Obesity drives adipose-derived stem cells into a senescent and dysfunctional phenotype associated with P38MAPK/NF-KB axis.

BACKGROUND: Adipose-derived stem cells (ADSC) are multipotent cells implicated in tissue homeostasis. Obesity represents a chronic inflammatory disease associated with metabolic dysfunction and age-related mechanisms, with progressive accumulation of senescent cells and compromised ADSC function. In this study, we aimed to explore mechanisms associated with the inflammatory environment present in obesity in modulating ADSC to a senescent phenotype. We evaluated phenotypic and functional alterations through 18 days of treatment. ADSC were cultivated with a conditioned medium supplemented with a pool of plasma from eutrophic individuals (PE, n = 15) or with obesity (PO, n = 14), and compared to the control. RESULTS: Our results showed that PO-treated ADSC exhibited decreased proliferative capacity with G2/M cycle arrest and CDKN1A (p21WAF1/Cip1) up-regulation. We also observed increased senescence-associated ß-galactosidase (SA-ß-gal) activity, which was positively correlated with TRF1 protein expression. After 18 days, ADSC treated with PO showed augmented CDKN2A (p16INK4A) expression, which was accompanied by a cumulative nuclear enlargement. After 10 days, ADSC treated with PO showed an increase in NF-κB phosphorylation, while PE and PO showed an increase in p38MAPK activation. PE and PO treatment also induced an increase in senescence-associated secretory phenotype (SASP) cytokines IL-6 and IL-8. PO-treated cells exhibited decreased metabolic activity, reduced oxygen consumption related to basal respiration, increased mitochondrial depolarization and biomass, and mitochondrial network remodeling, with no superoxide overproduction. Finally, we observed an accumulation of lipid droplets in PO-treated ADSC, implying an adaptive cellular mechanism induced by the obesogenic stimuli. CONCLUSIONS: Taken together, our data suggest that the inflammatory environment observed in obesity induces a senescent phenotype associated with p38MAPK/NF-κB axis, which stimulates and amplifies the SASP and is associated with impaired mitochondrial homeostasis.

Biochemical characterization of adenosine deaminase (CD26; EC 3.5.4.4) activity in human lymphocyte-rich peripheral blood mononuclear cells.

The conversion of adenosine to inosine is catalyzed by adenosine deaminase (ADA) (EC 3.5.4.4), which has two isoforms in humans (ADA1 and ADA2) and belongs to the zinc-dependent hydrolase family. ADA modulates lymphocyte function and differentiation, and regulates inflammatory and immune responses. This study investigated ADA activity in lymphocyte-rich peripheral blood mononuclear cells (PBMCs) in the absence of disease. The viability of lymphocyte-rich PBMCs isolated from humans and kept in 0.9% saline solution at 4-8°C was analyzed over 20 h. The incubation time and biochemical properties of the enzyme, such as its Michaelis-Menten constant (Km) and maximum velocity (Vmax), were characterized through the liberation of ammonia from the adenosine substrate. Additionally, the presence of ADA protein on the lymphocyte surface was determined by flow cytometry using an anti-CD26 monoclonal human antibody, and the PBMCs showed long-term viability after 20 h. The ADA enzymatic activity was linear from 15 to 120 min of incubation, from 2.5 to 12.5 µg of protein, and pH 6.0 to 7.4. The Km and Vmax values were 0.103±0.051 mM and 0.025±0.001 nmol NH3·mg-1·s-1, respectively. Zinc and erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) inhibited enzymatic activity, and substrate preference was given to adenosine over 2'-deoxyadenosine and guanosine. The present study provides the biochemical characterization of ADA in human lymphocyte-rich PBMCs, and indicates the appropriate conditions for enzyme activity quantification.

Biochemical characterization of adenosine deaminase (CD26 EC 3. 5. 4. 4) activity in human lymphocyte-rich peripheral blood mononuclear cells

The conversion of adenosine to inosine is catalyzed by adenosine deaminase (ADA) (EC 3.5.4.4), which has two isoforms in humans (ADA1 and ADA2) and belongs to the zinc-dependent hydrolase family. ADA modulates lymphocyte function and differentiation, and regulates inflammatory and immune responses. This study investigated ADA activity in lymphocyte-rich peripheral blood mononuclear cells (PBMCs) in the absence of disease. The viability of lymphocyte-rich PBMCs isolated from humans and kept in 0.9% saline solution at 4-8°C was analyzed over 20 h. The incubation time and biochemical properties of the enzyme, such as its Michaelis-Menten constant (Km) and maximum velocity (Vmax), were characterized through the liberation of ammonia from the adenosine substrate. Additionally, the presence of ADA protein on the lymphocyte surface was determined by flow cytometry using an anti-CD26 monoclonal human antibody, and the PBMCs showed long-term viability after 20 h. The ADA enzymatic activity was linear from 15 to 120 min of incubation, from 2.5 to 12.5 µg of protein, and pH 6.0 to 7.4. The Km and Vmax values were 0.103±0.051 mM and 0.025±0.001 nmol NH3·mg-1·s-1, respectively. Zinc and erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) inhibited enzymatic activity, and substrate preference was given to adenosine over 2′-deoxyadenosine and guanosine. The present study provides the biochemical characterization of ADA in human lymphocyte-rich PBMCs, and indicates the appropriate conditions for enzyme activity quantification.

Blockade of CD73 delays glioblastoma growth by modulating the immune environment.

Immunotherapy as an approach for cancer treatment is clinically promising. CD73, which is the enzyme that produces extracellular adenosine, favors cancer progression and protects the tumor from immune surveillance. While CD73 has recently been demonstrated to be a potential target for glioma treatment, its role in regulating the inflammatory tumor microenvironment has not yet been investigated. Thus, this study explores the immunotherapeutic value of the CD73 blockade in glioblastoma. The immuno-therapeutic value of the CD73 blockade was evaluated in vivo in immunocompetent pre-clinical glioblastoma model. As such, glioblastoma-bearing rats were nasally treated for 15 days with a siRNA CD73-loaded cationic-nanoemulsion (NE-siRNA CD73R). Apoptosis was determined by flow cytometry using Annexin-V staining and cell proliferation was analyzed by Ki67 expression by immunohistochemistry. The frequencies of the CD4+, CD8+, and CD4+CD25highCD39+ (Treg) T lymphocytes; CD11b+CD45high macrophages; CD11b+CD45low-microglia; and CD206+-M2-like phenotypes, along with expression levels of CD39 and CD73 in tumor and tumor-associated immune cells, were determined using flow cytometry, while inflammatory markers associated with tumor progression were evaluated using RT-qPCR. The CD73 blockade by NE-siRNA CD73 was found to induce tumor cell apoptosis. Meanwhile, the population of Tregs, microglia, and macrophages was significantly reduced in the tumor microenvironment, though IL-6, CCL17, and CCL22 increased. The treatment selectively decreased CD73 expression in the GB cells as well as in the tumor-associated-macrophages/microglia. This study indicates that CD73 knockdown using a nanotechnological approach to perform nasal delivery of siRNA-CD73 to CNS can potentially regulate the glioblastoma immune microenvironment and delay tumor growth by inducing apoptosis.

CD73 Downregulation Decreases In Vitro and In Vivo Glioblastoma Growth.

Glioblastoma is the worst and most common primary brain tumor. Here, we demonstrated the role of CD73, an enzyme responsible for adenosine (ADO) production, in glioblastoma progression. ADO increased glioma cell viability via A1 receptor sensitization. CD73 downregulation decreased glioma cell migration and invasion by reducing metalloproteinase-2 and vimentin expression and reduced cell proliferation by 40%, which was related to necrosis and sub-G1 phase blockage of cell cycle. Those effects also involved the stimulation of Akt/NF-kB pathways. Additionally, CD73 knockdown or enzyme inhibition potentiated temozolomide cytotoxic effect on glioma cells by decreasing the IC50 value and sensitizing cells to a non-cytotoxic drug concentration. CD73 inhibition also decreased in vivo rat glioblastoma progression. Delivery of siRNA-CD73 or APCP reduced tumor size by 45 and 40%, respectively, when compared with control. This effect was followed by a parallel 95% reduction of ADO levels in cerebrospinal fluid, indicating the role of extracellular ADO in in vivo glioma growth. Treatment did not induce systemic damage or mortality. Altogether, we conclude that CD73 is an interesting target for glioblastoma treatment and its inhibition may provide new opportunities to improve the treatment of brain tumors. Graphical Abstract ᅟ.

Relationship between nutritional status and histologic findings in small bowel mucosa of children presenting with diarrhoea of more than 14 days' duration.

In order to study the eventual effects of malnutrition on small intestinal mucosa, we evaluated 85 children with diarrhoea of more than 14 days' duration, aged from 4 to 114 months (median 17 months). A proximal small intestinal biopsy was obtained and villus height, crypt depth, mucosal thickness, and total mucosal thickness were measured. Gomez, Waterlow, and Z score criteria were applied. Statistical analyses were performed with the Spearman correlation test and the non-parametrical tests of Wilcoxon, Mann-Whitney, and Kruskal-Wallis. A value of p < 0.05 was considered significant. Average villus height was 269.2 microns (+/- 87.5 microns); crypt depth 113.0 microns (+/- 33.8 microns); mucosal thickness 210.5 microns (+/- 73.2 microns); total mucosal thickness 485.9 microns (+/- 111.8 microns); and villus height/crypt depth ratio 2.5:1 (+/- 0.8:1). Five children had kwashiorkor and 13 had marasmus. Villus height for kwashiorkor children ranged from 151 microns to 353.3 microns (average 286.7 microns), crypt depth from 90.3 microns to 154 microns (average 111.11 microns). According to Gomez criteria, as malnutrition increased, mucosal thickness and the villus/crypt ratio decreased. Waterlow criteria had no relation to mucosal sizes. When distributed in sequential decrease according to their nutritional state, the Z score for weight for age and weight for height indices showed a positive correlation with villus height, total mucosal thickness, and villus/crypt ratio.

Caroli's disease: report of 10 cases in children and adolescents in southern Brazil.

PURPOSE: The aim of this study was to describe the authors' experience with Caroli's disease in children and adolescents. METHODS: The authors reviewed the hospital charts of 10 children and adolescents with Caroli's disease diagnosed between 1968 and 1996. RESULTS: The median age at the onset of symptoms was 5.5 months and the median age at diagnosis was 12 months, both much lower than those reported in the literature. Clinical symptoms were compatible with the classical findings of Caroli's disease, but jaundice and hepatosplenomegaly occurred more frequently. There was an association with congenital renal malformation in eight cases (80%), congenital hepatic fibrosis in five cases, and choledochal cyst in two cases. One case presented the pure form of the disease.

Estudo citologico em nodulos da tiroide por biopsia de aspiracao. / Cytological study in thyroid nodules using aspiration biopsy

Os autores fazem um estudo comparativo entre os resultados dos exames citopatologicos de material obtido por puncao aspirativa, em 105 casos de nodulos isolados da tireoide, com os resultados dos exames anatomopatologicos por inclusao em parafina