Exploring the regeneration potential of salivary glands using organoids as a model

Radiotherapy is a potential life-saving treatment for head and neck cancer patients. However, despite improvements in precision of radiation dose delivered, the unavoidable co-irradiation of salivary gland still leads to irreversible diminishing of saliva secretion affecting among others the ability to speak, eat and sleep, drastically decreasing the quality of life of the patients. Existing treatments for this dry mouth syndrome, only provide short-term relief. Therefore, the development of new therapies, such as stem cell therapy, is crucial to alleviate this side effect caused by irradiation. Combining mouse injury models with 3D organoid cultures of mouse and human salivary gland derived cells, it is attempted to identify the optimal cell source for, as well as the regulatory mechanisms involved in, the salivary gland regeneration process. Thereby this work focused on the development and optimization of a potentially clinically relevant regenerative therapy approach to functionally restore the irradiated salivary gland tissue

The evolving definition of salivary gland stem cells

Dysfunction of the salivary gland and irreversible hyposalivation are the main side effects of radiotherapy treatment for head and neck cancer leading to a drastic decrease of the quality of life of the patients. Approaches aimed at regenerating damaged salivary glands have been proposed as means to provide long-term restoration of tissue function in the affected patients. In studies to elucidate salivary gland regenerative mechanisms, more and more evidence suggests that salivary gland stem/progenitor cell behavior, like many other adult tissues, does not follow that of the hard-wired professional stem cells of the hematopoietic system. In this review, we provide evidence showing that several cell types within the salivary gland epithelium can serve as stem/progenitor-like cells. While these cell populations seem to function mostly as lineage-restricted progenitors during homeostasis, we indicate that upon damage specific plasticity mechanisms might be activated to take part in regeneration of the tissue. In light of these insights, we provide an overview of how recent developments in the adult stem cell research field are changing our thinking of the definition of salivary gland stem cells and their potential plasticity upon damage. These new perspectives may have important implications on the development of new therapeutic approaches to rescue radiation-induced hyposalivation

Autophagy induction during stem cell activation plays a key role in salivary gland self-renewal

17 p.-6 figRelatively quiescent tissues like salivary glands (SGs) respond to stimuli such as injury to expand,replace and regenerate. Resident stem/progenitor cells are key in this process because, upon activation, they possess the ability to self-renew. Macroautophagy/autophagy contributes to and regulates differentiation in adult tissues, but an important question is whether this pathway promotes stem cell self-renewal in tissues. We took advantage of a 3D organoid system that allows assessing the self-renewal of mouse SGs stem cells (SGSCs). We found that autophagy in dormant SGSCs has slower flux than self-renewing SGSCs. Importantly, autophagy enhancement upon SGSCs activation is a self-renewal feature in 3D organoid cultures and SGs regenerating in vivo. Accordingly,autophagy ablation in SGSCs inhibits self-renewal whereas pharmacological stimulation promotes self-renewal of mouse and human SGSCs. Thus, autophagy is a key pathway for self-renewal activation in low proliferative adult tissues, and its pharmacological manipulation has the potential to promote tissue regeneration.F.R. is supported by Marie Skłodowska-Curie Cofund [713660], Marie Skłodowska-Curie ITN [765912], ALW Open Program [ALWOP.310]and ZonMW VICI [016.130.606] grants. R.C. and C.R. are supported by the Dutch Cancer Society [Grant number 5792 and 12458]. Patricia Boya is supported by the Ministerio de Ciencia, Innovación y Universidades (MCIU), the Agencia Estatal de Investigación (AEI),the Fondo Europeo de Desarrollo Regional (FEDER) [PGC2018-098557-B-I00] and a Marie Skłodowska-Curie ITN grant [765912]. Idil Orhon is a recipient of a FEBS postdoctoral fellowship and Beatriz Villarejo-Zori of a Fundacion Tatiana Perez de Guzman el Bueno predoctoral fellowhip.Peer reviewe

Purification and Ex Vivo Expansion of Fully Functional Salivary Gland Stem Cells

Hyposalivation often leads to irreversible and untreatable xerostomia. Salivary gland (SG) stem cell therapy is an attractive putative option to salvage these patients but is impeded by the limited availability of adult human tissue. Here, using murine SG cells, we demonstrate single-cell self-renewal, differentiation, enrichment of SG stem cells, and robust in vitro expansion. Dependent on stem cell marker expression, SG sphere-derived single cells could be differentiated in vitro into distinct lobular or ductal/lobular organoids, suggestive of progenitor or stem cell potency. Expanded cells were able to form miniglands/organoids containing multiple SG cell lineages. Expansion of these multipotent cells through serial passaging resulted in selection of a cell population, homogenous for stem cell marker expression (CD24hi/CD29hi). Cells highly expressing CD24 and CD29 could be prospectively isolated and were able to efficiently restore radiation-damaged SG function. Our approach will facilitate the use of adult SG stem cells for a variety of scientific and therapeutic purposes

Effect of starvation on brain glucose metabolism and 18F-2-fluoro-2-deoxyglucose uptake: an experimental in-vivo and ex-vivo study

Background: The close connection between neuronal activity and glucose consumption accounts for the clinical value of 18F-fluoro-2-deoxyglucose (FDG) imaging in neurodegenerative disorders. Nevertheless, brain metabolic response to starvation (STS) might hamper the diagnostic accuracy of FDG PET/CT when the cognitive impairment results in a severe food deprivation. Methods: Thirty six-week-old BALB/c female mice were divided into two groups: \u201ccontrol\u201d group (n = 15) were kept under standard conditions and exposed to fasting for 6 h before the study; the remaining \u201cSTS\u201d mice were submitted to 48 h STS (absence of food and free access to water) before imaging. In each group, nine mice were submitted to dynamic micro-PET imaging to estimate brain and skeletal muscle glucose consumption (C- and SM-MRGlu*) by Patlak approach, while six mice were sacrificed for ex vivo determination of the lumped constant, defined as the ratio between CMRGlu* and glucose consumption measured by glucose removal from the incubation medium (n = 3) or biochemical analyses (n = 3), respectively. Results: CMRGlu* was lower in starved than in control mice (46.1 \ub1 23.3 vs 119.5 \ub1 40.2 nmol 7 min 121 7 g 121 , respectively, p < 0.001). Ex vivo evaluation documented a remarkable stability of lumped constant as documented by the stability of GLUT expression, G6Pase activity, and kinetic features of hexokinase-catalyzed phosphorylation. However, brain SUV in STS mice was even (though not significantly) higher with respect to control mice. Conversely, a marked decrease in both SM-MRGlu* and SM-SUV was documented in STS mice with respect to controls. Conclusions: STS markedly decreases brain glucose consumption without altering measured FDG SUV in mouse experimental models. This apparent paradox does not reflect any change in lumped constant. Rather, it might be explained by the metabolic response of the whole body: the decrease in FDG sequestration by the skeletal muscle is as profound as to prolong tracer persistence in the bloodstream and thus its availability for brain uptak

Lack of Association between Nuclear Factor ErythroidDerived 2-Like 2

Oxidative stress involvement has been strongly hypothesized among the possible pathogenic mechanisms of motor neuron degeneration in amyotrophic lateral sclerosis (ALS). The intracellular redox balance is finely modulated by numerous complex mechanisms critical for cellular functions, among which the nuclear factor erythroid-derived 2-like 2 (NFE2L2/Nrf2) pathways. We genotyped, in a cohort of ALS patients ( = 145) and healthy controls ( = 168), three SNPs in Nrf2 gene promoter: −653 A/G, −651 G/A, and −617 C/A and evaluated, in a subset ( = 73) of patients, advanced oxidation protein products (AOPP), ironreducing ability of plasma (FRAP), and plasma thiols (-SH) as oxidative damage peripheral biomarkers. Nrf2 polymorphisms were not different among patients and controls. Increased levels of AOPP ( < 0.05) and decreased levels of FRAP ( < 0.001) have been observed in ALS patients compared with controls, but no difference in -SH values was found. Furthermore, no association was found between biochemical markers of redox balance and Nrf2 polymorphisms. These data confirm an altered redox balance in ALS and indicate that, while being abnormally modified compared to controls, the oxidative stress biomarkers assessed in this study are independent from the −65

Chloroquine inhibits autophagic flux by decreasing autophagosome-lysosome fusion

Macroautophagy/autophagy is a conserved transport pathway where targeted structures are sequestered by phagophores, which mature into autophagosomes, and then delivered into lysosomes for degradation. Autophagy is involved in the pathophysiology of numerous diseases and its modulation is beneficial for the outcome of numerous specific diseases. Several lysosomal inhibitors such as bafilomycin A(1) (BafA(1)), protease inhibitors and chloroquine (CQ), have been used interchangeably to block autophagy in in vitro experiments assuming that they all primarily block lysosomal degradation. Among them, only CQ and its derivate hydroxychloroquine (HCQ) are FDA-approved drugs and are thus currently the principal compounds used in clinical trials aimed to treat tumors through autophagy inhibition. However, the precise mechanism of how CQ blocks autophagy remains to be firmly demonstrated. In this study, we focus on how CQ inhibits autophagy and directly compare its effects to those of BafA(1). We show that CQ mainly inhibits autophagy by impairing autophagosome fusion with lysosomes rather than by affecting the acidity and/or degradative activity of this organelle. Furthermore, CQ induces an autophagy-independent severe disorganization of the Golgi and endo-lysosomal systems, which might contribute to the fusion impairment. Strikingly, HCQ-treated mice also show a Golgi disorganization in kidney and intestinal tissues. Altogether, our data reveal that CQ and HCQ are not bona fide surrogates for other types of late stage lysosomal inhibitors for in vivo experiments. Moreover, the multiple cellular alterations caused by CQ and HCQ call for caution when interpreting results obtained by blocking autophagy with this drug

Detección y caracterización preliminar de Escherichia coli O174 productor de toxina

El síndrome urémico hemolítico (SUH), una en­fermedad transmitida por alimentos, es la principal causa de insuficiencia renal aguda y la segunda causa de insuficiencia renal crónica y de trasplante renal en niños en la Argentina, con 400 a 500 notificaciones anuales de casos. Este síndrome origina los costos más elevados para el Sistema Nacional de Salud. Se considera que Escherichia coli productora de toxina Shiga (STEC) es el agente etiológico más frecuente del SUH. Dentro de este patotipo STEC asociado a SUH, se destaca el serotipo STEC O157:H7 que representa el 60 % de dichos aislamientos. Otros serogrupos STEC no-O157 representan el 40 % de los aislamientos de STEC provenientes de SUH. La preva­lencia de cada serotipo depende de cada país. Se informó que los cuatro serogrupos preva­lentes en la Argentina dentro de los no-O157 son O145, O121, O26 y O174. De ellos, el serogrupo O174 se destaca como proble­mática local y no está cubierto por protocolos de diag­nóstico europeos ni americanos. Las infecciones por STEC en la Argentina, por su naturaleza endémica, su alta incidencia y la presentación de brotes difusos, di­fieren de las de otras regiones referenciales en materia normativa. Para asegurar la calidad e inocuidad agroalimentaria, podría ser incluido el diagnóstico de O174 en alimentos. El objetivo de este proyecto es evaluar técnicas diagnósticas específicas de este serogrupo.Hemolytic uremic syndrome (HUS), a foodborne disease, is the main cause of acute renal failure and the second cause of chronic renal failure and kidney transplant in children in Argentina, with 400-500 cases each year. This syndrome provokes the highest costs to the National Health System. Shiga toxinproducing Escherichia coli (STEC) is considered the most frequent etiological agent of HUS. Among STEC serotypes associated with HUS, O157:H7 represents 60% of the isolates. Other non-O157 STEC serogroups account for the remaining 40%. The prevalence of each serotype varies from one country to other. It was reported that the four prevalent non-O157 serogroups in Argentina are O145, O121, O26 and O174. Among these, serogroup O174 stands out as a local problem and is not included in european or american diagnostic protocols. STEC infections in Argentina, due to their endemic nature, high incidence and occurrence of diffuse outbreaks differ from those taking place in countries whose regulatory standards serve for reference purposes. In order to ensure food quality and safety, diagnosis of O174 in food should be included. The aim of this study is to assess specific diagnostic techniques for this serogroup

MYC-containing double minutes in hematologic malignancies: evidence in favor of the episome model and exclusion of MYC as the target gene

Double minutes (dmin)—circular, extra-chromosomal amplifications of specific acentric DNA fragments—are relatively frequent in malignant disorders, particularly in solid tumors. In acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS), dmin are observed in ∼1% of the cases. Most of them consist of an amplified segment from chromosome band 8q24, always including the MYC gene. Besides this information, little is known about their internal structure. We have characterized in detail the genomic organization of 32 AML and two MDS cases with MYC-containing dmin. The minimally amplified region was shown to be 4.26 Mb in size, harboring five known genes, with the proximal and the distal amplicon breakpoints clustering in two regions of ∼500 and 600 kb, respectively. Interestingly, in 23 (68%) of the studied cases, the amplified region was deleted in one of the chromosome 8 homologs at 8q24, suggesting excision of a DNA segment from the original chromosomal location according to the ‘episome model'. In one case, sequencing of both the dmin and del(8q) junctions was achieved and provided definitive evidence in favor of the episome model for the formation of dmin. Expression status of the TRIB1 and MYC genes, encompassed by the minimally amplified region, was assessed by northern blot analysis. The TRIB1 gene was found over-expressed in only a subset of the AML/MDS cases, whereas MYC, contrary to expectations, was always silent. The present study, therefore, strongly suggests that MYC is not the target gene of the 8q24 amplification