Health is dependent on the homeostasis of both inner and external microenvironments. The microbiota as the external microenvironment plays a critical role in regulation of several organ systems in mammals. However, it is unclear whether the microbiota regulates homeostasis of the skeletal system and bone marrow mesenchymal stem cells (BMMSCs). Here, using a well-established germ-free (GF) mouse model, we show that the microbiota significantly alters the stemness of BMMSCs in comparison to specific-pathogen-free (SPF)-derived BMMSCs. Colonization of GF mice with SPF microbiota ) normalizes the proliferation and differentiation abilities of BMMSCs. On the other hand, normal microbiota is required to maintain immunomodulatory properties of BMMSCs through induction of activated T-cell apoptosis and cytokine secretion. GF-derived BMMSCs lose the capacity to ameliorate disease phenotypes in dextran sulfate sodium-induced experimental colitis mice. Mechanistically, single-cell RNA-sequencing analysis shows that ConvD BMMSCs have a similar gene expression pattern to SPF-derived BMMSCs, which have a distinct gene distribution from GF-derived BMMSCs.The online version of this article (doi:10.1186/s13287-017-0670-7) contains supplementary material, which is available to authorized users. This microorganism coexistence with their hosts is referred to as the microbiota. It is reported that the human microbiota contains as many as 10an cells , immune an cells , neuroloan cells , and bonan cells .Bone marrow mesenchymal stem cells (BMMSCs), a kind of adult stromal cell in bone marrow, both contribute to the bone turnover  and formTo examine whether BMMSCs are regulated by the microbiota, BMMSCs were isolated from germ-free (GF) and specific-pathogen-free (SPF) mice. PCR analysis showed no bacteria detected in GF feces by universal bacteria primers, while conventionalized feces from GF mice colonized with SPF microbiota showed a similar pattern to the SPF group Fig. . Flow cy, we performed micro-CT analysis to show that femoral BMD of GF mice was significantly increased compared to SFP mice Additional file 2: Table S1.Presenting pathway enrichment analysis. (DOCX 12 kb)Additional file 3:Supplemental materials and methods used in this study. (DOCX 24 kb)


Immune checkpoint inhibitors (ICI) targeting CTLA-4 and the PD-1/PD-L1 axis have shown unprecedented clinical activity in several types of cancer and are rapidly transforming the practice of medical oncology. Whereas cytotoxic chemotherapy and small molecule inhibitors (‘targeted therapies’) largely act on cancer cells directly, immune checkpoint inhibitors reinvigorate anti-tumour immune responses by disrupting co-inhibitory T-cell signalling. While resistance routinely develops in patients treated with conventional cancer therapies and targeted therapies, durable responses suggestive of long-lasting immunologic memory are commonly seen in large subsets of patients treated with ICI. However, initial response appears to be a binary event, with most non-responders to single-agent ICI therapy progressing at a rate consistent with the natural history of disease. In addition, late relapses are now emerging with longer follow-up of clinical trial populations, suggesting the emergence of acquired resistance. As robust biomarkers to predict clinical response and/or resistance remain elusive, the mechanisms underlying innate (primary) and acquired (secondary) resistance are largely inferred from pre-clinical studies and correlative clinical data. Improved understanding of molecular and immunologic mechanisms of ICI response (and resistance) may not only identify novel predictive and/or prognostic biomarkers, but also ultimately guide optimal combination/sequencing of ICI therapy in the clinic. Here we review the emerging clinical and pre-clinical data identifying novel mechanisms of innate and acquired resistance to immune checkpoint inhibition. Monoclonal antibodies targeting co-inhibitory immune checkpoints  have demonstrated clinical activity in several malignances, including melanoma, non-small cell lung cancer, renal cell carcinoma, bladder cancer, head and neck squamous cell carcinoma, MSI-high colorectal carcinoma, Merkel cell carcinoma, and Hodgkin lymphoma, and have changed the practice of medical oncology  those that respond initially and continue to respond (responders), (2) those that fail to ever respond (innate resistance), and (3) those that initially respond but eventually develop disease progression (acquired resistance)   insufficient generation of anti-tumour T cells, (2) inadequate function of tumour-specific T cells . Lack ofTumours can evolve to evade both innate and adaptive arms of the immune system ) can also lead to ICI resistance. Downregulation of HLA class I molecules and loss of (B2M) expression have been described. Loss of B2M expression results in impaired cell surface expression of MHC class I, which in turn impairs antigen presentation to cytotoxic T cells  or to enhance antigen presentation by stimulating innate immune responses and dendritic cell function  may promote formation or presentation of suitable neoantigens in tumours with a non-inflamed, immune cell poor TME , including DNA-methyltransferase inhibitors and histone modifiers, can act on tumour cells influencing expression of components of antigen-processing and presentation machinery , novel tumour-associated antigens , and cytokines  and JAK/STAT pathway components  resulted in resistance to PD-1 blockade   in response to PD-1 blockade has been demonstrated, and is more pronounced in patients responding to therapy (EM cells in anti-PD-1 action and clinical response (EM expansion following PD-1 blockade are not fully understood, however, recent studies have identified distinct transcriptional programmes associated with naive, acute effector, memory, and exhausted T-cell states (The most compelling clinical evidence for the success of ICI relates to the potential for long-term, durable clinical benefit. Thus, although ICI may temporarily re-invigorate CTLs to enhance tumour control, if formation of TSeveral clinical trials of combinations of immunotherapeutic agents with targeted agents, cytotoxic chemotherapy, and/or radiation are underway, all in the effort to provide long-lasting disease control to more patients . CombinaTo date, precision medicine has largely been synonymous with use of molecular and largely genomic features of the tumour to administer specific targeted small molecules and biologics; however, novel and functional precision medicine platforms may offer additional opportunities to tailor therapies for individual patients or patient populations (


The changes of miR-221 (baseline vs. after long-term exercise) were significantly correlated with AT VO2, peak work load and CK . Although further studies are needed, present findings set the stage for defining circulating miRNAs as biomarkers and suggesting their physiological roles in long-term exercise training induced cardiovascular adaptation.Emerging evidence indicates the beneficial effects of physical exercise on human health, which depends on the intensity, training time, exercise type, environmental factors, and the personal health status. Conventional biomarkers provide limited insight into the exercise-induced adaptive processes. Circulating microRNAs  are dynamically regulated in response to acute exhaustive exercise and sustained rowing, running and cycling exercises. However, circulating miRNAs in response to long-term basketball exercise remains unknown. Here, we enrolled 10 basketball athletes who will attend a basketball season for 3 months. Specifically, circulating miRNAs which were involved in angiogenesis, inflammation and enriched in muscle and/or cardiac tissues were analyzed at baseline, immediately following acute exhaustive exercise and after 3-month basketball matches in competitive male basketball athletes. Circulating miR-208b was decreased and miR-221 was increased after 3-month basketball exercise, while circulating miR-221, miR-21, miR-146a, and miR-210 were reduced at post-acute exercise. The change of miR-146a (baseline vs. post-acute exercise) showed linear correlations with baseline levels of cardiac marker CKMB and the changes of inflammation marker Hs-CRP (baseline vs. post-acute exercise). Besides, linear correlation was observed between miR-208b changes (baseline vs. after long-term exercise) and AT VO Systematic physical exercise is an effective clinical option to improve quality of life in many types of patients, and the beneficial effects of exercise on cardiac function and exercise capacity have been well documented. It is well established that regular physical exercise promotes the improvement of life quality and lengthens lifespan  are endogenous small noncoding RNAs and range from 18 to 25 nucleotides in length. miRNAs regulate gene expression at post-transcriptional levels through inhibition of translation or mRNA degradation in sequence-dependent manner  was collected into RNase/DNase-free tubes and immediately aliquoted and frozen at −80°C.A cardiopulmonary exercise testing was performed before and after the 3-month basketball season. All participants were refrained from any physical exercise for 1 day before testing and were conducted an overnight dietary fast. On the morning of testing, participants were encouraged to drink water (400–800 ml) without caloric or electrolyte content. Ten basketball athletes underwent a cardiopulmonary exercise test on a MasterScreen-CPX system  at Shanghai Tongji Hospital , CKMB, Troponin T, NT-proBNP, and Hs-CRP were measured by enzyme linked immunosorbent assays (ELISA) kits from Wuhan Xinqidi Biological Technology. Lactic dehydrogenase (LDH) was determined by ELISA kits (KeyGEN BioTECH).The total RNA extraction was performed using a mirVanaPARIS isolation kit  according to the manufacturer's instructions. To avoid variability of results, repeated freeze-thaw cycles of serum samples were minimized and all samples were extracted and analyzed in a single batch. Briefly, 400 μl of serum was used to extract the total RNA. After equal volume of denaturing solution was added, 50 pmol/L Caenorhabditis elegans miR-39 (cel-miR-39) was added to normalize the miRNA serum levels.TM (TliRnaseH Plus) were used to detect selected miRNA expressions. All qRT-PCR reactions were performed in triplicate using an Applied Biosystems 7900HT Fast Real Time PCR device. Fold-change of RNA species was calculated using the formula 2(−ΔΔ Ct) and Cel-miR-39 were used as spike-in control.To quantify circulating miRNA levels, Bulge-Loop™ miRNA qPCR Primer Sets (RiboBio) and quantitative reverse transcription polymerase chain reactions (qRT-PCRs) with Takara SYBR Premix Ex Taqt-test was performed to analyze the changes of miRNAs, general echocardiographic indexes, biochemical indexes and cardiorespiratory fitness before and after 3-month exercise. Correlation analyses were analyzed using the Spearman's or Pearson's method as appropriate for data distribution. Statistical significance is defined as P-values < 0.05.Subject characteristics, biochemical measurements and general echocardiographic indexes are presented as means ± standard error of the mean (SEM). Paired-samples 2 and peak VO2 were not changed  and the changes of inflammation marker Hs-CRP after acute exercise  , peak work load    have no changes after 3-month basketball match, which have been shown dynamically altered after different programs of long-term aerobic exercise training (Baggish et al., miR-21, miR-146a, and miR-210 regulate multiple functions relevant to cardiovascular diseases and exercise. It can be speculated that acute exercise might cause local short-term hypoxic conditions in skeletal muscle and cardiovascular systems. miR-21 plays anti-apoptosis roles in endothelial cells (Weber et al., Long-term exercise training causes various adaptations including angiogenesis, mitochondrial metabolism, skeletal muscle regeneration and myocardial remodeling, which will directly interfere with the exercise capacity and health outcomes. miR-208b is expressed in skeletal muscle and embryonic hearts, and its ectopic expression caused multiple cardiovascular diseases (Huang and Li, 2, peak VO2 and biochemical indexes. With the improvement of exercise capacities, we showed the changes of miR-208b after 3-month exercise training moderately correlated with AT VO2 at baseline and the changes of miR-221 correlated with AT VO2, peak work load and serum CK levels after 3-month exercise training. Thus, these results indicated the possibility of circulating miR-221 and miR-208b as potential biomarkers for assessing or revealing the muscle damage and adaptations of exercise capacity induced by long-term basketball exercise.In our study, after 3-month exercise training, echocardiography showed that EF% value and other cardiac indexes were not changed, whereas their exercise capacities are enhanced. These results indicate that echocardiography and biochemical measurements are unable to comprehensively evaluate the benefits of long-term exercise training. The biochemical measurements showed that the contributions of both acute and 3-month basketball match to tissue damage does not appear to be significant in the present study. To further determine the uniqueness of circulating miRNA changes, we next compared circulating miRNA profiles to those of conventional biomarkers such as EF%, AT VOAs an initial study, several limitations of this study were still existent. Firstly, considering the variations of exercise intensity and duration time of every subject during the basketball game, the number of enrolled athletes should be expanded. Secondly, in view of the difference of exercise abilities between male and female, it would be interesting to see the miRNA changes in response to long-term exercise training using female athletes as subjects. Thirdly, the current study was restricted to basketball athletes. Future study is required to determine whether the changes of circulating miRNAs are applicable to football or other similar exercise types. Fourthly, our exercise training program involved team-based basketball match. Thus, the volume or intensity of the exercise training could not be controlled and calculated. However, quantitative assessment was taken to document playing time and fitness time of every athlete. Finally, quantitative analysis was restricted to a subset of relevant miRNAs, and high-throughput screening is needed to obtain a more complete profile of circulating miRNA regulation during long-term basketball exercise training.In conclusion, serum circulating miRNA levels are dynamically regulated in response to acute cycling exercise and long-term basketball exercise training in basketball athletes. Future studies are highly needed to define these miRNAs as potential and useful biomarkers of long-term exercise training, and disclose their direct biological roles in exercise mode-specific training adaptations.YL: undertook the qRT-PCR experiments and contributed to the drawing up of figures and writing of the paper; MY: contributed to the drawing up of figures and ELISA experiments; QZ: undertook the qRT-PCR experiments and cardiopulmonary exercise test; YC, LC, and JiX: provided technical support in echocardiographic diagnosis and serum sample collections; JuX: was involved in discussion on experimental design and contributed to the writing of the paper; ZS and YB: designed the experiments and contributed to the writing of the paper.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.


People with schizophrenia and related psychotic disorders show abnormalities in several organ systems in addition to the central nervous system (CNS). It is not yet known how the magnitude of disturbance in non-CNS systems compares with the magnitude of CNS disturbance in FEP. Here, we statistically compare effect sizes (ES) for non-CNS and CNS dysfunction in FEP, and consider whether schizophrenia is a multi-system disorder.Pubmed was systematically searched from 1990 to May 2017 for meta-analyses examining non-CNS dysfunction in FEP, focusing on immune, cardiometabolic, and hypothalamic-pituitary-adrenal (HPA) systems. A parallel search was performed for meta-analyses examining representative CNS dysfunction in FEP, specifically neurophysiological, neurochemical and brain structural alterations. To statistically compare the magnitude of effect sizes (ES) between different CNS and non-CNS systems in FEP, data were extracted from case-control studies making up these meta-analyses, and meta-analyses repeated. For non-CNS parameters, random-effects meta-analyses were performed examining immune , cardiometabolic (glucose/insulin and lipids), and HPA parameters (cortisol and prolactin). For CNS parameters, meta-analyses were performed examining brain structural, neurophysiological , and neurochemical parameters (N-acetylaspartic acid levels). Standardized mean differences between patient and control cohort parameters were used as the ES (Hedges adjusted g). As well as individual meta-analyses being run for each parameter as described, 6 separate sub-group meta-analyses were performed examining data for overall immune, cardiometabolic, HPA, brain structural, neurophysiological, and neurochemical systems. Sub-group summary effect size magnitudes were calculated by running a combined analysis of all studies assigned to a sub-group . Summary effect sizes for these 6 individual systems were statistically compared in a fixed effects model using a Wald-type test. This was also used to compare overall summary CNS and non-CNS effect sizes. Antipsychotic naïve sensitivity analyses were performed.Data were extracted for 165 studies comprising a total sample size of 13,440. The summary effect size for immune alterations  was significantly greater than brain structural  and neurochemical alterations , and no different from neurophysiological alterations . The summary effect size for HPA alterations  was not significantly different from brain structural (P=.14), neurophysiological (P=.54), and neurochemical (P=.22) alterations. The summary effect size for cardiometabolic alterations  was significantly lower than neurochemical (P=.04), neurophysiological (P<.001) and brain structural alterations (P=.001). The overall summary effect sizes for non-CNS  and CNS  alterations were not significantly different (P=.28).These data indicate that there are robust alterations in non-CNS systems in psychosis, and that these are broadly similar in magnitude to a range of CNS alterations, indicating that psychosis involves multiple organ systems to a comparable degree from onset. We consider three models that could account for these findings and discuss implications for future research and treatment.


Antipsychotics (AP)s are the cornerstone of treatment for schizophrenia but cause serious metabolic side-effects. The hypothalamus is the primary brain region responsible for whole body energy regulation and disruptions in energy sensing  and inflammation in this brain region have been implicated in the development of peripheral insulin resistance and obesity. Thus, it is possible that hypothalamic inflammation and disturbed energy sensing could be involved in AP-induced metabolic disturbances. Data in relation to AP-associated changes in inflammatory markers in schizophrenia has been inconsistent, owing in part to confounds of illness-related factors  and secondary effects of weight gain. To our knowledge, direct effects of APs on hypothalamic cells in relation to insulin signaling and inflammation have not been examined.To examine direct, molecular effects of APs in the hypothalamus, an immortalized rat hypothalamic cell line, rHypoE-19, was treated with olanzapine (dose range between 0.25–100 uM), clozapine (2.5–100 uM) or aripiprazole (5–20 uM). Western blotting was used to detect changes in the energy sensing protein AMPK, components of the insulin signaling pathway , and components of the mitogen activated-protein kinase (MAPK) pathway , the latter which are linked to inflammation. Quantitative real-time PCR was performed to determine changes in the mRNA expression of interleukin (IL)-6, IL-10 and brain derived neurotrophic factor (BDNF).Both olanzapine (100 uM) and clozapine (100 uM) significantly increased pERK1/2 and pJNK protein expression, while aripiprazole (20 uM) only increased pJNK. Clozapine (100 uM) and aripiprazole (5 and 20 uM) significantly increased AMPK phosphorylation and inhibited insulin-induced phosphorylation of AKT. Olanzapine (100 uM) treatment caused a significant increase in IL-6 while aripiprazole (20 uM) significantly decreased IL-10. Olanzapine (100 uM) and aripiprazole (20 uM) increased BDNF expression.All the APs studied upregulated pJNK, along with olanzapine-associated increases in IL-6, and aripiprazole-associated decreases in IL-10, together suggesting AP-mediated upregulation of pro-inflammatory pathways in rHypoE-19 neurons. Aripiprazole and clozapine (but not olanzapine) inhibited insulin-stimulated AKT, suggesting impaired hypothalamic insulin action by some, but not all, APs. Clozapine additionally increased AMPK phosphorylation (activation), an orexigenic energy sensor, which would also be expected to disrupt energy homeostasis. Conversely, olanzapine and aripiprazole increased BDNF, a factor linked to the underlying etiology of schizophrenia, suggesting BDNF upregulation may be a mechanism of therapeutic action. Taken together, our findings suggest differential and pleotropic effects of APs on neuroinflammation and energy sensing in the hypothalamus, which do not necessarily align consistently with known metabolic liability of these agents (i.e. clozapine = olanzapine > aripiprazole). Our data warrants further exploration into the mechanism of these effects, including replication of these effects in an in vivo model.


During the last two decades, significant progress has been made in the identification of genetic defects underlying inherited arrhythmia syndromes, which has provided some clinical benefit through elucidation of gene-specific arrhythmia triggers and treatment. However, for most arrhythmia syndromes, clinical management is hindered by insufficient knowledge of the functional consequences of the mutation in question, the pro-arrhythmic mechanisms involved, and hence the most optimal treatment strategy. Moreover, disease expressivity and sensitivity to therapeutic interventions often varies between mutations and/or patients, underlining the need for more individualized strategies. The development of the induced pluripotent stem cell (iPSC) technology now provides the opportunity for generating iPSC-derived cardiomyocytes (CMs) from human material (hiPSC-CMs), enabling patient- and/or mutation-specific investigations. These hiPSC-CMs may furthermore be employed for identification and assessment of novel therapeutic strategies for arrhythmia syndromes. However, due to their relative immaturity, hiPSC-CMs also display a number of essential differences as compared to adult human CMs, and hence there are certain limitations in their use. We here review the electrophysiological characteristics of hiPSC-CMs, their use for investigating inherited arrhythmia syndromes, and their applicability for identification and assessment of (novel) anti-arrhythmic treatment strategies. Life-threatening ventricular arrhythmias and sudden arrhythmic death typically occur in the setting of common cardiovascular pathologies associated with structural cardiac abnormalities, including myocardial ischemia/infarction, heart failure, and cardiomyopathy. In a subset of patients, often relatively young and otherwise healthy individuals, ventricular arrhythmias are the consequence of an inherited arrhythmia syndrome. Here, mutations in genes encoding ion channels, transporters, interacting proteins, or regulatory pathways lead to potentially pro-arrhythmic alterations of cardiac electrophysiological properties , 2. SincXenopus oocytes, human embryonic kidney or HEK cells, and Chinese Hamster Ovary or CHO cells) -l-norleucine (ALLN) [The first report showing the capability of hiPSC-CMs to recapitulate the clinical phenotype of LQT2 was the study of Itzhaki and colleagues, who observed decreased mutation . PinacidiPSC-CMs . The potiPSC-CMs . However2-A561T) . Exposur2-A561T) . In the ing EADs . AP duraing EADs . It has ing EADs  tested siPSC-CMs . Of noteobserved , underlifedipine . In addiINa inactivation is compromised or channel reopening occurs, leading to INaL which persists throughout the entire AP and ultimately causes AP prolongation and pro-arrhythmia. Some drugs are known to preferentially inhibit this INaL, including mexiletine and ranolazine. In hiPSC-CMs from a patient with a de novo LQT3 mutation (SCN5A-F1473C) and a polymorphism (K897T) in KCNH2, analysis of the biophysical properties of INa and IKr demonstrated that increased INaL was responsible for the pro-arrhythmic phenotype and that the latter was not influenced by the KCNH2 polymorphism [INaL; when mexiletine was added, INaL was further reduced. This improved INaL block at the higher stimulation rate was associated with only a modest reduction in peak INa, ensuring minimal detrimental impact on conduction. Addition of a second INa blocker, flecainide, did not induce a significant further increase in INaL block at higher stimulation frequency but further reduced peak INa. Consistent with these in vitro findings, the most effective arrhythmia control in the patient was achieved by increasing atrial pacing via ICD and by mexiletine treatment [KCNH2-K897T on channel function, in hiPSC-CMs, this variant did not impact on anti-arrhythmic drug efficacy. In another study, mexiletine was found to shorten APD and FPD in LQT3 hiPSC-CMs and antagonized EADs in a dose-dependent manner [INaL blockers phenytoin and ranolazine also reduced APDs/FPDs in the same model (SCN5A-R1644H), with no effect on control hiPSC-CMs. These findings were in agreement with the pharmacological response profile of the underlying patient carrying the mutation and of other patients from the same family [INaL and APD in SCN5A-V1763M hiPSC-CMs, with little effect on control cells [SCN5A-1795insD mutation associated with both a gain (LQT3) and a loss  of Na+ channel function. The selective INaL blocker GS967 was able to reduce the prolonged AP duration (measured with in silico injection of IK1) induced by the increased INaL associated with the mutation in these hiPSC-CMs [INa consequent to the mutation, GS967 only caused a minimal reduction in AP upstroke velocity. Importantly, hiPSC-CMs displayed a similar response to GS967 as adult, freshly isolated CMs from mice harboring the mouse homolog of the mutation Scn5a-1798insD [Scn5a-1798insD hearts [Scn5a-1798insD CMs displayed EADs and delayed afterdepolarizations (DADs) which were reduced by GS967, but no such pro-arrhythmic features were observed in SCN5A-1795insD hiPSC-CMs [INaL.In LQT3, morphism . In the reatment . Importat manner . Moreovee family . Similarol cells . We receiPSC-CMs  , the most severe variant of LQTS because of its high mortality rate. Besides a marked QT interval prolongation and severe ventricular arrhythmia, LQT8 presents with congenital heart defects, AV block, syndactyly, autism, malignant hypoglycemia, and an abnormal immune system [. In 2011, Yazawa and colleagues showed that hiPSC-CMs from LQT8 patients recapitulated the electrical phenotype of the disease  [ICaL inactivation, reduced AP duration, restored the irregular Ca2+ transient, and decreased the frequency of abnormal depolarizations in LQT8 hiPSC-CMs [A single amino acid substitution (G406R) in nsients) . The expiPSC-CMs . HoweveriPSC-CMs .<40 years), typically during conditions of high vagal tone . On ECG analysis, a typical pattern is observed comprising ST-segment elevation in the right-precordial leads V1–V3 [SCN5A has been identified. In general, SCN5A mutations associated with BrS are “loss-of-function” mutations, leading to reduced Na+ channel availability, either through decreased membrane surface channel expression or through altered channel gating properties [SCN5A nonsense mutations (W156X and R1638X) which lead to premature termination of translation and production of truncated proteins [INa as compared to control hiPSC-CMs. In these hiPSC-CMs, the effects of two compounds were investigated, i.e., gentamicin and PTC124, both of which are considered capable of promoting translational readthrough of premature stop codons and hence restoring expression of the full-length, non-truncated protein. While the authors confirmed the readthrough efficacy of the two drugs in HEK293 cells, they did not observe rescue of the electrophysiological phenotype in these BrS hiPSC-CMs [Brugada syndrome (BrS) is an inherited disorder characterized by ventricular arrhythmias and sudden cardiac death occurring in otherwise healthy individuals at a relatively young age  or recessive mutations in CASQ2, the gene encoding for calsequestrin 2 (CPVT2) are the most common mutations linked to CPVT, resulting in spontaneous Ca2+ release from the SR and consequent DADs, triggered activity, and ventricular tachyarrhythmias [RYR2-M4109R mutation showed increased DADs incidence and Ca2+ transient irregularities as compared to control hiPSC-CMs, which were further exacerbated by β-adrenergic stimulation [RyR-L3741P hiPSC-CMs, where it improved calcium homeostasis and reduced DADs incidence; clinical efficacy of flecainide was subsequently observed in the mutation carrier [2+ release, which is effective on malignant hyperthermia and is commonly used in the treatment of CPVT patients, restored normal Ca2+ handling properties and rescued the arrhythmogenic phenotype in hiPSC-CMs from a CPVT1 patient carrying the S406L mutation in RyR2. In a successive study, the antiarrhythmic efficacy of dantrolene during exercise testing was assessed in six patients carrying various RyR2 mutations [RyR2 mutations in the N-terminal or cytosolic region, dantrolene abolished the majority of Ca2+ transient abnormalities, while it had minimal effect on Ca2+ transients in CMs carrying mutations in or near the transmembrane domain [RyR2-G2311A), the CamKII inhibitor KN-93 abolished isoprotenerol-induced DADs, thereby rescuing the arrhythmogenic phenotype [RyR2 knock-in CPVT mouse model [RyR2-I4587V hiPSC-CMs and showed that the compound S107, a 1,4-benzothiazepine derivate that stabilizes the closed state of RyR channels (thereby preventing Ca2+ leak) by strengthening the interaction between RyR and calstabin, decreased the incidence of isoproterenol-induced DADs [CASQ2 gene [CASQ2 by adeno-associated virus-mediated delivery restored calcium dysregulation and significantly reduced DADs incidence [CASQ2-D307H hiPSC-CMs and freshly isolated, adult ventricular CMs from mice carrying the same mutation [Casq2-D307H knock-in mice during rest and after exercise [TECRL, the gene encoding for the trans-2,3-enoyl-CoA reductase like protein, are associated with inherited arrhythmias characterized by clinical features of both LQTS and CPVT [TECRL (TECRLHOM hiPSC-CMs) revealed increased diastolic Ca2+ concentrations, smaller Ca2+ transient amplitudes, slower Ca2+ rise and decay, as well as prolonged APD compared to hiPSC-CMs generated from his heterozygous but clinically asymptomatic father (TECRLHET), and a healthy individual  [HOM hiPSC-CMs following exposure to noradrenaline was reduced by treatment with flecainide [Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited disease characterized by stress and exercise-induced ventricular arrhythmias  in young patients with structurally normal hearts. Autosomal dominant mutations in hythmias . The firmulation . Both flmulation . Similar carrier . In the  carrier , dantrolutations . Dantrole domain . In anothenotype . Of notese model . More reced DADs . Apart fSQ2 gene . Over-exncidence . More remutation . Moreoveexercise . Finallyand CPVT . hiPSC-Cl, CTRL)   cardiomyopathy, and hypertrophic cardiomyopathy. By studying electrophysiological characteristics of hiPSC-CMs from patients with inherited arrhythmia syndromes without an identified causal mutation, the underlying (pro-arrhythmic) disease mechanisms may be identified . ConversIK1 density facilitates accurate assessment of AP parameters. Improved definition and selection of hiPSC-CMs subtype  will furthermore allow investigation of disease mechanisms and therapeutic efficacy in a cell type-specific manner. These developments are expected to further strengthen the validity of hiPSC-CMs as models of inherited arrhythmia syndromes as well as other disease entities and their applicability in (future) development of disease-, mutation-, and patient-specific therapies.Despite their promising use in this wide range of applications, certain limitations intrinsic to hiPSC-CMs need to be considered and addressed. In particular, the issue of immaturity remains an important matter, and techniques aimed at enhancing maturity are continuously being developed and refined, including exposure to electrical stimulation, application of mechanical strain, and culturing hiPSC-CMs in three-dimensional tissue configuration. In addition, artificial enhancement of


This image illustrates a very unusual pattern of early and aggressive immature neo-intimal hyperplasia in a 52-year-old man with unstable angina, two months after deployment of a drug-eluting stent in the proximal left anterior descending artery. A 52-year old man was admitted with unstable angina two months after deployment of a drug-eluting stent (DES) in the proximal left anterior descending (LAD) artery. Five months prior to the current admission he had undergone percutaneous coronary intervention (PCI) with a DES to his proximal right, proximal circumflex and mid-LAD coronary arteries. The patient had no cardiovascular risk factors apart from a family history of premature coronary artery disease.Coronary angiography demonstrated in-stent restenosisof the proximal LAD stent . OpticalThis image illustrates a very unusual pattern of early and aggressive immature neo-intimal hyperplasia. Although immature neo-intimal hyperplasia has been described,1 to our knowledge this is the first image of such aggressive immature neo-intimal hyperplasia.


F8 in constructs) is costly, inconvenient, and incompletely effective. In addition, approximately 25 % of treated patients develop anti-factor VIII immune responses. Gene therapy that can achieve long-term phenotypic correction without the complication of anti-factor VIII antibody formation is highly desired. Lentiviral vector (LV)-mediated gene transfer into hematopoietic stem cells (HSCs) results in stable integration of FVIII gene into the host genome, leading to persistent therapeutic effect. However, ex vivo HSC gene therapy requires pre-conditioning which is highly undesirable for hemophilia patients. The recently developed novel methodology of direct intraosseous (IO) delivery of LVs can efficiently transduce bone marrow cells, generating high levels of transgene expression in HSCs. IO delivery of E-F8-LV utilizing a ubiquitous EF1α promoter generated initially therapeutic levels of FVIII, however, robust anti-FVIII antibody responses ensued neutralized functional FVIII activity in the circulation. In contrast, a single IO delivery of G-FVIII-LV utilizing a megakaryocytic-specific GP1bα promoter achieved platelet-specific FVIII expression, leading to persistent, partial correction of HemA in treated animals. Most interestingly, comparable therapeutic benefit with G-F8-LV was obtained in HemA mice with pre-existing anti-FVIII inhibitors. Platelets is an ideal IO delivery vehicle since FVIII stored in α-granules of platelets is protected from high-titer anti-FVIII antibodies; and that even relatively small numbers of activated platelets that locally excrete FVIII may be sufficient to promote efficient clot formation during bleeding. Additionally, combination of pharmacological agents improved transduction of LVs and persistence of transduced cells and transgene expression. Overall, a single IO infusion of G-F8-LV can generate long-term stable expression of hFVIII in platelets and correct hemophilia phenotype for long term. This approach has high potential to permanently treat FVIII deficiency with and without pre-existing anti-FVIII antibodies.Current treatment of hemophilia A (HemA) patients with repeated infusions of factor VIII (FVIII; abbreviated as  Deficiency of blood clotting factor VIII (FVIII) results in hemophilia A (HemA), a serious bleeding disorder. Current treatment of HemA patients with repeated infusions of FVIII is costly, inconvenient, and incompletely effective . In addiPrevious phase I gene therapy clinical trials –4, howevIt is demonstrated recently that in vivo gene transfer can be successfully carried out by direct intraosseous (IO) injection using several different vectors including adeno-, retro-, and lenti-viral vectors (LVs) –10. HSCsIn this concise review, we will discuss the recently developed novel approach of IO delivery of LVs to correct hemA . The benCurrent treatment of hemophilia involves repeated infusions of FVIII protein either as regular prophylaxis or treatment during bleeding episodes. For severe patients, the standard treatment consists of intravenous infusion of factor VIII concentrates three times per week or every other day , 7. In aRecently, clinical trials of gene delivery of factor IX mediated by adenovirus-associated viral (AAV) vectors into the liver generated encouraging results in hemophilia B patients –22. FurtAlthough ex vivo HSC transduction/transplantation protocols can successfully deliver FVIII into HemA mice , 31, theAs mentioned in the introduction, one of the major complications of hemophilia treatment is the formation of anti-FVIII inhibitory antibodies. For achieving successful gene therapy, it is essential to prevent or evade anti-FVIII immune responses. For this purpose, platelet has been considered as a potential gene transfer vehicle to ectopically express FVIII because: (1) FVIII can be released to promote clot formation when the circulating platelets are recruited to and activated at the injury sites, (2) when needed, ectopic FVIII can be provided by circulating platelets that are produced daily from megakaryocytes in BM, (3) in particular, platelet FVIII stored in α-granules is not neutralized by anti-FVIII antibodies in the circulation , 34. PreIn our recent study , it was 8 platelets, a level comparable to previously described transgenic animals expressing platelet FVIII by chromogenic assay [Following IO delivery, up to 3 % of platelets expressed FVIII driven by platelet-specific promoters  Fig. 3a3a. The aic assay . Our resic assay . Howeveric assay , 33–35. ic assay  clearly ic assay , 25, 39 Much effort has been devoted to investigate effective means to overcome the complication of inhibitory antibody formation to FVIII, including the development of immune modulation protocols (see recent Review ) or geneFurthermore, following IO delivery of LVs in immune-competent mice, we found lymphocyte infiltrates in BM and gradual decreases of transduced cells, which are similar to the innate and adaptive responses observed from LV transduction in hepatocytes –46. By aAlthough long-lasting factor concentrates have improved efficacy of protein replacement therapy, and FVIII-mimetic bispecific antibody is promising as an alternative treatment especially for patients with inhibitory antibodies, these therapies require frequent, at least weekly dosing of costly reagents. With the recent successes in clinical trials of AAV-mediated gene transfer, it is anticipated that gene therapy will be the next generation medicine for hemophilia treatment. The potential obstacles of AAV-mediated gene therapy include unpredictable events of transaminase elevation, potential requirement of repeated treatment that cannot be performed due to pre-existing anti-AAV immune responses, and the prohibitively high cost of high-titer vectors needed for hemA treatment. The recently developed novel strategy of IO delivery of F8-LVs  can effi


Membrane trafficking via the Golgi-localised KDEL receptor activates signalling cascades that coordinate both trafficking and other cellular functions, including autophagy and extracellular matrix degradation. In this study, we provide evidence that membrane trafficking activates KDEL receptor and the Src family kinases at focal adhesions of HeLa cells, where this phosphorylates ADP-ribosylation factor GTPase-activating protein with SH3 domain, ankyrin repeat and PH domain (ASAP)1 and focal adhesion kinase (FAK). Previous studies have reported extracellular matrix degradation at focal adhesions. Here, matrix degradation was not seen at focal adhesions, although it occurred at invadopodia, where it was increased by KDEL receptor activation. This activation of KDEL receptor at invadopodia of A375 cells promoted recruitment and phosphorylation of FAK on tyrosines 397 and 861. From the functional standpoint, FAK overexpression inhibited steady-state and KDEL-receptor-stimulated extracellular matrix degradation, whereas overexpression of the FAK-Y397F mutant only inhibited KDEL-receptor-stimulated matrix degradation. Finally, we show that the Src and FAK activated downstream of KDEL receptor are part of parallel signalling pathways. In conclusion, membrane-traffic-generated signalling via KDEL receptor activates Src not only at the Golgi complex, but also at focal adhesions. By acting on Src and FAK, KDEL receptor increases invadopodia-mediated matrix degradation. KDEL receptor (KDELR) is a seven-transmembrane-domain protein that is known to cycle between the endoplasmic reticulum (ER) and the Golgi complex to retrieve chaperones that have ‘escaped’ from the ER . KDELR aWe have recently reported that activation of KDELR controls extracellular matrix (ECM) degradation , which iA number of molecules that are located at invadopodia are also present at focal adhesions, which are sites of tight contact to the underlying ECM. These include the integrins, and cytoskeletal and signalling proteins, like focal adhesion kinase (FAK) , 9. FAK The specific role of FAK in the regulation of invadopodia formation and function in cancer cells remains under debate. Some studies have suggested that FAK is not targeted to invadopodia, such as in MDA-MB-231 and MTLn3 rat mammary adenocarcinoma cells . ConversHere we examined signalling triggered by membrane trafficking and Golgi-localised KDELR. First, we show that membrane trafficking activates Src at focal adhesions via KDELR signalling, whereby Src then phosphorylates ASAP1 and FAK. Indeed, direct stimulation of KDELR activates the same pathway. Then, prompted by these molecular players, we investigated degradation of the ECM using the A375 metastatic melanoma cell model. As previously shown [cis-Golgi, these chaperones move back to the ER. Furthermore, this binding of KDELR by chaperones triggers signalling cascades that lead to activation of Src on the Golgi complex [In previous studies, we showed that membrane trafficking from the ER to the Golgi transports small amounts of the ER-resident chaperones to post-ER compartments. Upon binding to KDELR in the intermediate compartment and the  complex . Thus, wSynchronisable secretory proteins have been exploited to deliver ‘pulses’ of traffic, such as with procollagen (PC)-IV, , 18–20. cis-Golgi marker GM130) and at the cell periphery  using an antibody that selectively recognises the phosphorylated tyrosine at position 419 in human Src . Duringy Figure . pSrc ley Figure  and the y Figure , as deteWe have previously shown that inhibition of KDELR function using a dominant-negative KDELR mutant (KDELR-D193N) impairs traffic-induced Src activation in the Golgi area . Thus, wIn these experiments, traffic pulses and the consequent KDELR stimulation were achieved using the temperature shift protocol 40°C–32°C). To rule out the possibility that the temperature shift itself contributes to the effects observed, KDELR was stimulated using an alternative approach. A soluble secreted variant of horseradish peroxidase (ssHRP) that bears the KDEL motif at its C-terminus (ssHRP-KDEL) represents a synthetic KDEL-containing polypeptide °C. To ru when expTo further validate the involvement of KDELR in the activation of Src in specific sub-cellular compartments, HeLa cells were co-transfected with ssHRP-KDEL in combination with KDELR-D193N-GFP, and GFP alone as a control. A second control was included with ssHRP instead of ssHRP-KDEL. The presence of KDELR-D193N-GFP inhibited Src activation both at the Golgi complex and at the cell periphery Figure . These dThe smaller signalling response observed in ssHRP-KDEL stimulation might be explained by the chronic (24 h) nature of this treatment as compared to acute stimulation by a traffic pulse. However, we cannot exclude that the temperature shift might partially contribute to pSrc activation.To better understand the relationships between activation of Src on the Golgi complex and at the cell periphery, we hypothesised that once activated on the Golgi complex, Src spreads to the cell periphery. This hypothesis stems from a previous study that demonstrated that active Src can be transported from the perinuclear recycling compartment to peripheral membrane structures through endosome-mediated trafficking .Thus, we monitored Src activation in the Golgi area and cell periphery as a time-course following the release of the temperature block during a PC-IV traffic pulse. HeLa cells were incubated at 40°C for 3 h, then shifted to 32°C, and fixed after 5, 10, 15 and 20 min. Src was already activated at the Golgi complex at 5 min after the release of the temperature block Figure , while iHere we investigated Src-dependent phosphorylation of focal adhesion proteins, as the above data indicated that membrane traffic and KDELR trigger activation of Src at focal adhesions. To address this issue, HeLa cells were exposed to a PC-IV traffic pulse, lysed, and analysed by Western blotting using phospho-specific antibodies that recognise Src phosphorylation sites on ASAP1 (pY782-ASAP1) and FAK (pY861-FAK). We observed marked increases in Src-dependent phosphorylation of ASAP1 and FAK during the active state of transport, compared to the resting state Figure .To determine whether ASAP1 and FAK are phosphorylated by Src at the focal adhesions or the Golgi complex, we carried out confocal IF analysis in traffic-activated HeLa cells. Strong increases in both pY782-ASAP1 and pY861-FAK signals were detectable at focal adhesions, as compared to the control cells Figure , with thThus, to determine whether this minor phosphorylation of pY782-ASAP1 and pY861-FAK at the Golgi complex has any detectable functional effects, the efficiency of cargo transport from the ER to the plasma membrane was determined in ASAP1 and FAK knock-down cells. The membrane-trafficking efficiency was investigated using the temperature-sensitive mutant of the vesicular stomatitis virus G glycoprotein (VSVG). This VSVG mutant is synchronisable according to temperature, and it has been widely used to investigate protein folding and efficiency of the secretory pathway. At 40°C, VSVG cannot fold completely in the ER, and consequently its exit from the ER is blocked . When the cells are then shifted to 32°C , VSVG can fold and leave the ER, and in this way it is synchronously transported through the secretory pathway to the plasma membrane. HeLa cells were transfected for 72 h with siRNAs targeting ASAP1 or FAK, and then infected with the temperature-sensitive mutant VSV. The transport of VSVG was monitored 90 min after the release of the traffic block. The same amount of VSVG reached the plasma membrane in ASAP1 and FAK interfered cells as well as in control cells , which iTo investigate whether traffic-induced phosphorylation of ASAP1 and FAK depend on KDELR, the levels of pY782-ASAP1 and pY861-FAK were monitored during traffic pulses in cells expressing the KDELR-D193N dominant-negative mutant. The expression of KDELR-D193N strongly reduced the levels of pY782-ASAP1 and pY861-FAK triggered by traffic pulses at focal adhesions, in comparison to mock-transfected cells Figure . These dTo confirm that in our experimental setting pY782-ASAP1 and pY861-FAK are indeed Src substrates, we examined whether their phosphorylation can be repressed by treatment with a Src inhibitor. To this end, HeLa cells underwent a PC-IV traffic pulses in the presence of the Src inhibitor SU6656  with celThese data thus indicate that ASAP1 and FAK are focal adhesion targets downstream of the KDELR–Golgi–Src signalling cascade.In the resting state, the tyrosine 397 autophosphorylation site of FAK is hidden by its N-terminal FERM domain. Classical integrin-mediated FAK activation induces a conformational change that displaces the FERM domain and allows autophosphorylation of tyrosine 397, which activate the kinase and creates a high-affinity binding site for Src , 26. SrcOf note, traffic-activated HeLa cells significantly increased pY397-FAK at the focal adhesions . This phPrevious studies have shown that focal adhesions are sites of ECM degradation, while we have reported that KDELR controls invadopodia-mediated ECM degradation , 17. ProIn analogy to HeLa cells, A375 cells endogenously express the synchronisable cargo PC-IV, and following the release of traffic pulses, they showed activation of Src at the Golgi complex and cell periphery . In addiKDEL, subjected to the degradation assay, and stained for FAK and cortactin (marker of invadopodia). These cells showed characteristic degradation activity underneath the invadopodia, but no degradation activity was revealed in proximity to the focal adhesion structures  and in combination with the KDELR ligand ssHRPr Figure . A similr Figure . These dKDEL), both FAK-WT and FAK-Y861F reduced ECM degradation, as compared to cells overexpressing ssHRPKDEL alone . Here, we measured UPR activation in A375MM cells transfected with KDELR2, ssHRPed cells .These data indicate that under conditions that result in Src activation and ECM degradation, KDELR activation does not result in the induction of the UPR, which makes a role of the UPR in KDELR-activated ECM degradation very unlikely.KDEL for 24 h, and subjected to the ECM degradation assay. The levels of pY397-FAK at the invadopodia of ssHRPKDEL-transfected cells was significantly increased compared to control cells ; KDELR-D193N-eGFP ; KDELR-D193N-myc from subcloning the KDELR coding sequence from KDELR-D193N-GFP into a myc-containing modified pCMV5 vector; FAK-WT and mutants .Antibodies used in this study were: anti-Src (Santa Cruz Biotechnology); anti-pSrc and anti-pY861-FAK (Invitrogen); anti-GM130 and anti-FAK ; anti-pY782-ASAP1 ; anti-ASAP1 (Transduction Laboratories); anti-cortactin ; anti-HRP antibodies (Abcam); anti-myc (Invitrogen); fluorophore-conjugated secondary antibodies (Molecular Probes). The expression vectors used were: ssHRP and ssHRPThe human melanoma A375MM and cervical adenocarcinoma HeLa cells were grown under standard conditions. The cells were plated at 50% confluence in 6-well plates and transfected using TransFast reagent  or JetPEI (Polyplus), according to the manufacturer instructions.The PC-IV and VSVG transport pulses were as previously described . FluoresImmunofluorescence microscopy was as described previously . ConfocaAreas of degradation were defined as dark patches in the fluorophore-conjugated gelatine matrix underlying the cells. Here, 50 cells per point per experiment were acquired, as reported above and in . The totTotal IF was acquired as described above, and IF intensity was calculated by integration of the IF signal within the region of interest divided by the area. All experiments were carried out at least twice. The data are shown as arbitrary units (AU).2 for a further 24 h. ECM degradation and invadopodia formation were evaluated as described above.The cells were transfected with 100 nM of the siGENOME SMARTpool reagents  containing four pooled siRNA duplexes against human FAK, using Lipofectamine 2000 , according to the manufacturer instructions. The cells were plated on gelatine-coated coverslips 48 h after siRNA treatment in the presence of 5 μM BB94, and incubated at 37°C in the presence of 5% COCells were lysed in RIPA buffer  plus protease and phosphatase inhibitors (Roche). Cell lysates were separated on 10% to 12% SDS-polyacrylamide gels and subjected to Western blotting.


Cultures derived from rat brains at different times during the latent period of brain-tumour induction by N-ethyl-N-nitrosourea (ENU) showed increased plasminogen activator (PA) activity before being able to form colonies in agar. Control cultures from buffer-exposed animals showed neither property at comparable passages. More detailed investigations, using a culture derived from foetal brains only 2 days after exposure to ENU and clones from this culture, showed a sequence of low PA activity, then increased activity, followed by the ability to form colonies in agar, suggesting progressive transformation of cells in culture. Continuous culturing in the presence of the mouse skin tumour promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), did not accelerate the rate at which these two properties were acquired, but did cause a much greater increase of PA activity once this started to rise. If included in the assay mixture TPA also increased the PA activity of the cells. It therefore appears that in this system TPA can modulate PA activity under certain circumstances.


N-methyl-N-nitrosourea (MNU) given i.v. to female rats of inbred strains induces mammary adenocarcinomas which are hormone-sensitive but do not spontaneously metastasize . Tissue culture and selection techniques have been used to derive metastasizing tumours from a mammary tumour induced with MNU in F344/N rats. Histologically, primary tumours and metastases in the lung and lymph nodes were similar. These systems may constitute useful models for the study of breast-cancer metastasis.


Of all the                        major organ systems in the body, the ovaries of females are the first to                        exhibit impaired function with advancing age. Until recently, traditional                        thinking was that female mammals are provided with a non-renewable pool of                        oocyte-containing follicles at birth that are depleted during postnatal                        life to exhaustion, driving ovarian failure. However, a growing body of                        evidence, including the isolation of germline stem cells (GSC) from adult                        mouse ovaries that produce developmentally-competent oocytes, has                        challenged this belief. In addition, rare germline stem-like cells capable                        of generating oocytes in vitro that undergo parthenogenesis to form                        blastocyst-like structures have recently been identified in postmenopausal                        human ovaries. Here we show that the germline-specific meiosis-commitment                        genes,Stimulated by retinoic acid gene 8 (Stra8) and Deleted                        in azoospermia-like (Dazl), are highly expressed in aged mouse                        ovaries. However, histological and marker analyses fail to demonstrate the                        presence of oocytes, supporting that Stra8 and Dazl are                        expressed in premeiotic germ cells that do not undergo further                        differentiation. Through the use of aged germline-specific GFP-expressing                        transgenic mice, we further show that these germ cells can generate                        GFP-positive oocytes that co-express the primordial oocyte marker NOBOX and                        form follicles when grafted into young adult wild-type female hosts. Thus,                        aged mouse ovaries possess a rare population of premeiotic germ cells that                        retain the capacity to form oocytes if exposed to a young host environment.                 In humans and laboratory rodent models                        (rats and mice), the ovaries exhibit age-related dysfunction relatively early                        in life, with failure noted long before aging-associated changes in other                        organs are manifest. In humans, this loss of ovarian function drives the                        menopause and its associated increased risk for development of diverse health                        complications, many of which are tied to disrupted ovarian hormone production .       INK4a and p19ARF, two senescence-associated proteins                        that contribute to stem cell failure during aging of the hematopoietic, neural                        and cardiac systems [For example, findings from gene mutant mice show that                        p16 systems -9, do no systems . However systems . Other s systems . When ma systems . AlthougDeleted in                                    azoospermia-like (Dazl) and Stimulated by retinoic acid gene 8                            (Stra8) - were consistently detected in aged ovaries                             and diplotene-stage                            meiotically-arrested oocytes (Msy2) were variably                             or not expressed; similarly, expression of a gene that marks growing oocytes (Zp3)                            was not detected in aged ovary tissue  and aged (20-month) C57BL/6 female mice for germline-specific gene                            expression. Genes associated with meiotic competence - namely s Figure .       e Figure . In conte Figure . Cells ee Figure .       e Figure  and 1C. Oct4-Gfp or TgOG2 transgenic) into the ovarian bursal                            sacs of young adult wild-type female recipients. In brief, the bursal sac                            surrounding a wild-type host ovary was opened, and one-half of the host ovary                            was removed prior to inserting one-half of an ovary from an aged TgOG2 female                            in its place. The tissue was then allowed to settle back into the peritoneal                            cavity and the incision was closed. The remaining half of each aged TgOG2 ovary                            not transplanted was fixed immediately and processed for pre-grafting GFP                            expression analysis. Six weeks later, the mice were given a single                            intraperitoneal injection of vehicle or trichostatin-A (TSA), the latter of                            which enhances oogenesis in young adult and middle-age female mice [To                            test if these quiescent germ cells in aged mouse ovaries still possess the                            ability to generate oocytes and form follicles, we grafted ovarian tissue                            harvested from aged female germline-specific GFP-expressing mice  germ cells in the ovaries after HU treatment,                        similar to that reported to occur in the testes of HU-treated males [Stra8 expression and regeneration                        of follicles following DXR treatment in young adult female mice  or the National                        Institute on Aging . Transgenic mice with GFP expression driven                        by a modified Oct-4 promoter (ΔPE-Oct4-Gfp or TgOG2 mice) to convey germline                        specificity of the transgene [                ransgene -28 were ransgene . DoxorubRT-PCR analysis.                 Total RNA was extracted from each                        whole ovary sample followed by DNase treatment to eliminate contaminating                        genomic DNA, and 1 μg was reverse transcribed  using oligo-dT primers. Amplification via 26-45 cycles of PCR was then performed using                        platinum Taq polymerase (Invitrogen) and Buffer-D . For each sample, mRNA encoded by the β-actin gene was                        amplified and used as a sample loading control for standardization. All PCR                        products were subcloned and sequenced for confirmation. Forward and reverse                        primers used were as follows, with GenBank Accession Number, size of amplified                        cDNA product and region of coding sequence amplified indicated:                                                   Oocyte (follicle) counts.                 Ovaries were fixed in a solution containing 0.34 N glacial                        acetic acid, 10% formalin and 28% ethanol, and embedded in paraffin. Serial                        sections were cut (8 μm), aligned in order on glass slides, and stained with                        hematoxylin and picric methyl blue. The number of non-atretic and atretic                        immature  follicles per ovary was then                        determined as detailed previously [                eviously ,30.    Immunodetection of STRA8, GFP and NOBOX.                Ovaries were fixed in 4% neutral-buffered paraformaldehyde at room                        temperature for 3-4 hours and embedded in paraffin. Tissue sections (6 μm) were                        cut and mounted on glass microscope slides. Sections were de-waxed in xylenes,                        re-hydrated in a graded ethanol series, and then boiled in 10 mM sodium citrate                        for antigen retrieval [                etrieval ,16,31. Aetrieval ,28. For etrieval , GFP detParabiosis.                 Each                        parabiont  was anesthetized and an incision was made from the                        olecranon to the knee joint of each mouse. The olecranon and knee joint were                        attached by a single 5-0 Polyglactin 910 suture and tie, and the dorsal and                        ventral skin flaps were approximated by staples and suture [                d suture . Seven wOvarian grafting.                For kidney capsule transplants, young (3-month-old), middle-age                        (12-month-old) or aged (24-month-old) wild-type female mice were anaesthetized                         to expose the left kidney in each mouse                        through a dorso-lateral incision. For each recipient animal, a small space was                        made under the kidney capsule, and ovaries collected from young adult                        (2-month-old) wild-type donor mice were placed into the space. At the same                        time, both host ovaries were removed. The kidney was then allowed to settle                        back into the peritoneal cavity and the incision was closed. Three weeks after                        surgery, grafted ovaries were removed from host kidneys and processed for                        follicle counts. For grafting into ovarian bursal sacs, similar surgical                        procedures were followed with the exception that the left ovary instead of the                        kidney of each recipient mouse was exposed. The bursal sac surrounding the                        ovary was opened, and one-half of the wild-type host ovary was removed prior to                        inserting one-half of an ovary from an aged TgOG2 female in its place,                        essentially as described [                escribed . The tisescribed ,16. TwenData                                presentation and analysis.                 All experiments were independently replicated at least                        3 times (see figure legends for details), using different mice in each                        experiment. Where appropriate, assignment of mice to each experimental group                        was made randomly. Quantitative data from experimental replicates were pooled                        and presented as the mean ± SEM. Representative outcomes from the RT-PCR and                        immunodetection analyses are provided for qualitative assessment.


Sensitisation to TNF-induced apoptosis was prevented by the broad caspase inhibitor zVAD-fmk, whereas overexpression of Bcl-2 had no effect. Mithramycin also potentiated cell death induced by Fas agonistic antibodies. In contrast, mithramycin reduced the percentage of cells undergoing apoptosis due to factor withdrawal. TNF-induced activation of NF-kappaB (NF-κB)-dependent gene expression was not modulated by mithramycin treatment. Concomitantly with the increased sensitivity, the protein level of the short-spliced cFLIP variant was downregulated. These results indicate that mithramycin enhances TNF-induced cell death in an NF-κB-independent manner, and suggest that the Fas-associated death domain protein plays a crucial role in the TNF-sensitising effect of mithramycin.In this report we show that mithramycin considerably increases the direct cytotoxic effect of tumour necrosis factor (TNF) on tumour cells  Mithramycin A (trade name Plicamycin) is an anticancer drug used to treat several types of cancer including chronic myeloid leukaemia and acute myeloid leukaemia (κB). Nuclear factor-kappaB is believed to be activated via TRADD and TNF receptor-associated factor-2(TRAF2), and/or receptor interacting protein (RIP) . Differential responses to this cytokine are thought to be due to different intracellular signalling pathways . One majin vivo, have limited the use of TNF as an antitumour agent  and 10% hGM-CSF-conditioned medium (complete medium). hGM-CSF-conditioned medium was obtained from stably transduced human embryonic kidney 293 cells with a pLHCX plasmid  encoding the human GM-CSF protein. The HL60 promyelocytic leukaemia and U937 human histiocytic lymphoma cell lines were maintained in RPMI 1640 medium containing 10% FCS. The GM-CSF producing HEK-293 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FCS. The caspase inhibitor zVAD-fmk, the transcription inhibitor actinomycin D and the antimitotic agent aphidicolin were supplied by Sigma, Dublin, Irl. Recombinant human TNF was purchased from R&D systems, Abington, UK. Anti-Fas antibodies  and anti-FLIP γ/δ antibodies were obtained from MBL, MA, USA and Calbiochem, Nottingham, UK respectively. Mithramycin A was purchased from Tocris, Bristol, UK.All human cell lines were obtained from the ATCC. TF-1, a human GM-CSF-dependent erythroleukaemic cell line  staining according to Darzynkiewicz  expressing a hrGFP gene controlled by a synthetic promoter that contains five binding sites for NF-κB. A measure of 5 μg of reporter plasmid was transfected with lipofectin  with complete protease inhibitor mix from Roche, East Sussex, UK. In total, 30 μg of protein were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (12% SDS–PAGE), transferred to a nitrocellulose membrane and immunoblotted with the indicated antibody. Immunoreactive proteins were detected by enhanced chemiluminescence .Proteins were extracted in RIPA buffer . We therefore investigated the expression levels of the short-spliced form of cFLIP in TF-1 cells. As shown in Figure 8Since TNF-induced apoptosis, but not TNF-mediated NF-This current report demonstrates that a combination of TNF and mithramycin enhanced apoptosis of TF-1 cells within 24 h, relative to single treatments of TNF or mithramycin alone. Apoptosis induced by TNF and mithramycin treatment was effectively blocked by treatment with z-VAD-fmk peptide, an inhibitor of caspases, indicating that caspases played a critical role in the execution phase of apoptosis induced by TNF in the presence of mithramycin. Studies on the involvement of the mitochondria in the regulation of apoptosis revealed that TF-1 cells overexpressing the antiapoptotic protein Bcl-2 were not protected against apoptosis induced by TNF and mithramycin. These results further indicate that in the presence of mithramycin, TNF induced apoptosis via a caspase-signalling cascade that executed apoptosis independently of the proapoptotic machinery of the mitochondria. These results support the hypothesis that mithramycin targets an inhibitor of apoptosis involved in TNF-induced apoptosis, at the level of death receptor signalling.Mithramycin also significantly increased apoptosis mediated by another member of the death receptor family, Fas (CD95/APO-1), and could not be suppressed by overexpression of Bcl-2, suggesting the existence of a common mithramycin-sensitive inhibitor of apoptosis. TNF- and Fas-induced signalling pathways leading to caspase activation and apoptosis converge at the level of FADD  FADD is a common mediator of Fas and TNF receptor-induced apoptosis, (ii) in the TNF-receptor signalling complex, FADD is immediately downstream TRADD, a molecule that allows TNF-mediated NF-The ability of mithramycin to enhance the direct cytotoxic effects of TNF (and TNF family members) has wide ranging therapeutic implications. Since the mithramycin/TNF combination is capable of inducing apoptosis in TF-1 cells independently of the mitochondrial pathway, combining TNF treatment with mithramycin could have potential for elimination of tumour cells that express large amounts of Bcl-2. In addition, apoptosis induced by TNF in the presence of mithramycin was enhanced in three cell lines with different p53 status. As loss of p53 function contributes to resistance of some tumour cells to TNF-induced cytotoxicity -induced cytotoxicity, thus representing a novel alternative in cancer therapy. Indeed, TRAIL or Apo-2L, a member of the TNF family, induces apoptosis preferentially in tumour cells also through binding to its cognate death receptors and recruitment of FADD (Recent data indicate that induction of tumour cell death by stimulation of death receptors such as Fas constitutes a more prominent mechanism in the defence against tumours than has been thought previously . As bloc of FADD . Unlike Since mithramycin has been used for many years in the clinic to treat several types of cancer, the sensitising effect of mithramycin is not only intriguing but merits further study to realize the full potential of mithramycin as an adjunctive therapy in death receptor-mediated apoptosis.


Elevated expression of matrix metalloproteinases (MMPs), a family of secreted proteinases that degrade matrix components of basement membranes and connective tissues, is strongly correlated with malignant expression in various human epithelial cancers and epithelial cancer cell lines. We have tested whether elevated levels of MMP expression are also associated with malignant progression in human cutaneous squamous cell carcinoma. Constitutive levels of expression of steady-state mRNA and of secreted protein encoded by three MMP genes  were compared in a unique in vitro model of human skin carcinogenesis. This model is composed of the parental immortalized non-tumorigenic human keratinocyte line (HaCaT), and three activated c-Harvey-ras-oncogene transfected variants . Although clone A-4 is non-tumorigenic, clones I-7 and II-4 exhibit benign and malignant tumorigenic phenotypes, respectively, after subcutaneous injection into athymic nude mice. Northern blot, Western blot, and zymogram analyses revealed three MMP-specific patterns of expression. Constitutive matrilysin mRNA expression was markedly increased in the I-7 cells compared with HaCaT, A-4 or II-4 cells. Secreted promatrilysin was distinctly increased in the tumorigenic I-7 and II-4 cells compared with the non-tumorigenic HaCaT and A-4 cells. Gelatinase A mRNA and secreted gelatinase A protein levels were increased in each transfectant compared with HaCaT. Both active and inactive forms of gelatinase A were detected. Gelatinase B transcripts were not detected, but an EDTA-inhibitable gelatinase activity comigrating with gelatinase B was moderately enhanced in both tumorigenic variants compared with the non-tumorigenic cells. Because promatrilysin and 92-kDa gelatinase secretion were increased in both benign and malignant tumorigenic cells, and not related to invasiveness in this model, it is concluded that enhanced constitutive expression of these two MMPs is associated with acquisition of the tumorigenic phenotype, before acquisition of the malignant phenotype.


BDNF (rBDNF) locus containing the gene, 13 kb of genomic sequences upstream of BDNF exon I, and 144 kb downstream of protein encoding exon IX, in which protein coding region was replaced with the lacZ reporter gene. This BDNF-BAC drove transgene expression in the brain, heart, and lung, recapitulating endogenous BDNF expression to a larger extent than shorter rat BDNF transgenes employed previously. Moreover, kainic acid induced the expression of the transgenic BDNF mRNA in the cerebral cortex and hippocampus through preferential activation of promoters I and IV, thus recapitulating neuronal activity-dependent transcription of the endogenous BDNF gene. genesis 48:214–219, 2010. © 2010 Wiley-Liss, Inc.Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family of neurotrophic factors, has important functions in the peripheral and central nervous system of vertebrates. We have generated bacterial artificial chromosome (BAC) transgenic mice harboring 207 kb of the rat  BDNF gene expression accompany and contribute to the development of various disorders of the nervous system (Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family of proteins, supports the survival and differentiation of certain neuronal populations during development . In the s system .BDNF gene contains multiple promoters that initiate the transcription of a number of distinct mRNAs, each of which contains an alternative 5′ untranslated exon spliced to a common 3′ protein coding exon. In addition, the protein coding exon employs two different polyadenylation sites that give rise to mRNA species with 3′ untranslated regions (UTRs) of different lengths. Alternative promoter usage, differential splicing, and the use of two different polyadenylation sites within each of the transcription units generate at least 22 different BDNF mRNAs in rodents and 34 BDNF mRNAs in human that encode the same mature BDNF protein  locus, encompassing the genomic region from 13 kb upstream of rBDNF exon I to 144 kb downstream of rBDNF coding exon. Neighboring genes of the rBDNF gene lie 151 kb upstream (Ifna4) and 190 kb downstream (Sqrdl) from it and therefore no additional genes/promoters were included in the BAC construct. To facilitate detection of transgene expression, we replaced the protein coding region of exon IX in the rBDNF-BAC with lacZ reporter gene (lacZ reporter gene in rBDNF-lacZ-BAC was detected by transient expression in COS-7 cells (data not shown).In the present study, we have generated a transgenic mouse line using a bacterial artificial chromosome (BAC) clone containing 207 kb of rat ter gene . This shrBDNF-lacZ-BAC transgenic line, the expression of rBDNF-lacZ mRNA was detected by RT-PCR in several brain regions and peripheral organs expressing endogenous mouse BDNF (mBDNF) mRNA  mRNA . SpecifirBDNF-lacZ-BAC transgenic mice, in situ hybridization analysis revealed intense labeling of both rBDNF-lacZ and endogenous mBDNF mRNAs in the cerebral cortex .In the adult brain of the l cortex  and 3g,hl cortex ,, hippocl cortex  and 3a–fl cortex , nucleusl cortex , and hypl cortex  includinl cortex . In the l cortex , caudatel cortex , high lelaustrum  and hypolaustrum , rBDNF-lubfields  mirroreds . These results suggest that β-galactosidase protein was either not translated from BAC-driven rBDNF-lacZ mRNAs or the levels of expression of the reporter protein remained below detection limits of the methods used in this study.We also analyzed the expression and enzymatic activity of β-galactosidase protein in BDNF mRNA expression in the adult rodent hippocampus and cerebral cortex .Kainic acid has been shown to induce mRNA see . The eleobserved . Quantitmoter VI . SimilarrBDNF gene have been described previously. rBDNF-CAT transgenic mice carrying 9 kb of genomic sequence comprising one or more BDNF 5′ untranslated exons were reported in  reporter gene expression in the heart  gene, 84 kb of upstream and 17 kb of downstream sequences gene) suggests that a heart-specific regulatory element is located within 18-144 kb 3′ of BDNF gene. However, this prediction should be treated with caution as regulatory regions of BDNF genes of different species are compared. On the other hand, neither hBDNF-EGFP-BAC  modified to replace rBDNF coding sequence with the lacZ reporter gene  (rBDNF genomic locus (GenBank: AC108236) including 50 kb of rBDNF gene, 13 kb of 5′ and 144 kb of 3′ flanking sequences  by injection of NotI-linearized rBDNF-lacZ-BAC into CBA x C57Bl/6 mouse pronuclei. One transgenic founder mouse was obtained and bred to establish a transgenic mouse line. Integration of two copies of rBDNF-lacZ-BAC transgene was estimated by slot-blot hybridization of genomic DNA with [α-32P]dCTP-labeled lacZ-specific probe.equences . PurifierBDNF-lacZ mRNA expression in transgenic mouse tissues with RT-PCR was performed as described ((r) Green I No ROX . qPCR reactions were processed in triplicate and all expression data were normalized to hypoxan-thine phosphoribosyltransferase 1 (HPRT1) mRNA levels. For primer sequences see 35S]UTP-labeled cRNA probes for rBDNF-lacZ and endogenous mouse BDNF mRNA was performed as described in rBDNF-lacZ-BAC mice weighing 20–25 g. Two kainic acid-treated and two vehicle-treated animals were used for qRT-PCR analysis. Four kainic acid-treated animals and one vehicle-treated animal were used for in situ hybridization analysis. Only animals with induced tonic-clonic seizures were selected for analysis and results are shown for individuals showing highest induction of transgenic and endogenous BDNF mRNA. All animal procedures were carried out in compliance with the local ethics committee.RNA isolation and analysis of


Somatic cell nuclear transfer (scNT)-derived piglets have high rates of mortality, including stillbirth and postnatal death. Here, we examined severe malformed umbilical cords (MUC), as well as other organs, from nine scNT-derived term piglets.Microscopic analysis revealed complete occlusive thrombi and the absence of columnar epithelial layers in MUC (scNT-MUC) derived from scNT piglets. scNT-MUC had significantly lower expression levels of platelet endothelial cell adhesion molecule-1 (PECAM-1) and angiogenesis-related genes than umbilical cords of normal scNT piglets (scNT-N) that survived into adulthood. Endothelial cells derived from scNT-MUC migrated and formed tubules more slowly than endothelial cells from control umbilical cords or scNT-N. Proteomic analysis of scNT-MUC revealed significant down-regulation of proteins involved in the prevention of oxidative stress and the regulation of glycolysis and cell motility, while molecules involved in apoptosis were significantly up-regulated. Histomorphometric analysis revealed severe calcification in the kidneys and placenta, peliosis in the liver sinusoidal space, abnormal stromal cell proliferation in the lungs, and tubular degeneration in the kidneys in scNT piglets with MUC. Increased levels of apoptosis were also detected in organs derived from all scNT piglets with MUC.These results suggest that MUC contribute to fetal malformations, preterm birth and low birth weight due to underlying molecular defects that result in hypoplastic umbilical arteries and/or placental insufficiency. The results of the current study demonstrate the effects of MUC on fetal growth and organ development in scNT-derived pigs, and provide important insight into the molecular mechanisms underlying angiogenesis during umbilical cord development. In the past decade, several species of animal, including goat, pig, sheep and cattle, have been cloned using scNT techniques . Howeverin utero, which can affect the survival, health and well-being of the newborn and the productivity of the animal at adulthood. Inadequate conceptus-maternal interaction is particularly acute in animal production using scNT. The incidence of abnormal placental development is very high in scNT-derived animals, and is largely responsible for the frequent fetal loss, late-term pregnancy complications and perinatal mortality associated with this technique . Slides were mounted in crystal mount  and visualized by microscopy.TUNEL assays were performed as described previously . BrieflyAll experimental data represent the means ± standard deviation (SD). Analysis of angiogenesis-related gene expression pattern in figure two were performed three times by using 10 scNT-N and 6 scNT-MUC piglets and the statistical significance was confirmed by using t-test. Wound healing and tubule formation in figures five and six were analyzed by using 2 controls, 3 scNT-N and 6 scNT-MUC samples with three replicates and the statistical significance was calculated by ANOVA and confirmed by Duncan's multiple range procedure. Protein and mRNA expression levels (figure seven) were examined in 3 controls, 6 scNT-MUC with three replicates and the statistical significance was confirmed by t-test.In this study, we carried out scNT using each donor fibroblast cell derived from Duroc, Berkshire and 3 way hybrids (Landrace × Duroc × Yorkshire). scNT embryos were transferred and successfully produced 65 cloned piglets from 24 surrogates Table . The numUpon histological examination, scNT-MUC exhibited severe tissue damage as compared to control and scNT-N, including tissue swelling Figure , arrows.When we examined the expression of PECAM-1 (CD31), which is abundantly expressed in ECs , scNT-MUEC activation is initiated by the binding of proangiogenic factors, such as vascular endothelial growth factor (VEGF), to their cognate receptors on endothelial cells, an event that induces the activation of angiogenic signaling pathways . VEGF isWe also observed several abnormalities in scNT-MUC-derived placentas, including villous hypovascularity and cytotrophoblast and syncytotrophoblast hypoplasia Figure . PlacentPUVECs were obtained from the veins of control, scNT-N and scNT-MUC, as described previously . We estaTo determine whether the changes in angiogenesis-related gene expression that we observed earlier in scNT-MUC affected EC motility, we performed a migration assay using PUVEC cell lines from control, scNT-N and scNT-MUC piglets. PUVECs were grown to confluence and an area of the dish  was scraped clear of cells by applying suction with a narrow tip Figure . Cells wIn addition to the migration and proliferation of ECs, proper vascularization requires vascular maturation, during which the rate of vascular sprouting is attenuated to prevent vascular collapse. Vascular maturation involves the recruitment of both perivascular and smooth muscle cells . We nextThe umbilical cords of rodents and large mammals, including humans, are glucose-dependent tissues that undergo limited mitochondrial respiration, and rely predominantly on the anaerobic conversion of glucose to lactate . We compp < 0.05) than in control. In particular, there was a pronounced difference in the mRNA expression level of destrin . When we directly compared protein and mRNA expression patterns, with the exception of alpha-centractin, all cytoskeletal and motility-related proteins exhibited similar patterns of down-regulation at both the protein and mRNA levels in scNT-MUC  polymerase (PARP) and pro/active caspases 3 and 8 were significantly up-regulated Figure  and 8D. To investigate the relationship between MUC and fetal malformation, we examined several postnatal fetal organs by microscopy Figure . We obseRecent studies from our laboratory suggest that the low success rate of scNT cloning is due to placental abnormalities rather than to cumulative genomic damage ,7,11,12.2, while diminishing O2 levels in fetal organs, stimulating apoptosis in the developing fetus. Thus, the functional consequences of MUC in scNT-derived animals are potentially severe, and include not only placental insufficiency, fetal abnormalities and mortality, but also fetal malformations, preterm birth, and low birth weight. Our data suggest that these effects are due to specific molecular defects that lead to the development of HUA and/or placental insufficiency. In summary, the results of the current study provide several clues to a better understanding of the molecular mechanisms of angiogenesis during umbilical cord development, as well as a robust model for the study of HUA syndrome in humans.Our observations suggest that due to MUC, the blood flow may be reduced between placenta and fetus, resulting in an increase in apoptosis in the umbilical cord. Because the umbilical cord plays a fundamental role in transporting metabolites between mother and fetus, MUC also contribute to placental insufficiency by preventing the removal of harmful materials from fetal circulation. MUC might also promote hypoxia and the accumulation of COAR: aldose reductase; Ang: Angiopoietin; CAPNS1: calpain small subunit; 2-DE: 2-dimensional gel electrophoresis; EC: Endothelial cell; HUAs: hypoplastic umbilical arteries; ILSM: inner longitudinal smooth muscle; MUC: malformed umbilical cords; OCSM: outer circular smooth muscle; PARP: poly (ADP-ribose) polymerase; PECAM-1: platelet endothelial cell adhesion molecule-1; Prx: peroxiredoxin; ROS: reactive oxygen species; PUVEC: porcine umbilical vein-derived endothelial cells; scNT: somatic cell nuclear transfer; SOD: superoxide dismutase; SUA: single umbilical artery; TMOD3: tropomodulin 3; TPM-1: tropomyosin 1 alpha; TPM-2: tropomyosin 1 beta; TPM-4: tropomyosin alpha 4 chain; TUNEL: TdT-mediated dUTP-X Nicked End labeling; VEGF: vascular endothelial growth factor; WD-1: WD-repeat protein 1HHP, SSP performed the experiments in Figure Table s1.Click here for fileMaterials and Methods for Supplementary Figures S1 and S2.Click here for fileFigure s1.Click here for fileTable s2.Click here for fileFigure s2.Click here for fileTable s3.Click here for file


Lymphoma of the mucosa-associated lymphoid tissue (MALT) type usually arises in MALT acquired through chronic antigenic stimulation triggered by persistent infection and/or autoimmune processes. Due to specific ligand–receptor interactions between lymphoid cells and high-endothelial venules of MALT, both normal and neoplastic lymphoid cells display a pronounced homing tendency to MALT throughout the body. In the case of neoplastic disease these homing properties may be responsible for lymphoma dissemination among various MALT-sites. According to this concept, we have standardized staging procedures in all patients diagnosed with MALT-type lymphoma. All patients with MALT-type lymphoma underwent standardized staging procedures before treatment. Staging included ophthalmologic examination, otolaryngologic investigation, gastroscopy with multiple biopsies, endosonography of the upper gastrointestinal tract, enteroclysis, colonoscopy, computed tomography of thorax and abdomen and bone marrow biopsy. Biopsy was performed in all lesions suggestive for lymphomatous involvement, and evaluation of all biopsy specimens was performed by a reference pathologist. 35 consecutive patients with histologically verified MALT-type lymphoma were admitted to our department. Twenty-four patients (68%) had primary involvement of the stomach, five (15%) had lymphoma of the ocular adnexa, three (8.5%) had lymphoma of the parotid, and three  of the lung. Lymph-node involvement corresponding to stage EII disease was found in 13 patients (37%), only one patient with primary gastric lymphoma had local and supradiaphragmatic lymph-node involvement (stage EIII). Bone marrow biopsies were negative in all patients. Overall, eight of 35 patients (23%) had simultaneous biopsy-proven involvement of two MALT-sites: one patient each had lymphoma of parotid and lacrimal gland, conjunctiva and hypopharynx, conjunctiva and skin, lacrimal gland and lung, stomach and colon, and stomach and lung. The remaining two patients had bilateral parotideal lymphoma. Staging work-up was negative for lymph-node involvement in all of these eight patients. The importance of extensive staging in MALT-type lymphoma is emphasized by the demonstration of multiorgan involvement in almost a quarter of patients. In addition, our data suggest that extra-gastrointestinal MALT-type lymphoma more frequently occurs simultaneously at different anatomic sites than MALT-type lymphoma involving the GI-tract. © 2000 Cancer Research Campaign