Eigenvalue estimates for submanifolds of warped product spaces

We give lower bounds for the fundamental tone of open sets in minimal submanifolds immersed into warped product spaces of type $N^n \times_f Q^q$, where $f \in C^\infty(N)$. We also study the essential spectrum of these minimal submanifolds.Comment: 17 page

Relato da Incidência da Cochonilha-do-Carmim (Dactylopius Opuntiae) em dois Gêneros de Cactáceas / Report of the Incidence of the Cochonylid (Dactylopius Opuntiae) in two Generals of Caccacies

A palma-forrageira apresenta-se como a principal fonte de alimento para os rebanhos bovinos, caprinos e ovinos nos longos períodos de estiagem no semiárido nordestino. No entanto, esta base de sustentação alimentar se encontra seriamente ameaçada por um inseto produtor do ácido carmínico, a cochonilha-do-carmim, Dactylopius opuntiae. Este trabalho objetivou avaliar o comportamento dessa cochonilha em relação à infestação cruzada, em três espécies de cactáceas, pertencentes a dois gêneros (Opuntia ficus indica - palma-forrageira, Opuntia dillenii - palmatória e Tacinga inamoena – gogoia ou quipá), A pesquisa foi conduzida no Instituto Federal de Educação Ciência e Tecnologia da Paraíba – IFPB/ Campus Picuí. O experimento foi instalado em uma estrutura móvel confeccionada com tubos de PVC e sombrite a 50%, com quatro tratamentos e cinco repetições: Tratamento 1: Palma não infectada + Gogoia infectada, Tratamento 2: Palma infectada + Gogoia não infectada, Tratamento 3: Palmatória não infectada + Gogoia infectada e Tratamento 4: Palmatória não infectada + Palma infectada. As raquetes infectadas e não infectadas foram dispostas lado a lado em vasos retangulares. A cochonilha-do-carmim presente em T. inamoena infestou a espécie O. ficus indica a partir de 14 dias até o final, não aparentado preferência por O. dillenii. Mesmo atacando a gogoia, a cochonilha da palma-forrageira não esteve presente nessa espécie em algumas avaliações. Entretanto, o inseto presente na palma-forrageira infestou a palmatória a partir da segunda avaliação (14 dias) até o final das observações. Contudo, constatou-se que houve contaminação inversa, o que demonstra certa preferência alimentar do inseto pelos dois gêneros de cactáceas estudados

Comparative Study of Immune Regulatory Properties of Stem Cells Derived from Different Tissues.

International audience: Allogeneic stem cell (SC)-based therapy is a promising tool for the treatment of a range of human degenerative and inflammatory diseases. Many reports highlighted the immune modulatory properties of some SC types, such as mesenchymal stromal cells (MSCs), but a comparative study with SCs of different origin, to assess whether immune regulation is a general SC property, is still lacking. To this aim, we applied highly standardized methods employed for MSC characterization to compare the immunological properties of bone marrow-MSCs, olfactory ectomesenchymal SCs, leptomeningeal SCs, and three different c-Kit-positive SC types, that is, amniotic fluid SCs, cardiac SCs, and lung SCs. We found that all the analyzed human SCs share a common pattern of immunological features, in terms of expression of activation markers ICAM-1, VCAM-1, HLA-ABC, and HLA-DR, modulatory activity toward purified T, B, and NK cells, lower immunogenicity of inflammatory-primed SCs as compared to resting SCs, and indoleamine-2,3-dioxygenase-activation as molecular inhibitory pathways, with some SC type-related peculiarities. Moreover, the SC types analyzed exert an anti-apoptotic effect toward not-activated immune effector cells (IECs). In addition, we found that the inhibitory behavior is not a constitutive property of SCs, but is acquired as a consequence of IEC activation, as previously described for MSCs. Thus, immune regulation is a general property of SCs and the characterization of this phenomenon may be useful for a proper therapeutic use of SCs

Prospective monitoring of Chronic Myeloid Leukemia Patients from Time of TKI Discontinuation: the fate of Peripheral Blood CD26+ Leukemia Stem Cells

Introduction: In chronic myeloid leukemia (CML), about half of the patients achieving a deep and stable molecular response with tyrosine kinase inhibitors (TKIs) may discontinue TKI treatment without disease recurrence. As such, treatment-free remission (TFR) has become an ambitious goal of treatment. Given the evidence that deepness and duration of molecular response are necessary but not sufficient requisites for a successful TFR, additional biological criteria are needed to identify CML patients suitable for efficacious discontinuation. Leukemia stem cells (LSCs) are supposed to be the reservoir of the disease. Previously, we demonstrated that residual circulating CD34+/CD38-/CD26+ LSCs were still detectable in a consistent number of CML patients during TFR.Methods: CML LSCs could be easily identified by flow-cytometry as they express the CD34+/CD38-/CD26+ phenotype. In this study, we explored the role of these cells and their correlation with molecular response in a cohort of 109 consecutive chronic phase CML patients prospectively monitored from the time of TKI discontinuation.Results: After a median observation time of 33 months from TKI discontinuation, 38/109 (35%) patients failed TFR after a median time of 4 months, while 71/109 (65%) patients are still in TFR. At TKI discontinuation, peripheral blood CD26+LSCs were undetectable in 48/109 (44%) patients and detectable in 61/109 (56%). No statistically significant correlation between detectable/undetectable CD26+LSCs and the rate of TFR loss was found (p = 0.616). The incidence of TFR loss based on the type of TKI treatment was statistically significant for imatinib treatment compared to that of nilotinib (p = 0.039). Exploring the behavior of CD26+LSCs during TFR, we observed fluctuating values that were very variable between patients, and they were not predictive of TFR loss.Discussion: Up to date, our results confirm that CD26+LSCs are detectable at the time of TKI discontinuation and during TFR. Moreover, at least for the observation median time of the study, the persistence of "fluctuating" values of residual CD26+LSCs does not hamper the possibility to maintain a stable TFR. On the contrary, even patients discontinuing TKI with undetectable CD26+LSCs could undergo TFR loss. Our results suggest that factors other than residual LSCs "burden" playing an active role in controlling disease recurrence. Additional studies evaluating CD26+LSCs' ability to modulate the immune system and their interaction in CML patients with very long stable TFR are ongoing

Residual peripheral blood CD26+leukemic stem cells in chronic myeloid leukemia patients during TKI therapy and during treatment-free remission

Chronic myeloid leukemia (CML) patients in sustained “deep molecular response” may stop TKI treatment without disease recurrence; however, half of them lose molecular response shortly after TKI withdrawing. Well-defined eligibility criteria to predict a safe discontinuation up-front are still missing. Relapse is probably due to residual quiescent TKI-resistant leukemic stem cells (LSCs) supposedly transcriptionally low/silent and not easily detectable by BCR-ABL1 qRT-PCR. Bone marrow Ph+ CML CD34+/CD38− LSCs were found to specifically co-express CD26 (dipeptidylpeptidase-IV). We explored feasibility of detecting and quantifying CD26+ LSCs by flow cytometry in peripheral blood (PB). Over 400 CML patients (at diagnosis and during/after therapy) entered this cross-sectional study in which CD26 expression was evaluated by a standardized multiparametric flow cytometry analysis on PB CD45+/CD34+/CD38− stem cell population. All 120 CP-CML patients at diagnosis showed measurable PB CD26+ LSCs (median 19.20/μL, range 0.27–698.6). PB CD26+ LSCs were also detectable in 169/236 (71.6%) CP-CML patients in first-line TKI treatment (median 0.014 cells/μL; range 0.0012–0.66) and in 74/112 (66%), additional patients studied on treatment-free remission (TFR) (median 0.015/μL; range 0.006–0.76). Notably, no correlation between BCR-ABL/ABLIS ratio and number of residual LSCs was found both in patients on or off TKIs. This is the first evidence that “circulating” CML LSCs persist in the majority of CML patients in molecular response while on TKI treatment and even after TKI discontinuation. Prospective studies evaluating the dynamics of PB CD26+ LSCs during TKI treatment and the role of a “stem cell response” threshold to achieve and maintain TFR are ongoing

Comparison of Epithelial Differentiation and Immune Regulatory Properties of Mesenchymal Stromal Cells Derived from Human Lung and Bone Marrow

Mesenchymal stromal cells (MSCs) reside in many organs including lung, as shown by their isolation from fetal lung tissues, bronchial stromal compartment, bronchial-alveolar lavage and transplanted lung tissues. It is still controversial whether lung MSCs can undergo mesenchymal-to-epithelial-transition (MET) and possess immune regulatory properties. To this aim, we isolated, expanded and characterized MSCs from normal adult human lung (lung-hMSCs) and compared with human bone marrow-derived MSCs (BM-hMSCs). Our results show that lung-MSCs reside at the perivascular level and do not significantly differ from BM-hMSCs in terms of immunophenotype, stemness gene profile, mesodermal differentiation potential and modulation of T, B and NK cells. However, lung-hMSCs express higher basal level of the stemness-related marker nestin and show, following in vitro treatment with retinoic acid, higher epithelial cell polarization, which is anyway partial when compared to a control epithelial bronchial cell line. Although these results question the real capability of acquiring epithelial functions by MSCs and the feasibility of MSC-based therapeutic approaches to regenerate damaged lung tissues, the characterization of this lung-hMSC population may be useful to study the involvement of stromal cell compartment in lung diseases in which MET plays a role, such as in chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Standardization of immune quality controls on clinical-grade mesenchymal stromal cells of different origin produced according to GMP rules

La versatile ed ampia applicazione delle Cellule Stromali Mesenchimali (MSC) per diverse applicazioni cliniche ha generato negli ultimi anni un grande e crescente interesse per utilizzo clinico delle MSC come terapia cellulare. La ricerca sulla biologia cellulare delle MSC e\u2019 in continuo sviluppo, come dimostrato dal crescente numero di trial clinici basati sull\u2019utilizzo clinico di MSC. Alcuni di questi trials sfruttano la capacita\u2019 immunosoppressiva delle MSC per la cura di patologie come la GvHD e la sclerosi multipla. Le MSC esercitano un\u2019azione immunosoppressiva potenzialmente diretta contro tutte le cellule del sistema immunitario. Di conseguenza, per quantificare le capacita' immunosoppressive di MSC espanse in accordo a regole GMP da diversi laboratori, e\u2019 di fondamentale importanza sviluppare test in vitro altamente standardizzati e riproducibili, senza i quali non sarebbe possibile ottenere risultati comparabili e riproducibili tra diversi gruppi di ricerca. Scopo del lavoro e\u2019 stato quello di comparare le capacita\u2019 immunoregolatorie di tre diversi tipi di MSC (espanse in accordo a regole GMP) in due laboratori indipendenti (Stem Cell Research Laboratory of the University of Verona and the INSERM Institute in Rennes, France). La standardizzazione di test funzionali ha permesso di dimostrare che MSC \u201cresting\u201d sono in grado di inibire la proliferazione di cellule T ed NK ma non di cellule B. MSC da tessuto adiposo coltivate in lisato piastrinico (ADSC-PL) hanno mostrato la piu\u2019 forte immunosoppressione nei confronti di cellule T, tramite l\u2019attivita\u2019, INF- \u3b3 dipendente, dell\u2019enzima indoleamina 2,3 diossigenasi. Le MSC non sono in grado di evocare una proliferazione di cellule T allogeniche, ma vengo efficacemente lisate da cellule NK attivate. L\u2019applicazione sistematica di test quantitativi e riproducibili ha messo in evidenza differenti proprieta\u2019 immunologiche delle MSC prodotte secondo differenti protocolli di espansione GMP. Tra le cellule testate, le ADSC-PL emergono come le piu\u2019 promettenti per l\u2019utilizzo in futuri trials clinici.The versatile and wide range application of Mesenchymal Stromal Cells (MSC) for a diverse set of clinical indications has generated a great and increasing interest in MSC as potential therapeutic agents during last years. Research on MSC biology is progressing rapidly, as illustrated by the growing number of clinical trials using MSC. Some of them are taking advance of the immunosuppressive ability of MSC to treat some immunological based disease like GvHD or Multiple Sclerosis. MSC exhibit a wide range of immunosuppressive properties that target virtually any cell of the immune system and these properties can be easily affected and hampered by culture conditions. Therefore, to assess the immunological properties of clinical-grade MSC obtained from different laboratories it is crucial to design fully standardized and reproducible in vitro assays; otherwise, only not comparable, and frequent contradictory results could be achieved. We aimed to compare immune modulatory properties of clinical-grade MSC using a combination of fully standardized in vitro assays. BMMSC expanded with FCS (BMMSC-FCS) or PL (BMMSC-PL), and ADSC-PL were analyzed in parallel in two independent laboratories (Stem Cell Research Laboratory of the University of Verona and the INSERM Institute in Rennes, France). Standardized functional assays revealed that resting MSC inhibited proliferation of T and NK cells, but not B cells. ADSC-PL were the most potent in inhibiting T-cell growth, a property ascribed to IFN-\u3b3-dependent indoleamine 2,3-dioxygenase activity. MSC did not stimulate allogeneic T cell proliferation but were efficiently lysed by activated NK cells. The systematic use of quantitative and reproducible validation techniques highlights differences in immunological properties of MSC produced using various clinical-grade processes. Among all cell tested, ADSC-PL emerge as the best promising candidate for future clinical trials