Related protein domains of a superfamily can be specified by proteins of diverse lengths. The structural and functional implications of indels in a domain scaffold have been examined.In this study, domain superfamilies with large length variations  were analyzed for the functional impact of such structural differences. Our delineated dataset, derived from an objective algorithm, enables us to address indel roles in the presence of peculiar structural repeats, functional variation, protein-protein interactions and to examine ‘domain contexts’ of proteins tolerant to large length variations. Amongst the top-10 length-deviant superfamilies analyzed, we found that 80% of length-deviant superfamilies possess distant internal structural repeats and nearly half of them acquired diverse biological functions. In general, length-deviant superfamilies have higher chance, than length-rigid superfamilies, to be engaged in internal structural repeats. We also found that ∼40% of length-deviant domains exist as multi-domain proteins involving interactions with domains from the same or other superfamilies. Indels, in diverse domain superfamilies, were found to participate in the accretion of structural and functional features amongst related domains. With specific examples, we discuss how indels are involved directly or indirectly in the generation of oligomerization interfaces, introduction of substrate specificity, regulation of protein function and stability.Our data suggests a multitude of roles for indels that are specialized for domain members of different domain superfamilies. These specialist roles that we observe and trends in the extent of length variation could influence decision making in modeling of new superfamily members. Likewise, the observed limits of length variation, specific for each domain superfamily would be particularly relevant in the choice of alignment length search filters commonly applied in protein sequence analysis. Diverse factors, such as the accumulation of sequence changes, gene duplications, gene combinations Length differences between domains are introduced through insertions and deletions (indels) into pre-existing domains. It has been shown that protein length expansions are 40–60% greater in eukaryotes than in prokaryotes and that such expansions correlate with the presence of introns and accretion of functional motifs that are involved in sophisticated regulatory networks albeit, discontinuous insertions In an analysis of length differences in 353 multi-membered PASS2 domain superfamily alignments Length variation in proteins has been the object of several analyses and many groups have performed independent studies on domain and protein length variations. Pascarella and Argos Here, we assess the functional and structural advantages of length variations amongst homologous members of 64 length-deviant domain superfamilies. The role of indels in mediating novel interaction interfaces through the formation of structural repeats, multi-domain combinations and higher order oligomers has been examined. The presence of distant internal repeats in length-deviant superfamilies has been carried out using computer algorithms, both using sequence and structural information. In addition to a manual comparison of the giant and dwarf representative domains in each length-rigid and length-deviant domain superfamily, literature has been consulted, where relevant, to support the structural observations and inferences on functional impact made here. Likewise, SCOP domain definitions and domain assignments have been consulted to understand the social contexts of domains. Further, the analysis has been extended to protein-protein interaction databases to examine if length-deviant domains are indeed social in functional contexts and associate with a high number of interacting partners. We have also reasoned whether additional lengths assist domains to interact with multiple copies of domains-either homologous or other. This would address if the ability to accommodate extra length reflects on the ‘social’ skills of a domain to interact with more neighboring domains.We have investigated the functional and structural implications of indels amongst related members of a domain superfamily. We applied the CUSP algorithm to identify indels in domain members of 353 multi-membered domain superfamilies etc., It is observed that several domain superfolds that are repeatedly re-used in protein evolution in diverse domain architectures Gene duplication is a method that facilitates evolution since it leads to the formation of phenotypically redundant genome portions that can be experimented for the generation of novel structural and functional products Interestingly, eight of the top-10 length-deviant domains occur as structural repeats . Full-leWe observe that domain repeats involving the entire structural domain can occur in single or multiple chains . The dometc. (data not shown). In these domain superfamilies, differences in the number of structural repeats can generate varied interaction interfaces that confer additional functional properties amongst the different members. This is also observed in some larger domains such as the pectin lyase, cupins and domain superfamilies such as the four-helical cytokines that harbor diverse copies of the Ig-like fold.The number and lengths of repeats across different members varies across related members as seen in proteins that contain repeating copies of the TPR, ARM, Ankyrin repeats, EF-hand domains etc. (data not shown).Duplications of entire domains result in tandem arrangements of the self-domain along the length of the protein in a beads-on-a-string arrangement as seen in cupredoxins and phospholipase D or result in discontinuous arrangements of the domain as in squalene hopene cyclase. In the former type of structural repeat, dwarf members function as homodimers that associate to generate an active site. Giant domains meet such functional requirements by possessing multiple copies of the same domain on a single chain and most likely involve gene duplication events. Such tandem arrangements of domains involve longer loops that serve as linkers in bringing domains together and are seen in length-deviant superfamilies such as the phospholipase D, trypsin-like serine proteases, actin-like ATPases In length-rigid domain superfamilies, only 33% (8 out of 24) are engaged in structural repeats suggesting that internal repeats are more common in length-deviant domain superfamilies  and S3. An important contribution to new structural interfaces/functional units is domain combination and shuffling resulting in new multi-domain architectures Domain assignments were tabulated for 1189 protein domains from length-deviant superfamilies and 268 domains from length-rigid superfamilies. The recurrent domains have more domain partners. Of the 1189 protein domains that we have examined in the 64 length-deviant superfamilies , 31.4% oDrosophila, yeast and other organisms and the number of known and predicted interactions was determined. We find that length- deviant domain superfamilies are highly interacting (1307), notably the domain superfamilies of SAM, cytochrome C and PRTase-like  specify a characteristic fold  that conn length   as the methyl donor are widespread and participate in a multitude of cellular processes through the methylation of a variety of substrates such as proteins, nucleic acids, phospholipids and small molecules. The domain superfamily includes ‘giant’ members such as the PRMT3 (321 residues) and VP39 (291 residues) and other ‘dwarf’ domains such as the ftsj and COMT that are only 180 and 213 residues in length, respectively , Table 4The lysozyme-like domain superfamily is a large multi-membered superfamily with at least seven different families in the SCOP database, all of them unified by the characteristic lysozyme-like fold. The ‘giant’ domain differs from other ‘dwarf’ domains of the lysozyme-like superfamily members in its acquisition of additional α-domain and β-domain extensions at its N- and C- terminal ends . AdditioThe protein members of the actin-like ATPase domain superfamily include a varied set such as sugar kinases, heat shock proteins and actins that perform distinct functional roles involving phosphoryltransfer . The ranThe collection of structure-derived domain superfamily alignments from PASS2 provides an opportunity to examine such alignments on a large scale in order to study domain length variations. Firstly, it has enabled a qualitative assessment of the extent of length variation in domain superfamilies and aided description of domains tolerant to length variation from a structural perspective. Secondly, by applying CUSP, we have examined the range of variations in diverse domain superfamilies by distinguishing structurally conserved blocks (of similar nature and lengths) from indels (regions susceptible to undergo length differences). We have found that the extent of length variation is not uniform across all classes. Thirdly, we have examined the role of such additional lengths in modifying/altering the general functions associated with a domain.Our investigations on the nature and typical lengths of indels showed that not all domains are uniformly tolerant to large variations in length and that certain domains are more susceptible. Indeed, of the 353 domain superfamilies considered, 64 domain superfamilies showed over 30% variation in length from their mean domain size.In the present analysis, we have addressed the functional and structural advantages conferred on a domain due to indels by considering the extreme cases, namely the giant and dwarf in the length-deviant domain superfamilies. It is possible that large insertions, such as whole-domain insertions, have arisen in the proteins considered in our dataset and are actually due to large gene insertions and not pointing to subtle functional changes brought about by small length variations. However, our current dataset is not biased by such occurrences since we perform our analysis at the domain level and do not consider whole domain insertions. In every case, the functional roles were considered in the light of increasing domain sizes and the effect of loss of indels or decrease in average domain length were excluded since the direction of indel evolution is not the focus of the analysis. Earlier findings have already projected that long insertions predominate over long deletions that are also less likely to occur in protein domain evolution We have also consulted GO annotations for each domain member of the length-deviant domain superfamilies and find that 40 of the 64 length-deviant domain superfamilies include proteins that are involved in catalytic activity, where additional lengths are perhaps required to confer varying themes in substrate specificity (data not shown). At least 14 length-deviant domains are involved in regulatory processes and others are involved in structural roles where protein-protein interactions would be the main functional theme.etc. By an examination of the functional and structural advantages in these most length-deviant domain datasets, we determine, at least in outline, the different contributions that additional lengths confer on a length-deviant domain although more may emerge with the determination of new structures. The descriptions given here attempt to discuss the salient roles of extra lengths in the most length-deviant superfamilies but do not undermine the important contributions of shorter length changes in variant domains. Indeed, in domain superfamilies such as the lipocalins and DNA polymerases, incremental additions in lengths are associated with substrate specificity. We have also briefly examined those length-rigid domain proteins that are functionally versatile. The strategies employed here are refreshingly different and include changes in the orientation of structures, modifications local to the active site to attain functional diversity despite such high structural integrity , we believe that this is a consequence of the high variability in the number of proteins in each dataset. Length-rigid protein domains are not as well-populated (270) as the number of length-deviant domains (1130) and this could affect the numbers projected for length-rigid domains. Here again, although the data is not discussed, we have observed that the number of interacting partners in length-deviant domains is far more than the length-rigid proteins and a more in-depth analysis is required to understand why this may be the case.The interesting trends that we have obtained on the nature and type of indels in protein superfamilies from different classes could affect the area of comparative modeling in structurally unconserved regions in newer superfamily members. Our analysis has shown that in a majority of the superfamilies that we have examined, the core structural scaffold is rarely affected, despite length differences. However, even within a superfamily, the extra lengths impacted differently on function for different members, and therefore, it may be difficult to generalize the exact role of additional lengths in newer members. Depending on their locations and lengths in the structure, we may be able to suggest an involvement in introducing substrate specificity, or in presenting newer protein interfaces for interaction with other proteins or promoters.What is the wealth component that dictates such vivid length variations observed in some protein superfamilies? We find that the ‘currency’ for versatility in length of domain superfamilies is not differential amino acid composition since both length-rigid and length-deviant domain superfamilies exhibit similar amino acid propensities. Could the complexities of domain architecture, nature of co-existing domains, need for internal symmetry, repeating structural themes and diverse quaternary arrangements dictate length variations amongst related protein members of a superfamily? Our analysis suggests that a multitude of these parameters operate to influence the structural revolts of length-deviant domains, imposing still a daunting exercise to predict such variations.In the current study, we have employed SCOP We have considered 353 multi-member superfamily alignments (with at least 3 distantly related members) from PASS2 We have also applied the CUSP algorithm To examine the occurrence of structural repeats in domain superfamilies, giant and dwarf domains were identified in each length-deviant and length-rigid domain superfamily. Full-length protein sequences for each of the giant and dwarf domains were retrieved from the SWISS-PROT database We define ‘domain context’ as the preferred mode of occurrence of a domain. Domain superfamily members differ in their associations. We attempted a correlation of the observed length variations with domain contexts and nature of domain associations for each length-deviant and length-rigid domain superfamily. For each length-rigid and length-deviant domain superfamilies in our dataset, all domain members were pooled together. If a domain member is available from multiple species, a representative sequence with the best resolution from any one species was selected. Full-length protein sequences were obtained from the SWISS-PROT database for representative domains from any one species. SCOP domain assignments were made for each sequence. Full-length proteins of known crystal structure were considered for domain assignments and structural databases alone were consulted for this preliminary analysis. In addition, the occurrence of domains singly (single domain in a single chain or single domain in multiple chains), repeating domains  and in their domain associations (single/multiple copies of a domain in association with neighboring/partnering domains in a single or multiple chains) was also noted.Functional interactions of length-deviant and length-rigid domain superfamilies were studied by examining the known and predicted protein-protein interactions for each domain member in searches in the STRING database We have analyzed the functional roles of indels for the 64 length-deviant domains by examining if indels are involved directly or indirectly in domain function in the giant (longest) and dwarf (shortest) domains of each length-deviant domain superfamily. Indels were identified by the CUSP method and alignments were projected through a graphical viewer, StructView, described elsewhere Text S1Functional variety in deviant and rigid domain superfamilies. This text file provides detailed description of eight types of functional attributes to length-deviant superfamilies and five superfamilies of length-rigid superfamilies by giving relevant examples.(0.14 MB DOC)Click here for additional data file.Figure S1DNA-glycosylase domain superfamily. The two domain scaffold of the DNA-glycosylase domain superfamily in Adenine glycosylase and Endonuclease III harbors a HhH motif (in pink) with active site residues (in red) to bind their respective substrates. Composition of residues in the active site is distinct for each member.(0.51 MB TIF)Click here for additional data file.Figure S2Interleukin-8-like superfamily. Interleukin-8 like chemokine superfamily shows high conservation of the core structure. Lymphotactin (a) and stromal cell derived factor 1 alpha (b) differ primarily in the N and C termini. Lower panel (c) shows a graphical projection of the alignments. The core structure involving the well conserved 310 helix and the three stranded sheet is well conserved across different members and structurally equivalent regions in the alignment are extensive.(0.15 MB TIF)Click here for additional data file.Table S1Structural repeats in length-deviant and length-rigid protein domain superfamilies(1.27 MB DOC)Click here for additional data file.Table S2The number of occurrences of domains singly, multiply and as repeats (tandem repeats of domain) in a single or multiple chain are provided.(0.31 MB DOC)Click here for additional data file.Table S3The number of occurrences of domains in single(SD), multiple(MD) and as R (tandem repeats of domain) in a single or multiple chain are provided. R+MD includes domain occurrences that show repeating copies of self domain in multidomain contexts.(0.07 MB DOC)Click here for additional data file.Table S4Number of Protein-protein interactions (known and predicted) in length-deviant and length rigid domain superfamilies after searching in STRING databaseS30.(0.05 MB DOC)Click here for additional data file.Table S5Structural and functional role of indels in the 64 length-deviant domain superfamilies(0.05 MB XLS)Click here for additional data file.


Previous studies in ovarian cancer have demonstrated that decreased expression and function of the T-cell receptor (TcR)-associated signal transducing zeta-chain correlates with deficient immune responsiveness of T cells. In this study, sera and ascitic fluids obtained from woman with advanced ovarian cancer were found to suppress the expression of TcR-associated zeta chain. This suppression of zeta chain expression was dose-dependent and was not observed with biologic fluids obtained from healthy women. © 2001 Cancer Research Campaign


PET (Positron Emission Tomography) is a nuclear medicine imaging method, frequently used in oncology during the last years. It is a non-invasive technique that provides quantitative in vivo assessment of physiological and biological phenomena. PET has found its application in common practice for the management of various cancers.Lung cancer is the most common cause of death for cancer in western countries.This review focuses on radiotracers used for PET scan with particular attention to Non Small Cell Lung Cancer diagnosis, staging, response to treatment and follow-up PET scan represents one of the most sophisticated nuclear medicine techniques of the last years. It was initially used to study the brain and the heart, but today it is used mainly in oncology. PET scanning is a non-invasive imaging method that differs from others because it observes "in vivo" metabolic activity using radio-isotopes with specific tissutal tropism. More in detail PET scans necessitate the injection of a small quantity of biologically important material like glucose or oxygen which have labelled with radio-nuclides such as carbon-11, nitrogen-13, oxygen-15 and fluoride-18.All the used isotopes are radioactive with a rapid time of decaying by positron emission: carbon-11 or 11C is a radioactive isotope of carbon with a half-life in the order of twenty minutes. Nitrogen-13 or 13N is an isotope of nitrogen with a half life of approximately ten minutes. Oxygen-15 or 15O is an isotope of oxygen having a half life of about two minutes.The most commonly used isotope in PET scans is fluorine-18. It is a fluorine isotope with a half life of approximately 110 minutes. This tracer is very useful because of its long half life and because it decays by emitting positrons having the lowest positron energy which contributes to a high-resolution imaging acquire.Most articles have considered the utility of FDG (fluorine-18 combined with deoxy-glucose) which is the most used radiotracer in clinical practice. Actually, a number of new compounds with promising prospective for PET imaging are also available to gain information about specific cellular and molecular tumour pathways.In the second part of this review a wide panoramic on the use of this technique will be conducted regarding an important oncologic pathology, both in term of incidence and mortality: lung cancer.FDG is a glucose analogue extensively used in oncology for staging, restaging and recently for the evaluation of tumour response to treatment . Cancer The basis for using FDG in oncology was demonstrated by Warburg who observed an increase in glycolytic activity in cancer cells, under both aerobic and anaerobic conditions -5. MoreoThe clinical utility of PET can be limited by FDG distribution in some normal tissues, which can result in a low or decreased tumor to background ratio Fig . In factWhile traditional radiological imaging techniques  make available structural information and identify disease states on the basis of cross anatomical changes, FDG PET imaging provides information on the biochemical processes which may precede cross anatomic changes. . The limThus functional scans obtained with FDG PET are not only complementary to those obtained with conventional modalities but also may be more sensitive because alterations in tissue metabolism generally anticipate anatomic alterations.According to EORTC guidelines for better evaluation of lesions, PET must be combined with Computed Tomography, but response criteria are still not available .To date newer PET/CT protocols have been developed, but there is no consensus about them or their standardization . This reTo correctly determine FDG PET activity it has been introduced the Maximum Standardized Uptake Value (SUV max). It is defined as maximum tumour concentration of FDG divided by the injected dose, corrected for the body weight of the patient: (SUV max = maximum activity concentration [injected dose/body weight]). It represents the metabolic activity for the tumor compared with that in surrounding tissue, corrected for injected dose and patient weight.18 F-Fluoro-L-dihydroxyphenylamine (18F-fluoro-L-DOPA). It is used to evaluate the in vivo activity of aromatic l-amino acid decarboxylase of dopaminergic system. For these characteristics this compound has been used in clinical practice for parkinsonisms diagnosis and to investigate subclinical lesions of the substantia nigra in Parkinson disease related disorders.[18F-fluoro-L-DOPA [Among the newest studied tracers there isisorders. MoreoverSomatostatin based radiotracers  are useful for diagnosis in patients with cancers (Neuroendocrine Tumours-NETs) which express the somatostatin receptor 2 (SSTR2). They are able to orient treatment on the basis of the quality and quantity of tracer uptake. In Fact well differentiated NET tumours do not necessarily shown an increased glucose metabolism, while they can be treated with new target drugs (Sunitinib) in addition to somatostatin analogues. On the other hand poor differentiated NETs and with high proliferation index are more commonly treated with cytotoxic drugs. ,17Choline is a precursor of biosynthesis of an essential element of phospholipids of the cell membrane (phosphatidil – choline). In neoplastic tissue there is an elevated level of phosphatidil-choline and an up-regulation of the enzyme that catalyzes the phosphorilation of choline ,19.11C-Choline) has been developed and is useful mostly to detect well differentiated tumours with low glucose uptake [18F-FDG. In particular the usefulness of this test is related principally to the detection of local tumour recurrence after radical prostatectomy or distant metastatic disease [Thus a new PET radiotracer , a structural analogue of 5-α-dyhidrotestosterone, can be useful to detect metastatic and recurrent prostate cancer lesions, binding affinity and selectivity for androgen receptors (ARs) [rs (ARs) .18F-3-Fluoro-3-deoxy-thymidine (18-F-FLT) is a pyrimidine analogue that reveals the activities of thymidine-kinase-1 during the phase S of mitoses [ mitoses . First i mitoses -2911C-Acetate is a metabolic substrate for synthesis of cholesterol and lipids.  This com18F-Fluoride PET uptake in bone lesions reveals both an increased bone turnover and blood flow.Fluorine-18-Fluoride is a PET tracer with elevated utility for detection of bone metastases in various tumors ,36. In fAnother studied tracer is represented by 11C-Methionine. Methionine is an important amino acid for protein synthesis process. The role of this tracer has been evaluated in various trials, mostly in brain tumors . becauseLung Cancer is one of the most important causes of tumour-related deaths in industrialized countries. NSCLC treatment includes surgery, radiation therapy, chemotherapy and molecular therapy. The choice of treatment alone or in combination is based on clinical and pathological tumour stage.PET scan plays an important role for tumour diagnosis and staging, providing functional information simultaneous to anatomical details when PET is combined with computed tomography (CT)The mainly point to establish a correct staging of NSCLC is to identify patients candidates to surgery versus those ones who are inoperable but can obtain benefits from chemotherapy, radiotherapy or both.In detail, patients with N0 – N1 disease (no metastatic lymph nodes or only intrapulmonary/hilar nodes) are generally candidates for surgical resection. On the contrary patients with N2 disease  could gain benefit from a combination of local and systemic treatments. Patients with N3 disease  are considered for non operability. Thus, an accurate loco regional lymph nodes assessment is mandatory to choose the best treatment options. This could be done either with invasive techniques  or with non-invasive exams such as CT or FDG PET.Among the invasive techniques, endobronchial ultrasound guided-fine needle aspiration (EUS-FNA) deserves a particular discussion. This new technique is now in development in many centers. First data confirmed a sensibility and specificity comparable to mediastinoscopy for staging malignant nodes in NSCLC . In a reOn the other hand it has been shown the relative frequency of metastatic N2 disease in the posterior mediastinal lymph nodes which are not accessible via mediastinoscopy but are easily and accurately sampled by EUS-FNA . Thus soThe increasing availability of FGD PET offers a noninvasive, accurate alternative for staging the mediastinum. Whether or not FDG-PET should be used as a routine procedure in mediastinal lymph node staging, replacing mediastinoscopy, is still a matter of debate .American College of Chest Physicians guidelines  recommenOn the one hand, of major clinical importance is the high negative predictive value of mediastinal FDG-PET (up to 97%),64,65. IOn the other hand, some conditions must be respected: sufficient FDG uptake in the primary tumor; use of a dedicated PET camera; absence of a central tumor or important hilar lymph node disease that may led to under-stage, obscuring coexisting N2 disease on PET ,55,69. APrevious studies have reported that PET positive uptake in a hilar node is a risk factor for occult N2 disease ,27,70. IAs reported by Verhagen, FDG-PET reduces the number of mandatory mediastinoscopy procedures by 46% with no increase in unexpected N2 involvement at thoracotomy . Even ifIn a cost-effectiveness analysis on use of PET for all N0 patients at CT scan, the results were encouraging. In fact the cost of PET scanning was almost counterbalanced by the more appropriate selection of patients for beneficial surgery .On the contrary, the use of cervical mediastinoscopy non-selectively among stage 1 NSCLC has been reported to be not cost-effective  as the iThus, on this latter prospective trial, Cerfolio et al suggest to reserve mediastinoscopy to only those patients with clinically staged N0 disease with a right upper lobe lesion (in which they found 10.4% positive rate for mediastinoscopy), and to limit EUS-FNA in patients staged N0 with tumors only in the right lower lobe (in which they found 15.4% positive rate for EUS-FNA). Moreover the authors recommend both mediastinoscopy and EUS-FNA in patients clinically staged as N1 after integrated PET/CT.However, as described in a recent study by Al-Sarraf et al.  16% of pOther pathological factors such as tumor subtype, grade of differentiation, tumor size, T stage and SUV max of primary tumor tend to be associated with the biological aggressiveness of tumors  and are max of 5.3 to assign malignanc, y highly improve PET/CT accuracy, thus significantly reducing the number of false positive results. The authors conclude that in the absence of other indications for mediastinoscopy, , mediastinoscopy can be omitted in patients who have a maxi-SUV of less than 5.3 in their N2 lymph nodes and low max-SUV primary tumor to mediastinal lymph node ratio, as found by integrated PET/CT analysis. [In another study by Lee et al the maximum standardized uptake value is a predictor of individual lymph node metastasis in non-small cell lung cancer. Using SUVnalysis. Albeit the positive predictive value is plausible, it must not be forgotten that false positive results can be obtained in the case of anthracosilicosis, infection, or granulomatous disorders . In thesKuehl et al. have described a well defined PET/CT staging protocol. According to the authors the procedure should include the analysis of the chest and abdomen in order to stage locally and to detect distant metastases. Moreover the field should include the neck to detect supraclavicular lymph nodes involvement. However, the general recommendation is that if PET scan result is negative, invasive mediastionoscopy could be avoided; on the contrary, a positive PET scan, makes mediastinoscopy necessary for lymph node sampling because of possibility of false-positive PET scan .Regarding the distant metastases detection, a recent randomized trial  suggestsThe use of PET can be applicable for selection or delineation of radiotherapy target volumes. In particular PET with 18F-FDG has taken on increasing importance for radiotherapy planning purposes. In fact because of the hight sensitivity of FDG PET for the staging of lymph node metastases in lung cancer implies that a negative PET examination could permit focusing on the primary neoplasm reducing target volumes .Few studies have prospectively evaluated PET with 18 FDG in radiotherapy planning in term of its role of local control and survival. It has been reported that PET based mediastinal lymph nodes radiotherapy does not affect the local control reducing the target volume. Omitting elective nodal irradiation of clinically uninvolved nodal station is a way of reducing toxicity in non small cell lung cancer.  This modIn particular in a study conducted on for 21 patients with N2/N3 NSCLC by van Der Wel et al. the use of PET/CT to define the radiotherapy target volume, demonstrated an increase in term of dose delivered to the tumour associated with a reduction of esophagus and lung toxicity. For assessing treatment with chemotherapy or radiotherapy in patients with non small cell lung cancer, PET and PET/CT play a major role than CT alone due to the fact that metabolism is a more sensitive marker for response to therapy than morphology. In patients undergoing therapy, imaging can play a crucial role and may aid in predicting the outcome of treatment regimens . After rDooms et al. in a very recent study evaluated the role of FDG PET in a particular subset of patients. This population was represented by resectable stage IIIA-N2 non small cell lung cancer subjects for whom therapy is often induction chemotherapy followed by resection. The authors concluded that FDG PET should select patients, after induction chemotherapy, who can be considered for surgical treatment . In factDespite the results of the various treatments it needs to consider that the radiotherapy could be responsible of inflammatory reactions that could give false positive to FDG PET examination. For this reason, to have a real evaluation of the response to the treatment is necessary to attend some weeks after chemotherapy administration and several months after radiotherapy .The clinically efficacy of the follow-up of lung cancer and the treatment of recurrence are the subject of controversy. RelapsiThus another possible application of PET/CT is evaluation of patients with the suspect of recurrence during the follow-up. This is supported by clinical advantage to detect not only local relapse but also distant metastatic spreading, with a significant impact on patient's management and selection. When considering suspected recurrences, it has been reported that PET may have a major impact on treatment. In one study, PET affected 63% of patients with possible relapse .In a recent study by Hellwig et al. the authHence, also in this field, fusion technology, gaining anatomical and physiologic data will give useful information, supporting the correct management of these patients.The authors declare that they have no competing interests.


Occult or latent hepatitis B virus (HBV) infection is defined as infection with detectable HBV DNA and undetectable surface antigen (HBsAg) in patients' blood. The cause of an overt HBV infection becoming an occult one is unknown. To gain insight into the mechanism of the development of occult infection, we compared the full-length HBV genome from a blood donor carrying an occult infection (d4) with global genotype D genomes.The phylogenetic analysis of polymerase, core and X protein sequences did not distinguish d4 from other genotype D strains. Yet, d4 surface protein formed the evolutionary outgroup relative to all other genotype D strains. Its evolutionary branch was the only one where accumulation of substitutions suggests positive selection (dN/dS = 1.3787). Many of these substitutiions accumulated specifically in regions encoding the core/surface protein interface, as revealed in a 3D-modeled protein complex. We identified a novel RNA splicing event (deleting nucleotides 2986-202) that abolishes surface protein gene expression without affecting polymerase, core and X-protein related functions. Genotype D strains differ in their ability to perform this 2986-202 splicing. Strains prone to 2986-202 splicing constitute a separate clade in a phylogenetic tree of genotype D HBVs. A single substitution (G173T) that is associated with clade membership alters the local RNA secondary structure and is proposed to affect splicing efficiency at the 202 acceptor site.We propose an evolutionary scenario for occult HBV infection, in which 2986-202 splicing generates intracellular virus particles devoid of surface protein, which subsequently accumulates mutations due to relaxation of coding constraints. Such viruses are deficient of autonomous propagation and cannot leave the host cell until it is lysed. Occult HBV infections are defined as the presence of HBV DNA and the absence of HBV surface antigen (HBsAg encoded by the S gene) in plasma or serum of HBV-infected patients . This inIn HBV sequences obtained from serum samples of HBsAg seronegative carriers, a plethora of mutations has been observed -10. Poineditorial). Posttranscriptional reduction of surface protein and mRNA expression to a background level was due to a single G458A substitution [Replication-defective mutants of HBV have been detected in the circulation of symptom-free individuals as early as 1987, and a notable example showed a deletion in to the pre-S region , which mtitution  and coultitution .Recently, we obtained sequence information for HBV strains present in occult infections . Based oEU155893, d4) takes the outgroup position in a bootstrapped phylogenetic tree based on JTT-estimates of amino acid replacements in genotype D surface proteins  remain unaffected, while sequences for large, middle and small surface protein gene expression in the overlapping reading frame are deleted. As a result of this post-transcriptional event, a virus genome may regularly replicate and be encapsidated inside the host cell, but cannot be enveloped and hence has lost the ability to exit the host cell and to enter new cells. These molecular properties match the characteristics of occult HBV infection.Splicing of HBV RNA is considered not to be essential for HBV propagation. Intriguingly, an association was reported between RNA splicing and the generation of replication-defective HBV variants -17. We atp domain of polymerase.Notably, the splice 2986 to 202 is rather unique in this virus-inactivation aspect. Other splice opportunities may not occur due to proximity 459 to 488), may induce a frame-shift (2986 to 488 or 734) or may affect essential viral functions . As shown by zu Putlitz and coworkers , deletio59 to 488Splice prediction in human mRNA by means of NetGene2 is a joint assignment method combining consensus sequence information with parameters of coding/non-coding transitions. It could be argued that an overlapping gene structure may interfere with these criteria. However, NetGene2 performs reliably in the prediction of splicing events that have been described to occur Fig . For insth mutation T173G displayed the ability of changing a grey phenotype (T) into a black one (G) and vice versa. This mutation is synonymous in the reading frame for surface protein (13Leu) and replaces a Ser (T) for an Ala (G) at the polymerase frame. The nucleotides A (Thr) and C (Pro) have not been found in this position. The T-to-G mutation interrupts a polypyrimidine tract that is likely to promote RNA splicing at the neighboring 202 splice acceptor site [The ability to promote 2986-202 RNA splicing may not be a special property of d4 HBV. In a collection of 104 HBV genotype D representatives, NetGene2 reported another 32 cases. Remarkably, 29 of these strains constitute a separate clade in a phylogenetic tree based on amino acid replacements in the polymerase protein of these viruses Fig . A tree tor site ,29. Alsotor site .In conclusion, a single nucleotide substitution brings on a bipartition among the genotype D HBVs causing a difference in tendency for 2986-202 RNA splicing and hence for the development of an overt into an occult HBV infection.We describe a thusfar unrevealed RNA splicing alternative (2986-202) that is prominent in a subset of genotype D HBV strains. Splicing of HBV RNA according to this scenario will suppress the expression of surface proteins and spares the functions dedicated to the core and X proteins and to the functional domains  of the viral polymerase. Consequently, virus genomes do replicate and are being encapsidated properly, but the virions are defective due to the absence of surface protein. These virus particles remain captured intracellularly and their propagation becomes dependent on liver cell division. Their release (without immune-reactive surface protein) to an individual's circulation and immune system depends on the turn-over of the infected liver cells. These properties are typical for HBV variants in blood samples of individuals with occult infection  like theExperimental evidence for a causal connection of 2986-202 RNA splicing with occult HBV infection is currently lacking, which is mainly due to the fact that collecting liver biopsies from healthy volunteers with occult HBV infection is much more complicated than obtaining blood samples. When analyzing occult HBV in blood samples, selection is inevitably in favor of HBV variants that have reached the patient's circulation. The results of splice prediction in HBV of frozen liver specimens . The C-May some of these surface protein deficient HBV variants reacquire the ability to initiate productive infection after a prolonged period of occult infection? Relevant scenarios must include a restoration of virus functionality damaged during the period of latency. From the evolutionary point of view, it is beyond expectation that these deficient viruses are able to achieve this solely by means of random mutation and natural selection within the duration of an individual's life, particularly because virus propagation approaches the zero level. Other options of the virus to regain infectivity and propagation are complementation and/or recombination catalyzed by superinfection of the host cell with another HBV strain. Also, it is likely that a single individual with occult HBV infection may carry quasi-species with different causes of latency waiting for superinfection or other triggers to become reactivated. This scenario gains improbability with time as inactivating mutations will accumulate in the surface protein genes. Finally, a small fraction of the liver cells may escape the scenario towards occult infection and may still continue to produce small amounts of infectious virions, which are effectively scavenged by the immune system of an alert host. These cells may induce a reactivation towards overt HBV infection under conditions of immunosuppression. The duration of occult HBV infection – in particular the impact of accumulated mutations – might be an important parameter in order to discern a superinfection from a reactivated existing HBV infection.A novel splicing opportunity of HBV mRNA prevents surface protein expression in HBV genotype D without affecting other gene functions . This splicing event may become dominant by intracellular evolution and selection. In this case, S-antigen is no longer produced and E-antigen is still secreted. A minute amount of HBV DNA can be detected in the patient's blood due to regular turn-over of infected cells. These criteria match the definition of an occult HBV infection.EU155889–EU155895), including a full-length genome  and six shorter sequences [Recently, we obtained HBV sequences from five donors with occult HBV infection  was empequences ,39 and tequences  in orderequences . The PDBequences  was the equences . Dockingequences . Predictequences ,43. Cutoequences  was usedequences .PDB: Protein Data Base (of X-ray structures); ALASCAN: (server performing) computational alanine replacement scanning.The authors declare that they have no competing interests.FJvH performed the analyses, HLZ provided materials, BB and VVL were supervisors and all authors were involved in writing the manuscript.A PDB formatted data file carrying the coordinates of the complex between core and surface protein of HBV genotype D.Click here for file


MYC signalling pathways. Importantly, the presence of these transcriptional signatures in primary ALL specimens significantly predicted patient outcome. We conclude that in lymphocytes the activation of bioenergetic pathways required for proliferation may suppress the apoptotic potential and offset the metabolic crisis initiated by GC signalling. It is likely that the link between GC resistance and proliferation in T-ALL has not been fully appreciated to date because such effects would be masked in the context of current multiagent therapies. The data also provide the first evidence that altered expression of wild-type MLL may contribute to GC-resistant phenotypes. Our findings warrant the continued development of selective metabolic inhibitors for the treatment of ALL.Glucocorticoids (GCs) are among the most important drugs for acute lymphoblastic leukaemia (ALL), yet despite their clinical importance, the exact mechanisms involved in GC cytotoxicity and the development of resistance remain uncertain. We examined the baseline profile of a panel of T-ALL cell lines to determine factors that contribute to GC resistance without prior drug selection. Transcriptional profiling indicated GC resistance in T-ALL is associated with a proliferative phenotype involving upregulation of glycolysis, oxidative phosphorylation, cholesterol biosynthesis and glutamate metabolism, increased growth rates and activation of PI3K/AKT/mTOR and  In children with acute lymphoblastic leukaemia (ALL) cellular drug sensitivity is a major component of clinical outcome. Event-free survival for these patients is now greater than 75% but a significant number continue to relapse and the outlook for these is dismal . Around L-asparaginase as a cornerstone of chemotherapy for ALL.Despite the clinical importance of this class of drug, the exact mechanisms involved in GC cytotoxicity and the development of resistance remain uncertain , the latOver the past 20 years, our laboratory has developed an authenticated panel of paediatric ALL cell lines that have been grown in the absence of drug selection (The cell line panel has been previously described  and compThe sensitivity of the T-ALL cell lines to methylprednisolone (MPRED) and DEX has been previously published . For serACTB and GAPDH.Briefly, RNA was extracted from cell lines in exponential growth phase and hybridised to Affymetrix HG-U133A microarrays  in accordance with our previously published protocols  as described . This tool identifies compounds that elicit transcriptional responses with similarities to the gene signature under test, and thus with potential for synergistic activity. Compounds significantly associated with GC-resistance signatures were identified using the CMAP permuted output. Drugs that demonstrated significant enrichment scores against both DEX and MPRED were tested for cytotoxicity against ALL cell lines in the presence and absence of DEX (see above).The top 100 positive and top 100 negative probe sets correlating with MPRED or DEX IC50 values were analysed using the CMAP software  to reduce the dimensionality of the data . The first three principal components from each gene set were then used in logistic regression to generate a binary classification of predicted outcomes  for the two patient cohorts. The log-rank statistics produced from these initial analyses were found to be highly  significant in almost all cases, an artefact of data overfitting by PCA and logistic regression. To control for this, the data were re-analysed 999 times using random permutations of the patient outcome labels to generate true (permuted) P-values for each gene set. Kaplan–Meier analyses to assess differences in relapse-free survival were generated using permutation testing to generate P-values for each gene set.Gene Set Enrichment Analysis gene sets associated with MPRED resistance were modelled for their ability to predict clinical outcome using previously published microarray data from 59 B-lineage and 17 T-ALL primary paediatric ALL diagnostic bone marrow specimens  vs both drugs . Of the genes listed in OPHN1 and NF1 as part of a gene signature that can distinguish diagnosis and relapse specimens from paediatric T-ALL patients (in vitro model is relevant to the in vivo situation.The resistance profiles of MPRED and DEX across the panel (IC50 scores) were correlated with gene-expression profiles as determined by HG-U133A microarray. The top 20 probe sets positively or negatively correlating with GC resistance are listed in cis-regulatory motifs) and C4 . To incorporate pathway information specifically related to the biology of leukaemia, we manually curated an additional database  for 63 genes known to be important in the biology of ALL resulting in 512 gene sets  database in this study for MPRED or DEX, indicating that genes associated with GC resistance were not confined to particular genomic loci.To interrogate the biological pathways represented by GC-resistance signatures, we used GSEA .One of the most prominent features associated with GC resistance in T-ALL in the present study involved upregulation of metabolic pathways. Closer inspection of these data revealed a compelling pattern of gene regulation associated with these changes . In the CROT, CRAT and SLC25A20 ; PDK3 ; LCAT ; DBT ; and GLUL (glutamine synthetase). These observations are consistent with an alteration in amino-acid metabolism and the downregulation of fatty acid β-oxidation. Together with the enrichment of gene sets associated with cell cycle, transcription, translation, MYC regulation and proteasomal pathways  and DEX IC50 values , see vs 10% fetal calf serum for 7 days inhibited the growth rate of HSB2 cells by 63%  or B-lineage (n=59) patient specimens are listed in P<0.005, P<0.05 GLUL  or multidrug-resistance pumps. To directly assess the contribution of these mechanisms to GC resistance in our T-ALL cell lines, we correlated the expression of 142 genes curated from the literature (corresponding to 253 probe sets) involved in canonical mechanisms of multidrug-resistance, apoptosis and survival signalling with MPRED and DEX resistance , CYP3A4 (downregulated), CCND1  and the multidrug transporter ABCC9 (upregulated). In contrast to previous work reporting an association between prednisolone sensitivity and expression of MCL1 or DAPK1 in ALL patient specimens , PARP (poly ADP-ribose polymerase), SMAC (homolog of DIABLO) and cytochrome c, all of which are key genes in the initiation of apoptotic signalling. Although initially counter-intuitive that expression of these critical pro-apoptotic factors might be associated with increased GC resistance in ALL cell lines, all of these genes have important functions in normal cellular function. PARP has multiple biological functions including regulation of gene expression, cell division, differentiation and DNA repair, whereas AIF and cytochrome c have important functions in mitochondrial metabolism and protection from oxidative stress. Thus, their positive correlation with GC resistance is likely to be indicative of biological processes other than initiation of apoptosis. The only anti-apoptotic factor significantly associated with resistance to both GCs was TRAF2 (TNF receptor-associated factor 2). In summary, there was no compelling signature at the level of gene expression of a coordinated deregulation of apoptotic pathways underpinning in vitro GC resistance.Aside from abnormalities in the activation or functionality of the GR, known resistance mechanisms for GCs include defects in apoptosis (sistance . Genes svs DEX (P<0.0001), confirming previous findings  CEM line (>250 μM) , a key component of the PI3K/AKT/mTOR signalling pathway. The PI3K inhibitor LY294002 also targets this pathway and thus might be expected to have similar effects. Similar to rapamycin, LY294002 had no direct cytotoxic effects on ALL-SIL cells  . AlthougIL cells  when addμM DEX resulted in an additive cytotoxic effect. This is the first assessment of the cytotoxic effects of these compounds in combination with GCs in ALL and suggests that their inclusion in therapeutic regimens may have important clinical benefits.Both quercetin and resveratrol demonstrated dose-dependent cytotoxicity in ALL-SIL cells in the absence of DEX . ImportaThe present study provides compelling evidence that cellular metabolism and proliferation are important aspects of GC resistance in ALL. It has long been recognised that an alteration in cellular metabolism is a fundamental phenotype of cancer  includinINK4A  and have confirmed the ability of this drug to sensitise even highly resistant ALL cell lines. Importantly, the expression of leading edge genes from the rapamycin-response signature in T-ALL diagnostic specimens could predict patient outcome; the same was true of the gene signatures associated with ex vivo DEX resistance, demonstrating the clinical relevance of this previously described signature , AKT and mTOR. Synergy between the AKT/mTOR pathway and GC sensitivity has previously been explored based on the finding that rapamycin responses show a significant relationship to DEX-resistance signatures in primary specimens from ALL patients . On the basis of our data, we hypothesise that the observed correlation with GC sensitivity in T-ALL cell lines is related to expression levels of wild-type MLL rather than MLL-translocation products. The relationship between MLL expression, proliferation and GC resistance is currently being explored in our laboratory.During the course of this investigation, we found an unexpected correlation between GC resistance and reduced expression of opoiesis . This coMLL rearrangements, induction failure or relapse. Since preparation of this paper, new observations to support our argument have been published by In summary, the present study provides compelling evidence that upregulation of cellular metabolism and proliferation is an important aspect of GC resistance in ALL and may contribute to patient outcome. We hypothesise that these changes simultaneously offset the adverse metabolic consequences of GC signalling whilst potentially suppressing apoptotic potential through the modulation of bioenergetic pathways. These observations support the continued development of selective metabolic inhibitors for the treatment of ALL, particularly for patients that are likely to be GC resistant, such as those with


The haematopoietic system is sensitive to cytotoxic damage and is often the site of dose-limiting toxicity. We previously reported that swainsonine, an inhibitor of protein glycosylation, reduced the bone marrow toxicity resulting from a single dose of anticancer drugs in otherwise healthy mice. However, more important questions are (1) can swainsonine protect tumour-bearing mice without interfering with the anti-tumour effects of the drugs, and (2) can swainsonine stimulate haematopoietic activity of human, as well as murine, bone marrow. We demonstrate here that swainsonine protects C57BL/6 mice bearing melanoma-derived tumours from cyclophosphamide-induced toxicity without interfering with the drug's ability to inhibit tumour growth. Similar results were obtained in vivo with 3́-azido-3́-deoxythymidine (AZT), a myelosuppressive agent often used in therapy for acquired immune deficiency syndrome. Swainsonine increased both total bone marrow cellularity and the number of circulating white blood cells in mice treated with doses of AZT that typically lead to severe myelosuppression. Swainsonine also increased the number of erythroid and myeloid colony forming cells (CFCs) in short-term cultures of murine bone marrow, restoring the number of progenitor cells to the control level in the presence of AZT doses that reduced CFCs by 80%. With respect to the sensitivity of human haematopoietic cells to swainsonine, we show that swainsonine protected human myeloid progenitor cells from AZT toxicity in vitro. These results suggest that swainsonine may be useful as an adjuvant in several types of human chemotherapy. © 1999 Cancer Research Campaign


We describe the role of intravitreal anti-vascular endothelial growth factor (VEGF) agents in Coats' disease in children. In a prospective, interventional, non-randomized case series, three patients (three eyes) aged 16, seven and two years were diagnosed to have Coats' disease. In Case 1  with macular edema, previous laser photocoagulation being unsuccessful, intravitreal pegaptanib sodium (Macugen™) was tried. Case 2  and Case 3  were diagnosed to have Stage 4 Coats' and underwent external needle drainage, laser photocoagulation, SF6 gas injection and intravitreal injection of bevacizumab (Avastin™). Reduction of exudation and attached posterior pole (Cases 2 and 3) was seen at a follow-up of six months and two months respectively. Intravitreal anti-VEGF agents may be successfully used as adjunct treatment in select cases of Coats' disease in childhood. George Coats in 1908 described an idiopathic retinal vascular disorder characterized by abnormal telangiectasia with a progressive deposition of intraretinal or subretinal exudates, potentially leading to exudative retinal detachment.The current treatment modalities aim at obliterating the affected retinal vessels by laser photocoagulation and cryotherapy. Laser photocoagulation is the treatment of choice in the early stages of Coats' disease. Cryotherapy is more effective for lesions in the far periphery and in the presence of exudation. Both techniques become less effective once the retina is detached and when more than two quadrants are affected. There is no universal recommendation for treatment in advanced Coats' disease and often the results are not optimum.Recently, anti-vascular endothelial growth factor (VEGF) agents have been tried in the management of diabetic retinopathy and proliferative vascular disorders such as Coats' disease with modest success.–5 We repA 16-year-old boy with Coats' disease in the right eye had prior trans-conjunctival cryotherapy and laser photocoagulation. His best corrected visual acuity (BCVA) at presentation was 20/400 in the right eye. On fundus examination, the affected eye showed diffuse macular edema with subretinal fluid (SRF) and exudation with telangiectatic vessels in the inferior quadrant . His optA seven-year-old male child presented to us with diminution of distance visual acuity since two and a half years in the left eye associated with inward deviation of the eye. On examination his left eye BCVA was perception of light. Fundus examination was normal for the right eye and showed total retinal detachment with subretinal exudation and cholesterol crystals with dilated vessels in the left eye  B Scan uA two-year-old female presented with leucocoria in the left eye since one year. Fundus examination showed telangiectatic vessels, subretinal exudates and total RD. B Scan ultrasonography of the left eye corroborated the exudative RD. CT (Computerized Tomography) scan or B scan did not reveal any calcification. External needle drainage of the SRF and laser photocoagulation followed by injection of SF6 and intravitreal bevacizumab (Avastin™) (1.25 mg/ 0.05 ml) was done. The histopathological examination of the SRF showed presence of amorphous acellular eosinophilic material with no malignant cells. Follow-up after two months showed a marked improvement in the retinal exudation and telangiectasia with reattachment of the posterior pole. The non-standard and off-label use of anti-VEGF drugs was clearly explained to the parents of all above patients.VEGF is a naturally occurring protein, which causes increased vascular permeability (important for the initiation of angiogenesis), endothelial cell migration and proliferation. The pathet al. showed elevated levels of VEGF in Coats' disease which rapidly reduced after injection of pegaptanib sodium. VEGF levels reduced from 908 pg/ml to 167 pg/ml  and an improvement of exudation, hemorrhage and near complete reattachment of the retina. Thus, they suggested that there may be a component of dysregulation of VEGF-mediated angiogenesis in Coats' disease.[Sun  disease.In our case series, we had not obtained consent for biochemical analysis except in Case 3 where we confirmed our diagnosis with light microscopy and histopathology of the drainage fluid.More recently, Venkatesh et al. reported two older children (14 years and 16 years) with Coats' disease treated with intravitreal bevacizumab (1.25 mg/0.05 ml). They concluded that despite reduction of macular edema, exudation and telangiectasia after injection, intravitreal injection may not alone suffice in all cases.[4Anti-VEGF being an adjunct treatment, the timing of this may be varied depending on the clinical judgment. In cases with extensive exudation and media haze, laser may be deferred while anti-VEGF agents may be the preferred treatment. Due to their anti-permeability role, they may be utilized to clear the exudation and media haze to visualize the vessels for ablation. Also, in cases such as Case 1, with previous unsuccessful laser treatment for macular edema, anti-VEGF agents may be the preferred treatment.Our communication highlights the merit of augmenting intravitreal injection with conventional treatment (laser/cryo/needle drainage). With increasing use of anti-VEGF agents in younger age patients for diseases such as retinopathy of prematurity (ROP) and Coats' disease, clinicians should also bear in mind the role of VEGF in the development of retinal vessels not only in the retinal periphery, but also in the macula. Long-term visual outcomes of the use of anti-VEGF agents in children are unknown. Until such time cautious use of anti-VEGF agents is advisable. Repeated injections of anti-VEGF agents are required to control the neovascular response in age-related macular degeneration (AMD). In Coats' disease, it is possible that with anti-VEGF therapy, repeated injections may be needed. However, with the use of concomitant therapeutic modalities such as laser, cryo and needle drainage, we can minimize the need for repeated injections.Due to the multiple procedures done before injection of anti-VEGF agents in our cases, it is difficult to determine if anti-VEGF agents alone were responsible for the anatomical success that was seen especially in Cases 2 and 3. We did not encounter any systemic or ocular side-effects (e.g.: increased intraocular pressure) in these three patients. A larger study would be needed to affirm the safety and efficacy of intravitreal injections in the pediatric population.


There are no effective interventions that significantly forestall or reverse neurodegeneration and cognitive decline in Alzheimer's disease. In the past decade, the generation of new neurons has been recognized to continue throughout adult life in the brain's neurogenic zones. A major challenge has been to find ways to harness the potential of the brain's own neural stem cells to repair or replace injured and dying neurons. The administration of hematopoietic growth factors or cytokines has been shown to promote brain repair by a number of mechanisms, including increased neurogenesis, anti-apoptosis and increased mobilization of bone marrow-derived microglia into brain. In this light, cytokine treatments may provide a new therapeutic approach for many brain disorders, including neurodegenerative diseases like Alzheimer's disease. In addition, neuronal hematopoietic growth factor receptors provide novel targets for the discovery of peptide-mimetic drugs that can forestall or reverse the pathological progression of Alzheimer's disease. Hematopoietic growth factors (HGFs) enhance proliferation, differentiation, and release of blood cell lineages into the circulation. Interestingly, these same cytokines have been shown to influence the proliferation of neural stem/progenitor cells. Review of the similarities and differences between the stem cells that give rise to neural cell lineages ('neuropoiesis'), and those that generate the various blood cell lineages (hematopoiesis), has led to the concept of the 'brain marrow' ,2. 'BraiMany of the cytokines and growth factors required for both hematopoeisis and neuropoiesis are constitutively expressed by bone marrow (BM) stromal cells ,2. Two cOther regulatory proteins that influence blood cell differentiation have effects on neuronal development. Erythropoietin, in addition to its function to promote the production of red blood cells, also has trophic effects on central cholinergic and cortical neurons . Erythrokit, the receptor for one of the major hematopoietic cytokines, stem cell factor (SCF). In hematopoietic cells, SCF and c-kit regulate a variety of developmental events, including cell proliferation, survival and differentiation [in vitro and in vivo [c-kit expressed in neurogenic zones of the brain, but ischemia has been reported to alter c-kit expression, which in turn stimulates neural stem cell (NSC) proliferation and differentiation into neurons [c-kit on NSCs also appears to play a key role in triggering NSC migration from the neurogenic zones to sites of brain injury [Neural stem/progenitor cells of the brain express c-ntiation . SCF has in vivo . Not onl neurons . The intn injury .Granulocyte-colony stimulating factor (G-CSF) is a potent growth factor that stimulates proliferation of hematopoietic stem cells and drives differentiation of myeloid lineage cells. It is used extensively in clinical practice to accelerate recovery of patients from neutropenia after cytotoxic therapy. G-CSF also impacts neural tissues . When adBoth neuronal G-CSF and its receptor are up-regulated in the ipsilateral forebrain hemisphere 2 hours after the occlusion of the middle cerebral artery and reperfusion in a rat model of stroke . In a siA growing focus of research has been on the discovery of multipotent stem cells in BM that are capable of giving rise to tissues of all embryonic germ layers . BM-deriin vitro, utilizing different sub-populations of marrow, and various combinations of cell culture media and growth factors. The combination of retinoic acid and brain-derived neurotrophic factor (BDNF) was effective in driving a BM stromal cell population into neuron-like cells [In vivo work on the problem of transdifferentiaton suggests that generation of new neurons from a bone marrow progenitor, if it really occurs, may be a rare phenomenon. Nevertheless, BM cells from the periphery are the source of a small proportion of the brain's immune cells (microglia) in the normal adult brain, and following various brain injuries, the trafficking of BM cells from blood to brain is markedly increased.The demonstration of transdifferentiation of BM cells has primarily been performed ke cells . More reke cells . In vivoMicroglia have been clearly observed in the core of amyloid plaques in transgenic mouse models of Alzheimer's disease (AD). Perhaps as many as 10% of these cells originate from the BM, and the amyloid-β (Aβ)-40 and Aβ-42 isoforms are able to trigger this chemoattraction. These newly recruited cells also exhibit a specific immune reaction to both exogenous and endogenous Aβ in the brain .Infiltration of the brain parenchyma by BM-derived cells in the course of cerebral amyloidosis has been demonstrated by tracking green fluorescent protein (GFP)-BM in the tg mouse model (APP23 mice) of AD . These m+ BM cells to create chimeric mice before the onset of AD-like pathology and their brains were analyzed 6.5 months later. The number of engrafted BM-derived cells was significantly higher than in age-matched wild-type mice. Moreover, the number of BM-derived cells associated with Aβ was significantly higher than in older transgenic mice transplanted after the establishment of AD-like pathology. Local inflammation caused by intrahippocampal lipopolysaccharide injection significantly increased the engraftment of BM-derived cells in old AD mice and decreased the hippocampal Aβ burden. These results suggest that infiltration of BM-derived monocytic cells into the brain contributes to the development of a microglial reaction in AD [In another study, young transgenic mice were transplanted with GFPon in AD .Most recently, administration of G-CSF into two different Aβ-induced AD mouse models substantially improved performance on the Morris water maze . In the ®), filgrastim (Neupogen®), and pegylated filgrastim (Neulasta®); the pegylation conferred a longer duration of action.A promising target for the development of a novel anti-AD therapy is the G-CSF receptor and its ligand G-CSF. The natural human glycoprotein exists in 174 and 180 amino acid forms of approximately 19,600 grams per mole. The more abundant and active 174 residue form has been used in the development of pharmaceutical products by recombinant DNA technology. The three proprietary recombinant G-CSF proteins include lenograstim  that plays a key role in hematopoiesis, acting both as a positive and negative regulator, often in synergy with other cytokines. It also plays a pivotal role in mast cell development, gametogenesis, melanogenesis and neurogenesis. The SCF receptor,  regions . When SC regions .Since G-CSF is already in advanced clinical trials for ischemic stroke , pre-cliin vitro, and in vivo studies have shown that BM cells migrate into the brain where they appear to differentiate into neurons and glia. The mechanism for transdifferentiation of BM to neural cells is not clear and may reflect the capacity of BM-derived cells to fuse with injured neurons. The in vitro demonstrations of transdifferentiation of BM cells has utilized different sub-populations of marrow and various combinations of cell culture media and growth factors. The combination of retinoic acid and BDNF were effective in driving a stromal BM cell population into neuron-like cells. More recently, a fragment of amyloid precursor protein has been reported to potentiate the nerve growth factor/retinoic acid-induced transdifferentiation of BM-derived adult progenitor cells into neural progenitor cells, and more specifically, enhances their terminal differentiation into a cholinergic-like neuronal phenotype. In vivo studies on the problem of transdifferentiation suggest that generation of new neurons from a bone marrow progenitor, if it really occurs, may be a rare phenomenon. Nevertheless, BM cells from the periphery are the source of at least 10% of the adult brain's immune cells (microglia) under normal conditions, and following various brain injuries, the trafficking of BM cells from blood to brain is markedly increased. Microglia have been clearly observed in the core of amyloid plaques in transgenic mouse models of AD. Perhaps as many as 10% of these cells originate from the BM and the Aβ-40 and Aβ-42 isoforms are able to trigger this chemoattraction. These newly recruited cells also exhibit a specific immune reaction to both exogenous and endogenous Aβ in the brain. G-CSF inhibits programmed cell death and promotes neurogenesis. Given that G-CSF administration in a mouse model of AD has been reported to improve performance in a hippocampal-dependent learning task and administration of G-CSF has been shown to be safe and well-tolerated in early clinical studies in patients with ischemic stroke, initiation of clinical trials to reverse dementia or forestall progression of AD is warranted.The application of HGF for the treatment of AD has not yet been undertaken but there is a strong rationale for initiating clinical studies. In the past five years it has become evident that BM houses more 'primitive', multipotent stem cells that are capable of giving rise to tissues of all embryonic germ layers. BM-derived cells have been shown by independent investigators to give rise to neural cells Aβ: amyloid-β; AD: Alzheimer's disease; BDNF: brain-derived neurotrophic factor; BM: bone marrow; BMP: bone morphogenetic protein; G-CSF: granulocyte-colony stimulating factor; GFP: green fluorescent protein; HGF: hematopoietic growth factor; HSC: hematopoietic stem cell; NSC: neural stem cell; SCF: stem cell factor.The authors declare that they have no competing interests.Each author contributed to the concepts discussed and participated in preparation of the manuscript.


Hepatic encephalopathy has a negative impact on health-related quality of life (QoL) in liver cirrhosis. There are scarce and conflicting data on whether type or etiology of liver cirrhosis could be related to hepatic encephalopathy in patients with cirrhosis. We aimed to determine the impact of cirrhosis etiology on hepatic encephalopathy and whether hepatic encephalopathy affects health-related QoL among patients with cirrhosis of different etiologies.A total of 156 cirrhotic patients were prospectively evaluated for the presence of hepatic encephalopathy according to the West-Haven criteria as well as by means of two psychometric tests. Patients with cryptogenic cirrhosis or cirrhosis due to mixed hepatocellular/cholestatic etiologies were excluded. Fasting plasma glucose levels were also measured. QoL was evaluated by means of a validated questionnaire (SF-36).Diabetes mellitus was more common in patients with hepatocellular cirrhosis compared to those with cholestatic cirrhosis but the two groups did not differ in cirrhosis severity or the prevalence of hepatic encephalopathy (p > 0.05). The groups of patients with cirrhosis due to alcohol, hepatitis C, or cholestatic liver disease did not differ in severity of liver cirrhosis or the prevalence of hepatic encephalopathy (p > 0.05). Patients with cirrhosis of different etiologies did not differ in any SF-36 domain (p > 0.05). In multivariate analysis, performance at neuropsychological testing was independently related only to age, diabetes mellitus, and the Child-Pugh score whereas the SF-36 physical component summary only to the Child-Pugh score and hepatic encephalopathy.Cirrhosis etiology does not seem to be related to hepatic encephalopathy or health-related QoL. Cognitive impairment is associated mainly with age, liver disease severity and diabetes mellitus. Patients with liver cirrhosis are prone to develop cognitive dysfunction termed hepatic encephalopathy (HE). The clinical manifestations of HE range from subtle intellectual and personality changes, that can be detected only by means of neuropsychological and neurophysiological tests , to coma . The patPublished data on cognitive impairment in cirrhosis due to cholestatic liver disease compared to cirrhosis due to hepatocellular disease are few and conflicting ,4. RecenPrevious studies have explored cognitive function in alcoholic patients as well as in patients with chronic hepatitis C and primary biliary cirrhosis (PBC), and all have been reported to have cognitive impairment in the absence of cirrhosis -12. HoweHealth-related quality of life (QoL) is impaired in patients with cirrhosis -20. LiveThe primary aim of the current study was to investigate the relation of the type (hepatocellular vs. cholestatic) and etiology of liver cirrhosis with the presence of hepatic encephalopathy. We also aimed to evaluate the relation of etiology of liver disease with health-related QoL .Consecutive adult patients with liver cirrhosis admitted to the gastroenterology ward or attending the outpatient clinic of the gastroenterology department at a transplant center in Sweden were prospectively enrolled. Inclusion criterion was liver cirrhosis of any cause. The diagnosis of liver cirrhosis was established histologically or based on the presence of at least 2 of the following: characteristic imaging features, esophageal or gastric varices, ascites, increased international normalized ratio (INR) that could not be attributed to any other cause. Patients unable to understand Swedish as well as those unable to give written informed consent or to fill in questionnaires  were excluded. Also excluded were patients with cryptogenic cirrhosis, mixed hepatocellular and cholestatic chronic liver disease (e.g. overlap syndrome), or with cirrhosis after liver transplantation. Patients with cryptogenic cirrhosis were excluded as the underlying etiology is unclear. Although a history of alcohol abuse is lacking in these patients it is difficult to exclude with certainty an alcoholic etiology in some. It has been recognized, in recent years, that a significant proportion of patients with cryptogenic cirrhosis might be due to a long-standing non-alcoholic steatohepatitis (NASH). Patients with NASH cirrhosis were included in the current study only if NASH had been appropriately diagnosed prior to cirrhosis development. Patients with unknown cirrhosis etiology despite extensive investigations and in the absence of a history of diagnosed NASH prior to cirrhosis development were excluded to allow proper allocation of enrolled patients to type and etiology groups. Out of 176 consecutive patients who fulfilled the inclusion criteria and were approached, 156 patients (87%) agreed to participate in the study and completed the questionnaires. Patients hospitalized because of acute diseases or complications related to liver disease were evaluated when stable clinical conditions were reached. No patient was on dialysis and none had hepatorenal syndrome. Patient data were collected from medical records, including etiology of liver disease, previous variceal bleeding, existing esophageal or fundic varices, and hepatocellular carcinoma. No patient was receiving interferon therapy at inclusion in the study. Serum albumin and bilirubin as well as fasting plasma glucose and the INR were measured by standard in-house methods. Patients were considered having diabetes if they were receiving antidiabetic treatment  or had elevated fasting plasma glucose levels (> 7 mmol/l). The severity of the liver disease was assessed according to the Child-Pugh classification and the Model for end-stage liver disease (MELD) score . The preth World Congress of Gastroenterology in 1998 [HE was evaluated according to the guidelines proposed by the 11 in 1998 . It was  in 1998 . Instruc in 1998 . Every e in 1998 . On the  in 1998 ,23.The generic health-related QoL instrument Short Form-36 (SF-36) was developed as a comprehensive measure of general health status for use in the Medical Outcomes Study, and has been thoroughly tested for validity and reliability -27. ThisData are expressed as mean (SD). The student's t-test or the Mann-Whitney test was performed as appropriate in order to compare continuous variables. The Chi-square or the Fisher's exact test was used as appropriate for comparisons between categorical variables. The Pearson's correlation coefficient was calculated for correlation analysis between continuous variables. For multiple comparisons among groups one-way ANOVA for continuous variables or chi-square test for categorical variables was performed. If p < 0.05 the Bonferroni test (continuous variables) or the chi-square test  was used for post-hoc comparisons. First, patients with different types of liver disease (hepatocellular vs. cholestatic) were compared as regards to the presence of hepatic encephalopathy as well as other clinical parameters. Subsequently, patients with one of the major three etiologies  were compared. In an attempt to explore independent predictors of NCT-A and NCT-B performance times as well as the SF-36 physical component summary, all parameters that were univariately correlated at p < 0.1 with these variables, were entered into stepwise linear multiple regression analyses. All tests were two-tailed and conducted at a 5% significance level.A total of 29 patients had cholestatic cirrhosis (11 with PBC and 18 with PSC) and 127 had hepatocellular cirrhosis ; 24 mixed alcoholic and viral; 6 autoimmune hepatitis; 4 NASH; one alpha-1-antithrypsine deficiency). Seventeen out of 79 patients with alcoholic or mixed alcoholic cirrhosis admitted to active alcohol overconsumption. The rest claimed to have been abstinent during the previous 3–6 months but only 28 these were followed-up by a specialized addiction center and had documented abstinence (by means of random urine or breath tests).Diabetes mellitus was less common among patients with cholestatic cirrhosis compared to those with hepatocellular cirrhosis but the two groups did not differ significantly in the prevalence of  hepatic encephalopathy nor in the prevalence of other complications or in severity of liver cirrhosis table . PatientPatients with compared to those without diabetes mellitus took longer time to perform NCT-A and B table . HoweverThe group of patients with hepatocellular cirrhosis was heterogeneous. In order to explore the potential effects of specific etiologies of liver disease on hepatic encephalopathy, patients with mixed etiologies or with etiologies represented in low numbers in the current cohort  were excluded from further analysis. Subsequently, the groups of patients with cirrhosis due to alcoholic liver disease, hepatitis C, or cholestatic liver disease were compared to one another table . DiabeteClinical parameters univariately related at p < 0.1 with the time needed to perform NCT-A or NCT-B were entered into stepwise multivariate regression analyses with NCT-A and NCT-B performance times as the dependent variables table . The timPatients with compared to those without  HE had lower SF-36 physical component summary (30.1 (12.8) vs. 40.6 (10.8), p < 0.001) and mental component summary (36.4 (12.8) vs. 41.8 (14.4), p < 0.05). However, etiology of liver cirrhosis was not related to health-related QoL  were used. Although neurophysiologic tools are often used in research studies there is no clear consensus as to the validity of these tests when used alone or in combination . In the on of HE ; clinicaWe conclude that the etiology of liver disease is not related to hepatic encephalopathy or health-related QoL in liver cirrhosis. Performance at psychometric testing is affected by age, diabetes mellitus, and cirrhosis severity. Although diabetes mellitus was more common among patients with hepatocellular compared to those with cholestatic cirrhosis, the prevalence of HE did not differ between the two groups.HE: hepatic encephalopathy; PBC: primary biliary cirrhosis; QoL: quality of life; INR: international normalized ratio; NASH: non-alcoholic steatohepatitis; MELD: model for end-stage liver disease; PSC: primary sclerosing cholangitis; NCT: number connection test; SD: standard deviation; SF-36: Short Form-36.The authors declare that they have no competing interests.EK participated in the design of the study, in data collection, analysis and interpretation, and wrote the manuscript. AJ participated in the design of the study and data collection/analysis, and revised critical the manuscript critically. EB participated in the design of the study and the interpretation of the results and revised the manuscript critically. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


The matrix invasion of lung carcinoma cells in the presence of the pleural fluid was significantly higher than that in the presence of culture medium alone or sera from normal subjects (P < 0.01). The invasion-stimulatory activity of the pleural fluid was strongly inhibited by HGF-neutralizing antibody. Positive correlation was found between the HGF level and invasion-stimulatory activity in the pleural fluids and normal sera (P = 0.0073). This is the first report demonstrating that the lung surgery induces a considerable amount of HGF, which is closely correlated with the invasion-stimulatory activity of the pleural fluid. © 1999 Cancer Research CampaignHepatocyte growth factor (HGF) is a stromal cell-derived cytokine that can stimulate matrix invasion by carcinoma cells. We analysed the concentrations of HGF and invasion-stimulatory activity in pleural fluid after lung surgery. The concentration of HGF in pleural fluids was measured by enzyme-linked immunosorbent assay in seven patients who underwent pulmonary resection for primary or metastatic lung cancer. The effect of the pleural fluid on cancer cell invasion across reconstituted basement membrane (Matrigel) was assessed with a Boyden chamber assay using a lung adenocarcinoma cell line, A549. HGF levels in the pleural fluid after lung surgery ranged from 6.0 to 23.0 ng ml


Gastric Leptin is absorbed by duodenal enterocytes and released on the basolateral side towards the bloodstream. We investigated in vitro some of the mechanisms of this transport. Caco-2/15 cells internalize leptin from the apical medium and release it through transcytosis in the basal medium in a time- temperature-dependent and saturable fashion. Leptin receptors are revealed on the apical brush-border membrane of the Caco-2 cells. RNA-mediated silencing of the receptor led to decreases in the uptake and basolateral release. Leptin in the basal medium was found bound to the soluble form of its receptor. An inhibitor of clathrin-dependent endocytosis (chlorpromazine) decreased leptin uptake. Confocal immunocytochemistry and the use of brefeldin A and okadaic acid revealed the passage of leptin through the Golgi apparatus. We propose that leptin transcytosis by intestinal cells depends on its receptor, on clathrin-coated vesicles and transits through the Golgi apparatus. Endocrine leptin is mainly produced by the white adipose tissue  and its Gastric chief cells synthesize and store leptin in their secretory granules which also contain pepsinogen . To ensuAt the level of the duodenal enterocyte apical membrane, leptin binds to its membrane-bound receptor triggering some signalling cascades , 11. In Interestingly, membrane-bound and soluble leptin receptors share a similar structure, have the same affinity for leptin and their competition modulates leptin action on the intestinal wall . FinallyMolecular events taking place for duodenal leptin transport have not been established. We have therefore undertaken the present study to characterize some aspects of the leptin intestinal transcytosis. Using human intestinal Caco 2/15 cells, we demonstrate that this transport across the cells is dependent upon the long isoform of the leptin receptor. We also show that as for other cell types the endocytotic process seems to be clathrin-dependent. In addition, our findings emphasize the role of the Golgi apparatus in the intracellular leptin trafficking and reveal that leptin released into the basal medium is bound to its protective soluble receptor.β-actin (clone AC15), fluorescein isothiocyanate (FITC), and albumin were from Sigma-Aldricht . Monoclonal mouse antiTNG38/42 (MA3-063) was obtained from ABR . Secondary antibodies with fluorescent tags Alexa 488 and Alexa 568 were from by Invitrogen . The secondary antibodies antirabbit/mouse for Western blot were part of the detection kit from Roche Diagnostics . Protein-A-HRP (Horse Radish Peroxidase) was from GE Healthcare limited . Human recombinant leptin was purchased from R&D systems . Recombinant insulin was from Eli Lilly .Rabbit antibodies against human leptin (A20) and human leptin receptor (H300) were purchased from Santa Cruz Biotechnology . Monoclonal mouse antiμg) and human recombinant leptin (1 mg) were solubilized in 300 μL of NaHCO3 buffer  and left overnight at 4°C with agitation, as previously reported ., 25.9, 2μg/mL) had no significant effect on leptin uptake while chlorpromazine (6 μg/mL) decreased leptin intracellular content by 57%  and nystatin  or okadaic acid (0.5 μM) prior to leptin addition. These agents are known to break down the Golgi apparatus and alter Golgi trafficking. Brefeldin A leads to the fusion of Golgi cisternae and rough endoplasmic reticulum, efficiently blocking protein trafficking [Since we previously demonstrated in in vivo studies that leptin is channelled through the Golgi apparatus in duodenal epithelial cells, we wanted to determine whether passage through the Golgi apparatus also represents a significant step along leptin transcytosis through Caco-2/15 cells. Immunocytochemistry was carried out on cells incubated for 30 minutes with leptin-FITC. Labelling for leptin-FITC revealed a positive signal within the cell cytoplasm in the form of small vesicular profiles particularly evident close to the nuclei (lization . To furtfficking  while okfficking . EndocytThe present study reveals the transcytotic transport of leptin across the polarized Caco-2/15 cells and highlights some of the characteristics of this transport. Leptin exposed to the apical side of the cells is internalized and transferred to the basolateral pole to be secreted into the basal medium though a transcytotic mechanism. We found that the long isoform of the leptin receptor is required in order to carry out what appears to be a clathrin-mediated endocytosis of the leptin present in the apical medium. We also show that the Golgi apparatus is involved in this intracellular transit of leptin. Leptin is eventually released into the basal medium bound to its soluble receptor. We have previously reported that leptin secreted by the gastric mucosa into the gastric juice in vivo and vehiculated to the duodenal lumen is captured by the intestinal mucosa, internalized, transcytosed, and secreted to be delivered into the systemic circulation . Herein,We have demonstrated that leptin introduced in the medium of the upper compartment of the cell culture crosses the cell monolayer to reach the lower compartment, in a time- and temperature-dependent and saturable manner, suggesting the involvement of a specific transporter, as reported for other proteins , 31. At Receptor-mediated transcytosis is a well-established system for protein transport . StudiesKnocking-down the long isoform was effective in decreasing both endocytosis and transcytosis of apical leptin. On the other hand, the invalidation of both the short and long isoforms had no cumulative effect, suggesting that the long isoform is mostly responsible for the transport of leptin through Caco-2/15 cell monolayer. These results differ from the situation reported in endothelial cells, where the short isoform of the leptin receptor seems to be the main carrier . HoweverIn blood and in gastric juice, leptin circulates bound to a protective protein which is the soluble isoform of its receptor . This soThe mechanism of leptin uptake was unravelled by the use of inhibitors of endocytosis. Chlorpromazin which among other effects is a clathrin-mediated endocytosis inhibitor inducing redistribution of AP-2 to endosomes ; nystatiDuring transcytosis through Caco-2/15 cells, internalized leptin was found in cisternae of the Golgi apparatus, similarly to observations made in situ on duodenal tissue . InvolveIn conclusion, we have shown that leptin follows a transcytotic pathway which includes clathrin-dependent endocytosis mediated by its membrane-bound receptors. After passage through the Golgi apparatus, leptin is released in the basal medium in a complex form bound to the soluble form of its receptor.