory ascertained elevated CSF/serum albumin ratio, indicating a disturbance of the blood-cerebrospinal fluid barrier.− reverse transcriptase  for 60 min at 37°C. The ubiquitously expressed β-actin, which proved amplifiable in each case with the specific primer pair, served as the internal control for the integrity of the transcribed cDNA.For reverse transcriptase polymerase chain reaction (RT-PCR), tissue samples from 5 different regions of the 7 donor brains were crushed in an agate mortar under liquid nitrogen and homogenized in 5 ml PeqGold RNA pure solution  with a Polytron homogenizer. The CSF samples were treated under the same conditions except for crushing under liquid nitrogen. Insoluble material was removed by centrifugation . Total RNA was isolated using RNeasy-Kit . Crude RNA was purified with isopropanol and repeated ethanol precipitation. Contaminating DNA was destroyed by digestion with RNase-free DNase I . The DNAse was heat-denatured for 15 min at 65°C. 500 ng RNA were used for each reaction: cDNA was generated with 50 ng/µl (20 pmol) oligo (dT)15 primer  and 0.8 µl superscript RNase H2O, 1 µl 50 mM MgCl2, 0.5 µl dNTPs, 2 µl 10x PCR buffer, 0.3 µl (5 U) Taq DNA polymerase (Invitrogen) and 0.5 µl (100 pmol) of each of the following primers  96°C for 60 s, (2) 57°C  for 60 s each, (3) 72°C for 60 s. 35 cycles were performed with each primer pair. The final elongation cycle consisted of 72°C for 4 min. The primers were synthesized by MWG-Biotech AG, Ebersberg, Germany. 10 µl of the PCR product solution was loaded on an agarose gel and after electrophoresis the amplified products were visualized via fluorescence. Base pair (bp) size values were compared with gene bank data. For verification and comparison, plasmids containing the open reading frame for the corresponding protein were used as a reference . PCR products were also confirmed by BigDye sequencing . To estimate the amount of amplified PCR product, we performed a β-actin PCR with specific primers  for eachAntibodies (displayed in *1 (1∶500), SP-B*4 (1∶200), SP-C*5 (1∶500), SP-D*6 (1∶500) at 4°C for 12 hours  of brainstem (BS), cerebellum (Ce), choroid plexus (CP), the circle of Willis (CW), subventricular cortex (Ve), pia mater (PM) from body donors were homogenized in RIPA buffer  on ice with a tissue homogenizer . Generally, 100 mg of each tissue sample was submerged in 300 µl of RIPA buffer. After homogenization the samples were incubated for 2 hours on ice and subsequently spun down at 21000 g for 20 min. The supernatant containing the proteins was used for further analysis. Two native CSF samples were used for Western blotting without prior protein isolation. The protein concentration of these samples was determined with a protein assay based on the Bradford dye-binding procedure . For SDS-PAGE, each well of a 15% Tris-HCL gel was loaded with 40 µg of total protein. Protein isolate from human lung tissue served as positive control. After separating the proteins in the gel under reducing conditions, the proteins were transferred electrophoretically onto nitrocellulose membranes. Unspecific binding of the primary antibody was prevented using 5% milk in PBS as blocking solution for 1 hour at room temperature. The blocking solution was removed and the membranes were incubated with the antibodies for SP-A*2, SP-B*3, SP-C*5 and SP-D*7  from brainstem, cerebellum, choroid plexus, pia mater, pineal gland, pituitary gland and Subventricular cortex were embedded in paraffin, sectioned (6 µm) and deparaffinized. Immunohistochemical staining was performed with antibodies to SP-ACommercially available enzyme-linked immunosorbent assay kits  were used to quantify the amount of SP-A , SP-B , SP-C  and SP-D  in samples of cerebrospinal fluid and protein extracts from human brain tissue. The analysis was performed using a microplate spectrophotometer (ELISA-reader) at a wavelength of 450 nm and 405 nm for measuring the absorbance. By comparing with the standard series and the determined values for antigen concentration (protein concentration), each sample was calculated as surfactant protein in ng/total protein in mg (ng/mg).After evaluating the values on normal distribution using Kolmogorov-Smirnov test we performed Mann-Whitney U test to compare the means among different groups. For the graphical representations we used box plots showing median value and 50% interquartile interval for SP-A, SP-B, SP-C, SP-D concentration in ng/mg for the different groups. The hinge values are ±1.5 standard deviation (SD) about the mean value, the data points indicated by unfilled circles are between 1.5 and 3 SD from the mean value and outliner data points (>3 SD) are shown as stars. The number next to the unfilled circle or star indicates to the case number in the raw data sheet. The PASW SPSS 18 (IBM) for windows® software package was used for the statistical analysis. A p value<0.05 was considered statistically significant.The analyzed CSF samples were sorted into one out of four main categories I) autoimmune diseased, II) infection diseased, III) cerebral infarction, IV) healthy. The medical history of every subject related to an analyzed CSF sample was studied; thereupon the CSF sample was categorized in one of these four groups.CSF samples were included into the ‘autoimmune diseased’ group if the sample was derived from a patient currently diagnosed with ongoing multiple sclerosis, acute disseminated encephalmomyelitis or neuromyelitisoptica.CSF samples were included into the ‘infection diseased’ – group if the sample was derived from a patient diagnosed with current CNS infection of if the suspected diagnosis was ‘encephalitis’, ‘meningitis’ or ‘meningoencephalitis’.CSF samples were included in the ‘infarction’ group if the sample was derived from a patient who was at the time diagnosed with current ‘cerebral infarction’ of either hemorrhagic or ischemic etiology.CSF samples were included in the ‘healthy’ group if the sample was derived from a patient who at the time showed no signs or findings of CNS-related diseases or was classified as ‘healthy’.Since examining m-RNA expression from tissues, donated post mortem and snap frozen 9–24 hours after death may have lead to results difficult to interpret, it was decided not to aim for quantitative PCR analysis. Standard RT-PCR was conducted to prove the expression of m-RNA corresponding to the surfactant proteins A, B, C and D only to provide evidence that surfactant proteins are inherent proteins of the CNS. Real time PCR analysis of human CNS tissue, obtained from the living individual or adequate ex vivo testing would provide a much better insight into the distribution and regulation of the surfactant protein system of the human CNS. The fact that unaltered, fresh human tissue specimens, especially samples of the CNS are almost impossible to obtain  and the lack of controllability of the post mortal interval were the causes for not conducting real time PCR instead of standard RT-PCR. Specific cDNA amplification products  were detected in tissue samples (n = 7 for each tissue) of brainstem (BS), cerebellum (Ce), chorioid plexus (CP), subventricular cortex (Ve), pia mater (PM) cerebrospinal fluid  see Protein isolates (n = 7 for each tissue) of brainstem (BS), cerebellum (Ce), choroid plexus (CP) the circle of Willis (CW), subventricular cortex (Ve), pia mater (PM) and cerebrospinal fluid  were tested for SP-A, SP-B, SP-C and SP-D using specific antibodies to each of the SPs, lung (Lu) served as positive control. Distinct bands were detected for SP-A , SP-B (40 kDa), SP-C (16 kDa) and SP-D (43 kDa) in each of the samples, see Paraffin-embedded 6 µm sections of choroid plexus, cerebral cortex, pineal gland and rhomboid fossa/brainstem were investigated for presence of SP-A, SP-B, SP-C and SP-D. Lung tissue was used as positive control and revealed positive immunoreactivity for all 4 SPs) not shown here, but already shown in previous work SP-A is located both diffuse in the cytoplasm and in more intensively stained granular structures in the ependymal cell layer in the ventricular region around the hippocampus. Some IR for SP-A is also present in the subependymal region  CSF samples derived from patients suffering from a CNS disease with autoimmune etiology were compared to 18  samples derived from patients suffering from a CNS condition with non-autoimmune etiology and 28  samples derived from healthy subjects.The level of SP-A concentration in CSF from patients with autoimmune CNS disease was statistically significantly decreased (p = 0.01) compared to the level of SP-A concentration in the healthy group.A total of 69 samples were analyzed. 20  CSF samples derived from patients suffering from a CNS disease with autoimmune etiology were compared to 21  samples derived from patients suffering from a CNS condition with non-autoimmune etiology and 28  samples derived from healthy subjects.Statistical analysis revealed that the level of SP-B in CSF from patients with an autoimmune CNS disease was significantly decreased (p = 0.015) compared to the level of SP-B in the healthy group.A total of 73 samples were analyzed. 20  CSF samples derived from patients suffering from a CNS disease with autoimmune etiology were compared to 25  samples derived from patients suffering from a CNS condition with non-autoimmune etiology and 28  samples derived from healthy subjects.The level of SP-C in CSF from patients with an autoimmune CNS disease was comparable to the level of SP-C in the healthy group (p = 0.415).A total of 74 samples were analyzed. 20  CSF samples derived from patients suffering from a CNS disease with autoimmune etiology were compared to 26 samples  derived from patients suffering from a CNS condition with non-autoimmune etiology and 28  samples derived from healthy subjects.The level of SP-D in CSF from patients with an autoimmune CNS disease was decreased in a statistically significant manner (p = 0.01) compared to the level of SP-D in the healthy group.A total of 64 samples were analyzed. 5  CSF samples derived from patients who had suffered a cerebral infarction were compared to 31  samples derived from patients suffering from a CNS condition not related to cerebral infarction and 28  samples derived from healthy subjects.The level of SP-A in CSF from patients with cerebral infarction was slightly decreased, but did not reach statistical significance (p = 0.083) compared to the level of SP-A in the healthy group.A total of 71 samples were analyzed. 7  CSF samples derived from patients who had suffered a cerebral infarction were compared to 36  samples derived from patients suffering from a CNS condition not related to cerebral infarction and 28  samples derived from healthy subjects. The level of SP-B in CSF from patients with cerebral infarction was statistically significantly increased (p = 0.002) in comparison to the level of SP-B in the healthy group.A total of 73 samples were analyzed. 7  CSF samples derived from patients who had suffered a cerebral infarction were compared to 38  samples derived from patients suffering from a CNS condition not related to cerebral infarction and 28  samples derived from healthy subjects. The level of SP-C in CSF from patients with cerebral infarction showed no difference (p = 0.967) compared to the level of SP-C in the healthy group.A total of 73 samples were analyzed. 7  CSF samples derived from patients who had suffered a cerebral infarction were compared to 39  samples derived from patients suffering from a CNS condition not related to cerebral infarction and 28  samples derived from healthy subjects. The level of SP-D in CSF from patients with cerebral infarction was statistically significantly decreased compared to the level of SP-D in the healthy group (p = 0.012).A total of 64 samples were analyzed. 9  CSF samples derived from patients diagnosed at the time with an infectious disease of the CNS were compared to 27  samples derived from patients suffering from a different disorder of the CNS than infection and 28  samples derived from healthy subjects. The level of SP-A in CSF from patients with a CNS infection was statistically significantly decreased (p = 0.044) compared to the level of SP-A in the healthy group.A total of 69 samples were analyzed. 10  CSF samples derived from patients diagnosed at the time with an infectious disease of the CNS were compared to 31  samples derived from patients suffering from a different disorder of the CNS than infection and 28  samples derived from healthy subjects.The level of SP-B in CSF from patients with a CNS infection was slightly increased, but did not reach statistical significance compared to the level of SP-A in the healthy group (p = 0.083).A total of 73 samples were analyzed. 11  CSF samples derived from patients diagnosed at the time with an infectious disease of the CNS were compared to 34  samples derived from patients suffering from a different disorder of the CNS than infection and 28  samples derived from healthy subjects. The level of SP-C in CSF from patients with a CNS infection was slightly decreased, but did not reach statistical significance (p = 0.072) compared to the level of SP-C in the healthy group.A total of 74 samples were analyzed. 12  CSF samples derived from patients diagnosed at the time with an infectious disease of the CNS were compared to 34  samples derived from patients suffering from a different disorder of the CNS than infection and 28  samples derived from healthy subjects. The level of SP-D in CSF from patients with a CNS infection was statistically significantly decreased (p = 0.001) in comparison to the level of SP-D in the healthy group.ELISA was performed with protein extracts from subventricular cortex, brainstem, cerebellum and choroid plexus from body donors with comparable postmortal intervals. The surfactant protein concentration in brain tissue (except the choroid plexus) was measured to be approximately twenty five percent of the surfactant protein concentration in the lungs. The surfactant protein concentrations of the cerebellum, the subventricular cortex and the brainstem were in equal range, whereas the tissue concentration of all SPs in the choroid plexus was approximately twofold higher compared to the concentration in brainstem, cerebellum and subventricular cortex.By use of RT-PCR we were able to verify the intrinsic production of messenger RNA for all investigated SPs by the central nervous system. The fact that we found mRNA for the SPs in the cerebrospinal fluid is unexpected. We hypothesize that the mRNA we found is a result of the cell damage due to the trauma of the lumbar puncture, which always leads to limited destruction of cells  in the area of the puncture. The cell damage results in the liberation of intracellular components like proteins and mRNA. We address this as the reason for the detection of SP-specific mRNA in the CSF. The results of Western blot and immunohistochemical analysis confirm our findings on protein level. Based on proof of both the production of SPs in the human CNS and the expression of SP-specific mRNA, our study reveals the existence of surfactant proteins as inherent proteins of the CNS.In the case of SP-C, the antibody used detected premature forms of the proteins at 12 kDa only in the non-CSF tissue. This issue is well known and a consequence of an intense posttranslational processing of the SPs. We therefore conclude that mature SP-C does not play an important role in host defense or amelioration of inflammation in CNS diseases.The collectin-type SPs A and D showed immunoreactivity in the tissue surrounding the microvasculature of the brain parenchyma, the capillaries of choroid plexus stroma and the small vessels at the base of the pineal gland. Both proteins were detected in the vascular layers (with the exception of the tunica intima) as well as in the adjacent brain tissue. Their volumetric abundance between the blood milieu of the cerebral vessels and the brain tissue, especially at the level of the glia limitans (formed by astrocytic endfeet) and the perivascular Virchow-Robin space, resembling the microanatomical correlate of the blood-brain barrier, indicates a participation in this convolute of CNS-protective mechanisms. In lung, SP-A and SP-D are known to be antimicrobially active proteins of the alveolar surface. They are localized in the surfactant layer lining the alveolar surface as well as within the apical portion of the type II alveolar epithelial cells. One of their tasks is to bind microbial invaders like Pseudomonas aeruginosa or Staphylococcus aureus, to mediate their elimination via direct bactericidal effects or indirectly by means of opsonization and subsequent phagocytosis by macrophages The lipophilic SPs show immunoreactivity in the epithelial layer of the choroid plexus, in the ependymal cells lining the four ventricles of the brain and in the ependyma of the spinal channel. The choroid plexus and the ependymal cells of the ventricles are the main production site of the cerebrospinal fluid The function and physiological importance of SP-B and SP-C has been studied in the lungs, the auditive tube, the tear fluid and the lacrimal system among other sites The choroid plexus and the ventricular ependyma also produce phospholipid transfer protein, another protein participating in the formation of the surfactant layer in the lungs The concentration of the collectin-type SPs in CSF samples from patients with autoimmune diseases, infectious conditions and CNS infarctions was significantly lower compared to samples derived from healthy subjects. It thus appears that both proteins are involved in inflammatory processes in the CNS. Considering the known functions of these proteins, we assume their contribution in the local immune response. SP-A and SP-D are involved in the clearance of pathogens and apoptotic polymorph nuclear neutrophils Polymorph nuclear neutrophils are among the first wave of innate immune cells at inflammation sites, they release a whole arsenal of anti-microbial molecules and their survival is closely regulated to prevent tissue damage caused by excessive neutrophil activity As a consequence of this, the collectin-type SPs may be removed from the CSF of patients with inflammatory CNS conditions like infarctions, infections or autoimmune diseases during the phagocytosis of (SP-) opsonized targets.The concentration of SP-B in CSF samples from patients with autoimmune CNS conditions was significantly lower compared to samples from healthy subjects. By contrast, the SP-B concentration in CSF samples from patients with infections and infarctions of the CNS was significantly higher than the concentration in samples from the healthy group.The phenomenon of high SP-B concentration in the CSF of patients with inflammation of non-autoimmune etiology and low SP-B concentration in the CSF of patients with autoimmune etiology probably has different underlying mechanisms.SP-B possesses anti-inflammatory properties and has the potential to reverse inflammation in the lungs Consistent with these findings, we propose that SP-B is upregulated during exogenously induced inflammation in order to limit and counterbalance the detrimental effects caused by the host's immune system. Specifically, we hypothesize that SP-B is a protein of the CSF, constitutively produced by the choroid plexus to facilitate CSF flow with a reactively increased production rate in case of pathogen-induced or ischemically triggered, acute neuroinflammation.The decreased concentration of SP-B in CSF samples from patients with autoimmune CNS conditions may be the consequence of a continuous depletion of SP-B due to the chronicity of autoimmune diseases. Autoimmune CNS conditions like multiple sclerosis are coupled with permanent inflammation, influx of immune cells and cell death in the CNS We were able to detect SP-C in all of the investigated CSF samples, but there was no difference in the concentration of SP-C between the different groups. We therefore conclude that mature SP-C does not play an important role in host defense or amelioration of inflammation in CNS diseases. Western blot analysis did not reveal the existence of the 12 kDa premature form of SP-C in the CSF. It is therefore conceivable that this premature form may be regulated differently in the investigated groups of CNS conditions, but the ELISA system we used may also not be capable of detecting the 12 kDa form.The ‘pulmonary’ surfactant proteins are also inherent proteins of the human CNS. The collectin-like SPs – SP-A and SP-D show a similar distribution pattern in the CNS, which varies from the distribution pattern of the hydrophobic SPs SP-B and SP-C. Their expression at different locations in the CNS indicates participation of the SPs in processes like host defense and maintenance of CSF flow, but the dimension of their importance and their exact function must be investigated in subsequent studies.The concentration of surfactant proteins in the CSF of healthy subjects deviates from patients with CNS conditions of autoimmune, ischemic and infectious etiology. This also speaks for the importance of the SPs in processes such as fine-tuning of inflammation and host defense. It is still not clear which cells are exactly responsible for production of the collectin-type SPs within the CNS. This remains to be investigated prior to more functional studies.


AbstractTrachypachus gibbsii LeConte, 1861 (family Trachypachidae), and family Carabidae: Pelophila rudis   and Ceroglossus chilensis  . The range and pattern of variation in structure of mandibles and labrum-epipharynx within the supertribe Cicindelitae was assessed using scanning-electron (SEM) images of these structures in nine exemplar taxa: Amblycheila baroni , Omus californicus  and Picnochile fallaciosa  (representing the Amblycheilini); Manticora tuberculata  (representing the Manticorini): Tetracha carolina  ; Pogonostoma chalybeum  (representing the Collyridini); and Therates basalis Dejean, 1826, Oxycheila species, and Cicindela longilabris Say, 1824 (representing the Cicindelini). An evolutionary transformation series was postulated for the mandibles and labrum-epipharynx, based on a reconstructed phylogenetic sequence, which, in turn, was based on morphological and DNAevidence.Principal features of the transformation series for the mandibles included development of a densely setose basal face; wide quadridentate retinaculum; a lengthened incisor tooth; a multidentate terebra , followed by subsequent loss of one or more such teeth; development of a diastema in the occlusal surface; development and subsequent loss of scrobal setae, and reduction and loss of the scrobe. Principal features of the transformation series for the labrum included evolution of form from transverse, sub-rectangular to elongate almost square, to triangular; position and number of setae evolved from dorsal to insertion on the apical margin, the number increased from 8-10 to as many as 36, and decreased to as few as four. The PageBreakepipharynx broadened evolutionarily, the pedium evolving in form from narrow, triangular and nearly flat, to broad, palatiform, and markedly convex; anterior parapedial setae both increased and decreased in number, and in orientation, from a row parallel to the parapedial ridge to a setal row extended forward at about a right angle to the latter.Using for comparison with, and as outgroups for, supertribe Cicindelitae, we describe and illustrate the mandibles and labrum-epipharynx of the basal geadephagans  Carabidae: Cicindelitae). Since then, Ball et al (1995: 302-311) provided the basis for inclusion of the epipharynx in systematic studies, but did not include the tiger beetles or other basal geadephagan lineages. We  decided to fill in these gaps in knowledge of geadephagan structure, and to do so, enlisted in the cause our willing and able colleague, Danny Shpeley.In their review of variation in mandibular structure within the coleopteran suborder Adephaga, Carabidae . We turned to analyses  of related basal stocks of geadephagans to seek the antecedents of the tiger beetle mandibles and labrum-epipharynx. Tiger beetles have been placed as a supertribe (Cicindelitae) within the arabidae  or as a ndelidae :31, or aindelini : 151. ReAmblycheilini, Manticorini, Megacephalini, Collyridini, and Cicindelini). We arrange them in a phylogenetic sequence, based on a postulated evolutionary pattern  microscopy, ), the putative adelphotaxon of the caraboid stock; and Carabidae— Carabitae-Ceroglossini ), and Nebriitae-Pelophilini ). These groups of Carabidae represent different feeding types .Specimen preparation. Standard techniques were used to prepare specimens for examination with the SEM. Each specimen was relaxed and cleaned in warm water and the sclerites of interest were removed from the head capsule and sputter coated with gold before SEM images were prepared. For light microscopy, specimens were relaxed, cleaned, and the mandibles spread to their fullest extent. For some but not all taxa, the labrum epipharynx was excised and point-mounted.SEM image preparation. Three aspects for both left and right mandibles are shown: dorsal, occlusal, and ventral; lateral aspects were noted, but not illustrated. For the labrum-epipharynx, the dorsal surface is the labrum, while the ventral surface is the epipharynx, keeping in mind that the epipharynx can also be thought of as the dorsal surface of the preoral cavity.Photographs. The mouthparts of 12 taxa examined are also illustrated in situ, with the mandibles widely spread, hopefully providing better perspective on the relativePageBreak coverage of the adducted mandibles by the labrum-epipharynx for each taxon, as well as some sense of potential occlusal relationships among mandible features, and bilateral asymmetry. Photographs were taken hand-held with a Nikon D300s camera set at ISO 800 and 1/200th of a second, equipped with an AF-S Micro-Nikkor 105mm lens set at f32, a TC-17EII (1.7X) teleconverter, and the Nikon R1C1macro flash system with two flash heads, each equipped with hand made, double-layered, frosted Mylar light diffusers. These images were presented on two color plates.Identification of structural elements. For tiger beetles ) Cicindela (sensu latissime)species. The major elements of the occlusal surfaces of adult geadephagan mandibles were identified by Acorn and Ball  as terebra  and retinaculum  short , in occlusal aspect broad, with short incisor tooth (it) and with small terebral tooth (tt). Retinaculum slightly posteriad terebral tooth; short, broad, with two ridges (rr and irr), and two teeth (art and prt), anterior tooth near ventral margin (2C-D), posterior tooth dorsal. Basal face not evident. Basal brush (bb) small. Ventral surface smooth except for the ventral groove (vg), extended anteriad anterior retinacular tooth, and with rather short and sparse microtrichia (vm).andibles .Trigonalandibles . Laterall groove , vg, extLabrum(las) near anterior margin, on dorsal surface (Labrum. Articul surface . Form ofEpipharynx (ped) trianguloid, apex posterior, slightly arched, with a short parapedial projection (pp), margined laterally each side by a thin parapedial ridge (pr); parapedial ridge anteriorly each side curved to lateral margin of labrum as a short lateral arm. Anterior parapedial setae (aps) in a row anterior and parallel to lateral arms of parapedial ridge. Posterior parapedial setae (pps) in a row.ipharynx . Pedium Ceroglossus type, and the particulate-feeding Pelophila type, evidently depending upon manipulation of food .5C-DStructural features.Mandibles (ss). Terebra (T) long , in occlusal aspect narrow, with short incisor tooth (it) and with small terebral tooth (tt). Retinaculum slightly posteriad terebral tooth, in dorso-ventral aspect (rr), and two teeth (art and prt), one at each end, in line with one another. Basal face not evident. Basal brush (bb) small. Ventral surface smooth except for the ventral groove (vg), extended anteriad anterior retinacular tooth, and with rather short and dense microtrichia (vm).andibles .Trigonalandibles . Laterall aspect  narrow wl groove , vg, extLabrum(las) near anterior margin, on dorsal surface (Labrum. Articul surface . Form ofEpipharynx(ped) broadly trianguloid, apex posterior, slightly arched, with a short parapedial projection (pp); margined laterally each side by a thin parapedial ridge (pr); parapedial ridge anteriorly each side curved to lateral margin of labrum as a short lateral arm. Anterior parapedial setae (aps) in a row anterior and parallel to lateral arms of parapedial ridge. Posterior parapedial setae (pps) few, in row along parapedial ridge.pipharynx. Pedium Comparisons. The prominent retinaculum seems to be a feature of the Nebriitae  as the adelphotaxon of the Cicindelitae; Liebherr and Will  include the Cicindelini as part of a quadritomy with three of the carabine tribes noted below.Cychrini; Ceroglossini; Pamborini; and Carabini. The tribe Ceroglossini is monogeneric, including eightspecies.The Carabitae includes 14genera, arranged in four tribes: Exemplar taxon.Ceroglossus chilensis .Plate 1AStructural features.Mandibles(T) long , in occlusal aspect (it) and with small terebral tooth (tt). Retinaculum slightly posteriad terebral tooth, short, oriented obliquely to long axis of mandible (rr), and two teeth (art and prt), one at each end of terebral ridge, offset from one another. Basal face long, densely setose (bfb), setae seemingly continuous with microtrichia of ventral groove (bb) small. Ventral surface smooth except for the ventral groove (vg), extended anteriorly nearly to base of the incisor tooth, and with long and dense microtrichia (vm).Mandibles.TrigonalMandibles, robust,Mandibles. Laterall aspect  broad; wmandible ,  near anterior margin, on dorsal surface (Labrum. Immovab surface . Form ofEpipharynx(ped) rather narrowly trianguloid, apex posterior; slightly arched, with a long parapedial projection (pp); margined laterally each side by a thin parapedial ridge (pr); parapedial ridge anteriorly each side curved gradually to lateral margin of labrum. Anterior parapedial setae (aps) in a row anterior and parallel to lateral portion of parapedial ridge. Posterior parapedial setae (pps) rather numerous, in row along parapedial ridge.pipharynx. Pedium Comparisons. The form of the mouthparts in Ceroglossus is in many ways reminiscent of that in cicindelites, and a more extensive survey of the Carabitae may well uncover additional shared features.Pasimachus Bonelli, Mouhotia Laporte de Castelnau) appear not to possess a retinaculum, whereas in Scarites the retinaculum is either reduced and confluent with the base of the terebra, or the basal portion of the terebra is multidenPageBreaktate and vaguely reminiscent of what we interpret here as the retinaculum of cicindelites. Some evidence exists for a relationship between tiger beetles and scaritines  the tiger beetles are arranged here in five tribes: Amblycheilini; Manticorini; Megacephalini; Collyridini; and Cicindelini. Gálian et al.  indicate the Megacephalini as polyphyletic, based on their study of multiple sex chromosomes in the cicindelites, the problem taxa being the oxycheiline genera Oxycheila and Cheiloxya. Based on their 18sRNA evidence and the DNA evidence of Vogler and Barraclough , these genera (and presumably Pseudoxycheila) belong in the tribe Cicindelini, and such a transfer obviates the taxonomic problem.Structural Features.Mandibles (ss). Terebra (T) elongate , in occlusal aspect broad to slender, with long incisor tooth (it) , and with or without terebral teeth (tt) . Retinaculum slightly posteriad basal terebral tooth or more widely separated by a distinct gap ; cusps of various sizes, in some taxa as large and prominent as terebral teeth. Basal face (bf) posteriad retinaculum, with long setae in form of a dense brush (bfb). Ventral surface smooth except for the ventral groove (vg) of various lengths, and with rather long and dense microtrichia (vm).andibles –16A-F.TrLabrum  palatiform, markedly arched, ventral surface concave; posteriorly broadly rounded, without a parapedial projection, margined laterally each side by a thin parapedial ridge (pr); parapedial ridge anteriorly each side curved to lateral margin of labrum as a short lateral arm. Parapedial setae in a row or cluster anterior (aps) to or posterior (pps) to lateral arms of parapedial ridge.ipharynx .Pedium , but increased to as many as four in other collyridine lineages and to five in some cicindeline lineages. The labrum-epipharynx is generally short in the amblycheilines, manticorines, and megacephalines, and sub rectangular or elongate and dorsally convex  among the collyridines and cicindelines.Synonymic note. Commonly known as the Omini W. AmblycheiliniClassification. This tribe includes three Western Hemisphere genera: the western Nearctic Amblycheila Say and Omus Dejean; and the southern Neotropical Picnochile Motschulsky . If the southern Afrotropical monobasic genus Platychile Macleay is placed in this group  ; Omus californicus  ; Picnochile fallaciosa  .Platychile pallida.Also examined but not treated in detail were two males of Structural features.Mandibles(ss) or glabrous. Diastema absent. Terebral teeth two, terebral tooth 1 tricuspidate . Retinacular cusps moderately long, extended about to middle of terebral tooth 1.Mandibles.— With mMandibles to moderMandibles. LateralMandibles, ss or gar cusps  rc 1 andl groove , vgmoderLabrum(las).Labrum. TransveLabrum or narroLabrum; or anteLabrum. Single Epipharynx (aps) extended laterally, parallel to lateral extensions of parapedial ridge (pr).ipharynx . Row of Comments.Amblycheila represented the “lowest.....and certainly the oldest line of descent...that probably diverged in the Mesozoic age”- a remarkably prescient line of thought, considering that it was developed more than a century ago.Platychile deserves special comment here. We examined two males of Platychile pallida (Fabricius) using light microscopy. A number of mandibular features  place the genus among the more basal lineages. On the other hand, the mandibles are so flat as to virtually eliminate the scrobe (and are thus reminiscent of the more derived tiger beetles), and they possess a single cusp terebral tooth 1 instead of the three cusps characteristic of the Western Hemisphere genera Megacephalini. Further, the body size and form is not unlike that of what could be expected in the megacephaline genus Phaeoxantha. The color pattern is also megacephaline-like, and a careful reading of Pearson and Vogler (2001: 53-57) indicates that such a feature may have importance in tiger beetle evolution. The unusual habitus of Platychile may also derive in part from convergence or mimicry, since these nocturnal beetles show a consistent ecological association with the PageBreakPageBreakdiurnal Eurymorpha cyanipes   , with whClassification. This tribe includes the southern Afrotropical genera Manticora Fabricius, 1792 and Mantica Kolbe, 1896.Exemplar taxon.Manticora tuberculata  .Other specimens examined: see Appendix.Structural features.Mandibles(ss). Diastema absent. Terebral teeth two  or three , terebral tooth 1 monocuspidate (tt 1); or bicuspidate, tt 1-2 moderately long, extended about to middle of terebral tooth 1.Mandibles. With maMandibles. LateralMandibles, ss. Diauspidate , tt 1; oar cusps  rc 1 andl groove , vgmoderManticora are pronouncedly sexually dimorphic, larger in males and asymmetric in form, the right mandible typically exhibiting greater elongation of the incisor region than the left. In Mantica, sexual dimorphism is slight, and some but not all males show larger left than right mandibles .Labrum . Transveur teeth : 300, meEpipharynx(aps) extended laterally, parallel to lateral extensions of parapedial ridge (pr).pipharynx.Row of aComments. See also Manticorini seems most similar to the Amblycheilini. However, the marked ventrad curvature of the mandibles is suggestive of the more derived megacephalines, cicindelines and collyrines. With the genera Amblycheila and Pogonostoma, members of Manticora share multisetose scrobes.Manticora, see Mantica PageBreakPageBreakPageBreak.For details about way of life, classification and relationships of Classification. This tribe includes more than 100 species, arrayed in eight genera  had indicated that DNA evidence showed the same thing for Oxycheila. No doubt, Oxycheila, Cheiloxya and putative close relative Pseudoxycheila belong in the tribe Cicindelini.t genera . Galián Exemplar Taxon.Tetracha carolina  .Other specimens examined: see Appendix.Structural Features.Mandibles (tt 1). Retinaculum with supplementary tooth anteriorly (srt) in Tetracha and Megacephala, very small in Phaeoxantha. Retinacular cusps moderately long, extended about to base of terebral tooth 2.andibles .With manandibles . Lateralandibles . Diastemandibles , tt 1. Rar cusps  rc 1 andl groove , vgmoderLabrum (las).Labrum . TransveEpipharynx (aps) extended anteriorly, at about right angle to lateral extensions of parapedial ridge (pr).ipharynx . Row of Comments. See also Tetracha carolina seems to be characteristic of males of that genus, contrasting markedly with the shorter t3 of the corresponding females. Number of labral setae in Megacephalini ranges from four to seven.Classification. This group is the equivalent of and co-extensive with Walther Horn’s (1908) “Phylum” Alocosternalia. This tribe includes seven genera arranged in two subtribes: Ctenostomatina, and Collyridina  .Ctenostomatina and Collyridina. See appendix.Other collyridines examined include representatives of subtribes Structural features.Mandibles (ss). Diastema (od) present. Terebral teeth various in size and number . Retinaculum without or with (Collyris only) supplementary tooth anteriorly (srt). Retinacular complex large, cusps (PageBreakPageBreak for details)Ventral groove (vg)moderately long, extended about to base of terebral tooth 2.andibles . With maandibles . Lateralandibles , ss. Diae, cusps  diagonall groove , vgmoderLabrum (las), each seta inserted in base of crenulation. For details, See “Variation”, below.Labrum . ElongatEpipharynx (ped) markedly concave. Row of anterior parapedial setae (aps) extended anteriorly, at about right angle to lateral extensions of parapedial ridge (pr). For details, See “Variation”, below.ipharynx . Pedium Variation. Terebral teeth two (each mandible), tt 1 monocuspidate (Pogonostoma), or only one tooth on each mandible (Collyris); or terebral teeth asymmetric, with two on right mandible and one on left mandible (Ctenostoma), or one on left mandible, two on right mandible (Tricondyla). Retinacular cusps five each mandible (Pogonostoma), or seven on left mandible, five on right mandible (Ctenostoma), or five on left mandible, four on right mandible (Tricondyla and Collyris).uspidate  .Exemplar taxa.Therates basalis Dejean (Theratina); Oxycheila species (Oxycheilina); and Cicindela longilabris Say (Cicindelina).Cicindela, principallyfor form, and for number of labral setae.Other cicindelines examined: see Appendix for names;plus numerous species of Nearctic Structural features.Mandibles (Dromica), and without setae. Diastema present (od) or absent (tt 2) to four (tt 1 – tt 4),with a maximum of five, observed in Oxygonia gloriola Bates, 1872. Retinaculum without or with . Retinacular complex large, cusps diagonally arranged, number of cusps four on each mandible (rc 1- rc 4).Ventral groove (vg)moderately long, extended about to base of terebral tooth 2, or shorter, extended only to base of retinacular complex  four to 12, on dorsal or apical surface, inserted at base of crenations.Labrum . MarkedlLabrum , as longLabrum , or sligLabrum . TrapezoEpipharynx(aps) in row parallel to lateral arm of parapedial ridge  showed sexual dimorphism in the Palaearctic species, Lophyridia angulata , as did Oberprieler and Arndt (2000: 86) for Manticora adults, and Franzen and Heinz (2005: 299) for Mantica adults. Although not well studied, it appears that in all but a few aberrant individuals, the left mandible adducts above the right , in his treatment of the Mexican species of Cicindela, illustrated striking differences in labra, particularly in form of the anterior margin of the labrum, proportions, and number of dorsal setae (from four to more than 30).Our three exemplar taxa differ markedly from one another in form and setation of the labrum. We note that the insertion of the labral setae on the dorsal surface of the labrum in ay, 1824  is a relTherates basalis Dejean, 1826 .The epipharynges of the three exemplar taxa are basically similar to one another, but differ in the anterior parapedial setation. The small number of such setae exhibited by an, 1826  is the mManticorini as the adelphotaxon of the Amblycheilini. Reference points on this diagram are the lineages indicated by the capital letters A to P.The following hypothesis of mandibular and labral-epipharyngeal evolution is illustrated in the reconstructed phylogeny  based onA represents the common ancestor of the Geadephaga, in which we assume the mandibles possessed a distinct terebra and retinaculum, of unknown form, but probably Pelophila-like. The labrum-epipharynx was likely movably articulated with the head capsule.Lineage B represents the evolution of the Trachypachidae, with mandible (bb) serving as the basal face setae. The retinaculum includes two long retinacular ridges , and is bicuspidate, teeth  short.Lineage mandible  and labrmandible  featuresThe labrum-epipharynx is movably articulated with the head capsule: labrum transverse, rectanguloid, anterior margin subtruncate, dorsal surface with a row of numerous setae ; epipharynx with short, trianguloid pedium, with few anterior and posterior parapedial setae, and a small parapedial projection. Diet yet to be determined: probably partially fluid and partially particulate matter.C The stem of the Carabidae. Evolution of the mandibles includes only simplification of the retinaculum to a single broad ridge and possibly lengthening of the terebra. The diet was probably generalized, including both solid and fluid food , and narrowing of the single ridged retinaculum (rr).  shortening of the retinaculum and its diagonal orientation (rr). The ventral groove (vg) became markedly lengthened to nearly the base of the incisor tooth, and the ventral microtrichia (vm) became markedly lengthened. The anterior margin of the labrum  describe the mode of feeding, unique among fluid-feeding carabids, in which prey is held, punctured and sheared by the incisor and terebral teeth, then passed posteriorly by the maxillary lacinia to the crushing teeth of the retinaculum. Within the preoral mill , a food bolus is rotated posterio-dorsally and anterio-ventrally, bathed in midgut enzymes. Partially digested fluid is drawn through the mouth by a powerful pharyngeal pump until all but fragments of cuticle have been ingested, at which point the bolus is ejected.G-N map the evolution of the mandibles and labrum-epipharynx in the Cicindelitae.Nodes and stems G Evolution of the amblycheiline mandibles involves principally enlargement of the terebral tooth 1 (tt 1-1), which becomes tricuspidate (tt 1-1 – tt 1-3). Within the Amblycheilini, the scrobal setae are retained in Amblycheila, but lost in the other genera  of the Cicindelitae. The basic mandibular and labral-epipharyngeal features are those of the Amblycheilini, outlined above, except terebral tooth 1 consists only of a single large cusp.Node I Evolution of the manticorine mandibles (Lineage I) involves marked ventral curvature of the terebra . Mandibles of this lineage develop a more or less extensive diastema between terebral tooth 1 and anterior margin of the retinaculum (od), and retain the scrobal setae in a few genera . The epipharynx undergoes slight differentiation with the row of the anterior parapedial setae extended anteriorly , and hence a longer retinaculum. Within this lineage, the terebral teeth differentiate in number (one to three) among taxa, and between sexes of the same taxon (cf. aps).Lineage ar tooth , srt, anaxon cf. . The epiaxon cf. , aps.L The common ancestor of the tribes Collyridini and Cicindelini. Compared to those of its adelphotaxon , the mandibles  mandible. Scrobal setae are lost in all collyridine lineages examined with the exception of Pogonostoma. Terebral teeth are reduced in number in, or lost from, the genera Ctenostoma Klug, Tricondyla Latreille, and Collyris Fabricius.Lineage al cusps . Within N The Cicindelini. . Also examined: Oxygonia gloriola Bates.Lineage Collyridini, the mandibles become markedly varied, as shown in Cicindela. The ancestral number PageBreakPageBreakof terebral teeth was probably three as in Cicindela (Oxycheila (tt 1 - tt 4), and reducing to one, as in Therates (tt 2). The supplementary retinacular tooth re-evolves in Therates (srt). The ventral groove, normally quite long (vg), becomes shortened in Cicindela longilabris (vg), and in other species of this genus (Cicindela. Also, in this genus, the number of labral setae in some species is markedly increased, and in others decreased from the ancestral cicindelite 10-12, giving an overall range of 4 to 36. The labrum-epipharynx, quite long in the earlier-evolved cicindeline lineages (Cicindela (Within this tribe, and as in the icindela , becominTherates , tt 2. TTherates , srt. Thite long , vg, becgilabris , vg, andlineages , becomesicindela .Megacephala, and collyridines except Pogonostoma; the supplementary retinacular tooth evolves twice, in the megacephalines and the cicindeline genus Therates; terebral teeth are lost in many collyridines and the cicindeline genus Therates, terebral teeth increase in number in many cicindelines; and the labrum acquires the plesiotypic shortened condition in some cicindelinesPageBreak.Building on increasingly sophisticated phylogenetic hypotheses for the Geadephaga, and the Cicindelitae e.g. , as wellCarabidae, in order to better address questions such as: is the Carabitae indeed the sister group of the Cicindelitae, and is Platychile more closely related to the amblycheilines or to the megacephalines? Incorporation of mouthpart features as character systems in phylogenetic analyses is recommended, as are further studies of the biomechanics of tiger beetle feeding and the use of mandibles during mating and mate-guarding, since the evolutionary changes we hypothesize are difficult to interpret without an appreciation of the functional consequences of changes in mouthpart configuration. In this light, further studies are also needed to adequately characterize sexual dimorphism, asymmetry, and chirality among tiger beetle mouthparts.Further research would benefit from the examination of additional taxa, both within the Cicindelitae and among other relatively basal lineages of the


Tau protein is a potential marker of neuronal damage. The aim of the study is to investigate its potential role as a marker of brain atrophy in multiple sclerosis (MS).181Thr-tau (p-tau) concentrations were assayed with commercially available INNOTEST® hTAU Ag and INNOTEST® phospho181Thr-tau(181P) and correlated with indices of brain atrophy in magnetic resonance imaging (MRI) and clinical characteristics of the study population.Cerebrospinal fluid (CSF) and blood samples were collected from 48 patients with multiple sclerosis. Total-tau (t-tau) and phosphoP = 0.01). Brain parenchymal fraction (BPF) was significantly decreased in SP patients (P = 0.002). BPF in the whole study population correlated inversely with Expanded Disability Status Scale (EDSS)  and Multiple Sclerosis Severity Score (MSSS) . T-tau in CSF in the whole patient group correlated inversely with EDSS .T-tau concentration in CSF was significantly higher in relapsing-remitting (RR) compared to secondary progressive (SP) MS patients (The results of our study suggest that total-tau concentration in CSF in a MS population decreases in the course of disease and reflects degree of parenchymal brain loss. One of An algorithm developed in 2005, the Multiple Sclerosis Severity Score (MSSS) , allows Magnetic resonance imaging (MRI) allows visualization of demyelinating lesions in the central nervous system and was incorporated in MS diagnostic criteria . The impOur working hypothesis was that the tau protein concentration in CSF reflects the degree of brain atrophy in a population of MS patients with relapsing-remitting (RR) and secondary progressive (SP) disease types.The aim of our study was to correlate indices of brain atrophy in brain MRI  with CSF and serum levels of total-tau (t-tau) and phospho181Thr-tau (p-tau) proteins. Additionally, T1 hypointense lesion (‘black holes') and fluid attenuated inversion recovery (FLAIR) hyperintense lesion volumes were calculated and correlated with indices of brain atrophy, levels of tau proteins, and clinical characteristics of the study population.CSF and blood samples were collected from 48 patients referred to our clinical ward with suspected demyelinating disease. Multiple sclerosis was diagnosed according to the McDonald criteria  with furg for 10 min. Clear serum was frozen at –80°C and stored for further assays. Lumbar punctures were performed in the morning to avoid diurnal variations in protein concentrations. A spinal catheter was introduced through the L3/L4 or L4/L5 interspace, and 5 mL of clear CSF were drawn. Samples were then centrifuged at 2000 g for 10 min, and supernatants were frozen in polypropylene tubes at –80°C for further examination. Samples were thawed just prior to laboratory processing. Total-tau (t-tau) and phospho181Thr-tau (p-tau) levels were assayed in duplicate with usage of commercially available enzyme-linked immunosorbent assay (ELISA) kits purchased from Innogenetics : INNOTEST® hTAU Ag and INNOTEST® PHOSPHO-TAU(181P) for t-tau and p-tau, respectively. The t-tau assay was designed to detect both phosphorylated and non-phosphorylated tau residues. The lowest detection threshold for total-tau was 60 pg/mL. The p-tau assay was designed to detect only molecules phosphorylated on threonine181. The lowest detection threshold was 15.6 pg/mL. ELISA procedures were performed at room temperature. For both assays the absorbance was measured with spectrophotometer at 450 nm. The standard blank was a sample of diluent alone. Intra-assay variability was <10%. T-tau and p-tau were assayed by an independent operator following instructions provided in the kit inserts.Blood samples were collected in polypropylene tubes and completely coagulated. Afterwards, they were centrifuged at 2000 In case t-tau protein was not detectable, measurement was rejected from further analysis. However, if t-tau protein level was detectable but below the threshold values mentioned above, we assumed lowest detection threshold for further analysis.Brain MRI was acquired as a part of routine hospital management with a Picker Eclipse GE Healthcare 1.5 Tesla scanner. MR T1-weighted images were acquired with gradient echo sequence, with TR = 300 ms and TE = 4 ms, flip angle = 80°. MR FLAIR images were acquired with spin echo sequence, with TR = 13,716 ms, TE = 112 ms, and TI = 1,800 ms, flip angle = 90°. In-plane resolution in all sequences was 1 mm, and slice-thickness was 5.0 mm.A first MRI examination was carried out in all patients at baseline simultaneously with serum and CSF sampling. In 25 patients brain MRI was performed twice after a mean period of 3.6 years (SD = 2.3).Brain morphometric measures were performed with Java Image (JIM) 5.0 software from Xinapse Systems with the use of ‘brain finder' and ‘multiple sclerosis lesion finder' tools, using axially orientated images.The ‘brain finder' tool was used to calculate brain volume, CSF volume, and brain parenchymal fraction (BPF). BPF was defined as the ratio of parenchymal volume to the total volume within the brain surface contour:After initial brain delineation, images were checked, and brain border was manually corrected where needed. Brain volume was calculated on the basis of pixel intensity. Each set of images was analysed visually to ensure appropriate calculation. Threshold pixel intensity, distinguishing brain from CSF, was set manually in all images to 300.Lesion load was calculated on T1-weighted and FLAIR MRI brain images with the ‘MS lesion finder' tool. Initially, during visual inspection, lesions were marked. A point marker was used in cases of well demarcated lesions. In cases of lesions with less distinct borders, the ‘irregular region of interest' marker was used. Before lesion volumes were calculated, the most appropriate settings were checked by trial and error. Finally, ‘weight on prior probabilities' was set to 0.6, and fuzzy threshold was set to 0.25. Lesion volume was calculated with use of ‘connect in 3D' option.R), and group comparisons were performed with the Mann–Whitney U non-parametric test; values were then presented as medians and range. If normality was assessed unpaired Student's t test was adopted, Pearson coefficient (R), was used, and values were presented as means and standard deviation. Linear regression coefficient (2R) with 95% CI was used to present graphical relations between variables in the Figures. P < 0.05 was regarded as statistically significant.All statistical calculations were accomplished with GraphPad Prism 5.01 software. Normality was assessed using the D'Agostino–Pearson test. In cases where normality could not be assumed, relations between variables were calculated as Spearman correlation coefficient (T-tau in CSF was detectable in 31/48 of cases (64.6%). P-tau in CSF was detectable in 48/48 of cases (100%). Total-tau in serum was detectable in 16/48 of cases (33%). P-tau in serum was detectable in 41/48 of cases (85%). Due to the low detection rate of t-tau in serum, these measurements were not included in further analysis.T-tau and p-tau concentrations in CSF, p-tau concentration in serum, brain volumes, CSF volumes, and BPF for RR and SP patients are summarized in U P = 0.01) with higher values in the RR group (t test P = 0.002). CSF volume differed significantly, with higher values in the SP group (Student's t test P = 0.006). There was no significant difference between the RR and SP groups regarding brain volume.T-tau concentration in CSF was significantly different in the RR and SP groups (Mann–Whitney RR group . No difft test P < 0.0001). A similar relation was detected with respect to MSSS which was significantly lower in RR patients (Student's t test P = 0.0001).EDSS was significantly lower in the RR group compared to SP patients  (T-tau in CSF in the whole patient group correlated inversely with EDSS (Spearman  = 0.04) .R = –0.51, P = 0.0002; linear regression 2R = 0.26, P = 0.0002  (Brain volume (in mL) in the whole study population correlated inversely with EDSS (Pearson = 0.007) .In the follow-up period of mean 3.6 years, in the population of 25 patients:P = 0.0008.1. BPF decreased significantly from a mean value of 0.87 (SD = 0.026) to 0.84 (SD = 0.049), P = 0.001.2. Brain volume (mL) decreased significantly from mean value of 1,351 (SD = 172) to 1,286 (SD = 176), P = 0.01.3. CSF volume (mL) increased significantly from mean value of 198 (SD = 32) to 245 (SD = 69), R = 0.63, P = 0.0008; linear regression 2R = 0.39, P = 0.0008).BPF decrement correlated with time of observation .Brain volume (mL) decrement correlated with time of observation .CSF volume (mL) increment correlated with time of observation  and FLAIR lesion load .EDSS correlated positively with T1-weighted lesion load (Spearman BPF did not correlate with T1-weighted lesion load or with FLAIR lesion load.U P = 0.0002) in the whole study population. There was a trend to show higher T1-weighted and FLAIR lesion load in the SP group compared to RR patients, but this difference did not reach statistical significance.FLAIR lesion load was significantly higher than T1-weighted lesion load  in subarachnoid spaces through arachnoid granulations and spinal nerve roots into dural venous sinuses . Experim181 moiety , can be easily explained by different detection thresholds assumed in available ELISA kits. In the work by Reiber mentioned above , compared to its phosphorylated on threonineTau protein can be detected in CSF and serum of healthy individuals of different ages without any apparent central nervous system (CNS) pathology . As the Moreover, we can assume that different pathological mechanisms result in various t-tau leakage patterns. In acute conditions, like stroke , head trOn the contrary, the RR type of MS is an autoimmune inflammatory disease with a pathological process driven by lymphocytic attack on myelin sheaths producing oligodendrocytes in the CNS. Demyelination with relative axonal preservation is the main pathological process. Despite clear evidence showing axonal loss being present from an early stage of the disease, basic MS pathophysiology remains extracellular . During In the SP stage, with predominant neurodegenerative mechanisms, the constant decrease in neuronal density results in loss of tau resources; thus a lower t-tau in CSF reflects degree of brain atrophy. A number of studies confirmed progressive neurodegeneration in the course of MS that begins in the early stage of the disease. BPF was found to be lower in MS patients compared to healthy controls and to decrease continuously in the course of the disease . In magnIn AD, continuous brain atrophy is accompanied by an increase of CSF t-tau and p-tau concentrations. The negative relation was strengthened in the context of regional cortical atrophy for t-tau , but alsThe most striking result of our study is the significant difference between CSF t-tau concentrations between RR and SP patients, with higher values in the RR group . To our Difference in t-tau concentration is consistent with BPF values, showing decrease of parenchymal fraction in the course of MS, thus indirectly reflecting degree of brain atrophy. Difference in BPF can be attributed to an increase in intracranial CSF volume, reflecting loss of parenchymal tissue. Accordingly, the degree of disability expressed in EDSS and disease severity in MSSS is higher in SP patients compared to RR population, and correlated inversely with BPF. Negative correlation of CSF t-tau with a degree of motor disability in EDSS merges theoretical assumptions, forming a pattern in which t-tau indirectly reflects degree of neuronal loss.Additional calculations performed on the basis of follow-up brain MRI in 25 patients confirmed BPF and brain volume decrement in the course of MS. Our study showed that the degree of disability (EDSS) correlated with T1-weighted and FLAIR lesion load. This result is confirmatory of previously published studies .In our material T1-weighted lesion load was significantly lower than FLAIR lesion load. This observation can be explained on the basis of the pathophysiological importance of various lesion types. Lesions appearing as T1 ‘black holes' represent irreversible neuronal loss and appear late as a result of profound parenchymal damage . A recenIn conclusion, the results of our study suggest that t-tau concentration in CSF in MS population decreases in the course of disease and indirectly reflects the degree of parenchymal brain loss. The influence that a disease subtype exerts on the t-tau concentration in CSF depends apparently on the predominant pathophysiological process (neurodegeneration in the SP stage and inflammation in the RR stage). It is important to note that our results are population-based. Due to a number of conceivable factors influencing the tau concentration in CSF and the relatively small concentration of this protein in CSF of MS patients , a single tau assay is not enough as an individual biomarker. Anyway, population measurements confirm constantly on-going neuronal loss in the course of MS and thus emphasize the need for future studies in search of MS aetiology and effective treatment.