Saccharomyces cerevisiae is an important microorganism for both industrial processes and scientific research. Consequently, there have been extensive efforts to characterize its cellular processes. In order to fully understand the relationship between yeast's genome and its physiology, the stockpiles of diverse biological data sets that describe its cellular components and phenotypic behavior must be integrated at the genome-scale. Genome-scale metabolic networks have been reconstructed for several microorganisms, including S. cerevisiae, and the properties of these networks have been successfully analyzed using a variety of constraint-based methods. Phenotypic phase plane analysis is a constraint-based method which provides a global view of how optimal growth rates are affected by changes in two environmental variables such as a carbon and an oxygen uptake rate. Some applications of phenotypic phase plane analysis include the study of optimal growth rates and of network capacity and function.The yeast Saccharomyces cerevisiae genome-scale metabolic network was used to formulate a phenotypic phase plane that displays the maximum allowable growth rate and distinct patterns of metabolic pathway utilization for all combinations of glucose and oxygen uptake rates. In silico predictions of growth rate and secretion rates and in vivo data for three separate growth conditions (aerobic glucose-limited, oxidative-fermentative, and microaerobic) were concordant.In this study, the Taken together, this study examines the function and capacity of yeast's metabolic machinery and shows that the phenotypic phase plane can be used to accurately predict metabolic phenotypes and to interpret experimental data in the context of a genome-scale model. The development of numerous high-throughput experimental techniques such as DNA microarrays, genome sequencing, and protein chips has revolutionized the analysis of biological systems and generated a catalog of information about a cell's components -3. EfforSaccharomyces cerevisiae. Flux-balance models of yeast have appeared for small-scale network reconstructions. Most of these studies are specific for growth conditions, such as anaerobic, glucose-limited metabolism [Several modeling approaches have been implemented in the study of tabolism , aerobictabolism  or growttabolism . In additabolism ,11, alontabolism ,13 and ttabolism . These mEscherichia coli [Haemophilus influenzae [Helicobacter pylori [Saccharomyces cerevisiae based on its annotated genome sequence and a thorough examination of online pathway databases, biochemistry textbooks, and journal publications [in silico gene deletion studies [Small-scale reconstructions can be limited in their prediction of cellular functions since these processes are typically dependent on the interaction of components at a whole-cell level. This has motivated the development of genome-scale models, several of which have already appeared for bacterial cells, including hia coli ,16, Haemfluenzae , and Helr pylori . We haveications . A total studies  and the  studies .The reconstruction and analysis of genome-scale microbial networks have advanced significantly in recent years ,23, as hThe constraint-based modelling approach is based on the assumption that organisms have developed control structures to ensure optimal growth in response to environmental constraints . Numerouin silico gene deletion simulations, and in vivo growth experiments. Finally, we evaluate the network's predictions for these growth states by comparing in silico biomass formation and by-product secretion rates to in vivo measurements.In this study, we formulate a glucose-oxygen phenotypic phase plane for yeast based on its recent genome-scale metabolic reconstruction . The groS. cerevisiae genome-scale metabolic network constructed by Forster et al. [1 - P7). The seven phases represent areas of the PhPP that have distinct metabolic phenotypes as defined by shadow price analysis, which identifies how changes in metabolite levels affect biomass formation [y-axis and the small square near the origin. Growth is infeasible in the region between the ordinate and P1 since yeast cannot use more than six oxygen molecules per glucose molecule. The two red lines in Fig. growth represents optimal aerobic glucose-limited growth of S. cerevisiae in which substrates are completely oxidized to produce biomass and LOethanol corresponds to maximum ethanol production under microaerobic conditions while growth is maximized.The r et al.  was usedr et al.  that desormation . There a3, sulfate and phosphate were introduced for the biomass synthesis. During anaerobic conditions (OUR = 0, on the x-axis), the growth rate was low and the respiratory quotient (RQ) was infinite by definition . This approach was especially useful for interpreting the physiological differences between growth states in phases 2 – 6 since their phenotypes were indistinguishable in terms of their secretion profiles.In phase 2, the ratio of oxygen uptake rate and glucose uptake rate (GUR) is lower than that on the line of optimality. As a result, the cell is oxygen limited and begins to ferment. Mitochondrial NAD+ is available in excess, meaning that the biomass synthesis would improve if its availability decreased. In order to maintain the cell's redox balance, the excess mitochondrial NAD+ must be reduced. This is done through the production of acetate and ethanol, which begin to be secreted in this phase. Thus it is the production of acetate and ethanol that makes the optimal growth rate less than that defined on the line of optimality.As the ratio of oxygen and glucose uptake rates is further decreased, three lower glycolysis reactions  become essential for growth in phase 3. Although these deletions severely hinder growth in phase 2 (reducing the growth rate by 55%, 19%, and 19%, respectively), the simulated growth rate is non-zero so these reactions were not considered to be essential. However, these reactions are essential in subsequent phases as the oxygen uptake rate is further decreased. Due to the limited oxygen, more carbons "overflows" into the fermentation pathway while at the same time oxidative metabolism becomes less effective.Shifting from phase 3 to phase 4, the pentose phosphate pathway is utilized to generate NADPH because not enough NADPH is produced through respiration at the lower oxygen uptake rate. The NADPH is then converted to NADH which is subsequently used for ATP production.Further lowering the ratio of oxygen and glucose uptake rates restricts the cell's ability to produce pyruvate in phase 5. Yeast can no longer utilize the oxidative pathways because an insufficient amount of cytosolic NAD+ is produced. When comparing phases 4 and 5, all of the metabolites with shadow price sign changes were folate intermediates. These are important energy carriers that are directly linked to the availability of both cytosolic and mitochondrial NAD+ and NADP+.As you enter phase 6, the acetate production is completely ceased. Ethanol is secreted as the only metabolic by-product to balance the redox potential of the cell.Three groups of experiments were conducted under different growth conditions in the PhPP Fig. . These t® Plus bioreactor during the cell cultivation. The system was controlled at the respiratory quotient of 1.06 for optimal S. cerevisiae growth by RQ-stat feeding strategy to maintain the glucose concentration at a low, stable level  growth experiments were conducted with a data acquisition and process control system. The dissolved oxygen (DO) level was maintained above 30% by sparging the compressed air into the CelliGenvel Fig. . The cel• Oxidative-fermentative (OF) batch growth experiments were carried out by allowing an essentially unlimited supply of oxygen and glucose. The DO level was maintained above 30% by sparging the compressed air into the shaker's flasks during the cell cultivation. Ethanol and acetate were accumulated in the aerated processes Fig. .S. cerevisiae were performed at a low dissolved oxygen level. The experiments were performed in side-arm flasks  batch cultivations with sks Fig.  in whichS. cerevisiae PhPP is a genome-scale model-based visualization platform which allows experimental data and simulation results to be displayed and compared. The three groups of batch and fed-batch experimental data are projected on Fig. in silico predictions are in good agreement.The S. cerevisiae genome-scale metabolic network constructed by Forster et al. [S. cerevisiae PhPP has led to clear interpretation and prediction of its metabolic capabilities. First, only a few distinct optimal S. cerevisiae growth phenotypes were found in silico, and these phenotypes correspond to well-defined phases of the PhPP. Second, two lines of optimality were identified in yeast's PhPP: LOgrowth, which represents optimal biomass production during aerobic, glucose-limited growth, and LOethanol, which corresponds to both maximal ethanol production and optimal growth during microaerobic conditions. The predictions of S. cerevisiae's PhPP and genome-scale model were compared to independent experimental data. The results showed that the agreement between the computed and observed growth rates, uptake rates, and secretion rates was close to the measured values or within the experimental error, and qualitatively the predictions agreed with published literature.In this study, the r et al.  was usedr et al.  that des1, the oxygen supply is sufficient for growth by aerobic respiration, resulting in carbon dioxide as the sole by-product. Phases P2-P6 correspond to states of oxidative-fermentative growth, which is characterized by secretion of oxidative and fermentative metabolic by-products, i.e., acetate and ethanol, respectively. These states are highly similar since the phases are essentially co-planar in the 3-dimensional PhPP . Cultures for aerobic, glucose-limited, fed-batch growth were initially grown in a batch mode, and a specific substrate limited after the culture reached particular biomass concentrations in each respective experimental condition. All batch culture experiments were performed in our multiple fermentation system which consists of acrylic enclosures filled with de-ionized water that can accommodate 32 cultures in parallel in batch operation mode at volumes ranging from 100 mL to 1500 mL capacity. We used this setup with either shaker's flasks as reactor vessels. A magnetic agitator (Bellco Glass, Inc., Vineland, NJ, USA) was used to continually mix flask contents at a speed of 225 rpm, and each flask was sealed with a rubber stopper containing apertures for probes, nutrient inlets/outlets, and sample harvesting. Temperature is strictly and uniformly controlled using a water circulator (model C10, Thermo Haake, Portsmouth, NH) with a temperature control module that drives a closed circuit of water to and from the controller to inlet and outlet drains on the water bath. Dissolved oxygen is measured and controlled using a polarographic electrode connected to DO meters/controllers. The fed-batch S. cerevisiae cultivations were automatically controlled in the 1-L bioreactor (CelliGen® Plus, New Brunswick Scientific Co., Inc., Edison, NJ, USA). It has its own controllers for temperature, pH and dissolved oxygen (DO). A Pentium II computer (233 MHz processor, Microsoft Windows 98) equipped with an AT-MIO-16E-10 Analog Input computer interface board  was used for data acquisition and process control for both the multiple fermentation system and CelliGen® Plus bioreactor. Data from the batch and fed-batch cell cultures, including pH, temperature, and dissolved oxygen concentration were acquired through the interface board. The real-time graphical data acquisition and process control programs was written in LabVIEW 6.0 . Media fed to the bioreactor was controlled by a feeding pump (Masterflex Computerized Drive 7550-90, Cole-Parmer Instrument Co., Chicago, IL, USA), with a RS 232 serial link accepting control signal from the computer, for fed-batch cell cultivation process. Acquisition of dissolved oxygen (DO) data was obtained with the aid of a respirometer [For experiments, 5 ml of overnight culture inoculated from single colonies grown on YPD agar was used to seed 50 ml of SD media pre-warmed to 30°C in a 250-ml Erlenmeyer flask, which was placed in a 30°C shaking incubator at 225 rpm for approximately 12 hours. This secondary seed was then used to inoculate either a 1.5-L Erlenmeyer flask with side arms for parallel batch fermentations or a 1.0-L bioreactor  to oxygen uptake rate (OUR) has previously proven useful in inferring a lack of substrates in the growth medium and in the calculation of feeding rates . For the2, in and CO2, in and O2, out and CO2, out are the oxygen and carbon dioxide fractions in % v/v in the inlet air and exiting gas measurements, respectively, Qin and Qout are the air flow rates, and Vm is the working volume of the bioreactor. When the estimated RQ reached 1.06, a peristaltic pump (Cole-Parmer) was utilized to begin feeding 10X concentrated growth medium into the bioreactor, and this quotient was maintained by an RQ-stat control strategy to limit the production of by-products or consumption of these by-products as an alternative energy source.where O600, A420, using a spectrophotometer (Beckman DU640, Beckman Coulter, Inc., Fullerton, CA, USA), and cell counts . The second aliquot was then filtered on a manifold containing a dry, pre-weighed, 0.2 μm pore-size filter to isolate a cell pellet, and was washed three times with 250 ml of sterile deionized water to ensure all salts were removed. After washing, the filters are placed in aluminum foil inside a 65°C incubator for 24 hours and subsequently weighed in an analytical balance to measure cell dry weight. The second aliquot was filtered through a 0.45 μm acrodisc syringe filter to separate cells from supernatant. The concentrations of metabolites in the supernatant such as glucose, acetate, ethanol, and glycerol were determined by using enzyme-based assay kits .During cultivations, two separate 1-ml aliquot samples were taken at early, mid, and late log-phase from both the bioreactor and flasks. The first aliquot was used to determine cell density by measuring the optical density AX = Xoeμt, where X is the cell concentration (gL-1), Xo is the initial inoculum cell concentration (gL-1), t is the time of inoculation, and μ is the specific growth rate (1 hr-1). A minimum of ten optical density measurements were needed for the growth rate determination for both batch and fed-batch cultures.All specific growth rate curves were obtained by a linear regression of all data points within the exponential growth phase using the following formula flask (OUR for the batch culture using flasks) were determined by fitting the dynamic mass balance equations for glucose, ethanol, acetate and DO measurements to the data points spanning the time period of the exponential cell growth phase. The specific uptake and production rates were then calculated by solving the dynamic mass balance equation within the culture medium using the following equation:The specific glucose uptake rate (GUR), ethanol and acetate formation rates, and OURV (L) is the culture volume, [S] (mM) is the substrate/product or DO concentration in the flask, q (mmole/g-dry weight/hr) is the substrate uptake rate or by-product formation rate or OURflask, and X(t) (g-dry weight/L) is the biomass concentration at time = t (hr). + is for the by-product formation and - is for the substrate consumption. The solution to this equation was fitted to the experimental data points using the nonlinear estimation routine in Statistica (StatSoft Inc, Tulsa OK) or the solver in Microsoft Excel. All data, to be considered valid and included in the analysis, must have correlation coefficients of 0.95 or greater. The data that were generated in this way represented the "pseudo-steady-state" [flask.where y-state"  of the bS. cerevisiae metabolic network has been reconstructed [x, and reaction activities, v, is described by the dynamic mass balance equation [A genome-scale structed . The netequation ,44:S is an m × n matrix of stoichiometric coefficients, x is an m × 1 vector of metabolite concentrations, and v is and n × 1 vector of reaction activities. Thus, the rows of S correspond to the internal metabolites and the columns represent the reactions in the network. Under steady-state conditions, the dynamic mass balance equation simplifies to:where S • v = 0     (Eq. 6)S. cerevisiae for various growth conditions [Since the number of reactions is often greater than the number of metabolites, Eq. 6 is underdetermined and contains multiple solutions. One approach to solving Eq. 6 for microbial networks is to define a set of inputs and outputs that correspond to the growth conditions and use linear optimization to maximize the cell growth . This apnditions .S. cerevisiae PhPP displays optimal growth rates for all possible variations in two constraining environmental variables, such as the carbon substrate and oxygen uptake rates. In this study, the glucose uptake rate (x-axis) was allowed to vary from 0 to 20 mmol/gDCW/hr and the oxygen uptake rate (y-axis) ranged from 0.1 to 20 mmol/gDCW/hr. The oxygen uptake rate was not allowed to reach zero because anaerobic simulations required additional supplements to maintain cell growth (ergosterol and zymosterol). Linear programming was used to calculate the optimal growth rate for all points in the x-y plane. Growth rate values were then plotted as the z-axis to form the surface of a three-dimensional PhPP  to changes in the availability of each metabolite:i is the ith metabolite and γi is the ith shadow price. Shadow prices were calculated for each point in the x-y plane during the linear programming simulations. By definition, phases were identified as regions of the PhPP in which all of the points have the same shadow prices. Lines of optimality, which represent the optimal ratio of glucose and oxygen uptake rates for maximal biomass synthesis, were also identified using shadow price analysis [where banalysis .e.g. the value of the objective function increases if the metabolite's net production increases or its net consumption decreases. Similarly, a positive shadow price indicates that a metabolite is available in excess and a shadow price equal to zero indicates that a change in the availability of the metabolite does not affect the objective value.To obtain a physiological interpretation of the differences between the oxidative fermentative phases (phases 2–6), we analyzed how the shadow prices of key metabolites changed across the phase boundaries. Simulations were run at a fixed glucose uptake rate of 5 mmol/gDCW/hr and an oxygen uptake rate ranging from 1.5 to 15 mmol/gDCW/hr. The sign of the shadow price was used to identify whether a small change in the metabolite's availability would positively or negatively affect the objective value. According to the convention defined in , a negatin silico . Single-5 and 1.27 × 10-4 mmol/gDCW/hr, respectively, were used for the calculation at OUR = 0). The simulations were then re-run with a fixed glucose uptake rate, oxygen uptake rate, and growth rate to determine the maximum and minimum secretion rates of each metabolite with a shadow price equal to zero.The first step in generating the secretion profile was to calculate the optimal growth rate for a given glucose and oxygen uptake rate. For the simulations in Figures NCD calculated the phenotypic phase plane Fig. , carried


Restriction enzymes (REases) are commercial reagents commonly used in recombinant DNA technologies. They are attractive models for studying protein-DNA interactions and valuable targets for protein engineering. They are, however, extremely divergent: the amino acid sequence of a typical REase usually shows no detectable similarities to any other proteins, with rare exceptions of other REases that recognize identical or very similar sequences. From structural analyses and bioinformatics studies it has been learned that some REases belong to at least four unrelated and structurally distinct superfamilies of nucleases, PD-DxK, PLD, HNH, and GIY-YIG. Hence, they are extremely hard targets for structure prediction and homology-based inference of sequence-function relationships and the great majority of REases remain structurally and evolutionarily unclassified.SfiI is a REase which recognizes the interrupted palindromic sequence 5'GGCCNNNN^NGGCC3' and generates 3 nt long 3' overhangs upon cleavage. SfiI is an archetypal Type IIF enzyme, which functions as a tetramer and cleaves two copies of the recognition site in a concerted manner. Its sequence shows no similarity to other proteins and nothing is known about the localization of its active site or residues important for oligomerization. Using the threading approach for protein fold-recognition, we identified a remote relationship between SfiI and BglI, a dimeric Type IIP restriction enzyme from the PD-DxK superfamily of nucleases, which recognizes the 5'GCCNNNN^NGGC3' sequence and whose structure in complex with the substrate DNA is available. We constructed a homology model of SfiI in complex with its target sequence and used it to predict residues important for dimerization, tetramerization, DNA binding and catalysis.The bioinformatics analysis suggest that SfiI, a Type IIF enzyme, is more closely related to BglI, an "orthodox" Type IIP restriction enzyme, than to any other REase, including other Type IIF REases with known structures, such as NgoMIV. NgoMIV and BglI belong to two different, very remotely related branches of the PD-DxK superfamily: the α-class (EcoRI-like), and the β-class (EcoRV-like), respectively. Thus, our analysis provides evidence that the ability to tetramerize and cut the two DNA sequences in a concerted manner was developed independently at least two times in the evolution of the PD-DxK superfamily of REases. The model of SfiI will also serve as a convenient platform for further experimental analyses. The enzymes that do not fit this definition or exhibit certain structural and functional peculiarities, have been classified into several subtypes (review: [kcat/Km for DNA cleavage by a factor ≥106 [Type II restriction endonucleases (REases) comprise one of the major families of endonucleases and one of the largest groups of experimentally characterized enzymes (comprehensively reviewed in: ). The "otor ≥106 .D-Xn-(D/E)-X-K (where X is any amino acid), together with a number of non-specific and structure-specific nucleases, suggesting that these proteins are evolutionarily related despite the absence of overall sequence similarity : BfiI iStreptomyces fimbriatus. It recognizes the interrupted palindromic sequence 5'GGCCNNNN^NGGCC3', where N denotes any base, and cleaves it as indicated by "^", leaving 3' extensions 3 nt long [SfiI is a REase isolated from  nt long . SfiI is nt long . However nt long -25, but In the absence of experimentally determined protein structures, homology-based models may serve as working models for the investigation of sequence-structure-function relationships between diverged enzymes . HomologThe sequence of SfiI showed no significant similarity to any other protein sequences. Also among the proteins reported by BLAST with sub-optimal scores, there were no proteins of known structure and no nucleases (data not shown) that could hint at potential relationships of SfiI to any previously characterized protein superfamily. Thus, in order to identify a template structure for modeling of SfI we used the threading approach, which allows to assess the compatibility of the target sequence with the available protein folds based not only on the sequence similarity but also on the structural considerations (match of secondary structure elements, compatibility of residue-residue contacts, etc.) (reviews: ,28). TheA homology model of SfiI was constructed based on the alignments reported by threading methods, using the "FRankenstein's Monster" approach  , followed by local geometry optimization. One of the adenine residues in the mismatched A/A base pair in the middle of the DNA molecule was "mutated" to T/A. The curvature of the DNA remained unchanged. Essentially, the global structure of the protein-DNA complex for SfiI remains exactly as in the BglI structure, as reliable modeling of macromolecular interactions remains beyond the capabilities of the existing methods. The model of SfiI dimer is shown in Figure .BglI belongs to the "EcoRV-like" β-class of PD-DxK nucleases. The most typical features of REases from this class are: antiparallel orientation of the 5elements ,9. Most elements . Brieflyelements  and "mutIn the proposed model of SfiI, the spatial configuration of the catalytic residues is typical for an active site architecture conserved among PD-DxK nucleases. We predict that the active site of SfiI comprises residues: E55, D79, D100 and K102, which superimpose well on the catalytic residues of BglI: E87, D116, D142 and K144, respectively Figure , backbonInterestingly, our model suggests that SfiI lacks the counterpart of a loop corresponding to aa 63–80 in BglI used by this enzyme to interact with the target site from the minor groove side. Thus, SfiI appears to recognize its target solely from the major groove side and to use fewer specific contacts than BglI to recognize its cognate site. This suggests that SfiI may be an easier target for the engineering of REases with new sequence specificities.The dimerization interface of SfiI is comparable to that of BglI. We predict that the following residues may be important for monomer-monomer interactions: Q59, Y60, E63, E66, R73, F74, G76 and that mutating them to change the volume of the side chain (for instance G76R) or introducing (or reversing) the charge (E63R, E66R, R73D, F74R) could disrupt the formation of the SfiI dimer and destroy the REase activity.SfiI is a Type IIF enzyme, i.e. a tetramer that binds simultaneously to two recognition sites and cleaves both sites concertedly , while BComparative analysis of nucleases from the PD-DxK superfamily suggests that they can be classified into two remotely related lineages: α (EcoRI-like) and β (EcoRV-like) -9. It waOur results strongly suggest that the archetypal Type IIF enzyme SfiI is closely related to a β-lineage member, an "orthodox" Type IIP REase BglI. Another well-characterized group of Type IIF REases comprises α-lineage members for which crystal structures were solved (NgoMIV, Cfr10I, Bse643I) -41. ThusSequence searches of the non-redundant (nr) database and of the putative translations from finished and unfinished microbial genomes were carried out at the NCBI using PSI-BLAST . SecondaThe alignments between the sequence of SfiI and the structures of selected templates (members of the fold identified by Pcons) were used as a starting point for modeling of the SfiI tertiary structure using the "FRankenstein's Monster" approach , comprisaa, amino acid(s); bp, base pair(s); nt, nucleotide; e, expectation; REase, restriction endonuclease; MTase, methyltransferase; ORF, product of an open reading frame, RM, restriction-modification;JMB carried out the fold-recognition analysis for SfiI, built the preliminary models, drafted the manuscript and coordinated the whole study. AC built the final models and identified functionally important residues. KS participated in interpretation of the data and writing the manuscript. All authors have read and accepted the final version of the manuscript.


Leishmania (L.) mexicana-infected neonatal BALB/c mice. DCs were isolated and purified from the spleens of the following donor groups: a) Adult BALB/c mice infected during adulthood with L. (L) mexicana; b) Adult BALB/c mice infected during neonatal life; c) Healthy neonatal BALB/c mice; d) Healthy adult BALB/c mice. A neonatal model of infection, generated after inoculation with 5 × 105 promastigotes of L. (L) mexicana, was used as the infection control group. Sixteen hours after intraperitoneal transfer of DCs (1 × 103, 1 × 105, or 1 × 106 cells/ml), neonatal recipient BALB/c mice were infected. The adoptive transfer of DCs diminished disease progression in neonatal mice. This reduction depends on the quantity and provenance of transferred DCs, since the effect was more evident with high numbers of DCs from adult mice infected during adulthood and healthy neonatal mice. Protection was significantly reduced in animals receiving DCs from healthy adult mice but it was absent in mice receiving DCs from adult mice infected during neonatal life. These results suggest that genetic susceptibility to Leishmania infection can be modified during neonatal life, and that the period of life when antigens are encountered is crucial in influencing the capacity of DCs to induce resistance or tolerance.We evaluated the adoptive transfer of DCs on  L. (L.) mexicana, and from healthy adult and neonatal mice. As in the L. major mouse model, we have shown that infection with L. (L.) mexicana strain MHOM/BZ/82/BEL21, generates a Th1 response associated to protective immunity in C57BL/6 mice, and a Th2 response related to non-healing disease in BALB/6 mice [Medawar et al.  showed aB/6 mice .Leishmania antigens to the lymph nodes and induce specific immune responses [Leishmaniasis is an excellent model to study the extremes of host/parasite relationships, particularly the diversity of the immune response associated to the genetic background of the host. In addition, mice can reproduce the distinct clinical forms observed in humans ,17. Thesesponses -24. Mollesponses .L. (L.) mexicana infection in neonatal BALB/c mice, after the inoculation with 5 × 104, 1 × 105, 2 × 105 or 5 × 105 promastigotes was determined by measuring the footpad thickness during 6 weeks. All 4 experimental groups developed lesions. Mice that received 1 × 105, 2 × 105 and 5 × 105 promastigotes respectively, showed a significant increase (p ≤ 0.05) on footpad thickness starting from the second week, reaching a maximal value on the sixth week of evaluation  mexicana-infected adult BALB/c mice inoculated with 1 × 106 promastigotes  two-tailed value and a very significant Spearman correlation (r = 1.000, p = 0.0014). The starting footpad thickness in neonatal and adult BALB/c mice was 1.67 mm and 1.85 mm, respectively.The progress of ion Fig. . This ineek Fig. . Moreovetes Fig. . The sta5 promastigotes as the optimal concentration for L. (L.) mexicana infection in all the subsequent experiments including the infection control group. This neonatal murine model of L. (L.) mexicana infection used half the numbers of promastigotes previously described to infect adult BALB/c mice [Leishmania infection. Other studies have shown a similar pattern of Th2-biased immune response in other models of neonatal infection [Leishmania infection [We used 5 × 10B/c mice . A signinfection ,11. We anfection . Susceptnfection -31. Seconfection ,33.DCs obtained by our purification method showed characteristic dendritic cell morphology, and a 97% purity as determined by CD11c immunostaining and flow cytometry. A minor fraction of about 3.5 % expressed CD3 and NK1.1 Fig. . The expSplenic DCs were isolated for our adoptive transfer experiments since they are mobile antigen-presenting cells that migrate to peripheral lymph organs where they stimulate naive T cells, thus initiating primary T cell responses -36. Furt3, 1 × 105 or 1 × 106 DCs from adult BALB/c mice infected during adulthood with L. (L) mexicana on neonatal recipient mice modified the course of infection, showing a delayed lesion growth from the second week onward  mexicana fail to modify the course of infection of neonatal recipient BALB/c mice as compared with infection control animals  on weeks 2, 3 and 4. This effect disappeared from the fifth week onwards  in lesion growth from the second week of infection  smaller than those observed in mice transferred with 1 × 105 DCs, which also initiated their lesions on the third week  in lesion growth from the second week of infection. This reduction in footpad thickness was very similar in both tested concentrations  mexicana, healthy adult BALB/c mice and healthy neonatal BALB/c mice. The reduction in these 3 groups was statistically significant (p ≤ 0.05) as compared with the infection control group. This reduction in footpad thickness was absent or considerably diminished in mice receiving DCs from adult BALB/c mice infected during neonatal life with L. (L) mexicana  mexicana infection in neonatal BALB/c recipient mice. These results contrast with those of Moll and Berberich [Leishmania infection. In this study, the observed protection depends on the quantity and provenance of the transferred DCs, since the effect was more evident with high cellular numbers of DCs from adult BALB/c mice infected during adulthood and healthy neonatal mice, where lesions were about 40% smaller than in the infection control group. DCs from these two groups have the intrinsic capacity to induce protective or resistant immune responses very early in life. That neonatal DCs appear to be more protective, on a per cell basis, than adults DCs is a very striking result since only Dadaglio et al.[L. (L) mexicana during neonatal life. This result confirmed recent studies by Adkins et al. showing that animals initially immunized as neonates are unable to develop the expected Th1 memory effector function observed in adults [Our results showed that the preceding intraperitoneal adoptive transfer of DCs diminished the progression of erberich  showing io et al. have shoio et al.. Also, aio et al.-48. Thisio et al.-51. Our n adults . These in adults .Our results show that tolerizing DCs from animals initially immunized as neonates play a key role in the attenuation of Th1 responses. The present results may have a considerable epidemiological impact on leishmaniasis, where infection at early stages of life may impose a tolerogenic state that favors the development of visceral or diffuse cutaneous leishmaniasis, both characterized by Th2-type responses.L. (L.) mexicana infection.In this study, we have shown that intraperitoneal adoptive transfer of splenic DCs is able to surpass the genetic bias of the mice, allowing the development of an immune response that modifies the progression of Adult (6 weeks) and neonatal (about 24 hour newborn) female BALB/c mice (Taconic, Germantown, NY, U.S.A.) were raised in the Animal House of the Instituto de Biomedicina, under appropriate conditions of temperature, water and feeding.d (MHC-II, clone AMS-32.1) and CD86 (B7.2, clone GL1) purchased from BD Pharmigen (San Diego, USA).The following rat monoclonal antibodies were used to isolate and characterize dendritic cells: CD19 (B cells, clone IBL-2), MOMA-2 (Macrophages/Monocytes), CD45R , CD3 , CD11c (dendritic cells and other leukocytes, clone N418), CD19 (clone 6D5) conjugated to phycoeritrine (PE), NK1.1 (clone PK136) conjugated to PE, Macrophages-Monocytes (MOMA-2) conjugated to fluorescein isothiocyanate (FITC), CD3 (clone KT3) conjugated to FITC. All were purchased from Serotec Ltd. (Oxford, United Kingdom) except CD205 (dendritic cells, clone NLDC-145, DEC205) donated by Georg Kraal, Vrije Universiteit, Amsterdam, The Netherlands; I-ALeishmania (Leishmania) mexicana (MHOM/BZ/82/BEL21) were extracted from footpad nodules of hamsters infected a month earlier with 1 × 106 amastigotes. The nodules were aseptically dissected out and washed in phosphate-buffered saline (PBS, pH 7.4) with 100 U/ml penicillin and 100 μg/ml streptomycin, and finely cut and ground in a Petri dish containing cold PBS. The suspension was filtered through a sterile sieve to remove large debris. These parasites were cultured on blood agar base (Sigma-Aldrich, St. Louis, U.S.A.) at room temperature for 7 days (the stationary growth phase) to obtain infective promastigotes. For an enriched population of parasites, free of erythrocytes and cellular debris, 100 μl of that sample were cultured in 2 ml Schneider's insect cell culture medium (Sigma-Aldrich, St. Louis, U.S.A.) for one week at room temperature. Promastigotes were isolated after 3 washes with sterile PBS and centrifugation at 1000 g at 4°C for 15 min. Pellets were resuspended in 1 ml of sterile PBS. Viable parasites were counted by trypan blue exclusion. Parasite concentration was adjusted to 5 × 104, 1 × 105, 2 × 105 and 5 × 105 per μl to be used in the different experimental groups.Amastigotes of L. (L.) mexicana infection to that established in adult mice [4, 1 × 105, 2 × 105, or 5 × 105 promastigotes suspended in 10 μl sterile PBS, applied with a tuberculin syringe (29-gauge needle) connected to a stepper repetitive pipette (Tridak, Danbury, U.S.A.). For comparison, adult BALB/c mice were infected the standardized optimal parasite load of 1 × 106 promastigotes of L. (L.) mexicana [A similar pattern of ult mice  was detemexicana . The cou7 cells/ml in complete RPMI-10 and 8 ml plated in tissue culture flasks. The flaks were incubated for 2 hr at 37°C in a 5% CO2 incubator (NuAire, Inc., Plymouth, U.S.A.), allowing DCs to adhere. Non adherent cells were carefully removed and placed in sterile 50 ml centrifuge tubes and spun at 250 g, 4°C for 10 min. Adherent cells were covered with 10 ml complete RPMI-10 and incubated as before for 16–18 hours, allowing DCs to detach. After gently washing the surface of the flasks with a plugged Pasteur pipette and complete RPMI-10, pools of the eluted cells were placed in sterile 15 ml centrifuge tubes and spun at 250 g, 4°C for 10 min. For each tube, the cell pellet was resuspended in 6 ml complete RPMI-10. This volume was carefully layered over a 3 ml NycoPrep™ density gradient (Nycomed Pharma AS, Torshov, Norway) and centrifuged at 600 g, 20°C for 20 min. Mononuclear cells were removed from the interface ring using a Pasteur pipette, transferred to a sterile 15 ml centrifuge tube and spun down in 10 ml complete RPMI-10 at 400 g, 20°C for 15 min three times. The final pellet was resuspended in 1 ml of cold (4°C) Hanks balanced salt solution supplemented with 10% decomplemented FBS and 2 mM HEPES. Cells were quantified and viability assessed by trypan blue exclusion.DCs from adult and neonatal BALB/c mice were isolated from the spleen. Under sterile conditions, spleens were minced on a metallic mesh with RPMI-1640 (Life Technologies, Rockville, U.S.A.) supplemented with 10% of decomplemented fetal bovine serum (FBS), 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 50 μM 2-mercaptoethanol and 100 U/ml penicillin (complete RPMI-10). The cell suspension was filtered on a nylon sieve and transferred to 15 ml centrifuge tubes (Corning Life Sciences, Acton, U.S.A.) and spun at 250 g at 4°C for 10 min. Viable cells were counted by trypan blue exclusion. Cell concentration was adjusted to 1 × 106 cells). After incubation, cells were washed three times in Hanks centrifuging at 250 g, 4°C for 10 min. The pellet was resuspended in 1 ml cold Hanks in a sterile 15 ml centrifuge tube and incubated under continuous agitation at 4°C for 1 hour with a secondary sheep anti-rat IgG polyclonal antibody coupled to magnetic microspheres  at a 7:1 sphere/target ratio. Non-dendritic magnetic-coated cells were removed by positive selection in three sequential depletions using a magnetic gadget  at 4°C for 6 min.The final purification stage consisted of an immunomagnetic negative selection of DCs. The cell suspension obtained above was incubated under continuous agitation at 4°C for 1 hour, with primary rat anti-mouse monoclonal antibodies recognizing B and T lymphocytes, NK cells and monocytes/macrophages (1.5 μg/ml antibody per 1 × 105 cells were suspended in PBS (1% FBS) and incubated with 10 μl primary monoclonal antibodies directly coupled to PE or FITC recognizing T and B lymphocytes, NK cells and monocytes/macrophages. DCs were characterized by an indirect method using primary monoclonal antibodies to CD11c and a secondary antibody, hamster anti-rat IgG1conjugated to FITC (clone MARG1-2, Serotec Ltd., Oxford, United Kingdom). The incubations were carried out in the dark at 4°C for 45 min, followed by 3 washes and centrifugation at 250 g, 4°C for 10 min. The cell pellet was resuspended in 500 μl PBS and the percentage of labeled cells determined in a flow cytometer (FACScan, Becton Dickinson, Franklin Lakes, U.S.A.). The control consisted of an antibody of irrelevant specificity conjugated to FITC.DC purity was determined by flow cytometry and immunocytochemistry. For flow cytometry, 1 × 105 cells were suspended in PBS (1% FBS) and spun down at 50 g in a Cytospin (Shandon Inc., Pittsburg, U.S.A.). Sample slides were hydrated in PBS, fixed in fresh acetone for 5 min. and sequentially incubated for 90 min with primary rat monoclonal antibodies to CD11c and CD205, biotinylated goat anti-rat IgG (50 μg/ml) (Vector Laboratories, Burlingame, U.S.A.) for 45 min., and Vectastain® Elite ABC kit (Vector Laboratories, Burlingame, U.S.A.) at 1:100, 30 min. Five-minute washes with PBS were done between incubations. The reactions were developed for 3 minutes in Vector® NovaRed™ substrate. The slides were then washed and counterstained with methyl green. Omissions of the primary antibody and incubation with an antibody of irrelevant specificity at the same protein concentration were used as controls.For immunocytochemistry, 1 × 10L. (L.). mexicana (n = 4); b) Adult BALB/c mice infected during neonatal life with L. (L.). mexicana (n = 4); c) Healthy neonatal BALB/c mice (n = 4); d) Healthy adult BALB/c mice (n = 4). The infection control group consisted of neonatal BALB/c mice infected with 5 × 105 promastigotes of L. (L) mexicana.DCs were isolated from the spleens of the following donor groups: a) Adult BALB/c mice infected during adulthood with 3, 1 × 105, or 1 × 106 cells/ml in sterile PBS for intraperitoneal transfer to neonatal recipient BALB/c mice. Cells, at the mentioned concentrations, were injected in 20 μl volumes using a tuberculin syringe (29-gauge needle) connected to a stepper repetitive pipette (Tridak, Danbury, U.S.A.). After sixteen hours of adoptive transfer, neonatal recipient BALB/c mice were infected with 5 × 105 promastigotes of L. (L) mexicana.DCs from the 4 experimental groups were adjusted to 1 × 10Isolation of DCs and adoptive transfer experiments were done in duplicates.).The results were expressed as mean ± standard error of the mean (SEM). Each experimental group consisted of 4–5 individuals. Comparisons between groups were made with Student t test and Welch t test for unpaired samples. Any value of p ≤ 0.05 was considered significant. All tests were performed using GraphPad InStat 3.02  declare that they have no competing interests.LVP carried out most of the experimental work and drafted the manuscript. JC developed the experimental design, carried out part of the experimental work and drafted the manuscript. NLD participated in the in experimental design and evaluated the progression of infection in the mice. FJT conceived the study, participated in the experimental design and coordinated the work. All authors read and approved the final manuscript.


Prosaposin is a potent neurotrophic molecule. Homozygous inactivation of We have recently reported that prosaposin is expressed at a higher level by androgen-independent (AI) prostate cancer cells as compared to androgen-sensitive prostate cancer cells or normal prostate epithelial and stromal cells. In addition, we have demonstrated that a synthetic peptide (prosaptide TX14A), derived from the trophic sequence of the saposin C domain of prosaposin, stimulated cell proliferation, migration and invasion and activated the MAPK signaling pathway in prostate cancer cells. The biological significances of saposin C and prosaposin in prostate cancer are not known.Here, we report that saposin C, in a cell type-specific and dose-dependent manner, acts as a survival factor, activates the Akt-signaling pathway, down-modulates caspase-3, -7, and -9 expression and/or activity, and decreases the cleaved nuclear substrate of caspase-3 in prostate cancer cells under serum-starvation stress. In addition, prosaptide TX14A, saposin C, or prosaposin decreased the growth-inhibitory effect, caspase-3/7 activity, and apoptotic cell death induced by etoposide. We also discovered that saposin C activates the p42/44 MAP kinase pathway in a pertussis toxin-sensitive and phosphatidylinositol 3-kinase (PI3K) /Akt-dependent manner in prostate cancer cells. Our data also show that the anti-apoptotic activity of saposin C is at least partially mediated via PI3K/Akt signaling pathway.We postulate that as a mitogenic, survival, and anti-apoptotic factor for prostate cancer cells, saposin C or prosaposin may contribute to prostate carcinogenesis at its early androgen-dependent or metastatic AI state. These mice also develop several abnormalities in their reproductive organs, such as atrophy and involution of the prostate gland and inactivation of MAPK and Akt in the prostate epithelium [Androgens, growth factors, neuropeptides, and other trophic agents are involved in normal and neoplastic growth of the prostate. Prosaposin is the intracellular precursor of four lysosomal glycoproteins, saposins A-D, that are involved in lysosomal hydrolysis of sphingolipids. These saposins, through their interaction with glycosphingolipid hydrolases and their substrates, increase lysosomal hydrolytic activities. Saposins and prosaposin are expressed by various cell types and as a secretory protein in body fluids including blood, seminal plasma, seminiferous tubular fluid, and prostatic secretions -5. Prosaes TX14A . Prosaptes TX14A -8. It haithelium ,10. The We have recently reported a higher expression of prosaposin in androgen-independent (AI) prostate cancer cells (PC-3 and DU-145) than in androgen-sensitive (AS) LNCaP or in normal prostate epithelial and stromal cells. In addition, we have found that prosaptide TX14A stimulates prostate cancer cell proliferation, migration, and invasion, activates the Raf-MEK-ERK-Elk-1 signaling cascade of the mitogen-activated protein kinase (MAPK) pathway, and inhibits the growth-inhibitory effects of sodium selenite administered at apoptogenic concentrations .In the present study, we show for the first time that saposin C also functions as a survival factor, activates PI3K/Akt-signaling pathway, and in a cell type-specific manner, modulates the expression of procaspase- and caspase-3, -7, and -9 in prostate cancer cells under serum-starvation stress. We demonstrated that prosaptide TX14A, saposin C, or prosaposin decreased the growth-inhibitory effects, caspase-3/7 enzymatic activity, and apoptotic cell death induced by etoposide. In addition, our data show that saposin C activation of a p42/44 MAPK in prostate cancer cells is not only pertussis toxin-sensitive, but also PI3K/Akt-dependent. Moreover, the PI3K-inhibitor, LY294002, restores the apoptogenic effect of etoposide in prostate cancer cells studied.We propose that as a survival and anti-apoptotic factor, saposin C or prosaposin may contribute to prostate carcinogenesis or to the development of hormone-refractory prostate cancer.The effect of saposin C as a survival factor was assessed under serum-starvation stress. Androgen-sensitive (AS) LNCaP cells did not maintain their viability when cultured in serum-free, 0.25% FBS-RPMI, or 0.5% FBS-RPMI media, for more than 36 h. These cells started to detach from tissue culture plates and cell viability was decreased to less than 40% as determined by the trypan blue dye-exclusion method. However, in the presence of 1% FBS, cells remained attached to the tissue culture plate and their growth increased 31% at day 4 and 20% on day 6 as compared with the control values at day 2  PC-3 and DU-145 cells Fig. . This rein vitro kinase assays were performed. After 5 or 10 min exposure of cells to saposin C, activated-Akt induced phosphorylation of glycogen synthetase kinase-3  in both AS and AI prostate cancer cells  at concentrations as low as 0.1 nM, was followed by a slight increase at higher concentrations  and non-cleaved (inactive) forms of the initiator caspase-9, its active downstream effectors caspases-3 and -7), and poly (ADP-ribose) polymerase (PARP, a nuclear substrate for caspase-3) in the cells after a 48 h serum deprivation period. Caspase-9 is closely coupled to proapoptotic signals and we found that the expression of procaspase-9 was not affected by saposin C; however, we were able to detect a reduction in its cleaved form at 10 nM saposin C in all cells investigated  of apoptotic cells in the three cell lines investigated. Saposin C decreased apoptotic cells by 47% in PC-3, 89% in DU-145, and 58% in LNCaP cells Fig. . This reWe also employed a sensitive fluorometric assay to measure caspase-3/7 (based on DEVDase) activity using the experimental conditions described above. Saposin C, at 1 nM concentration, demonstrated the highest reduction in caspase-3/7 activity in AS LNCaP (21%) and in AI PC-3 (35%) and DU-145 cells (30%) Fig. . ProsapoTo determine whether or not saposin C anti-etoposide apoptotic activity is PI3-kinase dependent, the effect of PI3K/Akt inhibitor (LY294002) on caspase-3/7 activity in the cells was examined in the presence or absence of saposin C ± etoposide. Through our initial studies, using trypan blue exclusion and MTS assays, we found 1.5 μM of LY294002 was a non-toxic and tolerable dosage for experimental period (3 days).Compared to DMSO-treated cells, LY294002 slightly increased (up to 10%) the caspase-3/7 enzymatic activity in PC-3 and DU-145 and showed almost no change in LNCaP Fig. . As descIn neuro-glial derived cells, neurotrophic activity, cell-death protection and the activation of MAPK by prosaptides i.e., TX14A), saposin C, or prosaposin are mediated by their binding to a pertussis toxin-sensitive GPCR ,7,12,13.A, saposiWe have previously demonstrated MAPK-activation by the TX14A peptide derived from a trophic sequence of saposin C . To examThe intensity of reduction of p42/44 activity and its cell type-specific pattern was very similar to PT plus saposin C- or PT-treated values. Interestingly, we observed a more profound reduction in p42/44 phosphorylative activity in cells pretreated with LY294002 and then treated with saposin C. To rule out the potential cytotoxic effect of the inhibitors, viability of cells was also determined by trypan blue dye-exclusion. We observed essentially similar results using the other structurally and mechanistically different PI3-kinase inhibitor (wortmannin). This experiment revealed that at the end of the pre-treatment incubation period, cell viability was equal to or more than 95%. These results indicate that MAPK activation by saposin C is at least partially mediated by saposin C-regulated PI3K/Akt pathways in prostate cancer cells. This result also provides additional proof for simultaneous activation of multiple (inter-related) signal transduction pathways by saposin C.Induction of apoptosis by androgen-ablation therapy significantly reduces androgen-dependent (AD) prostate cancer cells, but fails to cure the majority of patients due to the presence of apoptosis-resistant cancer cells that are androgen-independent . It is lprosaposin knock-out mouse model has revealed a number of interesting findings specifically in the male reproductive organs. Among these are atrophy of the prostate gland, epididymis, and seminal vesicles. Microscopic evaluation of affected tissues also shows undifferentiated phenotypes in prostate ventral and dorsal lobules and atrophy of the tubuloalveolar glands and their epithelial cell lining [The l lining . These din vitro and in vivo [In a variety of neuro-glial derived cells, synthetic peptides encompassing a trophic sequence of saposin C and/or prosaposin have been found to induce growth, survival, and/or differentiation, or to prevent apoptotic cell-death  in vivo ,18-20. F in vivo .Ser phosphorylative activity in the prostate cancer cells under investigation. This effect was substantially inhibited by LY294002, an inhibitor of the upstream Akt effector, PI3K , although the responses of androgen-sensitive (AS) LNCaP and AI PC-3 and DU-145 prostate cancer cells were different from each other, saposin C in a dose- and time-dependent manner, proved to increase proliferation and survival of both cell types , were able to decrease the growth-inhibitory effect of etoposide-treated prostate cancer cells Fig. . Under sInduction of mitogenic, survival, and anti-apoptotic signals in physiological and pathological conditions may begin from a wide array of extracellular stimuli and receptors, including receptor tyrosine kinases (RTKs) and G-protein coupled receptors (GPCRs). From a historical point of view, considerable attention has been given to the role of RTKs and their cognate polypeptide ligands in prostate cancer. However, accumulating evidence support the involvement of lysophosphatidic acids, neurotransmitters, and neuropeptides such as bombesin and neurotensin through GPCR signaling in the initiation or progression of prostate cancer ,33. GPCRHere, we showed that saposin C, in a pertussis toxin-sensitive manner, activated p42/44 MAPK Fig. . Our stuOur data for the first time show that by activation of multiple inter-related signaling pathways (PI3K/Akt and MAPK) and cell type-specific modulation of expression or activity of caspases, saposin C and/or its precursor (prosaposin) serve as a survival and anti-apoptotic factor for both AS- and AI-prostate cancer cells. Elucidation of such intricate mechanisms could potentially provide a therapeutic option that combines cytotoxic therapy and inhibition of survival/anti-apoptotic signals for AI metastatic prostate cancer. Finally, our observations provide novel insights into the diversity of biological activities of prosaposin in prostate cancer cells.Androgen-independent (PC-3, DU-145) and -sensitive (LNCaP) prostate cancer cell lines were obtained from the American Type Culture Collection (Manassas, VA) and grown in defined media (PC-3 and DU-145 in DMEM-10% FBS and LNCaP in RPMI-1640-10% FBS supplemented with 1 mM sodium pyruvate, 10 mM HEPES). Purified recombinant human saposin C and prurified human milk-prosaposin were characterized and provided by Dr. K. Sandhoff (University of Bonn, Germany) and Dr. M. Hiraiwa , respectively.Cells were initially grown in 100 mm plates in their respective culture media for 3 days, and after washing with PBS were incubated in serum-free DMEM (PC-3 and DU-145) or RPMI-1% FBS (LNCaP) in the presence or absence of saposin C (at 0.1, 1, or 10 nM) for 2, 4, or 6 days. Saposin C and culture media were replaced every 2 days. At the end of the incubation periods, cells were trypsinized and cell number was determined using a hemocytometer and the trypan blue exclusion method.2) supplemented with a protease inhibitor cocktail (Roche Diagnostic, Inc., Indianapolis, IN) and 1 mM sodium orthovandate, plus sodium dodecyl sulfate (SDS) at a final concentration of 0.1%. The lysates were then centrifuged (15 min, 4°C, 16,000 × g), aliquoted, and stored at -70°C until use. Protein concentration was determined by BCA assay (PIERCE, Rockford, IL). Each experiment was repeated at least two times. For western analysis, membranes were blocked with 5% BSA in the rinse buffer (150 mM NaCl, 20 mM Tris, 0.1% Tween 20) for 1 h, washed in rinse buffer for 10 min, and then incubated with the respective primary antibody at the indicated concentrations (see below). The membranes were then washed and incubated with the appropriate horseradish peroxidase-conjugated secondary antibody  for 1 h at room temperature, washed for 10 min and four more cycles of 5 min, and treated with an enhanced chemiluminescence (ECL) detection system (Amersham, Piscataway, NJ). In some cases, when the signal was very weak or undetectable, we used ECL-plus (Amersham).Protein expression analysis was performed according to standard procedures . BrieflyCells were grown in their respective complete culture media for 2–3 days (up to 60% confluency), washed with PBS, incubated in their serum-free  media for 20 h, and then fresh basal media was added to all plates for an additional 4 h. Various inhibitors [LY294002 (50 μM, 3 h), Wortmannin (10 μM, 15 min), U0126 (10 μM, 1.5 h), and Pertussis toxin (200 ng/ml, 4 h)] were added to the culture medium, just before treating cells with saposin C (at 0.1 nM, 5 min). We used 10% FBS as a positive control. Cells were lysed and 10 μg of clarified protein samples was subjected to SDS-PAGE under reducing conditions. Phospho-specific p42/44 antibody  was used as the primary antibody and as a loading control. Filters were also probed or reprobed with anti-p42/44 antibody. Additional tissue culture plates that had been treated with or without inhibitors were also tested for cell viability by trypan blue dye-exclusion assay.Cells were cultured up to 70% confluency in their complete media and after washing with PBS, they were serum-starved for 24 h, and then treated with 10% FBS or saposin C at 0.1, 1 or 10 nM for 10 min. A representative tissue culture plate was also pretreated with the PI3K-inhibitor (LY294002, 50 μM for 3 h) before treating cells with saposin C (at 1 nM for LNCaP and at 10 nM for PC-3 and DU-145 cells). After preparation of the cell lysate, 15 μg of protein per sample was subjected to SDS-PAGE under reducing conditions. Immunoblotting was performed using phospho-specific Akt antibodies against serine 473 or threonine 308 . A loading control was evaluated with anti-Akt antibody. Each experiment was performed in duplicate, and the assays were repeated three times.3VO4, and 10 mM MgC12 supplemented with 200 μM ATP and 1 μg glycogen synthase kinase-3 [GSK-3; a well characterized Akt/PKB substrate] of fusion protein (GSK-3α/β) and incubated 30 minutes at 30°C, allowing immunoprecipitated Akt (if activated) to phosphorylate GSK-3. The kinase reaction was terminated by adding 20 μl of 3 × SDS sample buffer. Phosphorylated GSK-3 was then detected by western analysis using phospho-GSK-3α/β (for Ser 21 of GSK-3α and ser 9 of GSK-3β) antibody. The above in vitro kinase assay is based on the fact that phosphorylated-Akt (active) regulates GSK-3α/β kinase activity via phosphorylation at ser 21/9. For control loading, 10 μg protein per sample from the same whole cell lysates were subjected to western analysis using monoclonal anti-Akt antibody or actin. Each experiment was performed in duplicate, and the assays were repeated three times.A non-radioactive Akt kinase assay kit  was used to determine whether saposin C treatment of cells under serum-starvation stress would lead to Akt-activation. For Akt-kinase assays, cells were grown up to 70% confluency in their maintenance media, serum-starved for 24 h, and then treated for 5 or 10 min in the presence or absence of saposin C at 0.1, 1, or 10 nM. Cells were washed once with ice-cold PBS and harvested under nondenaturing conditions using 1X ice-cold cell lysis buffer (from the Kit) supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF) on ice for 5 (or 10) minutes. Akt was selectively immunoprecipitated from 250 μg protein (whole cell lysates) using 20 μl of immobilized Akt 1G1 monoclonal antibody, and then incubated with gentle rotation for 4 h at 4°C. Samples were then centrifuged briefly (30 sec, 2000 × g) and pellets were washed twice with 1X lysis buffer and once with 1X kinase buffer. Immunocomplexes (pellets) were resuspended in 40 μl 1X kinase buffer [composed of 25 mM Tris (pH 7.5), 5 mM β-glycerolphosphate, 2 mM DTT, 0.1 mM NaCells were cultured up to 60% confluency in their complete culture media. After washing with PBS, they were incubated with their respective serum-free media in the presence or absence of saposin C at 0.1, 1, or 10 nM for 48 h; whole cell lysates were prepared as described above. Clarified protein samples (75 μg) were subjected to SDS-PAGE under reducing conditions. Western analyses were carried out using monoclonal antibodies against non-cleaved and cleaved caspases-3, -7, and -9 and poly (ADP-ribose) polymerase (PARP) provided in an Apoptosis Sampler Kit . Each experiment was performed in duplicate, and the assays were repeated three times.We next examined the effect of an apoptogenic agent, etoposide, on cell growth and caspase activity in the presence or absence of saposin C, prosaposin, prosaptide TX14A, or an analogous inactive mutant peptide . Cells were seeded at 1,000 per well in 96-well plates in their complete culture medium for 3 to 4 days. After this period, cells were treated in complete culture media for 3 days with etoposide (2, 20, or 200 μM) to find the lowest concentration that led to the highest growth inhibition, as measured by MTS assay (described above). Using the optimal cell type-specific etoposide concentration (20 μM for PC-3 and LNCaP and 2 μM for DU-145), cells were treated with the vehicle (DMSO), saposin C (0.1, 1, 10 nM), TX14A (10 nM), mutant peptide (769M, 10 nM), or prosaposin (at 1 ng/ml). Using the cell type-specific OD/cell number calibration curve as obtained by MTS assay (Promega, WI), cell number per well was determined for the above treatment conditions.Parallel tissue culture plates were also used to determine caspase-3/7 activity using the Apo-ONE™ Homogeneous Caspase-3/7 assay (Promega, WI). This assay provides a homogeneous Caspase-3/7 reagent  which performs a dual function, by rapidly and efficiently permeabilizing the cultured cells and at the same time exposing the intracellular space to the profluorescent caspase-3/7 substrate, rhodamine 110 (Z-DEVD-R110). After cleavage and removal of the DEVD peptides by caspase-3/7 activity, the fluorescence in each well was quantitated at an excitation wavelength of 485 + 20 nM and an emission wavelength of 535 + 25 nM and after correction based on blank control  or the homogeneous caspase-3/7 reagent. Final fluorescent intensity was depicted as endpoint relative fluorescent unit, RFLU.Using similar experimental conditions but as an independent study, the effect of LY294002 (a PI3-kinase inhibitor) was also evaluated on growth and caspase-3/7 activity of cells treated with saposin C ± etoposide. After initial studies to find the optimal concentration, we used a non-toxic tolerable dosage of 1.5 μM for LY294002. In addition, we chose the most effective  concentration of etoposide (20 μM for PC-3 and LNCaP and 2 μM for DU-145) and saposin C (1.0 nM for PC-3 and LNCaP and 10 nM for DU-145) for this study.In situ determination of apoptosis by Terminal dUTP nick-end labeling (TUNEL) was performed using an ApopTag Peroxidase In Situ kit as recommended by the manufacturer . The ApopTag Kit detects single- and double-stranded DNA breaks associated with apoptosis. Drug-induced DNA damage is not identified by the TUNEL assay unless it is coupled with the apoptotic response. Briefly, at the end of the incubation period, cells were fixed in 1% paraformaldehyde in PBS, pH 7.4 for 10 min at room temperature, washed with PBS-twice, and permeabilized in pre-cooled ethanol: acetic acid (2:1) for 5 min at -20°C. After washing twice in PBS, 5 min each time, endogenous peroxidase activity in the cells was quenched in 3% H2O2 in PBS for 5 min at room temperature, incubated with terminal deoxynucleotidyl transferase (TdT enzyme) and then with peroxidase-conjugated anti-digoxigenin antibody. Nuclear staining of the apoptotic cells was detected by 3',3'-diaminobenzidine tetrahydrochloride dihydrate substrate, as recommended by the manufacturer. Cells were then counterstained in 0.5% (w/v) methyl green and slides were mounted under a glass coverslip in permount mounting medium.Cells were cultured in multiwell chamber slides and treated with etoposide in the presence or absence of saposin C at 0.1, 1, or 10 nM as indicated above. For control staining, the enzyme incubation step was deleted. Microscopic examination of cells was carried out using a phase contrast microscope. Cells were counted by choosing ten random fields and the percentages of apoptotic cells were determined. Apoptosis was indicated by the presence of apoptotic bodies, exhibiting brightly labeled punctuated nuclei.p < 0.05 or 0.01. Statistical analyses were performed using GraphPad Prism version 3.00 for Windows (GraphPad Software, San Diego, CA).For cell survival and other quantitative data, a one-way analysis of variance (ANOVA) was employed to evaluate the influence of one variable on multiple independent groups. Bonferroni's corrections were also applied whenever a significant group effect was observed. To compare a control group with a single experimental group of interest, we used the Student's t-test. For cell survival studies, each treatment concentration was examined three times and in triplicate samples. The effect of saposin C or other effectors in the presence of etoposide on cell growth, apoptosis, or caspase-3/7 activity was studied in twelve replicates and repeated three times. Statistical significance was set at Author 1 (T-JL) carried out experiments described in figure


However, the α7 nicotinic receptor has been recently found in several non-neuronal cells and described as an important regulator of cellular function. Nicotine and ACh have been recently reported to inhibit tumor necrosis factor-α (TNF-α) production in human macrophages as well as in mouse microglial cultures. In the present study, we investigated whether the stimulation of α7 nicotinic receptor by the specific agonist nicotine could affect the functional state of activated microglia by promoting and/or inhibiting the release of other important pro-inflammatory and lipid mediator such as prostaglandin E2. Total RNA was assayed by RT-PCR for the expression of COX-2 mRNA.Expression of α7 nicotinic receptor in rat microglial cell was examined by RT-PCR, immunofluorescence staining and Western blot. The functional effects of α7 receptor activation were analyzed in resting or lipopolysaccharide (LPS) stimulated microglial cells pre-treated with nicotine. Culture media were assayed for the levels of tumor necrosis factor, interleukin-1β, nitric oxide, interleukin-10 and prostaglandin E2.Rat microglial cells express α7 nicotinic receptor, and its activation by nicotine dose-dependently reduces the LPS-induced release of TNF-α, but has little or no effect on nitric oxide, interleukin-10 and interleukin-1β. By contrast, nicotine enhances the expression of cyclooxygenase-2 and the synthesis of one of its major products, prostaglandin E2 modulates several macrophage and lymphocyte functions, which are instrumental for inflammatory resolution, our study further supports the existence of a brain cholinergic anti-inflammatory pathway mediated by α7 nicotinic receptor that could be potentially exploited for novel treatments of several neuropathologies in which local inflammation, sustained by activated microglia, plays a crucial role.Since prostaglandin E The inflammatory response is in the first instance a mechanism of self-defense, set by the innate immune system against endogenous and exogenous insults, and essential for the survival of the organism. Inflammation must be tightly regulated as deficiency as well as excess in its response will result in pathological conditions, such as immunodeficiency or chronic inflammatory diseases . In the Among the endogenous mechanisms that regulate the inflammatory response, cross-talk between the immune and nervous systems play an important role. In particular, it has been shown that electric stimulation of the vagus nerve attenuates the inflammation during endotoxemia in rats , and thaA, glycine and 5-HT3 receptors [Neuronal acetylcholine receptors (nAChRs) are ligand-gated ion channels, which belong to a large family of neurotransmitter receptors that includes the GABAeceptors . Each nAeceptors . At leaseceptors .2A-oligodendrocyte precursor cells but not in adult differentiated oligodendrocytes, suggesting that receptor expression is developmentally regulated [In addition to neurons and peripheral macrophages, several studies have demonstrated the expression of nAChRs in cell types both within and outside the nervous system . In the egulated . Cultureegulated  and cortegulated . A functegulated . In periegulated ,14. Acutegulated . Furtheregulated . Lymphocegulated . These o2 synthesis. Other important microglial products, such as interleukin-1β (IL-1β), nitric oxide (NO) and interleukin-10 (IL-10) were not affected or moderately decreased.Epidemiological studies indicate that nicotine, besides its immunosuppressive effects, may be protective against the development of neurodegenerative diseases such as Alzheimer disease (AD) and Parkinson's disease (PD) , in whicAll cell culture reagents were from Gibco (Grand Island, NY, U.S.A) and virtually endotoxin free (less then 10 E.U./ml as determined by the manufacturer). BCA protein assay was from Pierce (Rockford, Illinois). ELISA-kits for rat TNF-α and IL-10 were from Endogen Inc. (Woburn, MA). ED-1 monoclonal antibody was from Serotec (Oxford, UK). (±) Nicotine, α-bungarotoxin, FITC-α-bungarotoxin and lipopolysaccharide LPS (from Escherichia coli, serotype 026:B6) were from Sigma Chemical (St.Louis, MO). Rabbit polyclonal antibody against alpha 7 subunit was from Santa Cruz Biotechnology.5 cells/cm2. Cells were allowed to adhere for 20 min and then washed to remove non-adhering cells. After a 24 h of incubation, the medium was replaced with fresh medium containing the substance(s) under study. Cell viability was greater than 95%, as tested by Trypan Blue exclusion. Immunostaining, performed as previously described [Microglial cultures were prepared from 10–14 day mixed primary glial cultures obtained from the cerebral cortex of 1-day-old rats, as previously described  and in a2 content was quantified using a specific radioimmunoassay [2 with other prostaglandins less than 0.25%.At the end of the incubation time, cell supernatants were collected, centrifuged, and stored at -70°C until tested. The levels of TNF-α and IL-10 were assayed by specific ELISAs, following the manufacturer's instructions. The ranges of determination were: 31–2500 pg/ml for TNF-α, 10–1000 pg/ml and 8–500 pg/ml for IL-10. The production of NO by measuring the content of nitrite, one of the end products of NO oxidation, as previously described . PGE2 counoassay . The ass5 cells/cm2), allowed to adhere for 20 min and then washed to remove non-adhering cells. After a 24 h of incubation, the complete BME medium was replaced with fresh BME medium without serum. Cells were incubated at 4°C for 15 min with FITC-labeled α-bungarotoxin at 1.5 μg/ml. Where indicated, nicotine was added at the concentration of 500 μM for 10 min, in order to saturate all the binding sites before the addition of FITC-labeled α-bungarotoxin. Cells were washed 3 times with BME medium and then fixed with 4% paraformaldehyde at room temperature for 15 min. After fixation, coverslips were washed twice with PBS solution, mounted in PBS:glycerol and examined using a fluorescent microscope. Cell culture lysates from microglial cells and PC12 cells (used as positive control) were analyzed for α7 subunit expression. Total protein content was estimated using the Bio-Rad protein assay. An aliquot corresponding to 50 μg (microglia cells) and 20 μg (PC12 cells) of total protein for each sample was separated by sodium dodecyl sulphate polyacrylamide gel elecrophoresis (SDS-PAGE) and transferred electrophoretically to nylon membranes. Membranes were blocked with 10% non-fat milk and incubated with a rabbit policlonal antibodies against α7 subunit (1:2000) overnight at 4°C. Horseradish peroxidase conjugated anti-rabbit IgG (1:5000, 1 h at 25°C) and ECL reagents were used as detection system.Microglial cells were plated on uncoated glass coverslips  in a total reaction volume of 20 μl following the conditions provided by the manufacturer's protocol.Oligonucleotide primers with similar Tm were designed to generate a PCR fragment of 754 bp for the α7 subunit. PCR conditions (number of cycles and cDNA and primer concentration) that ensure the data to be obtained within the exponential phase of amplification of each template were carefully assessed. The amplification of the β-actin, COX-2 and α7 subunit within the exponential phase of amplification was achieved with 25, 30 and 40 cycles respectively.4 (2 mM), 1 Unit of Platinium Taq DNA polymerase High Fidelity (Invitrogen). The primers were: α7 subunit (Gene bank accession number S53987), sense 5'-TCT GTG CCC TTG ATA GCAC, antisense 5'-CTT CAT GCA ACC AGG ATC AG, product length 754; COX-2 [NM031144) sense 5'-GTC GAC AAC GGC TCC GGC ATG; antisense 5'-CTC TTG CTC TGG GCC TCG TCGC, product length 158 bp. A sample containing all reaction reagents except cDNA was used as PCR negative control in each experiment. The absence of genomic DNA was verified using 2 μg of RNA from microglia that was reverse-transcribed without the enzyme (-RT). The PCR conditions for COX-2 were as follows: initial denaturation at 94°C for 2 min followed by 30 cycles of 94°C for 30 sec, 58°C for 45 sec, 68°C for 1 min, and an additional cycle with extension at 72°C for 7 min. The PCR conditions for β-actin were as follows: initial denaturation at 94°C for 5 min followed by 25 cycles of 94°C for 30 sec, 68°C for 30 sec, 68°C for 45 sec and an additional cycle with extension at 72°C for 1 min. The PCR conditions for α7 subunit were as follows: initial denaturation at 94°C for 5 min followed by 40 cycles of 94°C for 30 sec, 57°C for 1 min, 68°C for 45 sec and an additional cycle with extension at 72°C for 7 min.Five μl, 15 μl and 40 μl of diluted cDNAs were amplified for β-actin, COX-2 and α7 respectively. PCR-amplification was done in a final volume of 50 μl containing 1x PCR buffer, the four dNTPs (0.2 mM), MgSO4; COX-2 , sense 5PCR products were analyzed by electrophoresis, stained with ethidium bromide and photographed. Transcript levels were analyzed by Fluor-STM Multimager analyser (Biorad). For each experiment, the ratio between optical density (arbitrary units) of bands corresponding to COX-2 and β-actin  was calculated to quantify the level of the transcripts for COX-2 mRNAs.t-test. A two-tailed probability of less than 5 % (i.e. p < 0.05) was taken as statistically significant.Data are expressed as mean ± SEM with the number of independent experiments, run in duplicate, indicated in figure legends. Comparison between treatment groups was made by Student's The expression of the mRNA for α7 subunit in rat microglial cells was investigated by RT-PCR. As shown in Figure Once we had demonstrated the presence of α7 subunit mRNA and protein in microglial cells, we studied the functional consequences of receptor activation using the specific agonist nicotine. Microglial cells were pre-treated for 30 min with increasing concentrations of nicotine and then incubated for 4 or 24 h in the absence or the presence of 10 ng/ml LPS. In resting microglial cultures nicotine did not affect the basal level TNF-α(data not shown).As previously demonstrated using mouse microglial cultures, nicotine pre-treatments dose-dependently inhibited the release of TNF-αFig. . At 1 μMTo verify that the effect of nicotine was mediated by α7 subunit, we measured the level of TNF-α in activated microglial cells exposed to nicotine in the presence or in the absence of α-Bgtx. The addition of 0.01 μM α-Bgtx almost totally prevented the inhibitory effect of nicotine Fig. .In addition to TNF-α, we also analyzed the release of two important microglial mediators such as NO and IL-1β and we found that nicotine pre-treatment only slightly reduced the release of NO  and did not modify the release of IL-1β (data not shown).2 (PGE2), two important local mediators with anti-inflammatory and immunoregulatory functions. Nicotine pre-treatment only moderately reduced  the level of IL-10 in the culture media of microglia cells stimulated for 24 h with LPS (data not shown).We then analyzed the effects of nicotine on the production of interleukin-10 (IL-10) and prostaglandin E2 in LPS-activated microglial cells. The presence of 0.01 μM α-Bgtx, blocked the nicotine-dependent increase of PGE2 released by LPS-activated microglia  positive fibers around β-amyloid plaques in transgenic APPSW mice suggests a close connection between cholinergic terminals and microglial cells [PGE in vivo ,37. We h and NGF ,28. In ticroglia , consisticroglia . It has g tissue . It is tal cells . A deficIn the last few years, several lines of evidence have suggested that activation of α7 subunits plays an important role in the maintenance of cognitive functions in several neurodegenerative disorders . EpidemiAs mentioned in the introduction, the presence of α7 subunit on immune cells as well as on other non-excitable cells has provided a molecular basis for a non-neuronal cholinergic pathway that might function as an essential regulator of inflammation as well as immune responses ,5. PrimaIt should be noted that although our study focused on the effects of nicotine on the process of microglial activation induced by LPS, our findings may have broader implications since other microglial activators, such as pro-inflammatory cytokines and fibrillogenic peptides, share some common signaling pathways with LPS ,53. In a2 up-regulation is under investigation. Shytle et al. [2 synthetic pathway [At present, the signaling pathways downstream to α7 subunit activation and leading, in particular, to COX-2 and PGEe et al.  have rep pathway ,56, thusActivation of α7 nicotinic receptors in microglial cells by nicotine controls some important microglial functions, thus preventing chronic inflammation. Since microglial activation and chronic inflammation have been associated with most neurodegenerative pathologies  the undeLipopolysaccharide (LPS)Acetylcholine (ACh)Neuronal acetylcholine receptors (nAChRs)Tumor necrosis factor-α (TNF-α)2 (PGE2)Prostaglandin EInterleukin-1β (IL-1β)Nitric oxide (NO)Interleukin-10 (IL-10)The author(s) declare that they have no competing interests.RDS conceived of the study, participated in its design and coordination, produced the primary microglial cultures, performed the ELISA and the immunofluorescence staining, was primarily responsible of the RT-PCR review the data and drafted the manuscript. MAAC participated in the design and coordination of the study, produced the primary microglial cultures, performed the ELISA and the immunofluorescence staining, was primarily responsible for western blot analysis and review the data. DC participated in the production of the primary microglial cultures and in RT-PCR. LM contributed to the design of the study, guided data interpretation and presentation and assisted in the preparation of the manuscript.


Alterations in carbohydrate metabolism are frequently observed in cirrhosis. We conducted this study to define the prevalence of diabetes mellitus (DM) and impaired glucose tolerance (IGT) in Iranian patients with chronic liver disease (CLD), and explore the factors associated with DM in these patients.One hundred and eighty-five patients with CLD were enrolled into the study. Fasting plasma glucose and two-hour plasma glucose were measured in patients' sera. DM and IGT were diagnosed according to the latest American Diabetes Association criteria.The subjects included 42 inactive HBV carriers with a mean age of 42.2 ± 12.0 years, 102 patients with HBV or HCV chronic hepatitis with a mean age of 41.2 ± 10.9 years, and 41 cirrhotic patients with a mean age of 52.1 ± 11.4 years. DM and IGT were diagnosed in 40 (21.6%) and 21 (11.4%) patients, respectively. Univariate analysis showed that age (P = 0.000), CLD status (P = 0.000), history of hypertension (P = 0.007), family history of DM (P = 0.000), and body mass index (BMI) (P = 0.009) were associated with DM. Using Multivariate analysis, age (OR = 4.7, 95%CI: 1.8–12.2), family history of DM (OR = 6.6, 95%CI: 2.6–17.6), chronic hepatitis (OR = 11.6, 95%CI: 2.9–45.4), and cirrhosis (OR = 6.5, 95%CI: 2.4–17.4) remained as the factors independently associated with DM. When patients with cirrhosis and chronic hepatitis were analyzed separately, higher Child-Pugh's score in cirrhotic patients (OR = 9.6, 95%CI: 1.0–88.4) and older age (OR = 7.2, 95%CI: 1.0–49.1), higher fibrosis score (OR = 59.5, 95%CI: 2.9–1211.3/ OR = 11.9, 95%CI: 1.0–132.2), and higher BMI (OR = 30.3, 95%CI: 3.0–306.7) in patients with chronic hepatitis were found to be associated with higher prevalence of DM.Our findings indicate that patients with cirrhosis and chronic hepatitis are at the increased risk of DM occurrence. Older age, severe liver disease, and obesity were associated with DM in these patients. Alterations in carbohydrate metabolism are frequently observed in liver cirrhosis. Glucose metabolism impairment in cirrhotic patients, both in fasting state and in response to oral glucose or meals has been widely documented in the literature -5. ThereIn this study, for the first time in Iran, we reported the prevalence of DM and IGT in patients with chronic liver disease (CLD) using the latest generally accepted criteria, and investigated independent correlation of CLD with DM, considering other known possible DM risk factors. Moreover, we explored the factors that might be potentially associated with DM in patients with cirrhosis and chronic hepatitis.From October 2002 to March 2003, all consecutive patients with chronic liver disease referred to "Tehran Hepatitis Center" were enrolled into the study. Tehran Hepatitis Center is a referral specialized clinic for liver diseases where many patients suffering from liver diseases and hepatitis from around Iran are referred to in order to receive consultation and clinical medical care. Pregnant females, patients under 20 years old, those who were on regular corticosteroid or hydrochlorothiazide therapy, known or suspected cases of hemochromatosis, or autoimmune disease were excluded. Having a clinical, biochemical (serum amylase and/or lipase elevation) or ultrasonographic evidence for chronic pancreatitis was also considered an exclusion criterion. No patient had a history of habitual drinking. All of the patients were Moslem and belonged to the white race.5 copies/ml (ROCHE Cobas Amplicore) and normal liver enzymes. The mean age in this group was 42.2 ± 12.0 years ranging between 23–84 years. Eight patients with nonalcoholic steatohepatitis (NASH) were initially enrolled but were not entered in analysis because of the low case number. Therefore analysis was finally performed for 185 patients.A total number of 193 patients who met the criteria were eligible to enter the study. First group included 41 cirrhotic patients. Mean age was 52.1 ± 11.4 years ranging between 26–74 years. Diagnosis of cirrhosis was confirmed by histology in 17 (41.5%) patients. The occurrence of signs or biochemical evidences of liver decompensation, ultrasound features of portal hypertension and/or esophageal varices in gastroscopy were used for the clinical diagnosis in the remaining cases. No cirrhotic patients had evidence of hepatocellular carcinoma, screened by serum alpha-fetoprotein level test and abdominal ultrasonography. Severity of liver cirrhosis was graded according to Child-Pugh's classification. Second group included 102 patients with chronic hepatitis B, or C. Mean age was 41.2 ± 10.9 years ranging between 22–67 years. All patients had adequate documentation of elevated serum aminotransferases for more than 6 months, and viral hepatitis molecular assays by qualitative RT-PCR. In 91 (89.2%) patients liver biopsy reports were accessible. The histological staging and grading in liver biopsy was scored according to Knodell scoring. Third group included 42 inactive hepatitis B virus (HBV) carriers who were hepatitis B s antigen (HBsAg) positive, quantitative HBV DNA level less than 10Informed consent in writing was obtained from each patient involved and the study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki as approved by institutional review board. Initial data were collected by chart review and interview. Then, two venous blood samples were taken: First sample in the morning after a 12-hour overnight fast to measure fasting plasma glucose (FPG) level, triglycerides (TG) level, and cholesterol (Chol) level; and a second sample following 2 hours after eating 75 gr glucose to measure two-hour post-loaded glucose (2-hr PG). FPG and 2-hr PG measurement were repeated on two other samples in second session.Patients receiving therapy with insulin or oral hypoglycemic medications were considered as diabetics. In other patients, FPG ≥ 126 mg/dl or 2-hr PG ≥ 200 mg/dl on more than one occasion was used as diagnostic for DM in agreement with the latest American Diabetes Association (ADA) criteria . FPG bet2).Body mass index (BMI) was calculated using the standard formula of weight in kilogram divided by square of height in meters (kg/mResults were expressed as mean ± standard deviation (SD). Comparison between diabetic and non-diabetic groups was made using Students T test for continuous variables and the Chi-square or Fisher exact test for categorical variables. At univariate analysis the factors possibly associated with DM development were evaluated. A multivariate analysis based on a stepwise logistic regression model was used to assess the independent effect of all variables found significant at the univariate analysis. P-value less than 0.05 was considered significant. All statistical analyses were performed using SPSS for Windows software .The sample population contained 150 (81.1%) males and 35 (18.9%) females. The overall mean age was 43.8 ± 12.0 years ranging between 22 and 84 years. In the total of 185 patients, DM was found in 40 (21.6%) patients, and IFG and/or IGT was found in 21 (11.3%) patients. Thirty patients were already aware of their problem whereas the diagnosis of DM was new in 10 patients.By univariate analysis a number of variables including the factors mentioned by ADA as risk factors for type II diabetes  in additAt multivariate analysis age (P = 0.008, OR = 4.7, 95%CI: 1.8–12.2), family history of DM (P = 0.000, OR = 6.6, 95%CI: 2.6–17.6), chronic hepatitis (0.000, OR = 11.6, 95%CI: 2.9–45.4), and cirrhosis (P = 0.000, OR = 6.5, 95%CI: 2.4–17.4) showed up as the only factors independently associated with DM prevalence rate  patients were diabetic and 7 (17.1%) patients had IFG and/or IGT. Univariate analysis showed that only two factors were associated with DM rate. Mean of age in diabetic cases was significantly higher than that of non-diabetic ones (P = 0.003). Moreover, DM was significantly more prevalent in patients with Child-Pugh's score B than score A (P = 0.04). Since there was no score C in study sample, we could not evaluate this class of cirrhosis. After applying logistic model, only Child-Pugh's score kept its significance (P = 0.04, OR = 9.6, 95%CI: 1.0–88.4) as an independent predictive factor for DM in cirrhotic patients (table In chronic hepatitis group including 102 patients, DM was found in 14 (13.7%) patients and IFG and/or IGT was found in 11 (10.8%) patients. There was no significant difference between diabetic and non-diabetic cases in regard with sex, etiology of chronic hepatitis, hypertension, interferon therapy, smoking, and serum TG and Chol levels. On the other hand, diabetic patients had higher mean age compared with non-diabetic cases (P = 0.001). More cases of diabetic patients compared with non-diabetic ones had a family history of DM (P = 0.01). The mean BMI of patients with DM was higher than that of patients without DM (P = 0.02). Furthermore, liver biopsy showed significantly more fibrosis activity in diabetic patients compared with non-diabetic cases (P = 0.003), whereas no difference in diabetic vs. non-diabetic patients was seen with respect to histological grading. Multivariate analysis revealed that older age (P = 0.04, OR = 7.2, 95%CI: 1.0–49.1), higher BMI (P = 0.004, OR = 30.3, 95%CI: 3.0–306.7), and more severe fibrosis activity  were the predictive variables for DM in patients with chronic hepatitis  declare that they have no competing interests.Dr. Alavian participated in the design of the study and coordination and drafted the manuscript. Dr. Hajarizadeh conceived of the study, participated in the design of the study, performed the statistical analysis and drafted the manuscript. Dr. Nematizadeh designed the questionnaire and helped in data collection. Dr. Larijani facilitated the study progress and participated in coordination. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here: