Sphingoid bases are formed from the precursors L-serine and palmitoyl-CoA-a reaction which is catalyzed by the serine-palmitoyltransferase (SPT). SPT metabolizes, besides palmitoyl-CoA also other acyl-CoAs but shows also variability towards the use of other amino acid substrates. The enzyme is also able to metabolize alanine, which results in the formation of an atypical deoxy-sphingoid base (DSB). This promiscuous activity is greatly increased in the case of the sensory neuropathy HSAN1, and pathologically elevated DSB levels have been identified as the cause of this disease. Clinically, HSAN1 shows a pronounced similarity to the diabetic sensory neuropathy (DSN), which is the most common chronic complication of diabetes mellitus. Since serine and alanine metabolism is functionally linked to carbohydrate metabolism by their precursors 3-phosphoglycerate and pyruvate, we were interested to see whether the levels of certain sphingoid base metabolites are altered in patients with diabetes.In a case-control study we compared plasma sphingoid base levels between healthy and diabetic individuals. DSB levels were higher in the diabetic group whereas C16 and C18 sphingoid bases were not significantly different. Plasma serine, but not alanine levels were lower in the diabetic group. A subsequent lipoprotein fractionation showed that the DSBs are primarily present in the LDL and VLDL fraction.Our results suggest that DSBs are a novel category of plasma biomarkers in diabetes which reflect functional impairments of carbohydrate metabolism. Furthermore, elevated DSB levels as we see them in diabetic patients might also contribute to the progression of the diabetic sensory neuropathy, the most frequent complication of diabetes. In this, the SPTLC3 subunit shows a higher affinity towards shorter acyl-CoAs (e.g. C12 and C14) whereas SPTLC2 shows a higher activity with C16 and C18 acyl-CoAs. Both C18 sphingoid and C16 sphingoid bases have been detected in significant amounts in human plasma [Sphingolipids comprise a heterogeneous class of lipids that contribute to plasma membrane and plasma lipoprotein formation. They are derived from the aliphatic amino-alcohol sphingosine, which is commonly formed from the precursors L-serine and palmitoyl-CoA. The condensation of serine with palmitoyl-CoA is a pyridoxalphosphate (PLP) dependent reaction and catalyzed by the enzyme serine palmitoyltransferase (SPT) EC 2.3.1.50). SPT is a heteromeric enzyme and composed of at least three subunits  [.50. SPT 1-hydroxyl group of SA and are therefore neither metabolized to complex sphingolipids nor degraded by the regular sphingolipid catabolism, since sphingosine-1P as a catabolic intermediate cannot be formed from DSBs.Moreover, SPT shows variability towards the use of other amino acid substrates. Besides L-serine, which is the preferred substrate, the enzyme also metabolizes L-alanine and to a certain extent glycine ,5. This in-vitro and in a disruption of the neuronal cytoskeleton structure [The activity of SPT with alanine and glycine is greatly increased in the presence of several SPT missense mutations which are associated with the inherited sensory neuropathy HSAN1 OMIM162400). HSAN1 is an autosomal dominantly inherited axonal neuropathy that is clinically characterized by a loss of pain and temperature sensation, usually starting in the lower limbs and often accompanied by neuropathic pain attacks and skin ulcers. The mutant SPT in HSAN1 shows a highly increased activity with alanine and glycine compared to the wildtype SPT. Consequently, these lipids are found at elevated levels in cells and plasma from HSAN1 patients [00. HSAN1Spisula polynyma as an investigational marine anticancer drug. It was shown to induce cell death in various breast cancer cell lines [Originally, doxSA  has been isolated from the arctic clam ll lines ,8 and toll lines .Clinically, HSAN1 shows a pronounced similarity to the diabetic sensory neuropathy (DSN), which is the most common chronic complication of diabetes mellitus. Both diseases have a late onset and slow progression and typically affect the distal extremities first. The degeneration of small sensory fibers results in the loss of pain sensation, which in turn leads to painless injuries .In this context it is important to note that DSBs are not exclusively formed by the mutant but also by the wildtype SPT - however to a lesser extent . CompariA total of 70 men and 29 women from Zurich volunteered to participate in the study. Written informed consent was obtained from all participants and the local Ethics Committees approved the study . The casPlasma sphingolipids were extracted and analyzed by LC-MS as described earlier Plasma was isolated from diabetic donors after overnight fasting. Three ml plasma was fractionated on a four step density gradient essentially as described . UltracePlasma amino acid concentrations were analyzed according to the method of Frank and Powers Significance levels were determined with an unpaired student t-test using Sigma Plot v10.0 . The correlation matrix was generated in SPSS . ROC analysis was performed with XLSTAT Sphingolipids in plasma are usually present in a broad variation of subspecies. Sphingoid bases can be found in the saturated  and unsaturated (sphingosine) state and are usually N-acetylated with a second fatty acid. Most of the plasma sphingolipids are also conjugated to O-linked head groups, which divert them analytically into various subgroups. However, for this study we were primarily interested in differences between the individual sphingoid base backbones. We therefore simplified the analysis by subjecting the sphingolipids to a sequential acid and base hydrolysis. Acid hydrolysis specifically breaks the N-alkyl chain, whereas alkaline conditions lead to a release of the O-linked head group. The resulting free sphingoid base metabolites were analyzed by LC-MS. The sum of the individual sphingoid bases reflects the total sphingolipid content in the analyzed samples.16-, C18-and deoxy-sphingolipid levels in a case-control setup comparing 50 patients with documented diabetes mellitus type II (D) and 49 healthy controls (C). The average age of the control group was slightly younger and included more females than the diabetic group. Results are summarised in table In this study we analyzed the C18-Sphingosine (C18SO) and C16-Sphingosine (C16SO). The concentrations of the dihydro-forms C18-Sphinganine (C18SA) and C16-Sphinganine (C16SA) were much lower and represented about 1-5% of the respective sphingosine forms. Deoxy-sphingoid bases were generally minor, representing 0.1-0.5% of the total plasma sphingoid bases. For the DSBs the saturated (doxSA) and unsaturated (doxSO) metabolites were found at similar concentrations. Deoxymethyl-SA or deoxymethyl-SO were not detected.As reported earlier, the most abundant sphingoid bases in plasma were C16 and C18 sphingoid base levels were not significantly different between the two groups. This indicates that certain sphingoid bases are specifically altered under diabetic conditions whereas others remain unchanged. Among the other variables total-and LDL-cholesterol was lower in the diabetic groups, which is explained by the general administration of cholesterol lowering drugs (e.g. statins) in this group. HDL cholesterol was lower in the diabetic groups whereas triglycerides were not different. Age and smoking had no influence.We found that the average doxSO levels were significantly higher in the diabetic than in the control group. Also doxSA levels were, although less pronounced, higher in the diabetic group table . On the Since doxSO showed the most significant changes between the groups we calculated the diagnostic values for this variable. A receiver operator curve (ROC) analysis figure  revealed16 and C18-sphingoid bases are functionally linked to serine metabolism whereas the DSBs are linked to alanine metabolism. We therefore also analyzed the plasma amino acid levels between the two groups. Total amino acid concentration was in general lower in diabetic patients. The most significant changes were seen for glycine, serine and threonine, whereas other amino acids like alanine, valine or histidine were not altered  and to a certain extent with LDL, but not with HDL.The correlation matrix table  for the 16-and the C18-sphingolipids were found in the LDL and HDL but not in the VLDL fraction. In contrast, the DSBs were predominantly found in LDL and VLDL but were nearly absent in HDL. This indicates that the plasma DSBs are of hepatic origin since LDL and VLDL but not HDL primarily originate from the liver.To gain further insight into the physiology of the DSBs we analyzed their distribution into plasma lipoprotein fractions. Lipoproteins were separated by ultracentrifugation, fractionated and the individual fractions analyzed for cholesterol, triglycerides (TG) and sphingoid bases figure . The C1616SA, C16SO, C18SA and C18SO were not different. This indicates that impairments in glucose metabolism as it is the base of diabetes are reflected in changes of some but not all sphingoid bases. Sphingolipid metabolism represents a metabolic cross point which interconnects lipid (acyl-CoA) and amino acid  metabolism. The cellular serine and alanine production is functionally linked to carbohydrate metabolism by their precursors 3-phosphoglycerate and pyruvate and thereby indirectly also to the carbohydrate metabolism. Therefore, the observed changes indicate a functional interaction between sphingolipid, glucose and fatty acid metabolism which results in an increased production of DSBs in diabetes.In this study we demonstrate that deoxy-sphingoid bases are elevated in patients with diabetes whereas the other analysed sphingoid base metabolites like CA limitation of this study is the overrepresentation of patients receiving cholesterol lowering drugs in the diabetic group compared to the controls. Thus an impact of statin treatment on the sphingoid base levels cannot be completely excluded. However, diabetic patients with and without statin treatment show no significant differences in the sphingoid base levels. This indicates that a statin treatment does not significantly influence sphingoid base levels. Nevertheless, the influence of statins needs to be addressed in more detail in further studies.16 and C18 sphingoid bases [18 and C18 sphingoid base levels. This was, by trend, indeed the case although the differences between the two groups were not significant for this cohort.Overall plasma amino acid levels were found to be lower in the diabetic group compared to controls. Among the most significantly lowered amino acids were threonine and serine. Plasma alanine levels, in contrast, were not significantly different between the two groups. It was recently shown that increasing L-alanine levels stimulate DSB formation, whereas the presence of L-serine suppresses DSB formation and increases the generation of Cid bases . The lowEarlier reports on amino acid metabolism in obese patients indicate that the situation might be more complex, however. The observation that DSBs are preferentially found in LDL indicates that the plasma DSB levels are primarily of hepatic origin. It has been reported that alanine uptake into the liver is significantly increased in obese individuals . IncreasBesides the direct uptake of alanine into the liver, another mechanism might also contribute to increased hepatic DSB generation. Hepatic glucose uptake is primarily mediated by GLUT2 and is hence insulin independent. Hyperglycemic conditions are therefore associated with high hepatic glucose levels, leading to increased glycolytic flux and production of pyruvate. Hepatic glucose overload might therefore lead to a build-up of pyruvate and its anaerobic conversion into either lactate or alternatively alanine which, in turn, is then converted to doxSA by the hepatic SPT.This mechanism could also lead to increased DSB generation in other tissues in which glucose uptake is insulin independent, e.g. in kidney, pancreatic beta cells or neurons.Especially in peripheral neurons elevated DSB levels could contribute to the pathology of diabetic sensory neuropathy (DSN). The inherited neuropathy HSAN1 is caused by a pathological overproduction of DSBs due to several missense mutations in SPT . InteresHowever, in the context of our results, further studies are necessary to validate the role of atypical sphingoid bases as novel biomarkers in diabetes and to address a potential involvement of these metabolites in the pathology of DSN.The authors declare that they have no competing interests.MB performed the lipids analysis; MF performed the amino analysis; AO performed the statistical analysis; JMJ and MH were involved in study samples collection; AE was involved in the clinical chemistry analytics; TH participated an the design of the study and wrote the manuscript.All authors have read and approved the final manuscript


A comparison has been made of the cachectic effects produced by the transplantable murine adenocarcinoma of the mouse colon (MAC16) with tumour necrosis factor-alpha (cachectin). Tumour necrosis factor-alpha  produced a dose-related weight reduction that was accompanied by a decrease in both food and water intake. The degree of weight loss was directly proportional to the decreased food and water intake. In contrast weight loss produced by the MAC16 tumour occurred without a reduction in fluid or nutrient intake. Both the MAC16 tumour and TNF-alpha produced hypoglycaemia and a reduction in the circulatory level of free fatty acids (FFA), but had opposite effects on the level of plasma triglycerides with the MAC16 tumour-induced cachexia causing a decrease and TNF-alpha producing an increase. The MAC16 tumour elaborated a lipolytic factor which caused an immediate release of FFA from adipose tissue. In contrast TNF-alpha had no effect on mobilization of adipose triglycerides over a short time period. Both TNF-alpha and extracts from the MAC16 tumour caused an enhanced release of amino acids from mouse diaphragm, which was suppressible with indomethacin and heat labile. No TNF was detected in the MAC16 tumour or in the serum of tumour-bearing animals. Both tumour and non-tumour-bearing animals responded with a similar elevation of their serum TNF levels 90 min after a single injection of endotoxin. It is concluded that weight loss produced by TNF-alpha arises from an anorexic effect and that this differs from the complex metabolic changes associated with cancer cachexia.


Herpes simplex virus (HSV) type 1 was identified in respiratory specimens from a cluster of eight patients on a surgical intensive care unit within 8 weeks. Six of these patients suffered from HSV-related tracheobronchitis and one from HSV-related pneumonia only. Our outbreak investigation aimed to determine the clinical relevance of and risk factors associated with HSV-related tracheobronchitis or pneumonia in critically ill patients, and to investigate whether the cluster was caused by nosocomial transmission.A retrospective cohort study was performed to identify risk factors for the outcomes of HSV-related tracheobronchitis or pneumonia and death using univariable analysis as well as logistic regression analysis. Viruses were typed by molecular analysis of a fragment of the HSV type 1 glycoprotein G.P < 0.0001). The interaction of longer duration of ventilation and tracheotomy was associated with HSV-related tracheobronchitis or pneumonia in multivariable analysis.The cohort of patients covering the outbreak period comprised 53 patients, including six patients with HSV-related tracheobronchitis and one patient with pneumonia only. HSV-related tracheobronchitis or pneumonia was associated with increased mortality (100% in patients with versus 17.8% in patients without HSV-related tracheobronchitis or pneumonia; Identical HSV type 1 glycoprotein G sequences were found in three patients and in two patients. The group of three identical viral sequences belonged to a widely circulating strain. The two identical viral sequences were recovered from bronchoalveolar lavages of one patient with HSV-related tracheobronchitis and of one patient without clinical symptoms. These viral sequences showed unique polymorphisms, indicating probable nosocomial transmission.HSV-related tracheobronchitis or pneumonia is associated with increased mortality in critically ill patients. Care should be taken to avoid nosocomial transmission and early diagnosis should be attempted. Herpes simplex virus (HSV) is a double-stranded DNA virus occurring in two types, HSV-1 and HSV-2. Transmission usually occurs by contact with infected saliva or cutaneous lesions . ClinicaAsymptomatic shedding of HSV in healthy individuals has been reported to occur in 2–10% of infected individuals ,5. HSV-1As more than 90% of adults have antibodies specific for HSV-1 , infectiEight patients in a surgical intensive care unit (SICU) had HSV-1 detected in their respiratory tract within 8 weeks. Tracheobronchitis was associated with HSV-1 detection in six patients and with pneumonia in four patients. This cluster prompted us to investigate the clinical impact of HSV-related tracheobronchitis or pneumonia and to identify risk factors predisposing to HSV-related tracheobronchitis or pneumonia and fatal outcome. As the cluster suggested the possibility of nosocomial transmission, molecular epidemiological studies were performed to type all viruses recovered from the patients.When a cluster of six patients with HSV-1-related tracheobronchitis occurred on a 15-bed cardiothoracic SICU .The institutional review board approved the outbreak investigation.n = 53). The analysed outcomes were death and HSV-related tracheobronchitis or pneumonia. The latter was defined as HSV detection in the respiratory tract concomitant with the presence of tracheobronchitis or pneumonia and an absence of other respiratory pathogens even though histopathology was not performed. Sampling for HSV detection in respiratory fluids was performed if clinically indicated .A retrospective cohort study was performed including the 8-week period that entirely covered the cluster episode. During this period all patients admitted to the SICU with a stay longer than 72 hours were included  because he did not show symptoms related to HSV Tables  and 2. TThe variables analysed as risk factors for HSV-related tracheobronchitis or pneumonia and fatal outcome are presented in Tables P < 0.05 was regarded as significant for univariable analysis. A significance level of 0.1 was chosen for inclusion of variables into a logistic regression model and for remaining included in the model.Univariable analysis and logistic regression with stepwise (forward and backward) variable selection were performed using SAS software . c index) is used to evaluate the predictive power of the logistic regression model. The c index represents the probability that the regression model equation assigns randomly chosen patients with HSV higher probabilities of acquiring HSV than randomly chosen patients without HSV [The area under the receiver operating characteristic curve  were submitted to direct immunofluorescence staining with antibodies specific for HSV. A sample (1–2 ml) of the specimen was added to cell cultures of Hep-2 and Vero cells, and was monitored twice weekly for up to 3 weeks for emergence of a cytopathic effect. DNA was isolated and a real-time PCR was performed, detecting a 254 bp fragment of the HSV UL27 gene . Melting curve analysis was used to differentiate HSV-1 and HSV-2.All viruses recovered from patients on the SICU during the time period of the cluster .The nucleotide sequences have been submitted to the NCBI database GenBank  and with the MEGA Software package (version 3.1) . The phyHSV-related tracheobronchitis was diagnosed in six patients on a SICU within 8 weeks. Endobronchial bleeding was life-threatening in three patients. HSV-1 was detectable in the respiratory specimens of all patients Tables  whereas All patients were HSV IgG-positive at the time of HSV detection, suggesting that none of them suffered from primary infection.In order to confirm that HSV-related tracheobronchitis or pneumonia was associated with a higher mortality, and to identify risk factors for this entity, a cohort study including 52 patients  was performed. Tables Fatal outcome was associated with longer duration of ventilation, with bronchoscopy and with higher number of thrombocyte concentrate transfusions in multiple logistic regression analysis Table . For matNo other examined variable showed a significant association with fatal outcome or with HSV-related tracheobronchitis or pneumonia in multiple logistic regression analysis.To address the question of endogenous reactivation versus nosocomial transmission, all cluster patients' viruses were subjected to molecular typing. Sequencing of the hypervariable region of the viral glycoprotein G revealed that patients' viruses fell into two groups, with viral sequences of patients C2 and C3 grouping together and with viral sequences of patients C1, C7 and C8 grouping together , although it was not associated with increased mortality in their patient cohort . InteresAntiviral therapy with acyclovir initiated after diagnosis of HSV-1 had been established in five of eight patients on average 2.9 days before death (data not shown). The therapy initiation, however, seems to have been too late to improve outcome. In case of endobronchial bleedings or bronchoscopic signs of haemorrhagic tracheobronchitis, therefore, acyclovir treatment should be initiated directly after bronchoscopy and specimen sampling (prior to diagnosis of HSV-1 infection), which would have been on average 8.6 days prior to death in our collective (data not shown).Surgical procedures and critical illness result in immune dysfunction ,19, whicP < 0.0001, Table The most significant risk factor for fatal outcome identified in univariable analysis was HSV-related tracheobronchitis or pneumonia  and not virologically (absence of HSV detection). HSV pneumonia or HSV-related tracheobronchitis was not confirmed by histopathology because, clinically, the presence of pneumonia or tracheobronchitis concurrent with HSV detection in respiratory fluids in the absence of other respiratory pathogens was regarded as sufficient for diagnosis and initiation of antiviral treatment. The size of the cohort is rather small, a limitation that is inherent to outbreak investigations.Worthy of note is the fact that two bronchoscope samples had HSV-1 DNA detected after routine disinfection procedures. Typing was successful in only one case , which had a sequence identical to patient isolate C6 and to a number of epidemiologically unrelated virus isolates Figures  and 3. TStudies examining HSV-1 detection in the respiratory tract of intensive care unit patients have assumed HSV-1 positivity to be caused by endogenous reactivation, and have not considered transmission although viruses were not typed ,7-9. HSVWe found three cluster patients with HSV-1 sequences that were not detected in other cluster patients' viral isolates (isolates C4, C5 and C6; Figure The group of three cluster patients with identical viral sequences also showed identical sequences to nine epidemiologically unrelated isolates and to the reference strain F Figure . FurtherAs HSV isolates are not routinely typed it is possible that nosocomial transmission is not a rare event but might often not be recognized as such.In the present article we have shown that HSV-related tracheobronchitis or pneumonia represents an important infectious complication in critically ill patients. This differential diagnosis has to be considered especially in the case of tracheobronchial haemorrhage or if the patient's condition does not improve with antibacterial and antifungal therapy. Whether acyclovir prophylaxis or early treatment in cases suspicious for HSV-related tracheobronchitis or pneumonia in critically ill patients has the potential of preventing fatal outcome should be addressed in future prospective studies.• HSV-related tracheobronchitis or pneumonia is associated with high mortality in critically ill patients.• Tracheobronchial haemorrhage should prompt diagnostics for HSV.• Molecular typing of virus isolates revealed one event of probable nosocomial transmission.bp = base pair; HSV = herpes simplex virus; SICU = surgical intensive care unit; PCR = polymerase chain reaction.The authors declare that they have no competing interests.IE participated in the design of the outbreak investigation, carried out the molecular genetic studies and the phylogenetic analysis, and wrote the manuscript. JG participated in the collection, interpretation and analysis of clinical data. AM participated in the collection, interpretation and analysis of clinical data. DS performed the statistical analysis. AR participated in the collection, interpretation and analysis of clinical data. CH-G participated in the phylogenetic analysis. PG participated in the study design, interpretation of data and drafting of the manuscript. TW participated in the interpretation of data and drafting of the manuscript. TFS participated in the study design, interpretation of the data and drafting of the manuscript. FM initiated the study, participated in the design of the study and in the interpretation and analysis of clinical data, and contributed to the writing of the manuscript.


Peripheral blood mononuclear cells will lyse antibody-treated human erythrocytes. Using Group A red cells and a hyperimmune anti-A1 serum, we have devised a microassay for the cytolytic capacity of mononuclear cell suspensions. The effector cells responsible for red-cell lysis are mononuclear, adherent and phagocytic, and their activity is blocked by aggregated IgG. Their presence correlates well with non-specific esterase-containing cells and we conclude that they are monocytes. Dose-response curves of red-cell lysis plotted against numbers of monocytes were used to derive a simple parameter expressing the number of monocytes needed to lyse 15% of the 51Cr-labelled red cells. The assay was applied to a group of 27 normal controls and 36 patients with a histologically proven diagnosis of malignant melanoma. The results indicate that monocytes from patients show significantly greater lytic activity than those from the controls. These data suggest that monocytes from cancer patients are in some way activated, and that other defects in monocyte function which have been detected in cancer patients (defective chemotaxis and maturation) may be associated with monocyte "activation".


Drosophila might also amplify neuronal proliferation by generating symmetrically dividing intermediate progenitors.In the mammalian brain, neural stem cells divide asymmetrically and often amplify the number of progeny they generate via symmetrically dividing intermediate progenitors. Here we investigate whether specific neural stem cell-like neuroblasts in the brain of Drosophila. These lineages are generated by brain neuroblasts that divide asymmetrically to self renew but, unlike other brain neuroblasts, do not segregate the differentiating cell fate determinant Prospero to their smaller daughter cells. These daughter cells continue to express neuroblast-specific molecular markers and divide repeatedly to produce neural progeny, demonstrating that they are proliferating intermediate progenitors. The proliferative divisions of these intermediate progenitors have novel cellular and molecular features; they are morphologically symmetrical, but molecularly asymmetrical in that key differentiating cell fate determinants are segregated into only one of the two daughter cells.Cell lineage-tracing and genetic marker analysis show that remarkably large neuroblast lineages exist in the dorsomedial larval brain of Drosophila that involves the amplification of neuroblast proliferation through intermediate progenitors. This type of neurogenesis bears remarkable similarities to neurogenesis in the mammalian brain, where neural stem cells as primary progenitors amplify the number of progeny they generate through generation of secondary progenitors. This suggests that key aspects of neural stem cell biology might be conserved in brain development of insects and mammals.Our findings provide cellular and molecular evidence for a new mode of neurogenesis in the larval brain of  Neural stem cells are primary precursors that have the ability to renew themselves at each division such that one of the two daughter cells retains stem cell identity, while the other enters a program of differentiation and contributes to a continuous supply of neural cell types. Understanding how neural stem cells maintain their pluripotent state and how their progeny differentiate into distinct neural fates is of central importance for understanding nervous system development . NeurDrosophila central nervous system is an excellent simple model system for analyzing the molecular mechanisms that control neural stem cell divisions , delaminate as single cells from the neuroectoderm and undergo repeated asymmetric cell divisions, each of which self-renew the NB while producing a smaller neural progenitor cell called a ganglion mother cell (GMC). Compared to the NB, the GMC adopts a radically opposite fate and undergoes a single neurogenic division to produce two cells that exit the cell cycle and differentiate . Drosiewed in -12). Duriewed in -16.Mechanisms involved in NB division and neural proliferation during embryogenesis have been studied in great detail . prospero in somatic clones results in massive overproliferation of cells that express molecular markers of NBs [During the postembryonic period of neurogenesis, the NBs of the central brain and thoracic ganglia are thought to undergo a similar proliferation program and express many of the asymmetric cell fate determinants that characterize embryonic neurogenesis ,30. Nucls of NBs -33. Addis of NBs ).Drosophila is an exceedingly complex structure with numerous highly organized neuropil assemblies, such as the mushroom bodies, central complex and antennal lobes, as well as other specialized neuropils and major fiber tracts required for complex behavioral functions [Controlled neuronal proliferation is especially important for the generation of the adult brain. The mature brain of unctions . Remarkaunctions ,37. GiveDrosophila bears remarkable similarities to mammalian neurogenesis, where neural stem cells as primary progenitors often amplify the number of progeny they generate via symmetrically dividing secondary progenitors . Using cell lineage-tracing and marker analysis, we show that remarkably large NB lineages develop in the dorsomedial (DM) area of the larval brain. Like any other lineages in the brain, they derive from unique NB precursors that remain associated with their post-mitotic neuronal progeny. In addition, they contain a large pool of cells that do not express neuronal differentiation markers, are engaged in the cell cycle, and show mitotic activity. While some of these mitotically active cells are GMCs, the others express NB-specific molecular markers and divide repeatedly to produce neural progeny, implying that they are IPs. The proliferative divisions of these IPs are morphologically symmetrical, but molecularly asymmetrical in that cell fate determinants such as Prospero and Miranda are segregated into only one of the daughter cells. The IPs are generated by a specific set of NBs that do not segregate Prospero to their smaller daughter cell, thereby allowing this cell to retain proliferative capacity instead of undergoing its final neurogenic division. The amplification of NB proliferation through IPs reported here for iewed in ). This sSince most of the secondary, adult-specific neurons of the brain are generated during larval development , we used3 (n = 12) and 424 ± 110 μm3 (n = 13), respectively. Preliminary analysis of the axonal tracts suggests that the large NB clones in the dorsal brain correspond to the pl and pm subgroups of the Dorsoposterior medial (DPM) lineages previously described (data not shown) [Prominent among these were unusually large clones recoverable at the DM margins of the brain hemispheres Figure . Six NBst shown) .To compare the proliferative capacity of the DM NBs with that of other NBs in the larval central nervous system, we quantified the number of cells in DM NB lineages, in mushroom body NB lineages, and in other NB lineages scored randomly in different brain and ventral ganglion regions of the late third instar larvae shortly before pupation (96 h ALH). The number of cells in the DM lineages had an average value of 450 (range 370–580). Remarkably, this was more than twice the average number of cells observed for the larval lineages of the mushroom body NBs  or for other larval NB lineages scored in other areas of the central nervous system Figure .To determine the rate of clone size increase during larval central nervous system development, we counted the number of cells in MARCM-labeled DM NB clones, mushroom body NB clones and other dorsal brain NB clones at various larval stages Figure . FollowiTo investigate this further, we cultured MARCM-labeled brain explants in 5-bromodeoxyuridine (BrdU) and then used anti-BrdU immunocytochemistry to determine the number of cells engaged in S-phase in DM clones compared to other NB clones of the central brain. Following a 90 minute pulse of BrdU incorporation in L3 brain explants, we found a markedly higher number of BrdU-positive cells in DM clones  than in the other NB clones scored at random in dorsal brain regions of the same specimens . These data indicate that a significant amplification of proliferation occurs in the DM lineages when compared to other NB lineages of the central brain (hereafter collectively referred to as 'non-DM' lineages).in situ detection of mitotically active versus post-mitotic cells in labeled NB lineages of the larval brain.The large number of cells found in the DM NB clones could, in principle, be due to an unusually high rate of mitotic activity of the DM NBs. However, immunodetection of mitotic DNA in MARCM clones (via the phospho-histone H3 (PH3) epitope) revealed a comparable mitotic frequency in these NBs  compared to NBs found in dorsal  or ventral  brain lineages. This prompted us to search for other types of progenitor cells in these lineages. To this aim, we first characterized molecular markers enabling Typically, in all NB clones examined, the majority of the labeled cells expressed the neuronal identity marker Elav. Prominent exceptions were the large NBs and a set of smaller cells closely associated with the NBs, all of which were Elav-negative Figure . Quantifmiranda gene has been reported to be expressed in larval NBs but not in their GMCs [Could these smaller Elav-negative cells associated with the NBs be GMCs? To investigate this, we first studied the expression of the coiled-coil protein Miranda. The eir GMCs ; Mirandaeir GMCs ,30). In In green fluorescent protein (GFP)-labeled non-DM NB clones, used as control, a small number of GMCs were observed as small NB-associated cells expressing either CycE or PH3 Figure . At 96 hContrasting with this simple pattern, DM lineages contained an average of 38 CycE-positive cells located around the NB, and many scattered mitoses, up to 14 per clone, were observed by PH3 immunoreactivity Figure . This stIf some of the mitotically active cells in DM NB clones are amplifying IPs, they might be expected to have cellular and molecular features in common with proliferating NBs. To investigate this, we first examined the expression patterns of Prospero, Miranda, and CycE in NBs of non-DM lineages, used as control, as well as in the small NB-associated progenitors of the DM lineages. For this, MARCM clones induced at larval hatching were scored at 96 h ALH. Importantly, we further restricted our analysis to cells engaged in mitosis (PH3-positive) in order to identify progenitor cells unambiguously and to obtain valid comparisons, since all markers showed cell-cycle dependent expression (see below). In non-DM clones, Prospero was specifically detected at the cellular cortex of the NBs, accumulating on one side during mitosis Figure , arrow. As expected, the adaptor protein Miranda formed prominent cortical crescents in dividing NBs of non-DM clones Figure , arrows.- nuclei; see also Figure To confirm the presence of both NB-like and GMC-like progenitors in the DM NB lineages, we searched for markers of cellular identity that did not rely on the conventional criteria of cell size and/or cortical polarity. Significantly, we found that in non-DM lineages (taken as reference lineages), CycE was detected in virtually all the self-renewing NBs during mitosis . In agreement with anti-PH3 staining on fixed tissue, we observed numerous cell divisions among the small cells that were closely associated with the NB in DM NB clones Figure . With thNext, we performed a more detailed analysis of the different types of MARCM clones that were recoverable in the DM lineages. To date, only two types of multicellular clones have been observed in the central brain following a somatic recombination event in a parental NB and the loss of the GAL80 repressor in one of the post-mitotic siblings. Thus, the NB clones described above derive from the proliferation of GAL80-minus NB founders, while two cell clones are obtained from GAL80-minus GMCs Figure . Other pMitotic recombination was randomly induced in progenitor cells at 24 h and 48 h ALH and progenies were examined in isolated GFP-labeled clones 48 hours later Figure . As expeThe observed variability in clone size could be due to intrinsic variations in the mitotic capacity of different IPs and/or may result from mitotic recombination occurring in an IP that had already completed a variable number of divisions after its birth. Interestingly, the distribution of clonal cell number appeared remarkably similar when FLP/FRT recombination was induced at 24 h or at 48 h ALH Figure . This suThese findings imply that IPs in DM lineages can divide several times and produce differentiated progeny in less than 48 hours. Thus, they allow considerable amplification of the number of neurons produced in comparison to the standard mode of division adopted by other lineages in the central brain.The experiments described above show that DM NBs generate multiply dividing daughter cells that produce neural progeny. Surprisingly, these amplifying IP cells appear to be restricted to the DM lineages. What might explain this restriction? DM and non-DM NBs are not morphologically distinguishable and both divide asymmetrically to generate smaller progeny cells  asymmetrical divisions that result in one daughter cell becoming a GMC while the other daughter cell self-renews as an IP. Alternative scenarios, such as one in which IPs first divide symmetrically to expand in numbers and then adopt a GMC fate to generate differentiating neurons, are unlikely given the spatiotemporal pattern of Prospero/Miranda expression and the stable ratio of IPs versus GMCs observed in DM NB clones throughout larval development.The experimental findings that support this novel model have implications for our understanding of neural stem cells and proliferation control. These are discussed in the following.The NBs of the developing central brain and ventral ganglia divide asymmetrically in a stem cell mode in which the larger NB self renews and the smaller daughter cell differentiates into a different cell type, usually a GMC . This sDrosophila is the NB. Our studies identify the IP cell as a second progenitor type with the capacity to undergo multiple rounds of divisions. This characteristic is coupled with several cellular and molecular features that are shared with NBs. Among these are the specific expression patterns of Prospero, Miranda and CycE during mitosis as well as the ability to asymmetrically segregate Prospero and Miranda during cell division. The number of divisions that IPs typically carry out is currently not known with precision. Our observations based on quantification of cell number in multicellular clones suggest an average of three-to five divisions as a conservative estimate. If, as assumed by our model, each IP cell division results in the generation of one GMC-like daughter cell, this estimate would predict a three- to five-fold amplification of the number of neuronal progeny in DM lineages compared with other lineages of the central brain and ventral ganglia. This prediction is in reasonable accordance with the amplified cell numbers observed in NB clones of DM versus non-DM lineages. The ultimate fate of the IPs is currently not known. The fact that almost all intermediate precursor-derived multicellular clones are composed exclusively of postmitotic neurons suggests that, after multiple divisions, these cells are either eliminated by programmed cell death or that they terminally divide and differentiate.The only repeatedly dividing progenitor cell type identified to date in the central nervous system of Although the DM NBs do not express and segregate Prospero to their daughter intermediate precursors, these daughter cells do express Prospero in a cortical and polarized manner during mitosis. The off/on state of Prospero must be kept under tight control for a controlled amplification of proliferation achieved in DM lineages since complete mutational loss of Prospero in brain clones leads to uncontrolled proliferative activity and brain tumor formation -33,44. IDrosophila brain neuroblasts during the postembryonic phase of neurogenesis. Unlike conventional GMC this intermediate progenitor express molecular markers of self-renewing neuroblasts and undergoes multiple divisions in absence of Prospero. In the dorsomedial brain these progenitors amplify the number of neurons that can be generated by the parental neuroblasts at each round of their divisions. The novel IP described here bears remarkable similarities to the IPs that have been identified recently in mammalian brain development . In tiewed by -47. The iewed by . The surDrosophila brain are molecularly diverse, and if the neuronal subpopulations they generate during development acquire distinguishable anatomical and functional characters. If this is the case, the expansion of neurogenesis and the generation of multiple neuronal subclasses may be intimately related in the brain development of animals as diverse as insects and mammals.In mammalian brain development, a two-step model has been proposed where, as a first step, asymmetric divisions of a primary neural stem cell generate a diverse set of molecularly different IPs and, as a second step, multiple symmetric divisions of each of these IPs generate large numbers of neurons of the same subtype . Given tDrosophila stocks were reared and maintained on standard yeast-cornmeal-agar medium and all experiments were performed at 25°C. Unless otherwise stated, fly stocks carrying transgenes and recombinant chromosomes were obtained from the Bloomington stock center and assembled using standard genetics. To generate positively marked MARCM clones y, w, hsFLP; FRT40A, tubP-GAL80LL10/CyO, ActGFPJMR1; tubP-GAL4LL7, UAS-mCD8::GFPLL6/TM6, Tb, Hu were mated to either w; FRT40A, UAS-mCD8::GFPLL5 (standard cell lineage labeling with membrane-tethered GFP), or UASp-cnnGFP26.1; FRT40A, UAS-cLacZBg4-1-2 , or w; FRT40A, UAS-cLacZBg4-1-2; UAS-tauGFP12/2/3 (gift of A Brand), for live imaging. Generation of MARCM clones and larval staging was performed as previously described [All escribed  for thisNervous systems were dissected from larvae, fixed and immunostained as previously described . PrimaryDrosophila Medium with 10% fetal bovine serum and mounted in 400-5 mineral oil  between a glass coverslip and a gas-permeable plastic foil .For live imaging, larval brains were dissected in Schneider's Fluorescently stained nervous systems were imaged using a Leica TCS SP scanning confocal microscope. Z stacks were collected with optical sections at 1–1.5 μm intervals. Pictures in this paper are presented as 'thick-section' merges projected as a flat image using ImageJ . FiguresFor time lapse, Z stacks made of 1 μm thick slices were collected at 4 minute intervals. Movies were processed and assembled using house made ImageJ plug-ins. Briefly, the sample motions were corrected in X and Y dimensions by manual reference point tracking. A single slice was arbitrarily selected per time point, allowing both some Z dimension drifting correction and the follow up of the most interesting cells within the sample.The author(s) declare that they have no competing interests.BB designed the study, wrote the manuscript and performed or participated in all experiments. NI performed the cell number quantification and BrdU labeling experiments. EC performed the time-lapse cell imaging experiments. HR participated in the overall design and coordination of the study and helped write the manuscript. All authors have read and approved the final manuscript.Time-lapse analysis of a proliferating dorsomedial NB clone. Cultured explant of larval brain. CD8::GFP and tau::GFP are used to mark cell membranes and mitotic spindles simultaneously. Each frame is a projection of a thick stack of confocal sections taken at 1 μm z-intervals. Frames were taken at 1-min intervals over an 810-min period. Two divisions of the large NB cell were observed during this period (red arrowheads) while many associated cells underwent mitosis giving rise to daughter cells of similar sizes (yellow arrowheads).Click here for file


Recent findings suggest that Alzheimer's disease (AD) neuropathological features (neuritic plaques and NFTs) are not strongly associated with dementia in extreme old (over 90 years of age) and compel a search for neurobiological indices of dementia in this rapidly growing segment of the elderly population. We sought to characterize transcriptional and protein profiles of dementia in the oldest-old.Gene and protein expression changes relative to non-demented age-matched controls were assessed by two microarray platforms, qPCR and Western blot in different regions of the brains of oldest-old and younger old persons who died at mild or severe stages of dementia. Our results indicate that: i) consistent with recent neuropathological findings, gene expression changes associated with cognitive impairment in oldest-old persons are distinct from those in cognitively impaired youngest-old persons; ii) transcripts affected in young-old subjects with dementia participate in biological pathways related to synaptic function and neurotransmission while transcripts affected in oldest-old subjects with dementia are associated with immune/inflammatory function; iii) upregulation of immune response genes in cognitively intact oldest-old subjects and their subsequent downregulation in dementia suggests a potential protective role of the brain immune-associated system against dementia in the oldest-old; iv) consistent with gene expression profiles, protein expression of several selected genes associated with the inflammatory/immune system in inferior temporal cortex is significantly increased in cognitively intact oldest-old persons relative to cognitively intact young-old persons, but impaired in cognitively compromised oldest-old persons relative to cognitively intact oldest-old controls.These results suggest that disruption of the robust immune homeostasis that is characteristic of oldest-old individuals who avoided dementia may be directly associated with dementia in the oldest-old and contrast with the synaptic and neurotransmitter system failures that typify dementia in younger old persons. The continued expansion of the elderly population resulting from recent advances in medical treatment, improved nutrition and reduced birth rate accompanied by elevated age-related risk of brain disorders such as dementia and Alzheimer's disease (AD) have prompted considerable interest in the study of the aging human brain. These advances in life expectancy have not only led to an increase in the proportion of the population that is aged, but also in the numbers of persons who can be classified as the oldest-old, variably defined as persons over the age of 85 or 90. US Census Bureau data and projections The high frequency of dementia incidence in the elderly with reported doubling every five years from ages 65 to 85 Neuropathological studies laid the foundation for our understanding of the development of the dementing processes in AD and helped define the relationship between neuropathological lesion severity and the magnitude of cognitive impairment. Neuritic plaques (NPs) and neurofibrillary tangles (NFTs), as well as synaptic loss in the temporal neocortex, the entorhinal cortex and the hippocampus were found to be associated with the severity of cognitive deficits in younger AD patients Significant progress has been made in understanding the molecular biological abnormalities in the brains of persons with AD The growing body of evidence showing dissociation between the neuropathological indices of AD from dementia in the extreme elderly prompted the current study of the molecular biological substrates of dementia in this rapidly growing segment of the elderly population. The presence, absence and severity of dementia was assessed by the clinical dementia rating (CDR) rating scale All samples in the microarray dataset were divided in two major age categories based on the median age (87 y.o.) of all of the subjects: youngest-old  and oldest-old  in order to analyze gene expression changes in clinically and neuropathologically distinct age groups of cognitively impaired subjects and controls . In ordeThe Ingenuity Pathway Analysis algorithm was used to determine which signaling pathways were affected in the groups of cognitively impaired subjects in each of the two age cohorts  and 3. AClear distinctions were observed in the oldest-old cohort with mild dementia. Changes in the expression of signaling pathways related to neural function as seen in the young-old subjects  were absBecause of the clear distinction in genes and pathways associated with cognitive impairment in young-old and oldest-old individuals, we sought to determine whether the gene expression profiles of cognitively intact oldest-old persons were also different from those of young-old cognitively intact persons. We compared cognitively intact young-old individuals to cognitively intact oldest-old persons. Under the same filtering conditions as for the cognitively impaired groups 332 probe sets showed significantly altered expression in oldest-old cognitively intact subjects relative to cognitively intact young-old persons. As shown in The same canonical immune function pathways that were identified in the oldest-old individuals with mild dementia  were affThe majority of the 75 probe sets  that werThe GeneChip U133AB platform data described above was normalized by MAS5. To further validate the microarray findings we re-analyzed the U133AB array raw dataset using a different normalization algorithm and performed microarrays of the same samples using the more advanced GeneChip U133 Plus2 platform. RMA normalization of the raw U133AB microarray data with subsequent CGEM based differential gene expression discovery using filtering condition similar to the MAS5 based analyses showed that although the results of the two analysis procedures differed with respect to the absolute number of differentially expressed genes identified , the corAcute phase response, which includes IL-6 signaling was among those affected in both microarray datasets in cognitively normal (CDR0) and severely cognitively impaired (CDR4–5) oldest-old subjects. Comparison of the genes involved in IL-6 signaling showed that more than half of the genes identified in U133Plus2 datasets were also detected as affected in U133AB datasets . As evidp(t-test)<0.001) in both MAS5 and RMA normalization algorithms in cognitively normal oldest-old individuals relative to the cognitively intact young-old donors in the microarray analysis. The results of this study are shown in ps≤0.013) in the temporal cortex of cognitively normal oldest-old individuals relative to the cognitively intact young-old donors. It is important to note that the microarray analyses were based on global changes across all brain regions examined, it is therefore not surprising that only 5 of 8 transcripts evidenced differential expression in the one brain region studied by qPCR. Furthermore, the expression of all eight tested genes was significantly downregulated in cognitively impaired oldest-old individuals with MD  significantly increased in cognitively intact oldest-old persons relative to cognitively intact young-old persons; and ii) impaired in cognitively compromised oldest-old persons relative to cognitively intact oldest-old controls.The notion that AD with its hallmark neuritic plaque, neurofibrillary tangle, and neurochemical system lesions is the preeminent cause of dementia in the elderly as been among the cornerstones of our conceptualizations of aging and dementia. This understanding has been supported by the nearly ubiquitous presence of AD pathological changes in age-related dementia as well as the near exponential increase of AD prevalence after 65 years of age. Increasingly, neuropathological and neuroanatomical studies see  have calAnalysis of a large microarray dataset identified a lack of significant overlap in differentially expressed gene sets associated with dementia in young-old vs. oldest-old persons. The top of the list of significantly altered signaling pathways in young-old persons with dementia was well represented with pathways associated with neurotransmission and synaptic function: GABA and glutamate receptor signaling, synaptic LTP and neural development, including genes participating in neuregulin, IGF1, VEGF signaling, axonal guidance and calcium signaling. As expected from a plethora of published studies, the majority of genes from these pathways were downregulated in cognitively impaired individuals. Other affected pathways included networks of genes previously implicated in brain aging: IGF1, glucocorticoid receptor, nitric oxide and NF-kB signaling, cell cycle regulation, oxidative stress response, and pathways associated with neurodegeneration: hypoxia, Huntington's and Parkinson's diseases and amyotrophic lateral sclerosis signaling. These data are consistent with previous genome-wide gene expression studies of different brain regions from persons with dementia and AD with designs that did not distinguish between young-old and oldest-old cases and utilized brain specimens from persons the majority of whom fall within the young-old age range of the current study In contrast to young-old persons, oldest-old subjects with mild dementia evidenced predominant abnormalities in pathways associated with the immune response system including antigen presentation, classic complement system, and oxidative phosphorylation. Also in contrast to young-old subjects, oldest-old subjects with dementia show under representation of pathways and gene ontology (GO) categories related to neuronal function and were limited only to LTP. These results are in agreement with data derived from neuropathology studies indicating that oldest-old individuals with AD display striking resistance to at least some neurodegenerative processes and only mild neuronal loss in the hippocampus We performed analyses of the microarray data sets to address the question of the expression of genes that are important to both healthy aging, i.e., as people age from young-old to oldest-old without dementia, and to dementia irrespective of age. The expression of 65–75 transcripts was changed in all 5 groups  and cognitively compromised oldest-old (mild and severe dementia)) relative to the cognitively intact young-old index group , Table 6Nine genes, members of major histocompatibility complex (MHC) classes I and II, were the most prevalent group among differentially expressed genes in cognitively normal oldest-old individuals. These molecules are central to antigen presentation processes to T-cells suggesting the presence of antigen presenting cells capable of initiating primary or secondary immune responses. In the CNS, MHC class II immunoreactivity is abundantly detected in activated microglia, in lymphocytes within the neuropil and scarcely in astrocytes in sporadic AD, and is also constitutively expressed by microglia in the white matter during normal aging The CX3CR1, fractalkine receptor is expressed by a wide spectrum of hemopoietic cells in the periphery. In the CNS, CX3CR1 was first described on microglia DAP12/TYROBP and LY96 were among the genes whose expression was significantly affected in successful aging and in dementia in oldest-old persons, albeit in different directions. DAP12/TYROBP is associated with a large family of receptors including those that recognize MHC class I molecules in hematopoietic cells: dendritic cells, monocytes, macrophages, natural killer cells, and some B and T cells. The importance of DAP12 in brain function is demonstrated by the fact that loss-of-function mutation of DAP12 is associated with a rare presenile dementia known as Nasu-Hakola disease Upregulation of two components of the classical complement system - C3 and C1Q suggests initiation of the complement cascade which ultimately leads to formation of the membrane attack complex (C5b-9) that targets affected cells for lysis The source of the immune related gene expression changes in oldest-old persons is not directly discernable from the current data set. The lack of detectable signals for T-cell receptors, which are central for antigen presentation to T cells in our microarrays, refutes the presence of infiltrating CNS peripheral T cells. Immune related gene expression changes could be attributable to expression changes in microglia, astrocytes, neurons or vascular elements. Only future detailed immunohistochemical studies can address this question fully. However, we note that there were no differences between levels of conventionally accepted markers for astrocytes , oligodendrocytes , neurons  or microglial  markers between young-old and oldest-old individuals.r≥0.7; p<0.01), a gene predominantly expressed in microglia The abundant expression level of four identified immune-related genes , their significant correlations with L-ferritin  the neurobiological substrates of dementia in young-old individuals are not identical to the neurobiological mechanisms that underlie dementia in oldest-old persons; 2) the predominant genes whose expression is affected in demented young-old persons comprise those associated with neurotransmission and synaptic function, whereas affected neurobiological pathways in oldest-old demented persons are more likely to be associated with immune/inflammatory responses of the CNS; and 3) the brains of cognitively intact persons who survive to advanced old age, the oldest-old, express a select set of immune/inflammatory genes at a higher level than young-old persons with intact cognition. These analyses also support the neuropathological study findings described above by suggesting that the neurobiological processes that subserve the development and progression of dementia in “conventional” young-old subjects have features that are fundamentally different from those associated with dementia in the oldest-old. One parsimonious interpretation of the expression of immune system marker genes in the oldest-old is that a robust immune system is essential for the maintenance of cognitive health to very old age Diagnostic and postmortem consent procedures were approved by the institutional review boards of Mount Sinai Medical Center, Jewish Home and Hospital and J.J. Peters VA Medical Center. Consents for brain donation were obtained in writing from the legal next of kin of all donors.3) limited to grey matter from the frontal cortex , 10 , 44 , 46 , 4 , anterior cingulate ), posterior cingulate cortex (BA: 23/31 – at the level of the pulvinar), parietal cortex , temporal cortex , 21 , 22 , 36/28 ), 38 (temporopolar) and occipital cortex  regions and from the hippocampus (at the level of the red nucleus) were dissected from flash frozen coronal sections, pulverized at −80°C and aliquoted. Aliquots (50 mg) from each region were used for microarray gene expression analysis. RNA isolation and preparation for the microarrays were as described previously Brain tissue specimens were derived from the Brain Bank of the Department of Psychiatry of the Mount Sinai School of Medicine /J.J. Peters VA Medical Center . The precise tissue handling procedures have been described in detail.All subjects died of natural causes with no history of licit or illicit drug abuse or neurological disease. Cognitively intact subjects (CDR0) with no evidence of neurological or neuropsychiatric diseases were matched with dementia subjects by age, postmortem interval (PMI) and brain pH . The preAll subjects were evaluated in detail for cognitive status during the last 6 months of life and the neuropathological assessment procedures were as previously described th percentile of all values per chip and median-centered per gene using GeneSpring normalization. Statistical comparisons were made using GeneSpring's Cross Gene Error Model (CGEM), based on the deviation from 1.0 algorithm. The following filtering criteria were used in these GeneSpring/RMA/CGEM analyses: expression level ≥1; single factor ANOVA with confidence (p≤0.05) and fold change (≥1.4).Microarray analysis was performed using Affymetrix  HG-U133AB set as well as HG-U133 Plus2 GeneChip® as described in the standard protocol outlined in the GeneChip® Expression Analysis Technical Manual  and reported previously p≤0.05) were overlaid onto a global molecular network in the Ingenuity Pathways Knowledge Base  to identify biological functions and canonical pathways. Ingenuity pathway analysis algorithmically generates networks of genes based on their connectivity. Fischer's exact test was used to calculate a p-value determining the probability that each biological function assigned to that dataset, or the association between genes in the dataset and the canonical pathway, was not due to chance alone. Significant (ps<0.05) canonical pathways were additionally ranked according to the ratios between the numbers of genes from the dataset that mapped to the pathway to the total numbers of genes in the canonical pathway. The scores for the networks were calculated based on the number of network eligible genes and the size of the network to approximate how relevant any particular network was to the total list of eligible genes.Datasets containing identifiers for the genes expressed (presence ≥80%) in the samples from the comparison analyses and corresponding differential expression scores  obtained from comparison analysis  were uploaded into the Ingenuity Pathway Analysis application. Differentially regulated genes .For relative quantification of mRNA expression, relative values of examined genes calculated using the standard curve method were further normalized to geometric means (GMs) of endogenous control-genes as described previously Protein abundance was measured in the inferior temporal cortex, BA20 , from seTissue specimens  were homogenized in Urea/Tris solution: 50 mM Tris/HCl pH 7.4; 6 M Urea; 2% CHAPS containing 1 mM PMSF and cocktails of proteinase/phosphatase inhibitors . Total protein concentration in the tissue homogenates was determined with a CBQCA Quantitation Kit . Aliquot samples of 15 µg of total protein in triplicates were loaded onto pre-cast 4–12% Tris-glycine gel  under reduced conditions. A “standard-calibrator”  was used as a calibrator between the gels and run on each gel in triplicate. Blots were incubated with antibodies: rabbit anti-human CX3C chemokine fractalkine receptor 1, CX3CR1 (1∶500 v/v) from LifeSpan Biosciences Inc. ; mouse anti-human HLA-DPA1 (1∶100 v/v) from Abnova ; mouse mAb against human CD74 antigen protein (1∶1000 v/v), which recognizes cytoplasmic tail of CD74  and mouse anti-human β tubulin  – from Santa Cruz Biotech. Inc.  or rabbit anti-human TUBB . Electrophoresis, blotting, immunostaining, and infra-red (IR) fluorescence detection  were performed under standard conditions. Multiplex western blots were scanned on Odyssey Infrared Imaging System . The linearity of the dose responses for the antibodies used was established in preliminary experiments. Images were analyzed and quantitated with Odyssey software ver.3 . To account for gel to gel variability, the relative expression value (REV) of analyzed proteins in each sample was calculated as a ratio between the averaged intensities of the band in the experimental sample and in the “standard-calibrator”. Finally, relative values for examined proteins were normalized to endogenous control - TUBB.t-scores, Pearson correlation coefficients and corresponding p-values (ANOVA) p≤0.01) and Pearson correlation coefficients meant that the pattern of variation of expression values between sample sets closely follows the pattern represented by the contrast vectors, indicating upregulation of gene expression. Large negative t-score values together with significant (p≤0.01) negative Pearson correlation coefficients meant that the pattern of variation is the inverse to the pattern represented by the contrast vectors, indicating downregulation of gene expression. Finally, t-scores close to zero meant that the gene's expression pattern matches neither the contrast pattern nor its inverse, or that the amount of variation between sample sets is comparable to or smaller than the variation within sample sets.Multiple statistical procedures were employed for different aspects of the study. Max t-test was used to compare relative mRNA expression of analyzed genes in qPCR experiments and relative abundance of proteins in Western blots. Student's t-test and correlation analyses were performed using Statistica (release 6.0).Differences in expression between CNTs and different groups of demented persons in the GeneSpring/RMA/CGEM analyses were examined using a one-way analysis of variance (ANOVA) with Benjamini and Hochberg multiple testing/false discovery rate corrections.Figure S1Heat map of differentially expressed 75 common probe sets in YO and OO cognitively intact (CDR0) groups and cognitively impaired OO groups. Depicted are 75 probe sets  identifi(0.19 MB PNG)Click here for additional data file.Figure S2Western blots of CX3CR1 and beta tubulin -TUBB in inferior temporal cortex (BA20). Signal for CX3CR1 appeared as a doublet/triplet band ∼50kD (under reducing conditions). Extended boiling (up to 5 hours) of protein extract in the presence of 2-mercaptoethanol eliminate these bands, and CX3CR1 appears as a single band ∼50kD. Optical density was measured for all of the bands.(0.45 MB TIF)Click here for additional data file.Figure S3Western blots of HLA-DPA1 and beta tubulin -TUBB in inferior temporal cortex (BA20). Signal for HLA-DPA1 appeared as a doublet band ∼29kD (under reducing conditions). Extended boiling (up to 5 hours) of protein extract in the presence of 2-mercaptoethanol eliminate doublet, and HLA-DPA1 appears as a single band ∼29kD. Optical density was measured for all of the bands.(0.14 MB TIF)Click here for additional data file.Figure S4Western blots of CD74 and beta tubulin -TUBB in inferior temporal cortex (BA20). Signals for HLA-DPA1 appeared in multiple bands ranging from 11 to 72kD with the main bands corresponding to MWs: doublet 33/35, 41 and 11 kD (under reducing conditions).(0.73 MB TIF)Click here for additional data file.


Alterations in the expression of Fas (CD95/APO-1) and its ligand (FasL) have been demonstrated in various types of cancers as a mechanism for tumour cell to escape from the immune system. In the present study, we evaluated the expression of the Fas and FasL genes in a wide range of primary gynaecological carcinomas. These included 31 ovarian, 29 cervical and 25 endometrial carcinoma tissues as well as four ovarian and three cervical carcinoma cell lines. Our real-time quantitative reverse transcription polymerase chain reaction analysis revealed that down-regulation of Fas expression is more prominent than the up-regulation of FasL expression in all types of gynaecological cancer studied. This down-regulation of Fas expression was also true for the seven carcinoma cell lines. Only one cervical carcinoma cell line, DoT, exhibited a high level of FasL expression. These results indicated that down-regulation of Fas expression is a common abnormality in many types of cancers including gynaecological cancers, whereas an increase in FasL expression is not a common phenomenon in these cancers. © 2000 Cancer Research Campaign


A number of non-malignant diseases that share similar morphological features as gastrointestinal stromal tumor (GIST) have been reported. Co-existence of GIST with these other diseases is rarely recognized or reported.We report a case of a 62 year-old man with long-term stable control of metastatic GIST with systemic therapy, presented with an apparent intra-abdominal progression but not supported by imaging with positron emission tomography. Subsequent resection of the intra-abdominal tumor identified a non-malignant fibroid.Differentiating localized progression of GIST from other diseases has important prognostic and therapeutic implications. The potential for co-existence of non-malignant soft tissue neoplasm should always be considered. The finding of gain-of-function mutation of KIT has revolutionized the treatment of advanced gastrointestinal stromal tumor (GIST). This has subsequently led to development of effective systemic therapy utilizing tyrosine kinase inhibitors (TKI). Imatinib is the prototype TKI that was initially reported to achieve a partial response rate of 53.7% and stable disease rate of 27.9%. With thA number of soft tissue neoplasm share many similarities in the morphological and immunophenotypic profiles with GIST. Aggressive fibromatosis (AF) and keloid type fibromatosis scar tissues are distinct soft tissue tumors. AF is a fibroproliferative disease with a propensity for intra-abdominal presentation; it may In this report, we present a case of a man who had been treated with imatinib and achieved long-term stable advanced GIST, but presented with localized proliferation of soft-tissue neoplasm mimicking GIST.A previously healthy, 62 year old man was diagnosed with a gastric antral tumor after investigations for symptomatic anemia. A barium swallow confirmed the presence of tumor causing subacute gastric outlet obstruction. Laparoscopy identified a gastroduodenal tumor and synchronous bilobar liver metastases. No peritoneal disease was identified. The primary tumor was completed excised and a liver biopsy was performed intra-operatively. Histopathology was consistent with metastatic malignant gastrointestinal stromal tumor, with typical spindle cell features on light microscopy. C-KIT was positive and the mitotic rate was 60/50 per high power fields Figure . SubsequPost-operatively, this man recovered well from his surgery and was commenced on imatinib 600 mg daily; he subsequently required dose-reduction to 400 mg daily due to grade 3 neutropenia. He still had residual hepatic metastatic disease which was visible on computerized tomography [CT] scan, and was FDG-avid on positron emission tomography [PET] evaluation. For a period of eighteen months after surgery, imatinib was tolerated well and achieved good disease control. CT and PET imaging during this period revealed regression of size of the liver metastases. After two years of therapy, however, a CT scan revealed an increase in size of a dominant segment VI hepatic metastasis which was treated with radiofrequency ablation. He was then maintained on imatinib at 600 mg daily with subsequent disease control.At four and half years from diagnosis, an asymptomatic infrapyloric mesenteric mass was identified on a surveillance CT which progressively increase in size over the next two months Figure . A PET sAt subsequent laparotomy, the tumor was found to be lying within the peritoneal leaves of the mesocolon extending from the origin of the superior mesenteric vessels to the inferior pancreatico-duodenal vessels. Histopathology showed a tumour mass composed of spindle shaped fibrocytic/fibroblastic like cells amongst intervening collagen Figure  with lowImatinib was continued 600 mg daily, with brief cessation during the peri-operative period, as metastatic GIST remained radiologically stable. No specific adjuvant therapy for the soft-tissue tumor was employed post-operatively.In this patient with metastatic GIST, the development of the mesenteric tumor four years after the institution of imatinib initially suggested disease relapse. Debulking surgery remains a recognized standard practice in the case of local progression where such procedure is associated with prolonged survival with the elimination of imatinib resistance clones. HoweverThis patient underwent surgery with the pre-surgical diagnosis of a localized progression; surgery was aimed to achieve disease control with the elimination of a presumably localized imatinib resistance tumor. Post-surgery, the histopathologic findings revealed a tumor with reduced cellularity and low mitotic activity consistent with the pre-operative non-glucose avid PET findings. Collaborative pathologic review was obtained and excluded diagnosis of a recurrent GIST. However, a definitive uniform diagnosis could not be made. The possible differential diagnoses of this soft-tissue tumor include aggressive fibromatosis (AF) or intra-abdominal keloid type fibrocollagenous scar.Immunohistochemistry was positive for C-KIT, which is unusual in AF or intra-abdominal keloid type fibrocollagenous scar. Mutation analysis that was performed on the mesenteric tumor further clarify that this mass, which was absent of Exon 11 C-KIT mutation, was different from the Exon 11 C-KIT mutation positive of the original resected antral GIST.APC or CTNNB1). Some studies [Histologically, AF lies on a spectrum of disorders characterized by excess proliferation of fibroblast-like spindle cells. These c studies -11 have There is very limited literature on intra-abdominal keloid type fibrocollagenous scar. The scar in this patient was presumably formed from previous surgical laparotomy. There is growing evidence to suggest that Transforming Growth Factor β is impliThe immunophenotypic profiles of GIST, AF and keloid may overlap. Fibromatoses may stain for vimentin, smooth muscle actin, and desmin. In some series, fibromatosis did not stain for CD34 or S-100 protein, while CD34 staining and occasional S-100 protein positivity were seen in GIST,14. It hThe long stable disease control, the absence of glucose avidity of pre-operative PET and the absence of the C-KIT mutation in the mesenteric tumor were all features that might have suggested the possibility of an alternative diagnosis to GIST recurrence. Although surgical resection of this mesenteric mass may remain necessary, a correct diagnosis has important implication for his future systemic therapy. This case report highlights the importance of recognizing the coexistence of other diseases in patients with chronic GIST.Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.CL and AH drafted the original manuscript, with subsequent further contributions from KD, GS and DG. KD reported the histopathological findings and supplied the photomicrographs used in this manuscript. GS reported the surgical findingsAll authors read and approved the final manuscript.


The recently developed cDNA expression array technique can be used to generate gene-expression fingerprints of tumour specimens. To gain insight into molecular mechanisms involved in the development and progression of cancer, this cDNA expression array technique could be a useful tool, however, no established methods for interpreting the results are yet available. We used the Atlas cancer cDNA expression array  for analysing total RNA isolated from four human endometrial carcinoma samples (two cell-lines and two tissue samples), one benign endometrial tissue sample and a human breast cancer cell-line, in order to develop a method for analysing the array data. The obtained gene-expression profiles were highly reproducible. XY-scatterplots and regression analysis of the logarithmic transformed data provided a practical method to analyse the data without the need of preceding normalization. Three genes  were identified to be differentially expressed between the benign endometrial tissue sample and the endometrial carcinoma samples (tissue and cell-lines). These three genes may potentially be involved in cancer progression. A higher degree of similarity in gene-expression profile was found between the endometrial samples (tissue and cell-lines) than between the endometrial samples and the breast cancer cell-line, which is indicative for an endometrial tissue-specific gene-expression profile. © 2000 Cancer Research Campaign


Methamphetamine (METH) is an illicit drug which is neurotoxic to the mammalian brain. Numerous studies have revealed significant decreases in dopamine and serotonin levels in the brains of animals exposed to moderate-to-large METH doses given within short intervals of time. In contrast, repeated injections of small nontoxic doses of the drug followed by a challenge with toxic METH doses afford significant protection against monoamine depletion. The present study was undertaken to test the possibility that repeated injections of the drug might be accompanied by transcriptional changes involved in rendering the nigrostriatal dopaminergic system refractory to METH toxicity. Our results confirm that METH preconditioning can provide significant protection against METH-induced striatal dopamine depletion. In addition, the presence and absence of METH preconditioning were associated with substantial differences in the identity of the genes whose expression was affected by a toxic METH challenge. Quantitative PCR confirmed METH-induced changes in genes of interest and identified additional genes that were differentially impacted by the toxic METH challenge in the presence of METH preconditioning. These genes include small heat shock 27 kD 27 protein 2 (HspB2), thyrotropin-releasing hormone (TRH), brain derived neurotrophic factor (BDNF), c-fos, and some encoding antioxidant proteins including CuZn superoxide dismutase (CuZnSOD), glutathione peroxidase (GPx)-1, and heme oxygenase-1 (Hmox-1). These observations are consistent, in part, with the transcriptional alterations reported in models of lethal ischemic injuries which are preceded by ischemic or pharmacological preconditioning. Our findings suggest that multiple molecular pathways might work in tandem to protect the nigrostriatal dopaminergic pathway against the deleterious effects of the toxic psychostimulant. Further analysis of the molecular and cellular pathways regulated by these genes should help to provide some insight into the neuroadaptive potentials of the brain when repeatedly exposed to drugs of abuse. METH  is an illicit drug whose abuse prevalence has reached greater proportion than the combined use of heroin and cocaine in the world. The clinical history of METH abuse is characterized by the user initially taking small doses of the drug followed by consumption of progressively larger doses of the psychostimulant Studies conducted in the 1970's were the first ones to document significant decreases in the levels in DA in the brain of nonhuman primates that had been exposed to repeated injections of METH and sacrificed two weeks after cessation of drug exposure Several groups of investigators have suggested that METH pretreatment might cause inhibition of METH-induced changes in body temperature, vesicular DA uptake, free radical production, and microglial activation The acute METH challenge also caused substantial decreases in striatal 5-HT concentrations in the saline- (−53%) and METH-pretreated (−24%) rats . METH prMicroarray analysis has become an important tool in toxicological research because it allows investigators to obtain a better panoramic view of drug-induced transcriptional changes after exposure to pharmacological agents and toxins One interesting observation among the response profiles occurs between the saline- and METH-pretreated rats after injections of toxic doses of METH. The Venn diagram showed that only 7 genes overlapped between these two sets of comparisons . These iWe used quantitative PCR to validate the expression of some of the genes identified by the microarray analyses using RNA from individual animals from the four groups. The primers are listed in We also sought to confirm the METH-induced changes in the expression of thyrotropin-releasing hormone (TRH) observed in the microarray data because TRH is widely distributed in the rat brain As shown in We also confirmed METH-induced increases in Ptprm in the presence of METH preconditioning . We alsoMicroarray analyses may sometimes underestimate changes in gene expression We also measured the expression of brain derived neurotrophic factor (BDNF) which is thought to be a mediator of ischemic preconditioning Finally, we measured the expression of several antioxidant genes that have been proposed as potential mediators of ischemic preconditioning . In what follows, we discuss the potential protective role of differential changes in gene expression in the model of METH preconditioning.The major findings of our study are that a challenge with toxic doses of METH caused marked reductions in the levels of DA and 5-HT in the striatum but significant decreases only in 5-HT concentrations in the VTA/SN of rats. Pretreatment of the animals with progressively higher but nontoxic doses of METH caused complete protection against METH challenge-induced DA depletion but partial protection against 5-HT depletion in the striatum. In contrast, the pretreatment did not afford any protection against METH-induced decreases in 5-HT levels in the ventral midbrain. The observations on the protective effects of METH preconditioning on drug-induced monoamine depletion are consistent with those reported by several groups of investigators  Also of interest are the increases in Hmox-1 expression observed after the METH challenge in the METH preconditioned group. Hmox-1 is a phase 2 enzyme that is induced by oxidative stress and cellular injury In summary, this is the first demonstration that prior repeated injections of nontoxic doses of METH, which cause protection against METH-induced striatal DA depletion in the rat, are also associated with differential transcriptional responses to toxic METH doses in the ventral midbrain of rats. These findings suggest that METH preconditioning protects against striatal DA depletion, in part, by suppressing injurious mechanisms while also augmenting neuroprotective pathways in the nigrostriatal dopaminergic pathway. Thus, the protective effects observed after METH preconditioning are not solely dependent on changes in one specific biochemical or molecular pathway but on multiple endogenous protective systems working in concert. Our observations further suggest that METH preconditioning-induced transcriptional alterations might be the results of epigenetic switches that affect promoter regions of genes in such a way that changes in their transcriptional regulation become manifest only in the presence of challenges with toxic doses of the psychostimulant. Finally, because our results are consistent with observations reported in models of brain preconditioning mediated by ischemia or pharmacological agents, it will be of interest to test if METH preconditioning might exert neuroprotective effects against other models of neurodegeneration.Guide for the Care and Use of Laboratory Animals and were approved by the local Animal Care Committee.Male Sprague-Dawley rats , weighing 330–370 g in the beginning of the experiment were used in the present study. Animals were housed in a humidity- and temperature-controlled room and were given free access to food and water. All animal procedures were performed according to the National Institutes of Health Following habituation, rats were injected intraperitoneally with either (±)-METH-hydrochloride  or an equivalent volume of 0.9% saline for a period of three weeks as described in 4 and centrifuged at 14, 000×g for 15 min. DA, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) levels were analyzed in the brain tissue extracts using HPLC with electrochemical detector as previously described For monoamine analysis, the brain regions were homogenized in 0.01 M HClOTotal RNA was isolated using Qiagen RNeasy Midi kit  according to the manufacturer's instructions. RNA integrity was assessed using an Agilent 2100 Bioanalyzer  and showed no degradation.Microarray hybridization was carried out using Illumina's RatRef-12 Expression BeadChips arrays  . In brief, a 600 ng aliquot of total RNA from each striatal sample was amplified using Ambion's Illumina RNA Amplification kit . Single-stranded RNA (cRNA) was generated and labeled by incorporating biotin-16-UTP . 750 ng of each cRNA sample were hybridized to Illumina arrays at 55°C overnight according to the Illumina Whole-Genome Gene Expression Protocol for BeadStation . Hybridized biotinylated cRNA was detected with Cyanine3-streptavidin  and quantified using Illumina's BeadStation 500GX Genetic Analysis Systems scanner as described previously The microarray data reported in the manuscript are in accordance with MIAME guidelines. The raw data for the analyses of the four groups of animals have been deposited in the NCBI GEO database: Accession number GSE17665. The Illumina BeadStudio software was used to measure fluorescent hybridization signals. Data were extracted by BeadStudio  and then analyzed using GeneSpring software v. 7.3.1 . Raw data were imported into GeneSpring and normalized using global normalization. The normalized data were used to identify changes in gene expression in these 3 group comparisons: MS vs SS, SM vs SS, and MM vs MS. A gene was identified as changed if it showed increased or decreased expression according to an arbitrary cut-off of 1.7-fold changes at p<0.05.Total RNA extracted from a midbrain region that encompasses the ventral tegmental area and substantia nigra of the rat was used to confirm the expression of genes of interest by real-time RT-PCR as previously described Statistical analysis was performed using analysis of variance (ANOVA) followed by Fisher's protected least significant difference post-hoc comparison . Values are shown as means ± SEM. The null hypothesis was rejected at p<0.05.Table S1(0.06 MB DOC)Click here for additional data file.