Class-switzched B cells display response to therapeutic B-cell depletion in rheumatoid arthritis

Introduction Reconstitution of peripheral blood (PB) B cells after therapeutic depletion with the chimeric anti-CD20 antibody rituximab (RTX) mimics lymphatic ontogeny. In this situation, the repletion kinetics and migratory properties of distinct developmental B-cell stages and their correlation to disease activity might facilitate our understanding of innate and adaptive B-cell functions in rheumatoid arthritis (RA). Methods Thirty-five 'RTX-naïve' RA patients with active arthritis were treated after failure of tumour necrosis factor blockade in an open-label study with two infusions of 1,000 mg RTX. Prednisone dose was tapered according to clinical improvement from a median of 10 mg at baseline to 5 mg at 9 and 12 months. Conventional disease-modifying antirheumatic drugs were kept stable. Subsets of CD19+ B cells were assessed by flow cytometry according to their IgD and CD27 surface expression. Their absolute number and relative frequency in PB were followed every 3 months and were determined in parallel in synovial tissue (n = 3) or synovial fluid (n = 3) in the case of florid arthritis. Results Six of 35 patients fulfilled the European League Against Rheumatism criteria for moderate clinical response, and 19 others for good clinical response. All PB B-cell fractions decreased significantly in number (P < 0.001) after the first infusion. Disease activity developed independently of the total B-cell number. B-cell repopulation was dominated in quantity by CD27-IgD+ 'naïve' B cells. The low number of CD27+IgD- class-switched memory B cells (MemB) in the blood, together with sustained reduction of rheumatoid factor serum concentrations, correlated with good clinical response. Class-switched MemB were found accumulated in flaring joints. Conclusions The present data support the hypothesis that control of adaptive immune processes involving germinal centre-derived, antigen, and T-cell-dependently matured B cells is essential for successful RTX treatment.

Italia: La chiusura della Casa dell’Emigrante come spunto riflessione sui rientri degli emigrati italiani

In the literature on migration, as well as in social policies regarding this phenomenon, the situation of returning emigrants receives scant attention. This essay establishes an intricate connection between attitudes and policies that prevail in a country regarding emigration and those concerning immigration. The case of Italy provides a prime example for this as it once was a classical country of emigration, only to turn, in recent decades, into a country that appears highly attractive (and relatively accessible) to immigrants. The essay traces the pervasive ambiguity that characterizes this country’s attitudes towards emigration from the beginning of mass emigration shortly after the unification of Italy in 1868 to the emigration policies of the fascist regime of Mussolini and the post-World War II waves of emigration right through to the corresponding ambiguity concerning the status of immigrants in contemporary society, including the indifferent treatment of returning Italian emigrants who constitute a considerable numerical phenomenon. These reflections take their origin from the impending closure of a reception centre in Lazio, the Casa dell’Emigrante near Sant’Elia Fiumerapido, Province of Frosinone, ostensibly for financial reasons. This centre had been the only one of its kind in the whole of Italy dealing officially with the needs of repatriated Italians. It had assisted returning emigrants both with practical matters, such as negotiating the labyrinth of Italian bureaucracy , and with psychological implications of a return, which are often considerable given the time lag of experiences with current social realities and the frequently unrealistic expectations associated with the return. Questions of identity become highly acute in those circumstances. The threatened closure of the centre illustrates the unwillingness of the state to face up to the factual prevalence of migratory experiences in the country as a whole and as a core element of national history, experiences of migration in both directions. The statistics speak for themselves: of the 4.660.427 persons who left Italy between 1880 and 1950, 2.322.451 have returned, almost exactly 50%. To those have to be added 3.628.430 returnees of the 5.109.860 emigrants who left Italy between the end of World War II and 1976 for Europe alone. Attitudes towards people leaving changed ostensibly over time. In the first two decades after Unification parliament on the one hand wanted to show some concern over the fate of its citizens, not wanting to abandon those newly created citizens entirely to their own destiny, while on the other portraying their decisions to emigrate as expressions of individual liberty and responsibility and not necessitated by want and poverty. Emigrants had to prove, paradoxically that they had the requisite means to emigrate when in fact poverty was largely driving them to emigrate. To admit that publicly would have amounted to admission of economic and political failure made evident through emigration. In contrast to that Mussolini’s emigration policies not only enforced large population movements within the territory of Italy to balance unemployment between regions and particularly between North and South, but also declared it citizen’s duty to be ready to move also to the colonies, thereby ‘turning emigration as a sign of social crisis into a sign of national strength and the success of the country’s political agenda’ (Gaspari 2001, p. 34). The duplicity continued even after World War II when secret deals were done with the USA to allow a continuous flow of Italian immigrants and EU membership obviously further facilitated the departure of unemployed, impoverished Italians. With the growing prosperity of Italy the reversal of the direction of migration became more obvious. On the basis of empirical research conducted by one of the author on returning emigrants four types of motives for returning can be distinguished: 1. Return as a result of failure – particularly the emigrants who left during the 1950-1970 period usually had no linguistic preparation, and in any case the gap between the spoken and the written language is enormous with the latter often being insurmountable. This gives rise to nostalgic sentiments which motivates a return into an environment where language is familiar 2. Return as a means of preserving an identity – the life of emigrants often takes place within ghetto-like conditions where familiarity is being reproduced but under restricted conditions and hence not entirely authentic. The necessity for saving money permits only a partial entry into the host society and at the same time any accumulating savings add to the desire to return home where life can be lived fully again – or so it seems. 3. Return of investment – the impossibility to become fully part of another society often motivates migrants to accumulate not so much material wealth but new experiences and competences which they then aim to reinvest in their home country. 4. Return to retire – for many emigrants returning home becomes acute once they leave a productive occupation and feelings of estrangement build up, in conjunction with the efforts of having invested in building a house back home. All those motives are associated with a variety of difficulties on the actual return home because, above all, time in relation to the country of origin has been suspended for the emigrant and the encounter with the reality of that country reveals constant discrepancies and requires constant readjustment. This is where the need for assistance to returning emigrants arises. The fact that such an important centre of assistance has been closed is further confirmation of the still prevailing politics of ambiguity which nominally demand integration from nationals and non-nationals alike but deny the means of achieving this. Citizenship is not a natural result of nationality but requires the means for active participation in society. Furthermore, the experiences of returning immigrants provide important cues for the double ambivalence in which immigrants to Italy live between the demands made on them to integrate, the simultaneous threats of repatriation and the alienation from the immigrants’ home country which grows inexorably during the absence. The state can only regain its credibility by putting an end to this ambiguity and provide to returning emigrants, and immigrants alike, the means of reconstructing strong communal identities.

Bevezető: utak és úton lévők

A kvalitatív módszerekkel nyert kutatási eredményeink értelmezése során a transznacionális tér, a transznacionális és az etnikai migráció elméleti és szemléleti kereteit egyaránt figyelembe vettük. Az általunk vizsgált migrációs folyamatok transznacionális térben zajlanak, és a transznacionális irodalomban leírt migráns élethelyzetek, gyakorlatok – különböző nemzetállamokban elhelyezkedő lokalitásokhoz való egyidejű, bár eltérő intenzitású kötődés, kapcsolatok – több példájával is találkoztunk. Ludger Pries nyomán a transznacionális migrációt és a transznacionális migráns alakját olyan ideáltípusnak tekintettük, amelyhez az egyes migráns utak és helyzetek csupán közelítenek, és empirikus eredményeink alapján azt mondhatjuk, hogy a valóban plurilokális, vagyis a két helyhez való egyidejű, intenzív és tartós kötődés s az ehhez kapcsolódó gyakorlatok csupán a migránsok kisebbségét, illetve a migrációs életpályák egy-egy szakaszát jellemzik. A vizsgált migrációs folyamatokban az etnicitás strukturális tényezőként és a migráns tapasztalatok értelmezési kereteként egyaránt perdöntő szerepet játszik. Az etnikai migráció szakirodalomban tárgyalt mindhárom magyarázó modellje – az anyaországba való hazatérés, a gazdasági okokból való, illetve a kisebbségi létben elszenvedett sérelmek által ösztönzött migráció – alkalmas a migrációt kiváltó és mozgató okok elemzésére, a migráns narratívák értelmezésére, azt azonban nem állíthatjuk, hogy bármelyikük kizárólagos érvényre tehet szert. Más kutatókhoz hasonlóan Rogers Brubaker meghatározását tartjuk a leginkább gyümölcsözőnek, aki az etnikai migráció tág értelmezését használva minden olyan vándorlási folyamatot etnikai migrációnak tekint, amelyben az etnicitás kulturális és szimbolikus tőkeként szabályozó szerepet játszik. This special issue of Tér és Társadalom presents some results of an international research project carried out by researchers from Switzerland, Hungary and Serbia between 2010 and 2012. The topic of the research was “Integrating (Trans-)national Migrants in Transition States” (TRANSMIG) and was financed by the Swiss National Science Foundation (SNSF). The research aimed to explore and interpret migration flows from the Vojvodina (Serbia) to Hungary and from ex-Yugoslav republics to the Vojvodina. In the first period of the last twenty years, wars which contributed to the disintegration of Yugoslavia and the formation of new national states have caused migration flows. After the change of the millennium, educational migration of Vojvodina Hungarian youth can be considered the most important migratory movement from the Vojvodina to Hungary. Labour (economic) migration also occurs, but this cannot be understood as a one-way movement, since in the Hungarian–Serbian border zone migrants from the Vojvodina who already resettled to Hungary commute to the Vojvodina. While interpreting the qualitative research data the theoretical frameworks and approaches of transnational space, transnationalism and ethnic migration were taken into consideration. The migration movement in question occurs in a transnational social space where migrants are in constant motion. By their movements and actions that space is continually recreated. With Ludger Pries we see a transnational migrant as an ideal type to whom individual migratory movements and positions only approximate. Based on our empirical results we can conclude that real pluri-local, intensive and long-lasting bonding to two places at the same time and the relating practices only characterise a minority of migrants and certain sections of migratory careers. In the migration processes studied, ethnicity as a term is needed as a “structural factor” and frame of interpretation to approach migrant experiences. All three explanatory models for ethnic migration – return migration, economic migration, migration motivated by grievances suffered in a minority situation – are suitable to analyse the reasons that initiated migration and kept it in motion. They are helpful in interpreting migrant narratives. However, none of the reasons can claim exclusive validity. Agreeing with other researchers, we find Roger Brubaker’s definition the most useful: Ethnic migration should be comprehended in a broad sense. In addition, every migration can be considered as “ethnically” motivated where ethnicity plays a dominant role as a cultural and symbolic capital.

Sphingosine 1-phosphate (S1P) induces COX-2 expression and PGE2 formation via S1P receptor 2 in renal mesangial cells

Understanding the mechanisms of sphingosine 1-phosphate (S1P)-induced cyclooxygenase (COX)-2 expression and prostaglandin E2 (PGE2) formation in renal mesangial cells may provide potential therapeutic targets to treat inflammatory glomerular diseases. Thus, we evaluated the S1P-dependent signaling mechanisms which are responsible for enhanced COX-2 expression and PGE2 formation in rat mesangial cells under basal conditions. Furthermore, we investigated whether these mechanisms are operative in the presence of angiotensin II (Ang II) and of the pro-inflammatory cytokine interleukin-1β (IL-1β). Treatment of rat and human mesangial cells with S1P led to concentration-dependent enhanced expression of COX-2. Pharmacological and molecular biology approaches revealed that the S1P-dependent increase of COX-2 mRNA and protein expression was mediated via activation of S1P receptor 2 (S1P2). Further, inhibition of Gi and p42/p44 MAPK signaling, both downstream of S1P2, abolished the S1P-induced COX-2 expression. In addition, S1P/S1P2-dependent upregulation of COX-2 led to significantly elevated PGE2 levels, which were further potentiated in the presence of Ang II and IL-1β. A functional consequence downstream of S1P/S1P2 signaling is mesangial cell migration that is stimulated by S1P. Interestingly, inhibition of COX-2 by celecoxib and SC-236 completely abolished the migratory response. Overall, our results demonstrate that extracellular S1P induces COX-2 expression via activation of S1P2 and subsequent Gi and p42/p44 MAPK-dependent signaling in renal mesangial cells leading to enhanced PGE2 formation and cell migration that essentially requires COX-2. Thus, targeting S1P/S1P2 signaling pathways might be a novel strategy to treat renal inflammatory diseases.

Size-Dependent Uptake of Particles by Pulmonary Antigen-Presenting Cell Populations and Trafficking to Regional Lymph Nodes

The respiratory tract is an attractive target organ for novel diagnostic and therapeutic applications with nano-sized carriers, but their immune effects and interactions with key resident antigen-presenting cells (APCs) such as dendritic cells (DCs) and alveolar macrophages (AMs) in different anatomical compartments remain poorly understood. Polystyrene particles ranging from 20 nm to 1,000 nm were instilled intranasally in BALB/c mice, and their interactions with APC populations in airways, lung parenchyma, and lung-draining lymph nodes (LDLNs) were examined after 2 and 24 hours by flow cytometry and confocal microscopy. In the main conducting airways and lung parenchyma, DC subpopulations preferentially captured 20-nm particles, compared with 1,000-nm particles that were transported to the LDLNs by migratory CD11blow DCs and that were observed in close proximity to CD3+ T cells. Generally, the uptake of particles increased the expression of CD40 and CD86 in all DC populations, independent of particle size, whereas 20-nm particles induced enhanced antigen presentation to CD4+ T cells in LDLNs in vivo. Despite measurable uptake by DCs, the majority of particles were taken up by AMs, irrespective of size. Confocal microscopy and FACS analysis showed few particles in the main conducting airways, but a homogeneous distribution of all particle sizes was evident in the lung parenchyma, mostly confined to AMs. Particulate size as a key parameter determining uptake and trafficking therefore determines the fate of inhaled particulates, and this may have important consequences in the development of novel carriers for pulmonary diagnostic or therapeutic applications.

Directed differentiation and functional maturation of cortical interneurons from human embryonic stem cells

Human pluripotent stem cells are a powerful tool for modeling brain development and disease. The human cortex is composed of two major neuronal populations: projection neurons and local interneurons. Cortical interneurons comprise a diverse class of cell types expressing the neurotransmitter GABA. Dysfunction of cortical interneurons has been implicated in neuropsychiatric diseases, including schizophrenia, autism, and epilepsy. Here, we demonstrate the highly efficient derivation of human cortical interneurons in an NKX2.1::GFP human embryonic stem cell reporter line. Manipulating the timing of SHH activation yields three distinct GFP+ populations with specific transcriptional profiles, neurotransmitter phenotypes, and migratory behaviors. Further differentiation in a murine cortical environment yields parvalbumin- and somatostatin-expressing neurons that exhibit synaptic inputs and electrophysiological properties of cortical interneurons. Our study defines the signals sufficient for modeling human ventral forebrain development in vitro and lays the foundation for studying cortical interneuron involvement in human disease pathology.

Temporal genetic structure and relatedness in the Tufted Duck Aythya fuligula suggests limited kin association in winter

Conspecific aggregation of waterfowl in winter is a common example of animal flocking behaviour, yet patterns of relatedness and temporal substructure in such social groups remain poorly understood even in common species. A previous study based on mark-recapture data showed that Tufted Ducks Aythya fuligula caught on the same day were re-caught together in subsequent winters more often than expected by chance, suggesting stable assortments of ‘socially familiar’ individuals between wintering periods. The genetic relationships within these social groups were not clear. Based on 191 individuals genotyped at 10 microsatellite markers, we investigated the temporal genetic structure and patterns of relatedness among wintering Tufted Ducks at Lake Sempach, Switzerland, in two consecutive winters. We found no evidence of genetic differentiation between temporal groups within or between winters. The average levels of relatedness in temporal groups were low and not higher than expected in random assortments of individuals. However, Mantel tests performed for each sex separately revealed significant negative correlations between the pairwise relatedness coefficients and the number of days between the capture dates of pairs of wintering Tufted Duck in males and females. This pattern suggests the presence of a small number of co-migrating same-sex sibling pairs in wintering flocks of Tufted Ducks. Our findings provide one of the first genetic analyses of a common duck species outside the breeding season and contribute to the understanding of social interactions in long-distance migratory birds.

Downregulation of Rap1GAP contributes to Ras transformation.

Although abundant in well-differentiated rat thyroid cells, Rap1GAP expression was extinguished in a subset of human thyroid tumor-derived cell lines. Intriguingly, Rap1GAP was downregulated selectively in tumor cell lines that had acquired a mesenchymal morphology. Restoring Rap1GAP expression to these cells inhibited cell migration and invasion, effects that were correlated with the inhibition of Rap1 and Rac1 activity. The reexpression of Rap1GAP also inhibited DNA synthesis and anchorage-independent proliferation. Conversely, eliminating Rap1GAP expression in rat thyroid cells induced a transient increase in cell number. Strikingly, Rap1GAP expression was abolished by Ras transformation. The downregulation of Rap1GAP by Ras required the activation of the Raf/MEK/extracellular signal-regulated kinase cascade and was correlated with the induction of mesenchymal morphology and migratory behavior. Remarkably, the acute expression of oncogenic Ras was sufficient to downregulate Rap1GAP expression in rat thyroid cells, identifying Rap1GAP as a novel target of oncogenic Ras. Collectively, these data implicate Rap1GAP as a putative tumor/invasion suppressor in the thyroid. In support of that notion, Rap1GAP was highly expressed in normal human thyroid cells and downregulated in primary thyroid tumors.

ANALYSIS OF β8 INTEGRIN IN NEUROGENESIS AND NEUROVASCULAR HOMEOSTASIS

Neurogenesis in the adult mouse brain occurs within the subventricular zone (SVZ) of the lateral ventricle. In the SVZ, neural stem cells (NSC) reside in a specialized microenvironment, or vascular niche, consisting of blood vessels and their basement membranes. Most NSCs in the SVZ differentiate into progenitor cells, which further differentiate to generate neuroblasts, which then migrate from the SVZ to the olfactory bulbs (OB) along the rostral migratory stream (RMS). ECM-mediated adhesion and signaling within the vascular niche likely contribute to proper NSC self-renewal, survival, differentiation and neuroblast motility. The mechanisms that control these events are poorly understood. Previous studies from our group and others have shown that loss of the ECM receptor, αvβ8 integrin, in NSCs in the embryonic mouse brain leads to severe developmental vascular defects and premature death. Here, the functions of αvβ8 integrin in the adult brain have been examined using mice that have been genetically manipulated to lack a functional β8 integrin gene. This study reveals that loss of β8 integrin leads to widespread defects in homeostasis of the neurovascular unit, including increased intracerebral blood vessels with enhanced perivascular astrogliosis. Additionally, β8 integrin dependent defects in NSC proliferation, survival, and differentiation, as well as neuroblast migration in the RMS were observed both in vivo and in vitro. The defects correlated, in part, with diminished integrin-mediated activation of TGFβ, an ECM ligand of β8 integrin. Collectively, these data identify important adhesion and signaling functions for β8 integrin in the regulation of neural stem and progenitor cells in the SVZ as well as in neuroblast migration along the RMS in the adult brain.

ELUCIDATING THE ROLE OF CD44 EXPRESSION ON MESENCHYMAL STEM CELLS WITHIN THE TUMOR MICROENVIRONMENT

The tumor microenvironment is comprised of a vast array of heterogeneous cells including both normal and neoplastic cells. The tumor stroma recruitment process has been exploited for an effective gene delivery technique using bone marrow derived MSC. Targeted migration of the MSC toward the tumor microenvironment, while successful, is not yet fully understood. This study was designed to assess the role of CD44 in the migration of MSC toward the tumor microenvironment and to determine the implications of CD44-deficient MSC within the tumor stroma. Inhibition of MSC migration was evaluated through a variety of methods in vitro and in vivo including CD44 receptor knockdown, CD44 antagonists, CD44 neutralizing antibodies and small molecule inhibitor of matrix metalloproteinases. Blocking CD44 signaling through MMP inhibition was characterized by lack of intracellular domain cleavage and lead to the decrease in Twist gene expression. A functional relationship between CD44 and Twist expression was confirmed by chromatin immunoprecipitation. Next, a series of murine tumor models were used to examine the role of CD44 deficient stroma within the tumor microenvironment. Labeled transgenic CD44 knockout (KO) MSC or wild type (WT) C57/B6 MSC were used to analyze the stromal incorporation within murine breast carcinomas (EO771 and 4T1). Subsequent tumors were analyzed for vessel formation (CD31), and the presence of tumor associated fibroblast (TAF) markers, α-smooth muscle actin (α-SMA), fibroblast activation protein (FAP), and fibroblast specific protein (FSP). The tumors with CD44KO MSC cells had less vessel formation than the tumors with WT MSC. The lack of fibroblastic TAF population as defined by FAP/FSP expression by the CD44KO MSC admixed tumors suggest that the bone marrow derived population of MSC were unable to contribute to the fibroblastic stromal population. Subsequently, a bone marrow transplantation experiment confirmed the endogenous migratory deficiencies of the CD44KO bone marrow derived stromal cells toward the tumor microenvironment in vivo. WT mice with CD44KO bone marrow had less CD44KOderived tumor stroma compared to mice with WT bone marrow. These results indicate that CD44 is crucial to stromal cell migration and incorporation to the tumor microenvironment as TAF.

Sex identification and PIT-tagging: tools and prospects for studying intersexual differences in freshwater fishes

This study evaluated a technique to allow the long-term monitoring of individual fishes of known sex in the wild using sex confirmation in close proximity to the reproductive period combined with individual tagging. Hundreds of partially migratory roach Rutilus rutilus were tagged with passive integrated transponders (PIT) following sex determination in spring and various performance measures were compared with fish tagged outside the reproductive period in autumn. Short-term survival was > 95% for R. rutilus sexed and tagged under natural field conditions. Total length (LT) did not affect the probability of survival within the size range tagged (119–280mm), nor were there differences in timing of migration the following season between individuals sexed and tagged in spring and individuals tagged in autumn (i.e. outside the reproductive period). Also, a similar per cent of R. rutilus sexed and tagged in spring and tagged in autumn migrated the following season (34·5 and 34·7%). Moreover, long-term recapture data revealed no significant differences in body condition between R. rutilus individuals sexed and tagged in spring, individuals tagged in autumn and unmanipulated individuals. The observed sex ratio of recaptured fish did not differ from the expected values of equal recapture rates between males and females. Hence, there is no observable evidence for an adverse effect of tagging close to the reproductive period and therefore this method is suitable for studying intersexual differences and other phenotypic traits temporarily expressed during reproduction at the individual level in fishes.

There and back again: migration in freshwater fishes

Animal migration is an amazing phenomenon that has fascinated humans for long. Many freshwater fishes also show remarkable migrations, whereof the spectacular mass migrations of salmonids from the spawning streams are the most well known and well studied. However, recent studies have shown that migration occurs in a range of freshwater fish taxa from many different habitats. In this review we focus on the causes and consequences of migration in freshwater fishes. We start with an introduction of concepts and categories of migration, and then address the evolutionary causes that drive individuals to make these migratory journeys. The basis for the decision of an individual fish to migrate or stay resident is an evaluation of the costs and benefits of different strategies to maximize its lifetime reproductive effort. We provide examples by discussing our own work on the causes behind seasonal migration in a cyprinid fish, roach (Rutilus rutilus (L., 1758)), within this framework. We then highlight different adaptations that allow fish to migrate over sometimes vast journeys across space, including capacity for orientation, osmoregulation, and efficient energy expenditure. Following this we consider the consequences of migration in freshwater fish from ecological, evolutionary, and conservation perspectives, and finally, we detail some of the recent developments in the methodologies used to collect data on fish migration and how these could be used in future research.

Cell surface galactosyltransferase structure and function during mesenchymal cell migration

In order to more fully understand the function of surface GalTase on mesenchymal cells, anti-GalTase IgG was used to (a) examine the role of surface GalTase during mouse mesenchymal cell migration on laminin and fibronectin; (b) define the plasma membrane distribution of GalTase by indirect immunofluorescence on migrating cells; (c) quantitate the level of surface GalTase on migrating cells; and (d) determine whether GalTase is associated with the cytoskeleton.^ Results show that anti-GalTase IgG was able to inhibit migration (48-80% as compared to basal rate) when cells were migrating on laminin-containing matrices. Monovalent Fab fragments inhibited migration on laminin by 90% after 4 hours. On the other hand, anti-GalTase IgG had no effect on cells migrating on fibronectin. This illustrates the substrate specificity of GalTase mediated-migration. When anti-GalTase IgG was used to localize surface GalTase on cells migratory on laminin, the enzyme was restricted to the leading and trailing edges of the cell. Assays indicate that GalTase is elevated approximately 3-fold when cells are migrating on laminin-containing matrices as compared to migratory cells on plastic or fibronectin, or as compared to stationary cells on any substrate. Laminin appears to recruit GalTase from preexisting intracellular pools to the growing lamellipodia.^ Double-label indirect immunofluorescence studies indicate that there is an apparent co-localization between some of the surface GalTase and some actin filaments. This relationship was explored by extracting cells prelabeled with anti-GalTase IgG and quantitated by radiolabeled second antibodies. Results show that 79% of the surface GalTase is associated with the cytoskeleton (as judged by detergent insolubility) when monovalent antibodies (Fab) are used. However virtually all (80-100%) of the surface GalTase can be induced to associate with the cytoskeleton when cross-linked with bivalent antibodies. Furthermore, when cells in suspension are incubated with divalent antibodies, an additional 66% of the surface GalTase can be induced to associate with the cytoskeleton. The elevated levels of surface GalTase detectable on cells migrating on laminin also appear to be associated with the cytoskeleton.^ Several lines of evidence suggest that GalTase is associated with F-actin. Data suggest that laminin induces the expression of surface GalTase to the growing lamellipodia where it becomes associated with the cytoskeleton leading to cell spreading and migration. (Abstract shortened with permission of author.) ^

Targeting class IA PI3K isoforms selectively impairs cell growth, survival, and migration in glioblastoma.

The phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway is frequently activated in human cancer and plays a crucial role in glioblastoma biology. We were interested in gaining further insight into the potential of targeting PI3K isoforms as a novel anti-tumor approach in glioblastoma. Consistent expression of the PI3K catalytic isoform PI3K p110α was detected in a panel of glioblastoma patient samples. In contrast, PI3K p110β expression was only rarely detected in glioblastoma patient samples. The expression of a module comprising the epidermal growth factor receptor (EGFR)/PI3K p110α/phosphorylated ribosomal S6 protein (p-S6) was correlated with shorter patient survival. Inhibition of PI3K p110α activity impaired the anchorage-dependent growth of glioblastoma cells and induced tumor regression in vivo. Inhibition of PI3K p110α or PI3K p110β also led to impaired anchorage-independent growth, a decreased migratory capacity of glioblastoma cells, and reduced the activation of the Akt/mTOR pathway. These effects were selective, because targeting of PI3K p110δ did not result in a comparable impairment of glioblastoma tumorigenic properties. Together, our data reveal that drugs targeting PI3K p110α can reduce growth in a subset of glioblastoma tumors characterized by the expression of EGFR/PI3K p110α/p-S6.

Accelerated wound closure in vitro by fibroblasts from a subgroup of cleft lip/palate patients: role of transforming growth factor-α.

In a fraction of patients surgically treated for cleft lip/palate, excessive scarring disturbs maxillary growth and dento-alveolar development. Since certain genes are involved in craniofacial morphogenesis as well as tissue repair, a primary defect causing cleft lip/palate could lead to altered wound healing. We performed in vitro wound healing assays with primary lip fibroblasts from 16 cleft lip/palate patients. Nine foreskin fibroblast strains were included for comparison. Cells were grown to confluency and scratch wounds were applied; wound closure was monitored morphometrically over time. Wound closure rate showed highly significant differences between fibroblast strains. Statistically, fibroblast strains from the 25 individuals could be divided into three migratory groups, namely "fast", "intermediate", and "slow". Most cleft lip/palate fibroblasts were distributed between the "fast" (5 strains) and the "intermediate" group (10 strains). These phenotypes were stable over different cell passages from the same individual. Expression of genes involved in cleft lip/palate and wound repair was determined by quantitative PCR. Transforming growth factor-α mRNA was significantly up-regulated in the "fast" group. 5 ng/ml transforming growth factor-α added to the culture medium increased the wound closure rate of cleft lip/palate strains from the "intermediate" migratory group to the level of the "fast", but had no effect on the latter group. Conversely, antibody to transforming growth factor-α or a specific inhibitor of its receptor most effectively reduced the wound closure rate of "fast" cleft lip/palate strains. Thus, fibroblasts from a distinct subgroup of cleft lip/palate patients exhibit an increased migration rate into wounds in vitro, which is linked to higher transforming growth factor-α expression and attenuated by interfering with its signaling.