Clostridium thermocellum (CtCBM35) in polysaccharide recognition. CtCBM35 was cloned into pET28a (+) vector with an engineered His6 tag and expressed in Escherichia coli BL21 (DE3) cells. A homogenous 15 kDa protein was purified by immobilized metal ion chromatography (IMAC). Ligand binding analysis of CtCBM35 was carried out by affinity electrophoresis using various soluble ligands. CtCBM35 showed a manno-configured ligand specific binding displaying significant association with konjac glucomannan (Ka = 14.3×104 M−1), carob galactomannan (Ka = 12.4×104 M−1) and negligible association (Ka = 12 µM−1) with insoluble mannan. Binding of CtCBM35 with polysaccharides which was calcium dependent exhibited two fold higher association in presence of 10 mM Ca2+ ion with konjac glucomannan (Ka = 41×104 M−1) and carob galactomannan (Ka = 30×104 M−1). The polysaccharide binding was further investigated by fluorescence spectrophotometric studies. On binding with carob galactomannan and konjac glucomannan the conformation of CtCBM35 changed significantly with regular 21 nm peak shifts towards lower quantum yield. The degree of association (Ka) with konjac glucomannan and carob galactomannan, 14.3×104 M−1 and 11.4×104 M−1, respectively, corroborated the findings from affinity electrophoresis. The association of CtCBM35with konjac glucomannan led to higher free energy of binding (ΔG) −25 kJ mole−1 as compared to carob galactomannan (ΔG) −22 kJ mole−1. On binding CtCBM35 with konjac glucomannan and carob galactomannan the hydrodynamic radius (RH) as analysed by dynamic light scattering (DLS) study, increased to 8 nm and 6 nm, respectively, from 4.25 nm in absence of ligand. The presence of 10 mM Ca2+ ions imparted stiffer orientation of CtCBM35 particles with increased RH of 4.52 nm. Due to such stiffer orientation CtCBM35 became more thermostable and its melting temperature was shifted to 70°C from initial 50°C.This study investigated the role of CBM35 from  CBMs are found in the protein both as appended with hydrolytic enzymes such as cellulase, mannanase, xylanase, as non hydrolytic functions, as scaffoldings and as peptide with non catalytic functions with variety of hydrolytic enzyme complex (cellulosome) Hydrolytic enzymes and their enhanced polysaccharide specificity often improve by appended non catalytic carbohydrate binding module either at their N or C terminal ends. A major portion of plant carbohydrate reservoir is composed of hemicelluloses such as the polymer and oligomer of xylose, mannose, arabinose etc. apart from celluloses. Polysaccharide recognition, binding and enhanced catalysis of hydrolytic enzymes truly facilitates by non catalytic modular carbohydrate binding modules. Carbohydrate binding modules (CBMs) are classified into 67 distinguished families based on sequence similarity (Clostridium thermocellum ATCC 27405 (CtCBM35) upon polysaccharide and cation binding. CtCBM35 usually display the specificity towards manno-configured polysaccharides. Mannose has single stereochemical difference from glucose (at 2′-OH site) in the manno-configured polysaccharides which made it less rigid structural conformation. CtBM35 has varying degree of affinity for lesser and higher galactose and glucose substituted polysaccharides. The affinity electrophoresis, fluorescence measurements and dynamic light scattering were employed to analyze both qualitative and quantitative binding of CtBM35 with manno-configured polysaccharides. The structural variations of CtBM35 due to Ca2+ ion binding and the alterations of domain conformations were described in this work.Our work is focused on the functional and conformational properties of CBM35 from CtCBM35 was amplified from the genomic DNA of Clostridium thermocellum ATCC 27405 using a forward primer containing NheI restriction site: GCTAGCGCATATTCCCTTCCTGCAC and a reverse primer with XhoI restriction site: CTCGAGTTAAAGTTCATCCAAGCTGCAC. The PCR conditions were followed as Mg2+ ions (2.5 mM), dNTPs (2 mM), primers (1.5 µM), 1.0 µl of Taq DNA polymerase (1 µl of 1Unit µl−1) and 1 µl of genomic DNA of C. thermocellum. The PCR amplification cycles used were: denaturation at 94°C for 4 min followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 60 s and extension at 72°C for 2 min and final extension at 72°C for 10 min. The amplified products were run on 0.8% agarose gel and further purified by gel extraction (Qiagen kit.) The amplified products were then digested with NheI-XhoI restriction enzymes and cloned into NheI/XhoI restricted pET-28a (+) expression vectors containing kanamycin as resistant marker respectively which resulted in cloned plasmids (pCBM35). Thereafter, E. coli DH5α cells were transformed with above cloned plasmids. The transformed cells were grown on LB −1) at 37°C for growth of recombinant clones. Positive clones were selected by restriction digestion analysis of the resulting cloned plasmids.The open reading frame (ORF) of E. coli BL-21(DE3) (Novagen) cells were used for expressing CtCBM35 as described elsewhere E. coli BL-21 (DE3) cells were transformed with pCBM35. The cells were grown similarly as described in Section 2.1, with incubation at 37°C and shaking at 180 rpm till mid-exponential phase (A600nm≈0.6). The cells were induced with 1.0 mM isopropyl-1-thio-β-D-galactopyranoside (IPTG) for hyper-expression of recombinant protein at optimized expression conditions of 24°C at 180 rpm for 16 h. The cells were harvested at 9,000 g and the resulting pellet was resuspended in Sodium phosphate buffer . Then the cells were sonicated  on ice for 16 min (9 s on/off pulse) with further centrifugation  to get the crude cell free protein in the supernatant. Hybrid protein containing CtCBM35 appended by His6 tag were purified in a single step by immobilized metal ion affinity chromatography (IMAC) using Ni-Sepharose columns  as recommended by the manufacturer. The purity and molecular mass of recombinant proteins were verified by SDS-PAGE CtCBM35 (18 µg) was assayed with soluble polysaccharides such as carob galactomannan, konjac glucomannan, carboxymethyl cellulose, rye arabinoxylan, birchwood xylan, oatspelt xylan and glucouronoxylan. The polysaccharide samples were prepared subsequently by diluting in filtered water from  polysaccharide stock. Native polyacrylamide gels (7.5%) were prepared containing varying polysaccharide concentrations ranging from 0.0 to 1.5 . Bovine serum albumin (BSA) sample (1.0 mg ml−1) was run in native gel for non-specific binding interaction. Binding study of CtCBM35 with carob galactomannan and konjac glucomannan was carried out in absence and presence of Ca2+ ions. 10 mM Ca2+ ion was incorporated in 7.5% native polyacrylamide gel before CtCBM35 electrophoresis. In absence of Ca2+ ions 7.5% native gels were prepared as control and the relative mobilities of CtCBM35 were calculated.The polysaccharide binding capability of the non-catalytic modules was determined by visualizing the adsorption to soluble polysaccharides using gel retardation in native polyacrylamide gel electrophoresis containing the polysaccharides as described earlier by Takeo, (1990) CtCBM35 binding was carried out with insoluble mannan, avicel and wheat arabinoxylan. Thirty microgram of CtCBM35 in 50 mM sodium phosphate buffer, pH 7.0, was mixed with 1 mg of mannan in a final reaction volume of 200 µL. The reaction mixture was incubated for 2 h at 4°C with gentle shaking. After that the insoluble ligand was precipitated by centrifugation at 13000 g for 5 min. The supernatant, containing the unbound proteins, was removed and the pellet was washed three times with 200 µl of 50 mM sodium phosphate buffer pH 7.0. The bound protein from the washed pellet was eluted by boiling the polysaccharides in 200 µl of 10% (w v−1) SDS containing 10% (v v−1) β-mercaptoethanol for 10 min. The pellets of bound protein and the supernatant of unbound protein were analysed by 12% SDS-PAGE. A Bovine Serum Albumin (1 mg ml−1) control was set in parallel to check for any non specific binding. All the gels containing protein and no polysaccharide and the electrophoresis were performed in parallel to ensure also that no precipitation of protein occurred. For quantitative analysis the free or unbound protein concentration in un-bound fraction obtained after centrifugation was determined by Bradford method aK is the association constant then at equilibrium adsorption were calculated as described earlier by Gilkes et al (1992) The quantitative and qualitative assessment of CtCBM35 was incubated with polysaccharides viz., carob galactomannan and konjac glucomannan of varying concentrations. Polysaccharide concentrations  from 0.5% (w v−1) stock solution in 100 µl reaction mixture were prepared in 50 mM sodium phosphate buffer, pH 7.0. The samples were incubated at 4°C for 2 h. The fluorescence measurements were carried out using a fluorimeter . Emission and excitation slits were kept at 3.00 and 1.00, respectively, with 0.5 s integration time. Three scans were taken per sample along with a control to reduce the noise created by buffer and polysaccharide. All the samples were excited at λmax = 295 nm with observance of emission spectra between λmax = 320–400 nm. The emission spectra of all the solutions were corrected against buffer and polysaccharide solution without CtCBM35 before setting the interaction study. Relative fluorescence intensities  were plotted against polysaccharide concentration. The association constants −1) of CtCBM35 complex with carob galactomannan and konjac glucomannan were derived using modified Stern Volmer equation On binding to polysaccharides, carbohydrate binding modules undergo conformational changes and behave differently than in its unbound native form CtCBM35 was studied by dynamic light scattering (DLS). In a dynamic environment, the particles of ligand and protein molecules diffuse randomly. DLS essentially measures fluctuation in scattered light intensity due to diffusion of particles, the diffusion coefficient of the particles can be determined. The diffusion coefficient D is then related to the radius R of the particles by means of the Stokes-Einstein Equation where, k = Boltzmann-constant, T = temperature and η = viscosity.Polysaccharide binding greatly influences the protein conformational changes. These changes may lead to more dispersion in the dynamic environment leading to higher hydrodynamic area. The binding of polysaccharide with H) of CtCBM35 in presence of 0.1% (w/v) polysaccharides such as carob galactomannan and konjac glucomannan were measured by Zetasizer Nano ZS  spectrophotometer. Refractive index and viscosity of 50 mM sodium phosphate buffer were adjusted to 1.3206 and 8.945×10−3 g (cm s)−1, respectively by using SEDNTERP tool package (http://www.jphilo.mailway.com). The hydrodynamic radius of CtCBM35 was also studied in presence and absence of 10 mM Ca2+ ions. CtCBM35 was incubated at 25°C for 2 h and the extra unbound Ca2+ was removed by dialysis against water. The hydrodynamic radius of bound CtCBM35 with Ca2+ conjugate was measured and compared against Ca2+ free CtCBM35. The instrument was set to measure the absorbance at a fixed angle (θ = 90°). All the measurements were derived by deconvolution of intensity and sample autocorrelation function. Deconvolution of measured intensity was obtained using non negative least square analysis (NNLS) The hydrodynamic diameters  by measuring in UV-absorption spectrophotometer  at 280 nm following the method of Dvortsov et al. (2009) CtCBM35 was incubated with 10 mM CaCl2 at 25°C for 2 h and dialyzed against water to remove additional Ca2+ ion.Binding assays with soluble polysaccharides showed that an increase in enzyme affinity could be attributed to the presence of family 35 carbohydrate binding module. To investigate whether the presence of Ca2+ ion in altering protein conformations a model of CtCBM35 from Clostridium thermocellum was generated in presence of Ca2+ ion based on the crystal structure of closest homolog of CBM35 from Amycolaptosis orientalis (PDB ID: 2VZPA) by Modeller9v8 program. The homolog was identified using Blast PDB (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi). The structure was energy minimized with GROMACS4.0.7 package (http://www.gromacs.org/) using steepest descent algorithm with GROMOS96 43a1 force field and simple point charge (SPC) water model CtCBM35 model was carried out with the periodic boundary conditions applied in three dimensions to analyse the stability of the protein model. The net charge of system was neutralized by the addition of eleven sodium ions by replacing water molecules that are at least 3.50 Å from the protein surface To investigate the role of CaCtCBM35 was amplified by polymerase chain reaction and successfully ligated to pET28a (+) expression vector and transformed E. coli DH5α cells. The colonies appeared in the LB plates supplemented with 50 µg ml−1 kanamycin were screened for positive clones by digestion with Nhe I and Xho I restriction enzymes. Few positive clones with an insert of 420 bp and a vector fragment of 5.4 kb were obtained in 1% agarose gel electrophoresis. The transformed E. coli BL21 (DE3) cells by recombinant plasmids of positive clones after IPTG induction were screened for expression. The expression of CtCBM35 (15 kDa protein) band was observed on 12% SDS-PAGE by comparing with long range (10–200 kDa) prestained molecular weight marker (Fermentas). The recombinant CtCBM35 was purified by Ni2+-NTA  and the elution was accomplished with 300 mM Imidazole. A purified homogenous single band 15 kDa of CtCBM35 appeared on 12% SDS-PAGE  2+ ion the affinity of CtCBM35 for carob galactomannan was increased to approximately 2.5 folds and the association constant  form and the total purified protein  software using non-linear regression analysis based on one binding site equation. The estimated values of relative equilibrium constant Kr and concentration of binding sites [No] were 0.49±0.2 l g−1 and 0.04±0.002 µmole g−1, respectively  of CtCBM35 with carob galactomannan and konjac glucomannan were derived from Hill plot (4 M−1 and with konjac glucomannan 14.3×104 M−1 (CtCBM35 confirmed the results of affinity electrophoresis. The number of binding site concentrations (n) were derived from Stern Volmer equation and with carob galactomannan n = 0.79 and whereas with konjac glucomannan n = 0.80 , T = Temperature in Kelvin, −1). The free energy binding of CtCBM35 with carob galactomannan was −22.0 kJ mole−1 and with konjac glucomannan −25.0 kJ mole−1  of CtCBM35 was found to be 4.25 nm which was in the acceptable range as this value is higher than theoretical RH, 2 nm for a 15 kDa protein. The RH augmentation of CtCBM35 was seen in presence of 0.1% (w v−1) carob galactomannan from 4.25 nm to 6 nm (−1) exhibited much broader RH of 8 nm  the hydrodynamic radius  value of 1.08 Å as compared to unbound state of 1.8 Å. This reduced RMSD value due to the stiffer binding with Ca2+ ion prevented less free movement at their bound state Melting of  at 50°C . In the mpletely . Thus, CCa2+ ion  displayeCa2+ ion , it was CtCBM35) from Clostridium thermocellum preferred binding with manno-configured polysaccharides. CtCBM35 discriminated during carbohydrate selection showing its affinity only with manno-configured ligands among the manno and xylo-configured polysaccharides. Instead of β-1,4-mannose chain affinity the variance of ligand selection within the manno-configured polysaccharides was often observed. CtCBM35 has higher affinity with konjac glucomannan than carob galactomannan. Rationale behind this selective affinity was due to the galactose unit in carob galactomannan likely interferes with the CtCBM35 binding. Carob galactomannan is composed of a 1,4-β-linked D-mannan backbone to which single D-galactosyl units are attached to C-6 of D-mannosyl residues. Whereas glucomannan is a linear polysaccharide comprising 1,4-β-linked D-glucosyl and D-mannosyl residues. Therefore, CtCBM35 was more glucomannan specific than galactomannan. This finding gained a new insight into the CBM35 family when compared with other mannan specific CBM35 from Clostridium thermocellum and Cellvibrio japonicasCtCBM35 with insoluble polysaccharides was quite insignificant. Low affinity against insoluble mannan was due to the inability of trans (as a discrete fold) form of CtCBM35 to disrupt the inter chain interactions in mannan. This in contrast to some CBMs has the ability to disintegrate the surface of crystalline insoluble polysaccharides that potentiates higher binding to some soluble fractions of insoluble polysaccharides CtCBM35 for konjac glucomannan masks the available tryptophan for fluorescence emission as compared to carob galactomannan. Thus, gradual fall in peak intensities were coupled with peak shifts (∼21 nm) due to altered conformation of native CtCBM35. In dynamic light scattering, the larger particle size of CtCBM35 is due to the polysaccharide binding. The cationic interaction of aromatic residues with carob galactomannan and konjac glucomannan insists CtCBM35 domain alteration to more compact form reducing the random diffusion of the particles between polysaccharide and amino acid residues. Due to simpler structure of konjac glucomannan, the interaction with aromatic residues in the binding pocket of CtCBM35 uphold strong binding as compared to carob galactomannan with substituted galactose side chain. The structure of CtCBM35 gains its particle size in the conjunction with polysaccharide and the Brownian motion of light measured between the larger particles and ends up in a larger hydrodynamic area. Melting of a protein module is a cooperative process 2+ ion makes polar contact with the amino acid residues away from the polysaccharide binding sites. This imparts stiffer orientation of the residues dragging more towards the ion and holding into a proper position of the residues even at varying physical parameters such as higher temperature, pH change etc with reduced RMSD value. Ca2+ ion attacks on the negatively charged amino acid side chains and do not allow their mobility at harsh environmental conditions due to which the protein folding remain intact Clostridium thermocellum may be useful to enhance the activity by appended to a mannanase for higher degree of hydrolysis of complex manno-configured polysaccharides into simple sugars. Moreover, these findings might lead to comprehend both glucomannan and galactomanan specific CBM35 from Clostridium thermocellum which may play a potential role in biofuel production in conjunction with mannanase from mannan rich polysaccharides in future.The cloned family 35 Carbohydrate binding module (


Three authors were indicated as having the incorrect affiliations. These authors and their correct affiliations are as follows:Marta Rusmini: Laboratory of Molecular Genetics, IRCCS, Giannina Gaslini Istitute, Genoa, Italy, Unit of Clinical and Experimental Immunology, Humanitas Clinical and Research Center, Rozzano, Milan, ItalyIvana Matera: Laboratory of Molecular Genetics, IRCCS, Giannina Gaslini Istitute, Genoa, Italy, Unit of Clinical and Experimental Immunology, Humanitas Clinical and Research Center, Rozzano, Milan, ItalyRoberto Ravazzolo: Laboratory of Molecular Genetics, IRCCS, Giannina Gaslini Istitute, Genoa, Italy, DINOGMI Department, University of Genova, Genova, Italy


While there has been much debate about the coverage and quality of Wikipedia . In contrast, contributions to Wikipedia can be anonymous and do not count for much in the current system of academic advancement. We hope to help to resolve this disparity in PLoS Computational Biology publishes a version that is static, includes author attributions, and is indexed in PubMed. In addition, we intend to make the reviews and reviewer identities of Topic Pages available to our readership. Our hope is that the Wikipedia pages subsequently become living documents that will be updated and enhanced by the Wikipedia community, assuming they are in keeping with Wikipedia's guidelines and policies, either by individuals, or, perhaps as is already happening in medicine and molecular and cell biology, by something more organized, or with a more formal review structure. We also hope this will lead to improved scholarship in a changing medium of learning, in this case made possible by the Creative Commons Attribution License that we use.This month, we have published our first Topic Page on “Circular Permutations in Proteins” by Spencer Bliven and Andreas Prlić ploscompbiol@plos.org. We are looking for topics in computational biology that are of interest to our readership, the broader scientific community, and the public at large, and that are not yet covered, or only poorly so , in Wikipedia: http://en.wikipedia.org/wiki/Wikipedia:WikiProject_Computational_Biology. Our guidelines for Topic Pages are available here: http://www.ploscompbiol.org/attachments/topicpages.pdf. Wikipedia is the world's most widely used knowledge source, and computational biology should be appropriately represented—please help. New uses of Wikipedia are being explored, as a recent example illustrates Our Editorial Board has been enthusiastic in its support of this initiative and a number of Topic Pages are under development. We hope you will contribute too; please send ideas for Topic Pages to


Rapamycin/Sirolimus™ leads to the regression of transplant-associated Kaposi sarcoma (KS). It also leads to disease stabilization in HIV-associated KS. Case reports and a wealth of preclinical studies support rapamycin’s efficacy also in AIDS associated lymphoma, such as primary effusion lymphoma (PEL). Rapamycin inhibits the mammalian target of rapamycin (mTOR) and papamycin derivatives are approved for the treatment of mantle cell lymphoma and other cancers. It is not universally effective against all solid tumors. Even within this group of clinically responsive cancers, there are exceptions of cases or cell models in which this drug or its derivatives  fail.We hypothesized that genetic alterations of the tumor will determine the response to rapalogs. We used a novel KS TMA (from the AIDS cancer specimen resource (ACSR) to evaluate the PI3K/Akt/mTOR pathway in KS and identified a unique pattern of protein activation that is associated with sustained expression and phosphorylation of the PTEN tumor suppressor protein. We used Affymetrix array-based comparative genome hybridization (CGH) and targeted sequencing to query genomic loci of members of the mTOR pathway, which confirmed the absence of genetic alterations, which in other cancers have been associated with mTOR pathway activation. We conclude that in KS, PEL and perhaps other virus-associated cancer the mTOR pathway is activated post-translationally, which is not as permanent and does not have the same impact as for instance a deletion in PTEN. This may explain the unique sensitivity of these tumor types to rapalogs.We established a novel animal model of KS (L1T2 cells), which develops tumors with short latency and in which the KS associated herpesvirus (KSHV) is maintained in each tumor cell. We evaluated multiple rapalogs , Tacrolimus/FK506 and Doxil™ in this model. We found sustained, but reversible tumor suppression by the rapalogs, which was associated (a) with inhibition of mTOR activity and (b) with reduced neo-angiogensis. In this model rapalogs outperformed Doxil™, presumably because of a combined effect on endothelial lineage KS tumor cells and endothelia lineage tumor vasculature. This represents the first animal test of rapalogs in a human KS tumor model, it validates novel biomarkers and provides a strong rational for further clinical development and testing of rapalogs in AIDS-associated Kaposi sarcoma.As rapamycin has recently become affordable and is available in developing countries, where the majority of KS-associated mortality occurs, it is one of the few new anti-cancer drugs with a cost/benefit profile that is affordable for global cancer treatment.


The aim of this paper is to investigate the topological properties of a class of fullerene molecules containing 60 + 12n carbon atoms.A molecular graph M is a simple graph in which atoms and chemical bonds are the vertices and edges of M, respectively. The molecular graph M is called a fullerene graph, if M is the molecular graph of a fullerene molecule. It is well-known that such molecules exist for even integers  G are denoted by V(G) and E(G), respectively. Molecular graphs are graphs with vertices representing the atoms and edges representing the bonds. A bi-connected graph is a connected graph in which, by removing any vertex, the graph will remain connected. A graph in which all vertices have degree three is called a cubic graph. A fullerene graph is a cubic bi-connected planar graph whose faces are pentagons and hexagons. From Euler’s theorem, one can easily see that such graphs have exactly 12 pentagonal and (n/2 − 10) hexagonal faces, where 20 ≤ n. It is not so difficult to prove that there is no fullerene with exactly 22 carbon atoms. After the discovery of buckminsterfullerene C60 by Kroto and Smalley in 1985  and Sze(G) = ∑e=uvmu(e)mv(e), respectively. Interested readers can consult [Suppose  consult –6 and re consult . Some ma consult –9. One o consult  proposedm0(e) denotes the number of vertices equidistant from u and v. In [where nd v. In , some maFn of fullerenes with exactly 60 + 12n carbon atoms , mv(e) and m0(e) = 90 + 18n – mu(e) – mv(e) for these exceptional cases are recorded. We notice that there are two cases, that is, when n is odd or even. If n is even then we have 12 different types of edges, n is odd then there are 13 different types of edges (In of edges .n ≤ 7 are recorded.In mu(e), mv(e) and m0(e) are calculated. By these tables and a case-by-case investigation on the molecular graph of Fn led to the following observation: The PI, edge Szeged and edge revised Szeged indices of C60+12n fullerenes can be computed by the following formulae:In Fn.It is possible to find a proof for this observation by a tedious calculation on the molecular graph of In this paper a computational method for computing PI, edge Szeged and edge revised Szeged indices of fullerene graphs is presented. In –19, the


Aim: To compare exenatide and sitagliptin glucose and glucoregulatory measures in subjects with type 2 diabetes.Methods: An 8-week, double-blind, randomized, crossover, single-centre study. Eighty-six subjects  received either exenatide 10 µg (subcutaneous) twice daily or sitagliptin 100 mg  daily for 4 weeks and crossed to the other therapy for an additional 4 weeks. Main outcome was time-averaged glucose during the 24-h inpatient visits.Results: Both treatments decreased average 24-h glucose, but exenatide had a greater effect . Both treatments decreased 2-h postprandial glucose (PPG), area under the curve of glucose above 7.8 mmol/l (140 mg/dl) and 11 mmol/l (200 mg/dl) and increased the time spent with glucose between 3.9 and 7.8 mmol/l (70 and 140 mg/dl) during 24 h, but exenatide had a significantly greater effect (p < 0.05). Both treatments decreased postprandial serum glucagon, with exenatide having a greater effect (p < 0.005). Both treatments decreased fasting blood glucose to a similar degree (p = 0.766). Sitagliptin increased, while exenatide decreased, postprandial intact glucagon-like peptide-1. Both drugs improved homeostasis model assessment of β-cell function (HOMA-B), with exenatide having a significantly greater effect (p = 0.005). Both exenatide and sitagliptin decreased 24-h caloric intake, with exenatide having a greater effect (p < 0.001). There was no episode of major hypoglycaemia. Adverse events were mild to moderate and mostly gastrointestinal in nature with exenatide. No study withdrawals were due to an adverse event.Conclusion: Compared to sitagliptin, exenatide showed significantly lower average 24-h glucose, 2-h PPG, glucagon, caloric intake and improved HOMA-B. Advances in the treatment of hyperglycaemia include glucagon-like peptide-1 (GLP-1) receptor agonists, such as exenatide and liraglutide, and dipeptidyl peptidase-4 (DPP-4) inhibitors, such as sitagliptin and saxagliptin. Exenatide binds and activates the GLP-1 receptor, while sitagliptin increases endogenous GLP-1 concentrations by inhibiting DPP-4 degradation of circulating GLP-1 . Exenatiβ-cell function, α-cell function and 24-h caloric intake.A recent clinical study by DeFronzo et al.  comparin2, stable body weight for at least 3 months prior to screening visit, haemoglobin A1c (HbA1c) ≥7% and ≤11%, fasting glucose <280 mg/dl and subjects must have been on a stable dose of metformin or thiazolidinedione for at least 60 or 120 days, respectively. Exclusion criteria were females of childbearing potential; treatment with insulin, exenatide (or any GLP-1 receptor agonist), sulphonylureas, drugs affecting gastrointestinal motility, weight loss drugs, corticosteroids, α-glucosidase inhibitors; history of organ transplant; history of liver or renal disease; fasting triglycerides >400 mg/dl; blood pressure >165/90 mm Hg. All subjects provided written informed consent prior to undergoing any study procedure or receiving any study treatment. The study was performed in accordance with the principles of the Declaration of Helsinki  in addition to their prestudy medication(s). At the end of the first 4 weeks, subjects were switched to the alternative treatment. Subjects received their injections and a capsule before the breakfast test meal during the postrandomization 24-h inpatient visits.β-cell function (HOMA-B), calculated using the updated HOMA model [The primary efficacy measure was the time-averaged glucose during the 24-h inpatient visits . The 24-MA model . Safety During the 24-h inpatient visits, three similar caloric and macronutrient content meals were given, beginning with the evening meal. All meals were individualized for each subject based on gender and weight ,12. The α level. To account for multiplicity, p-values for postbreakfast serial measurements were reported only if <0.005 (0.05/10) instead of <0.05, as there were 10 or fewer values for each measurement. Baseline characteristics were described by mean, standard deviation (SD) or n (%) and postbaseline efficacy values were described by least-squares (LS) mean and standard error (SE).Statistical analyses were performed using SAS version 9.1. All analyses were based on two-sided tests at the 0.05 Analysis of efficacy variables and hypoglycaemia included all subjects who received at least one dose of study medication and completed the first treatment period. Baseline for both treatment periods was defined as the randomization visit. Adverse events were analyzed on all randomized subjects. Analyses for continuous variables used Grizzle's model , includiEighty-six subjects were randomized but three subjects discontinued before receiving the study drug. Of those 83 subjects, 82 were receiving metformin and 1 was receiving thiazolidinedione . The treAt baseline, the 24-h glucose profiles were similar between treatment groups . After 4Both exenatide and sitagliptin significantly (p < 0.001) reduced fasting glucose and mean 2-h PPG from baseline . A signiAt endpoint, both treatments significantly (p < 0.001) decreased the average exposure to hyperglycaemia as measured by AUC for glucose values above 7.8 mmol/l and above 11 mmol/l, with a significant (p ≤ 0.01) treatment difference favouring exenatide , Table 2At endpoint, the PPG concentrations were significantly lower (p < 0.005) with exenatide compared to sitagliptin at most time points following the test meal . PostpraBoth exenatide and sitagliptin significantly (p < 0.001) increased HOMA-B, with exenatide having a greater effect  was similar between treatment sequences . At endpoint, the caloric intake was significantly lower (p < 0.001) over 24 h in both groups, with exenatide having a greater effect . At endpoint, the calorie intake was not significantly different between groups at the morning  or evening meal . Calories consumed were significantly less (p < 0.001) with exenatide compared to sitagliptin at the midday meal .Both treatments led to decreases in weight .The most common adverse events were nausea , vomiting , headache  and diarrhoea . Two subjects experienced serious adverse events (SAEs) while on exenatide . Neither SAE was assessed to be related to the study drug. In the opinion of the investigator, neither event was related to exenatide treatment. No subject discontinued the study due to an adverse event. Three subjects on exenatide and one subject on sitagliptin experienced minor hypoglycaemia. There was no episode of major hypoglycaemia.β-cell function with exenatide treatment. The predominant glucodynamic difference between exenatide and sitagliptin in the current study was the observation that exenatide substantially decreased PPG concentration, with no significant difference on fasting glucose between the two drugs. This observation can be explained at least in part by multiple mechanisms, the most important of which may be glucagon suppression [Several glucoregulatory mechanisms showed key differences between exenatide and sitagliptin in lowering blood glucose levels. Our study showed significant decreases in the average daily glucose, glucagon concentrations, caloric intake and improved pression . The curpression . Shah etpression . Cerverapression . Thus, dpression . Using apression . In the pression . The difβ-cell function in type 2 diabetes [β-cell function than sitagliptin, as measured by HOMA-B . DecreaA recent open-label study by Arnolds et al. showed no difference in PPG excursions over 6 h between exenatide and sitagliptin when either drug was added to treatment for 48 subjects with type 2 diabetes receiving insulin glargine . HoweverThe current study confirms previous observations that sitagliptin increases endogenous intact GLP-1 in subjects with type 2 diabetes . In contA limitation of this study is the lack of a washout period between the two treatments. However, the short half-lives of sitagliptin and exenatide  make a carryover effect unlikely . FurtherIn conclusion, the 24-h glucose profiles obtained during treatment with either exenatide or sitagliptin revealed that the predominant difference between the drugs lies in the ability of exenatide to substantially decrease PPG concentration. The effect of exenatide to improve 24-h glucose profile relative to sitagliptin may be explained, at least in part, by a greater effect of exenatide on suppressing PPG concentration, decreased appetite, decreased caloric intake, and improved insulin response to the ambient glucose. The overall effects of exenatide in the current study may be because of a greater molar concentration of exenatide compared to the molar concentration of intact GLP-1 during sitagliptin therapy .


Kallmann syndrome (KS), comprised of congenital hypogonadotropic hypogonadism (HH) and anosmia, is a clinically and genetically heterogeneous disorder. Its exact incidence is currently unknown, and a mutation in one of the identified KS genes has only been found in ~30% of the patients.Herein, we investigated epidemiological, clinical, and genetic features of KS in Finland.p = 0.02). The reproductive phenotype of 30 probands  ranged from severe HH to partial puberty. Comprehensive mutation analysis of all 7 known KS genes  in these 30 well-phenotyped probands revealed mutations in KAL1 (3 men) and FGFR1 , but not in other genes.The minimal incidence estimate of KS in Finland was 1:48 000, with clear difference between males (1:30 000) and females (1:125 000) , possibly implying that, in addition to CHD7, there may be other genes associated with phenotypes ranging from KS to CHARGE.Our results suggest that Finnish KS men harbor mutations in gene(s) yet-to-be discovered with sex-dependent penetrance of the disease phenotype. In addition, some KS patients without  Kallmann syndrome KS; MIM# 147950), a combination of congenital hypogonadotropic hypogonadism  and decreased/absent sense of smell, results from disturbed intrauterine migration of gonadotropin-releasing hormone (GnRH) neurons from the olfactory placode to the hypothalamus 47950, a . PatientKAL1  were PCR-amplified and screened by direct sequencing. In FGFR1, both exons 8A and 8B, generating isoforms FGFR1-IIIb and FGFR1-IIIc by alternative splicing ); the brother also had cleft lip and palate, and limb malformations manifested as a fusion of 3rd and 4th metatarsal bones, and missing of the 2nd and 3rd toe in both feet  , had a sKAL1 (Table KAL1 mutation.Three probands had mutations in CHD7 (c.7988C > T [p.A2663V), not present in 100 controls. This variant was predicted to be benign by PolyPhen [http://genetics.bwh.harvard.edu/pph/), and it is therefore unlikely that this is a disease-causing mutation. CHD7 was also analyzed by MLPA because intragenic CHD7 deletions have been found in up to 22% of CHARGE patients [One proband carried a novel missense change in PolyPhen   were found in probands carrying an FGFR1 mutation . However, these were also present in the controls .In addition, novel missense changes in FGFR1 missense mutations G48S, R209H, and E670A, we compared total and cell-surface expression, receptor maturation, and signaling activities of WT and mutant receptors.To examine the functional consequences of the f) treatment, which removes only high-mannose N-linked sugars typical for immature forms of the receptor, resulted in similar maturation patterns for G48S, R209H, and E670A, as compared to WT, as only the minor 120 kDa bands changed mobility  FGFR1 at approximately 140 kDa and 120 kDa Figure . RemovalCell-surface expression of the WT receptor and the mutants G48S, R209H, and E670A were examined in COS-1 cells. Consistent with the results of deglycosylation experiments, the G48S, R209H, and E670A mutants had similar cell-surface expression levels as WT Figure .The signaling activities of the FGFR1 mutants G48S, R209H and E670A were assayed in L6 myoblasts, a cell line largely devoid of endogenous FGFRs and FGFs . Cells wCHD7.All 18 probands without an identified molecular genetic cause underlying KS were men. A subset of 7 probands had a history of micropenis, and 4 of them also displayed cryptorchidism, consistent with deficient hypothalamic-pituitary-gonadal (HPG) axis activation in fetal life and/or during infancy. Pubertal development was absent in 15 probands, whereas 3 men had displayed partial puberty. In addition, three probands had CHARGE syndrome-associated features. One of them had hypoplastic lateral and superior left semicircular canals, as well as absent right posterior canal  was considerably lower than reported among male military conscripts in France (1 in 10 000) , and higFGFR1 mutations are not the sole cause of KS in women worldwide [FGFR1 mutation, a proportion significantly higher than in men. A similar sex-specific difference between FGFR1 mutation carriers was also observed among a large series of normosmic HH patients [In addition to sex-specific difference in the incidence of KS, the molecular genetic diagnoses also differed between sexes. Although orldwide ,22,23,37patients . From thpatients .FGFR1 mutations are located in different domains of the receptor . However, two probands with an FGFR1 defect did also harbor novel variants in NELF (G94S and T505M), but given that the incidence of KS in Finland was 1 in 48 000 and these variants were also present in 6% and 1% of the controls, it is apparent that these variants are not causing congenital HH. Of note, lack of mutations in PROK2 and PROKR2, now known to cause autosomal recessive KS [In rare occasions, a congenital HH patient may carry mutation(s) in more than one HH gene; Falardeau et al.  showed thenotype ,12, but ssive KS ,17,37,46ssive KS .WDR11 has been analyzed in a series of KS patients after Kim et al. identified this gene by positional cloning of a translocation breakpoint in a KS patient, and discovered heterozygous missense variants in this gene in 6/201 (3%) of patients with KS or normosmic HH [WDR11 are rarely involved in congenital HH, at least in the Finnish population.To the best of our knowledge, this is the first study where osmic HH . HoweverIn contrast to women, the majority of male probands remained without identified mutations, implying the existence of still undescribed gene(s) underlying KS in Finnish men. Indeed, sex-dependent penetrance of the mutations in KS genes yet-to-be discovered could contribute to the higher incidence of KS in men worldwide.KAL1 and FGFR1, and all women had loss-of-function mutations in FGFR1. Some KS patients without CHD7 mutations display CHARGE syndrome-associated phenotypic features, implying that, in addition to CHD7, there may be other genes associated with both syndromes.KS is a rare condition with a nationwide minimal incidence estimate of 1:48000 in Finland. In Finnish men, mutations were detected in only The authors declare that they have no competing interests.EML recruited and phenotyped the patients, performed the statistical analyses, and drafted the manuscript. KV performed most of the molecular genetic analyses, performed the functional analyses, and drafted the manuscript. JT participated in the study design and the molecular genetic analyses, and drafted the manuscript. EE and MT participated in the molecular genetic part of the study. LV planned, provided, and interpreted the MRI analyses. TR carried out the study design, participated in recruiting and phenotyping the patients, and drafted the manuscript. All authors read and approved the final manuscript.


Comparison of the mean σo values with the stdev values results in a specific triangular distribution of data points for all spatial domains. Analysis of the mean σo and stdev with respect to in-situ soil moisture measurements demonstrates that this triangular shaped distribution can be explained by soil moisture dynamics during monsoon and winter periods. This shows that the relationship between the spatial mean soil moisture and variability is not uniquely defined and may change throughout seasons. Downscaling of coarse resolution soil moisture products should, therefore, be ideally based on additional near real time data sources. In this context, the presented results could form a basis for the development of SAR-based soil moisture downscaling methodologies.This paper reports on the analysis of a 2.5 year-long time series of ASAR wide swath mode (WSM) observations for characterizing the soil moisture dynamics. The employed ASAR WSM data set consists of 152 VV-polarized scenes acquired in the period between April 2005 and September 2007 over the Naqu river basin located on the Tibetan Plateau. For four different spatial domains, with areas of 30×30 km Satelet al. .For this investigation the top 4-cm soil moisture measured at Naqu station are used. These soil moisture measurements have been recorded using a 10-cm long impedance probe (type: ECHsat = 1.2 m d-1) positioned on top of an impermeable rock formation. Precipitation is, therefore, not able to drain deeply into the ground and will runoff towards the lower parts in the landscape. In these local depressions, wetland vegetation is the dominant land cover. The higher parts are covered by sparse vegetation, which consist of grasses and mosses. Soils in the wetlands have high organic matter content and can be classified as peat, while in the grasslands soils are sandy. In spite of the overall high altitude, on average 4,500 meters above sea level, the terrain is relatively smooth with rolling hills varying tens of meters in elevation. The top soils have a high saturated hydraulic conductivity  has been determined for all images within the ASAR WSM time series. The stdev is used, here, as a statistical variable representing the spatial σo variability. In σo is plotted against the σo stdev for the four domains. The distribution of data points for each of the four spatial domains in Over each of the four domains σo and σo stdev represent dry and frozen conditions. Because drought and freezing conditions have an impact on large areas, the spatial σo variability is small and is primarily influenced by speckle and spatial variations in surface roughness. Comparison of the minimum stdev's obtained from the different domains shows that the spatial σo variability increases with the size of the domain. This might be expected because over larger areas the variety of roughness conditions may be higher.The lowest mean σo value increases under conditions where liquid soil moisture is present. When thaw/freeze cycles and precipitation are homogeneously distributed throughout the study domains, the σo variability remains relatively low. The σo variability increases due to spatial differences in soil thermal and hydraulic properties, and precipitation inputs.The mean σo and maximum stdev at specific σo levels and its slope could be seen as measure for the surface heterogeneity of a specific domain. Steeper slopes indicate a larger surface heterogeneity. For the 5×5 km2 domain, the slope is steepest and its surface heterogeneity may be considered to be the largest of the four domains. This is, however, also influenced by the distribution of wetlands and grasslands in the selected areas, because differences in land surface conditions between wetlands and grasslands persist, specifically during the monsoon. The similarity between the slopes in plots of the 30×30 km2, and 1×1 km2 wetland and grassland domains is striking and suggests a similarity in the surface heterogeneity between these areas. Additionally, it should be noticed that for the grassland domain the number of data points with a high stdev is small. Soils in the grasslands are sandy and have a high hydraulic conductivity. Over short time periods, soil moisture is transported from the top to deeper soil layers. Spatial variations due to dry-down cycle diminish, therefore, quickly and are difficult to capture by observations acquired at a 6-day temporal resolution.For each of the four domains, a well-defined and linear relationship exists between the mean σo values. Simultaneous to these higher σo observations, its spatial variability decreases for three reasons. Firstly, high σo values are only obtained when the entire domain is at or near saturation. Secondly, vegetation attenuates the soil surface scattering contribution and reduces the σo sensitivity to soil moisture changes. Thirdly, the σo response is less sensitive to soil moisture changes under wet than under dry conditions [σo sensitivity to soil moisture changes due to either vegetation or soil wetness reduces the impact of spatial soil moisture variations on the σo variability.During monsoon periods, land surfaces are wetter and vegetation grows, which both lead to an increase in the average nditions . The decσo variability is higher under saturated than under dry conditions, which is caused by a combination of spatial variations in the porosity and vegetation. The increase in the spatial variability is stronger for larger domains (30×30 km2 and 5×5 km2) than the 1×1 km2 domains, which is expected because the variability in the porosity and vegetation tend to increase over larger distances.Further, the plots show that the σo and maximum stdev at specific σo levels under wet conditions is non-linear and is influenced by spatial variations in the soil hydraulic behavior and vegetation. Binding forces between the water molecules and soil particles determining the capillary force are, in general, smaller under wet than under dry conditions. A large amount of moisture is, therefore, transported relatively fast to deeper soil layers initiating the dry-down cycle. The time scale over which this process occurs depends strongly on the water retention capacity and hydraulic conductivity of the soil, which are both spatially variable. In addition, vegetation covering the soil surface reduces the σo sensitivity to soil moisture and destroys the spatial variability induced by the soil hydraulic behavior.The relationship between the mean 5.σo and the stdev is described. Within this discussion the magnitude of the mean σo is implicitly assumed to be representative for the local soil moisture conditions and the σo stdev is utilized as indication for the spatial soil moisture variability. A robust validation of these assumptions would require intensive soil moisture sampling across the spatial domains over a long time period. Unfortunately, such data set is not available for the selected study area. However, at Naqu station an almost continuous time series of soil moisture measurements has been collected during the period in which ASAR observations were collected. These soil moisture measurements are plotted against the mean σo and stdev for the four spatial domains, which is shown in oσ = a · soil moisture+ b have been computed and are presented in the plots. Statistics related to these regression functions are given in In the previous section, the impact of land surface processes on the relationship between the mean σo and stdev with the measured soil moisture the SAR observations have not been corrected for the effects of surface roughness and vegetation. The objective of this investigation is, however, not to present (or apply) a methodology to correct the σo observations for the surface roughness and vegetation effects, but to analyze the relationship between the spatial backscatter statistics and in-situ soil moisture measurements. For the description (and application) of methodologies that correct σo observations for the effects of surface roughness and vegetation the reader is referred to previous investigations [i.e. It should be acknowledged for the comparison of mean ons i.e. -21.σo is compared to soil moisture measured only at a single location and no correction for the effects of vegetation has been applied, still positive relationships are observed for all domains. Somewhat surprising is that for the 30×30 km2 and 5×5 km2 domains the coefficient of determination (R2) between the mean σo and soil moisture is higher than for the 1×1 km2 wetland and grassland domains. Because the size of the domains is smaller, the spatial soil moisture variability is smaller for the 1×1 km2 domains. Therefore, it would be expected that uncertainties due to imperfect representation spatial soil moisture variability are lower for the 1×1 km2 than for the 30×30 km2 and 5×5 km2 domains, which should result in better defined relationships between the mean σo and the measured soil moisture. Apparently, soil moisture dynamics measured at Naqu station is a better representation of the temporal soil moisture evolution observed over the larger 30×30 km2 and 5×5 km2 domains.Despite the mean 2 grassland domain low mean σo values are observed even when the measured soil moisture is near saturation. Furthermore, the overall σo response observed over the 1×1 km2 grassland domain to the measured soil moisture is lower than for the other domains, while for the 1×1 km2 wetland domain the σo sensitivity to soil moisture is higher. This suggests that measured soil moisture at Naqu station is systematically higher than actual soil moisture in 1×1 km2 grassland domain and systematically lower than the actual soil moisture 1×1 km2 wetland domain. These observations supports that the soil moisture evolution measured at Naqu station represents better the soil moisture dynamics of the 30×30 km2 and 5×5 km2 areas, which explains the higher R2 for those domains.As is shown in σo stdev to the measured soil moisture for the four domains results in similar triangular distributions of the data points as is observed in σo and stdev can be considered to be representative for the temporally varying spatial soil moisture distributions in the selected domains.The comparison of the σo stdev and measured soil moisture towards dry conditions is not as well defined as relationship between the mean σo and the stdev in σo stdev is compared in 3cm-3), the σo stdev varies between 1.19 and 2.07 (dB). Since wetlands are to be systematically wetter than Naqu station , it can be expected that the actual soil moisture condition in the wetlands are wetter for the data points with a high stdev.However, also some differences are observed in the distribution of data points between 6.σo and stdev of four spatial domains obtained from a time series of 152 ASAR WSM images acquired over the Naqu river basin have been analyzed. The selected spatial domains have areas of 30×30, 5×5 km2 and 1×1 km2 (two domains). The 30×30 km2 and 5×5 km2 domains are covered by a mixture of grasslands and wetlands, while for the 1×1 km2 domain a grassland and wetland have been selected. Comparison of the mean σo values with the stdev values results in very specific triangular data point distributions for all spatial domains. The decrease of σo stdev as the mean σo decreases is observed because dry and freezing conditions have an impact on large areas and are, typically, homogeneously distributed across spatial domains. During the monsoon, however, intensive rain showers may saturate large areas. A decrease in the σo stdev is, therefore, observed as the mean σo increases. This impact of the soil moisture dynamics during monsoon and winter periods on the σo signature and its spatial variability is consistently observed for all four selected spatial domains. These findings are confirmed through a comparison of the mean σo and stdev with in-situ soil moisture measurements at a single station.In this paper, the spatial mean A consequence of the reported results is that the relationship between mean soil moisture and the spatial variability is not uniquely defined over the Tibetan Plateau and varies during the monsoon and winter periods. In downscaling coarse resolution soil moisture products changes in the relationship between the mean soil moisture and spatial variability should be considered, and should ideally be based on additional near real time data sources. SAR observations could be utilized to provide soil moisture information within passive microwave and scatterometer footprints.


The hypocretins/orexins are comprised of two neuroexcitatory peptides that are synthesized exclusively within a circumscribed region of the lateral hypothalamus. These peptides project widely throughout the brain and interact with a variety of regions involved in the regulation of arousal-related processes including those associated with motivated behavior. The current review focuses on emerging evidence indicating that the hypocretins influence reward and reinforcement processing via actions on the mesolimbic dopamine system. We discuss contemporary perspectives of hypocretin regulation of mesolimbic dopamine signaling in both drug free and drug states, as well as hypocretin regulation of behavioral responses to drugs of abuse, particularly as it relates to cocaine. The hypocretin/orexin system consists of two neuroexcitatory peptides (hypocretin-1 and hypocretin-2) that are synthesized within neurons restricted to the lateral hypothalamus and adjacent regions. These neurons project to a vast number of brain regions and interact with two known G-protein-coupled receptors, the hypocretin 1 and hypocretin 2 receptors  where a large population of dopamine neurons resides. The first evidence for this came from anatomical studies showing a significant hypocretin innervation of the VTA where both hypocretin 1 and hypocretin 2 receptors are found on dopamine neurons  via the dopamine transporter. In initial studies, rats were pretreated with vehicle or hypocretin-1 (0.5 nmol) directly into the VTA and dopamine signaling was recorded for 20 min prior to additional manipulations (see cocaine results below). In contrast to what was observed with the microdialysis studies, hypocretin-1 significantly increased evoked-dopamine release within 5 min of administration without affecting dopamine uptake rate . In contrast, the high temporal resolution afforded by fast scan cyclic voltammetry allows for rapid sampling of phasic changes in both dopamine release and uptake. Using voltammetry in anesthetized rats we further explored the possibility that hypocretin regulates dopamine signaling  and hypocretin knockout (KO) mice to examine whether a complete loss of hypocretin signaling would produce deficits in dopamine signaling. Hypocretin KO and WT mice were sacrificed and recording and stimulating electrodes were positioned within the NAc core to allow for measurement of locally-evoked dopamine release  directly into the VTA and dopamine responses were monitored for 40 mins  in dopamine neurons in animals that had received non-contingent cocaine injections. Importantly, these effects were blocked with SB-334867  directly into the VTA 20 min prior to receiving a single 10 mg/kg injection of cocaine. In rats treated with vehicle, injections of cocaine produced expected increases in extracellular dopamine levels. In contrast, hypocretin-1 significantly augmented the effects of cocaine to nearly double of what was observed with vehicle Figure . The oppin vitro voltammetry in brain slices from WT and hypocretin KO mice demonstrate that in addition to baseline disruptions in dopamine signaling, a loss of hypocretin also reduces the effects of cocaine on evoked dopamine release and dopamine uptake inhibition  such that dopamine neurons will fire and release dopamine at levels appropriate for typical responses to drugs of abuse. Under these circumstances cocaine can exert its typical effects and elevate dopamine levels in target regions. However, when hypocretin neurotransmission is enhanced or compromised, dopamine neuronal activity and release are affected. In the case of reduced hypocretin signaling, , dopamine neuronal activity state would be compromised and thus dopamine release and sensitivity to glutamate would similarly be reduced leading to dysregulation in dopamine transporter function and alterations in the ability for cocaine to exert its effects.The neurochemical studies described above provide compelling evidence for the hypothesis that hypocretin neurotransmission influences dopamine signaling in the NAc core, particularly in response to cocaine. Moreover, given that many of the described experiments used infusions of agents directly into the VTA, these studies indicate that the actions of hypocretin on dopamine release and uptake involve signaling within the VTA and possibly enhancement of glutamatergic signaling within dopamine neurons in this region.The mesolimbic dopamine system, including the projection from the VTA to the NAc, is hypothesized to play an integral role in the reinforcing properties of various stimuli including food, sex, and drugs of abuse such as cocaine  schedule of reinforcement in which single lever presses resulted in cocaine delivery  schedule of reinforcement is designed to assess an animal's motivation to work for a reinforcer. In the initial stages of a PR session, single cocaine injections are obtained with relatively low effort (few lever responses) and thus like an FR1 schedule, animals can readily attain preferred blood levels of cocaine. However, as the PR session continues, lever response requirements increase for subsequent cocaine injections and thus rats must exert greater effort to obtain cocaine. The point at which animals discontinue to work for the reinforcer is termed the “breakpoint” and is a measure of the effort that an animal is willing to expend to obtain drug  = 6.82, p < 0.001]. Nevertheless, a Tukey's post-hoc test indicated that this effect was only significant at 30 mg/kg SB-334867 (p < 0.001). More importantly, however, when rats were challenged with an i.p. dose of 10 mg/kg cocaine, there were no significant differences in locomotor activity across doses of SB-334867 . In fact, rats treated with 30 mg/kg showed the highest levels of activity when compared to all other groups, suggesting that when cocaine is on board, rats treated with SB-334867 do not display overall disruptions to arousal or deficits in locomotor activity.Many observations indicate that the hypocretin system regulates arousal-related processes, including locomotor activity and sleep-wake behavior (Hagan et al., These observations indicate that while hypocretin manipulations may affect some aspects of arousal, they do not produce sedation or locomotor deficits that would explain reductions in behavioral responses to drugs of abuse. Indeed, blockade of hypocretin 1 receptors does not reduce locomotor responses to cocaine, has no effect on lever responding under an FR1 or sucrose-reinforced PR schedule in highly-motivated rats. When considered together, these data suggest that the pharmacological effects of SB-334867 on motivated responding for cocaine cannot be solely attributable to sedation or disruptions to motor activity.In less than a decade, nearly a hundred articles have focused on the involvement of the hypocretin system in regulating natural and drug reward. Studies using a variety of electrophysiological, neurochemical, molecular, and behavioral approaches have shown that the hypocretin system is important for normal neurotransmitter and behavioral responses to drugs of abuse. At a gross level, it is evident that enhancement of hypocretin signaling enhances behavioral responses to the rewarding properties of drugs, promotes the motivation for animals to work for drugs of abuse, and increases the propensity for drug seeking and drug taking. By comparison, disruptions in hypocretin signaling reduce drug reward, decrease the motivation to work for drugs, and reduce drug intake and drug seeking.The effects of hypocretin on behavioral responses to drugs of abuse are likely to be associated with alterations in dopamine neuronal activity in the VTA. Many observations indicate that under normal conditions hypocretins influence dopamine cell firing and dopamine release across regions in the brain that are known to participate in reward and reinforcement processes. Furthermore, it is clear that manipulations to hypocretin neurotransmission also affect dopamine responses to drugs of abuse with enhancement of dopamine release observed with increased hypocretin signaling and reduced dopamine release seen when hypocretin neurotransmission is disrupted. Whether hypocretin regulation of dopamine signaling is a principal participant in the alterations observed with behavioral responses to drugs of abuse is unclear. Nevertheless, given the importance of dopamine systems in regulating reward and reinforcement-related behaviors, it appears that the hypocretin system may be a viable target for pharmacotherapy development to treat drug dependence and relapse without the abuse potential, or intolerability, associated with many of the current treatments for drug addiction. Furthermore, given the evidence that hypocretins also modulate the motivation to work for palatable foods when rats are hungry, but not when they are sated, suggests that the hypocretins may also serve as targets for the treatment of addiction to natural reinforcers, in general, and not only for addiction to drugs of abuse.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Such experiments led to the formulation of the general concept of negative reinforcement and specific theories attempting to explain escape and avoidance behavior, or why animals choose to either terminate or prevent the presentation of an aversive event. For example, the two-factor theory of avoidance holds that cues preceding an aversive event begin to evoke conditioned fear responses, and these conditioned fear responses reinforce the instrumental avoidance response. Current neuroscientific advances are providing new perspectives into this historical literature. Due to its well-established role in reinforcement processes and behavioral control, the mesolimbic dopamine system presented itself as a logical starting point in the search for neural correlates of avoidance and escape behavior. We recently demonstrated that phasic dopamine release events are inhibited by stimuli associated with aversive events but increased by stimuli preceding the successful avoidance of the aversive event. The latter observation is inconsistent with the second component of the two-factor theory of avoidance and; therefore, led us propose a new theoretical explanation of conditioned avoidance: (1) fear is initially conditioned to the warning signal and dopamine computes this fear association as a decrease in release, (2) the warning signal, now capable of producing a negative emotional state, suppresses dopamine release and behavior, (3) over repeated trials the warning signal becomes associated with safety rather than fear; dopaminergic neurons already compute safety as an increase in release and begin to encode the warning signal as the earliest predictor of safety (4) the warning signal now promotes conditioned avoidance  Conditioned avoidance is an acquired behavioral response that results in the prevention of an aversive event. Conditioned avoidance was first described by one of Ivan Pavlov's chief scientific rivals, Bechterev  before bWhen experimental psychologists began considering the phenomenon of conditioned avoidance in the middle of the twentieth century, they were relatively unsatisfied with Watson's interpretation that the avoidance response is simply a conditioned reflex resulting from classical conditioning Bolles, . AlternaBefore we discuss a role for subsecond dopamine release in conditioned avoidance, it is important to first briefly overview the neural circuitry involved in centrally representing incentive salience. One of the most studied components of the motivational circuitry of the brain is the nucleus accumbens. This brain region has been referred to as a limbic-motor . These high frequency bursts of dopaminergic neural activity produce transient increases in dopamine concentration in terminal fields . Dopamine concentration transients are detectable at the neurochemical level within terminal fields of the mesolimbic dopamine system using fast-scan cyclic voltammetry, an electrochemical technique that allows for the detection of dopamine on the millisecond timescale. Importantly, only neurochemical techniques like fast-scan cyclic voltammetry provide the temporal resolution necessary to measure dopamine release events evoked by a warning signal in a standard conditioned avoidance procedure.The mesolimbic dopamine system is a neural pathway that originates from A10 dopamine neurons in the ventral tegmental area of the midbrain and projects to the brain's motivational circuitry, most prominently the nucleus accumbens, amygdala and prefrontal cortices Swanson, . During Pharmacological, lesion, genetic and microdialysis studies have been conducted over the last few decades to demonstrate a general role for dopamine in conditioned avoidance. Animals fail to acquire avoidance following 6-hydroxydopamine lesions of midbrain dopamine neurons, a deficit that is reversed by the restoration of dopamine levels using L-dopa treatment  becomes more prominent. These findings support other recent work demonstrating that the representation of a conditioned cue can switch between appetitive and aversive stimuli over repeated pairings ; later in training, the safety signal remains associated with the alleviation/avoidance of shock while the warning signal becomes associated with the successful avoidance of foot shock through a reinforcement learning mechanism. The role that temporal difference reinforcement learning may play in transition of cue-evoked dopamine from the safety signal to the warning signal during conditioned avoidance has been previously discussed in detail . It should be noted that this model is only intended to apply to operant signaled shock avoidance tasks. We still believe the mesolimbic dopamine system may function in Sidman operant avoidance tasks, where operant avoidance is maintained without an exteroceptive warning signal Sidman, , as an aThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Complement Factor I (CFI) is a serine protease with an important role in complement alternative pathway regulation. Complete factor I deficiency is strongly associated with severe infections. Approximately 30 families with this deficiency have been described worldwide.We have studied five new Spanish families suffering from CFI deficiency. From 19 screened people, 7 homozygous, 10 heterozygous and 2 healthy subjects were identified. Clinical, biochemical and genetic descriptions are included.Molecular studies demonstrated 4 novel mutations in the screened individuals; amongst them, we describe here the first great gene deletion reported in the CFI locus, which includes full exon 2 and part of the large intron 1.CFI deficiency is possibly an underestimated defect and the eventual existence of this deficiency should be tested in those patients exhibiting low C3 and recurrent bacterial infections. We propose a simple diagnostic flowchart to help clinicians in the identification and correct diagnosis of such patients. The human complement system consists of more than 35 plasma or membrane-bound proteins whose activation is tightly controlled through the action of complement inhibitors. It is involved in host defense against infectious agents, in the removal of apoptotic cells and immune complexes, and in the modulation of the adaptive immune system . Complem*217030) comprises 13 exons localized on chromosome 4q25 [One of the most important complement activation regulators is Complement Factor I (CFI), a serine protease that circulates in a zymogen-like state  at a conome 4q25 ,10 and iCFI inactivates C3b by cleaving it into iC3b, C3d and C3dg and, in an analogous way, C4b into C4c and C4d. To properly perform its functions, CFI requires the presence of several cofactor proteins such as C4b-Binding Protein (C4BP), Complement Factor H (CFH), Complement Receptor 1 (CR1/CD35) and Membrane Cofactor Protein (MCP/CD46) . CompletIn this article we describe five new Spanish families with complete CFI deficiency, an underestimated complement defect mainly characterized by recurrent, severe infections, low C3 and normal C4 levels in serum. The fact that this persistent biochemical profile is shared by other relatively infrequent complement abnormalities leads us to propose a simple scheme to help clinicians in the diagnosis of patients presenting this complement phenotype.Members from five unrelated Spanish families with CFI deficiency were studied. Serum and DNA samples were obtained under standard conditions after written informed consent of the participants.Patient I.1 was a 37 year old woman with a long-standing history of recurrent rhino-conjunctivitis. At the age of 4, she had several episodes of otitis, sinusitis and bronchitis. At age 16, she had an episode of meningococcal septicaemia with disseminated intravascular coagulation (DIC) and skin complications. Later, she suffered from an acute arthritis episode. Infections severity declined with age. Her mother (I.2), sister (I.4) and brother (I.5) were also studied.Patient II.1 was an 8 year old boy, the only child of healthy parents (II.2 and II.3). He had a history of recurrent pneumonia, an episode of meningococcemia and other infections such a balanitis or oral thrush.Patient III.1 was a 6 year old boy. His parents  and brother (III.4) were also studied. He had recurrent episodes of bronchitis. At the age of 5 he had a bacteraemia episode and a meningococcal sepsis with meningitis when he was 6. His recurrent episodes of otitis made the implantation of ear tubes necessary.Patient IV.1 was a 5 year old girl. She had recurrent episodes of pneumonia and one episode of facial cellulitis. At the age of 5 she had a hypocomplementemic vasculitis episode. Her parents and a sister, aged 8 months, were also studied .Patient V.1 was a 6 year old girl with a history of meningitis and pneumonia. At the age of 4, she began suffering from recurrent Henoch-Schönlein purpura outbreaks (15 in two years). Her parents (V.2 and V.3) and her half sister (V.4) were included in the study.The functional activity of the classical pathway was measured by CH-50 haemolytic assay, according to standard procedures. C3 and C4 were measured by Nephelometry . Ouchterlony analysis: Initial screening for the qualitative estimation of the remaining complement components was performed by double immunodifussion with polyclonal antibodies against most of the soluble C components . Serum levels of CFH and CFI proteins were measured by standard methods . Anti CFFour microliters of serum from patients and healthy controls were separated by 8% gel electrophoresis under non-reducing conditions, transferred to a PDVF membrane and detected with polyclonal anti-CFI antibodies, as described by Gonzalez-Rubio et al. .The 13 exons and their flanking intronic regions in the CFI gene were amplified and sequenced using primers and conditions described by Ponce-Castro et al. . For mutIn order to detect eventual deletions or insertions in the CFI locus, copy number variation was measured with the SALSA MLPA kit P296 aHUS  following manufacturer instructions. The kit contains probes for the amplification of the 13 CFI exons, their flanking regions and several other reference chromosomal locations. Data normalization and analysis were done with the Coffalyser v9.4 MLPA data analysis software (MRC-Holland).A genomic fragment spanning approximately 9Kb (intron 1 to intron 3) was amplified using the GeneAmp XL PCR kit . Reaction products were resolved in 0.7% ultrapure agarose gels, purified with the QIAquick Gel Extraction kit (Qiagen) and sequenced. The sequence of the primers used for long range amplification and sequencing were as follows:5’GCACCAGTAAGAACCAATCCC3’ (Forward);5’GAAAGGTTAGGTAATCAAAAAGC3’ (Reverse)The clinically affected index cases from the five families studied have complete deficiency of CFI protein. Two of the relatives, III.4 and IV.4, brother and sister of III.1 and IV.1 respectively, were also completely CFI deficient, although they had no clinical signs related with this defect by the time the study was performed. All the CFI homozygous deficient had very reduced or no detectable CFI antigen in serum by ELISA and Western Blot . Their C3, CFB and CFH levels were also diminished as a consequence of the spontaneous activation and uncontrolled amplification of the complement alternative pathway. In contrast, partial CFI deficiency in some patients’ relatives was characterized by diminished levels of CFI but normal amounts of all other complement proteins analyzed. In all cases, other possible causes of uncontrolled complement consumption, like the presence of C3NeF or anti CFH auto antibodies (CFHA), were ruled out. Complement profiles are shown in Figure A genetic cause of the deficiency was found in all families. The mutations identified in this series , demonstrating a deletion size of approximately 5-6Kb (c.266-?_536 + ?del) in patient III.1, his mother III.2 and his brother III.4. The resulting fragments were resolved in 0.7% ultrapure agarose gel  was identified in families IV and V. The C > T transition converted residue R187 into a premature termination codon. No consanguinity was reported affecting families IV and V, but their close geographic origin hints at a founder effect as a probable cause for the co-occurrence of this mutation in these families.In family IV, a novel AT insertion in exon 13 between nucleotides 1610 and 1611 was also identified. The resultant frameshift change leads to a premature termination codon shortly downstream of the insertion site.The second mutation found in family V was another unreported mutation. Patient V.I, her mother and half sister, were found to bear an AT deletion (c.80_81delAT) in a region of exon 2 in the N-terminal domain. This deletion converts D27 into an alanine residue and leads to a frame-shifting situation that causes a premature stop codon (p.D27Afs*18).de novo alteration, as both brother and sister of the proband do not carry this change. The father was deceased, so no further investigations could be performed on the segregation of this c.485 G > A change.The remaining mutations found in the patients that we studied had been previously described elsewhere: c.485 G > A , c.772 GAll the mutations reported here are shown in a schematic CFI protein model based on Protein Data Base accession 2XRC Figure .As part of innate immunity, the complement system plays an important role in the defense against pathogens, especially in early childhood. Spontaneous and continuous activation of the complement cascade  must be via complement receptors), enhancement of antigen solubility and immunogenicity, non-inflammatory removal of cell debris and immune complexes by phagocytes and promotion of the inflammatory environment required for correct immune response [*217030) is a very rare primary immunodeficiency disease [The C3 fragments generated by complement activation are involved in many important functions, such as opsonisation  as well as to aseptic meningitis [Defective opsonization in these patients makes them susceptible to recurrent pyogenic infections  and thus can guide the clinician in diagnostic tasks. The presence of C3NeF can cause excessive C3 consumption, but clinical signs associated with this autoantibody fit in better with Dense Deposit Disease (DDD) and partial lipodystrophy (PLD) ,30. AutoComplete C3 or CFH deficiency presents clinical outcomes similar to those of CFI deficiency so that biochemical and/or functional studies are needed to distinguish among these deficiencies. Identification of the underlying molecular defect can be accomplished following the diagnostic algorithm depicted in Figure On the other hand, partial defect of C3, CFH or CFI is a susceptibility factor to develop aHUS, one of the most severe human diseases related to alternative pathway deregulation. When CFI deficiency has been established, family studies are needed in order to predict the consequences arising from the segregation of the mutated allele in the family. Multiple concurrent factors, both environmental and genetic, may be necessary in an individual for disease manifestation of aHUS . EnvironCFI locus.Complement Factor I (CFI) is the major complement inhibitor that degrades activated complement components C3b and C4b in the presence of specific cofactors. Its complete deficiency results in secondary complement deficiency due to uncontrolled spontaneous alternative pathway activation and causes susceptibility to infections. To date, mutations in several CFI domains have been described worldwide. Here, we report four novel mutations and the first large gene deletion in the We propose a general screening for this complement immunodeficiency whenever suggestive clinical features are present in the patient. C3 and C4 quantification are available to most laboratories, thus guiding the clinician at an early stage of the study  declare that they have no competing interests'.MA-D carried out the molecular genetic studies, participated in sequence analysis, performed the immunoassays and drafted the manuscript. AL-L carried out the molecular genetic studies, took part in sequence analysis and drafted the manuscript. SG carried out the molecular genetic studies and immunoassays. IGG, JM, PS-P, CC and PN conceived the study and helped to draft the manuscript. ML-T conceived the study, took part in its design and helped to draft the manuscript. All authors read and approved the final manuscript.


Midbrain dopaminergic neurons send numerous projections to cortical and sub-cortical areas, and, in a manner dependent upon their activities, diffusely release dopamine (DA) to their targets. In particular, in the ventral tegmental area (VTA), the release of dopamine is thought to be associated with reward-related information . The DAe


Antibody mediated rejection (AMR) poses a significant and continued challenge for long termgraft survival in kidney transplantation. However, in the recent years, there has emerged anincreased understanding of the varied manifestations of the antibody mediated processes inkidney transplantation. In this article, we briefly discuss the various histopathological and clinicalmanifestations of AMRs, along with describing the techniques and methods which have made iteasier to define and diagnose these rejections. We also review the emerging issues of C4dnegative AMR, its significance in long term allograft survival and provide a brief summary of thecurrent management strategies for managing AMRs in kidney transplantation.  Antibody-mediated rejection is an important cause of acute and chronic allograft dysfunction and graft loss. Although hyperacute  rejection has been recognized since the 1960s, the role of antibodies in other forms of rejection was not clear until new diagnostic methods became available. Our knowledge about the role of antibodies in allograft rejection, particularly in some forms of chronic allograft rejection, has been evolving rapidly over the last decade.Antibodies directed against donor antigen can cause different types of rejection that can vary in acuity and severity.It occurs due to preformed donor specific antibodies (DSA) present in high titers and presents as graft failure that can occur within minutes (but sometimes may be delayed for a few days) after transplantation . The occIt is characterized by graft dysfunction manifesting over days and is a result of DSAs, that may either be preformed or develop denovo after transplantation . Acute AIt is now well recognized that antibodies can mediate chronic allograft injury which is characteristically seen as transplant glomerulopathy (TG) on kidney biopsies . TG /major-histocompatibility-complex (MHC) Class I and II antigens . HLA claThe major mechanism involved is activation of classical complement pathway by the antigen-antibody complex, leading to formation of the membrane attack complex resulting in cellular injury. The target antigens in AMR are most often situated on the endothelium resulting in the histological findings of acute  and chronic (transplant glomerulopathy) vascular injury. Endothelial damage also results in platelet activation and microthrombi formation. The byproducts of complement activation  act as chemokines resulting in inflammatory cell infiltration and amplification of the inflammatory process. Long standing inflammation results in cell proliferation, basement membrane duplication, and mesangial interposition which can be easily seen on light and electron microscopy as glomerular BM splitting and PTC BM multilayering, respectively. The ability of different IgG subclasses to fix complemsent also varies. IgG1 and IgG3 have strong complement fixing properties compared to the IgG2 and IgG4 subclasses, which fix complement weakly. The significance of this was studied in a series of 74 patients with pretransplant anti-HLA antibodies. Only 4 patients in this series had exclusively weak or no complement fixing HLA antibodies (IgG2 or IgG4) . Of the Based on the increasing evidence for the role of antibodies in allograft dysfunction and the strong correlation with C4d-staining and DSA, the Banff committee updated its renal allograft biopsy classification to involve a separate antibody-mediated rejection diagnosis . AccordiBased on the histopathology, AMR can be classified into three subtypes as below.Class I: Presence of acute tubular necrosis (ATN) only, with minimal inflammation.Class II: glomerulitis, peritubular capillaritis, and microthrombosis.Class III: Arteritis.Chronic AMR according to the Banff criteria involves demonstration of C4d, DSA, and at least one feature of morphologic evidence of chronic tissue injury, such as glomerular double contours, peritubular capillary basement membrane multilayering, interstitial fibrosis/tubular atrophy, and/or intimal thickening of arteries . HoweverC4d is a complement split product that is formed during breakdown of C4b into C4d and C4c. C4d has a thioester moiety that enables strong covalent bonding with the endothelial cells and basement membrane. It is constitutively expressed in all normal kidneys in the mesangium and the vascular pole owing to the constant complement turnover. This can extend into glomerular capillaries in cases of immune-mediated glomerulopathies, but peritubular C4d deposition is noted mostly in the transplanted kidney, with rare reports of C4d presence in PTC of native kidneys , 22.There are two methods for C4d detection in biopsy specimens . It can n = 14) and 5 of the 6 biopsies with cyclosporine toxicity [The association of C4d with AMR was initially described by Feucht et al. in 1991 when they showed a significant association of C4d with preformed anti HLA antibodies in patients with acute rejection . This watoxicity . Crespo toxicity .The data for C4d-staining in chronic AMR is more variable, with a few studies showing strong correlation while others showing poor correlation. Mauiyyedi et al. reported presence of C4d in 23 of 38 biopsies with features of chronic AMR  but no C4d in the control group without features of chronic AMR . SimilarProtocol biopsy studies have also demonstrated another important characteristic of C4d which is variability of staining over time, suggesting a constant flux between states of positive to negative C4d . In a siThe techniques to identify anti HLA antibodies have improved significantly with the development of single-bead antigen testing methods which have very high sensitivity for antibody detection. The complement-dependent cytotoxicity (CDC) still remains the gold standard test for the detection of preformed antibodies prior to transplantation. The addition of antihuman globulin enhances the sensitivity of the assay by cross-linking the antibodies (AHG-CDC). Flow cytometry crossmatch (FXM) assay is more sensitive than the CDC assay and detects antibodies via fluorochrome-tagged antihuman Immunoglobulin antibody. The newer solid phase assays use purified single HLA antigens to detect anti-HLA antibodies by ELISA or flow cytometry techniques. These tests have increased sensitivity to detect presence of anti-HLA antibodies even with a negative FXM –36. The Although the current diagnosis of AMR requires the concomitant presence of DSA, C4d, and histopathological evidence of AMR, treatment may be initiated in circumstances where the above criteria may not be fulfilled in entirety. This depends on the risk factor profile of the patient  and presence or absence of organ dysfunction. Treatment is often initiated in situations of diffuse C4d positivity with allograft dysfunction even in the absence of DSA or histological evidence of AMR. The inability to measure DSAs in these cases may be related to the presence of non anti-HLA antibodies, to antigen not present on the single-bead assays, or to the possibility that the DSAs may be completely adsorbed onto the allograft . SimilarThere is a paucity of randomized controlled trials in the treatment of AMR. Many of the studies have used historical controls to compare the effectiveness of therapies. There is also bound to be some publication bias related to selective publication of positive studies.Even with these inadequacies, there has been good progress made in developing treatments for AMR. The primary goal in AMR treatment involves targeting the reduction/removal of DSAs and elimination of the B-cell/plasma cell population responsible for the production of these antibodies.A number of treatment modalities have been employed for the treatment of AMR as characterized below.Antibody removal/neutralization: plasmapheresis, immunoadsorption, intravenous immunoglobulin, and splenectomy. Anti B-Cell therapies: Mycophenolate mofetil, Rituximab, IVIG, and splenectomy. Antiplasma cell therapy: Bortezomib. Anti-T-cell therapies: T-cell depleting agents such as Antithymocyte globulin (ATG). Conversion to tacrolimus-based regimens. Terminal-complement pathway inhibitor: Eculizumab.The presence of a vast array of therapeutic modalities signifies the ineffectiveness of one drug or one particular combination therapy to reverse or treat AMR successfully in all scenarios. All of these treatments have been used in different combinations by different groups without a good control arm, resulting in poor evidence to argue for the superiority of one treatment regimen. These treatment modalities are also used for pretransplantation desensitization protocols to abrogate positive crossmatch in highly sensitized patients.This is the most commonly used agent either alone or often, in combination with plasmapheresis. Although the exact mechanisms involved are not clear, they appear to involve multiple processes such as neutralization of complement fixing antibodies, alteration in the activity of complement, modulation of Fc receptor activation and function, and regulation of T and B lymphocytes . Recent Plasmapheresis is very effective in reducing the antibody load but needs to be used in conjunction with other therapies that target the antibody producing mechanisms. The most common type of Plasmapheresis performed is plasma exchange, with albumin being the most common replacement fluid used. It is usually performed on alternate days with a 1–1.5 volume exchange with albumin (commonly) or fresh frozen plasma. Most institutions also follow each PP session with low-dose IVIG (100 mg/kg) . DSAs arA retrospective study analyzed one-year graft outcomes of 16 patients with AMR treated with PP and IVIG and 43 ACR patients and found similar overall graft survival of 81% and 84%, respectively, indicating the effectiveness of these therapies in improving outcomes of acute AMR . Plasmapheresis is generally well tolerated. Side effects are relatively uncommon and are related to the use of vascular access , volume removal, type of replacement fluid used , hypocalcemia, and side effects related to use of anticoagulants .IA is currently not used in the United States. IA was studied in a randomized controlled trial in Europe where it proved very successful in reversing severe AMR . Both arThe study was stopped early because of significant success rate in the IA group (80% versus 20%). It was, however, a small study (5 patients in each group), with a higher prevalence of diffuse C4d in the control group. Considering the widespread acceptance of IVIG and PP for treatment of AMR and unavailability of IA in the USA, a head-to-head study of IA with PP and IVIG will be necessary to prove its superiority, prior to its acceptance as an alternative to IVIG and PP.Rituximab is an anti-CD20 monoclonal antibody that induces profound depletion of B-cells and was initially approved for the treatment of B-cell lymphoma. It has since been tested in multiple immune-mediated disorders with varying degrees of success. Rituximab has been used to treat AMR in a number of uncontrolled studies.2/wk for 2–4 weeks. Rituximab results in prolonged and profound B-cell depletion which may cause reactivation of latent viruses such as hepatitis B, C, cytomegalovirus (CMV), and also mycobacterium tuberculosis. It also carries a boxed warning for progressive multifocal leukoencephalopathy (PML) caused by JC virus. Most of the studies reported so far with the use of Rituximab have reported favorable outcomes. Becker et al. treated 27 patients with AMR with a single dose of rituximab. Twenty-two of these patients also received ATG and plasmapheresis . At a mePatients can also manifest acute infusion reactions, which usually occur within 30–120 minutes and may be mild or severe, such as bronchospasm, angioedema, acute respiratory distress syndrome, cardiogenic shock, and anaphylaxis. These have often been reported in leukemic patients with high pretherapy leukocyte counts .Initiation or augmentation of anti B-cell maintenance therapy is routinely done when AMR is identified. The most commonly used agent for this purpose is mycophenolate mofetil. It is also common practice to change to a calcineurin-based immunosuppression, specifically to tacrolimus, if patients are not on a calcineurin inhibitor (CNI).2/week, with 4 doses given over 2 weeks.Bortezomib is a novel proteosome inhibitor that is approved for the treatment of multiple myeloma. Proteasomes are involved in breakdown of ubiquitinated proteins and are present both in the nucleus and cytoplasm. Inhibition of proteasomes can lead to decreased nuclear factor-Kappa B activation, cell cycle arrest, endoplasmic reticulum stress, and increased cell apoptosis . This acn = 26). The incidence of AMR at 3 months was significantly less compared to an historical control group (7.7% versus 41.2%), although the presence of C4d in patients with DSA did not differ between the study and control group, thus providing evidence for the downstream activity of eculizumab in blocking the complement pathway. The use of eculizumab also resulted in a reduced need for PP. The one-year TG prevalence was also low with only one (6.7%) of the 15 patients in the eculizumab group developing TG compared to 15 (35.7%) of the 42 controls. Most patients in the treatment group received weekly eculizumab for 4–8 weeks while one patient needed a year of eculizumab because of persistent FXM positivity [Eculizumab is a humanized monoclonal antibody directed against complement protein C5. It binds to the C5 protein with high affinity, thereby inhibiting conversion of C5 to C5b and preventing formation of the membrane attack complex (C5–9). Initially approved for use in paroxysmal nocturnal hemoglobinuria (PMH), it was also recently approved for use in atypical hemolytic-uremic syndrome. Prior vaccination against meningococcus and pneumococcus is necessary. One dose of Eculizumab was used with IVIG and rituximab in a patient with severe AMR, who recovered from the AMR but died of a fatal pulmonary hemorrhage a few months later . A prospsitivity . No signSplenectomy It has also been used in resistant AMR patients with good success rate [ess rate , 68. HowAcute cellular rejection frequently coexists with AMR and needs to be treated aggressively with either steroids or T-cell depleting therapies such as Antithymocyte globulin (ATG). The role of these T-cell depleting therapies in AMR has not been clearly studied in patients with pure AMR or AMR with low grades of ACR. The use of these agents may reduce the T-cell stimuli that are driving the B-cell-mediated antiallograft responses, thereby helping to gain better control of the AMR process. Indirect proof can be obtained from one study that reported no significant change in graft outcomes between C4d-positive and negative cases. However, there was aggressive use of antilymphocyte therapy to treat C4d-positive cases which might have improved outcomes in this group of patients .It has been shown in multiple studies that AMR portends worse outcome in terms of graft survival at one and five years. Some of these studies were reported prior to the utilization of aggressive therapies that are currently in use for AMR and often serve as historical controls for trials of newer therapies for AMR. Lederer et al. reported a 4 year 50% graft survival for C4d+ patients compared to a 8 year 50% graft survival for C4d-patients . PoduvalThe significance of C4d+ allograft biopsies appears to differ based on whether the transplantation was ABO or HLA incompatible . MultiplThe significance of focal C4d on biopsies is currently still being evaluated and is not entirely clear. A significant number of these biopsies may not be associated with histologic evidence of AMR, but its presence has still been associated with inferior graft outcomes , 76. GraImprovements in HLA typing and DSA identification have increased our ability to identify high-risk recipients who may be at risk for antibody-mediated rejection posttransplantation. For patients who are highly sensitized or those with ABO or HLA incompatible living donors, there are at present four options available for successful transplantation. Patients can undergo desensitization protocol followed by a kidney transplant provided they can achieve sufficient reductions in DSA titers and become crossmatch negative. Even with successful transplantation after desensitization, these patients remain at increased risk for AMR. They also have reduced graft survival compared to nonsensitized patients. However, their outcomes are still superior when compared to remaining on dialysis . PatientAntibody-mediated rejection is an important cause of acute and chronic graft failure. Improvements in HLA technology along with the recognition of the role of C4d in AMR have revolutionized the understanding of this important entity. New research is attempting to elucidate the mechanisms and epidemiology of C4d-negative antibody-mediated rejection processes. Further research should help clarify the identification, prognosis, and treatment of these C4d-negative AMRs. Therapies for AMR are still not optimal with high rates of graft loss leading to poor patient outcomes. Newer therapies, such as bortezomib and eculizumab that target novel pathways in the AMR process are promising but will need further randomized studies before becoming widely used. Studies will need to be performed to determine the best use, either alone or in combination, of the myriad number of therapies currently available. Transplantation of sensitized patients remains a difficult problem. However, developments such as paired kidney donation and desensitization protocols are continuously improving the rates of transplantation in this difficult to transplant population.