An Extended Flow Cytometry Evaluation of ex Vivo Expanded NK Cells Using K562.Clone1, a Feeder Cell Line Manufactured in Brazil.

Natural killer (NK) cells play a crucial role in the immune system's response against cancer. However, the challenge of obtaining the required quantity of NK cells for effective therapeutic response necessitates the development of strategies for their ex vivo expansion. This study aimed to develop a novel feeder cell line, K562.Clone1, capable of promoting the ex vivo expansion of NK cells while preserving their cytotoxic potential. he K562 leukemic cell line was transduced with mbIL-21 and 4-1BBL proteins to generate K562.Clone1 cells. NK cells were then co-cultured with these feeder cells, and their expansion rate was monitored over 14 days. The cytotoxic potential of the expanded NK cells was evaluated against acute myeloid leukemia blasts and tumor cell lines of leukemia and glial origin. Statistical analysis was performed to determine the significance of the results. The K562.Clone1 co-cultured with peripheral NK showed a significant increase in cell count, with an approximate 94-fold expansion over 14 days. Expanded NK cells demonstrated cytotoxicity against the tested tumor cell lines, indicating preservation of their cytotoxic characteristics. Additionally, the CD56, CD16, inhibitory KIRs, and activation receptors were conserved and present in a well-balanced manner. The study successfully developed a feeder cell line, K562.Clone1, that effectively promotes the expansion of NK cells ex vivo while maintaining their cytotoxic potential. This development could significantly contribute to the advancement of NK cell therapy, especially in Brazil.

Scientists of Tomorrow/ Cientistas do Amanhã : a project to inspire, stimulate scientific thinking, and introduce scientific methodology for young students.

The Scientists of Tomorrow/ Cientistas do Amanhã project is an immersive science training program developed by the Program of Post-Graduation in Health Sciences at Hospital Israelita Albert Einstein. This program was conducted in partnership with Volunteering and Escola Municipal de Ensino Fundamental Professor Paulo Freire in Paraisópolis, São Paulo, Brazil. The Scientists of Tomorrow Program comprised a short training period conducted in May 2022 involving 37 students, and a long training period from August to December 2022, which included 15 students. It aimed to popularize science through practical activities; transfer knowledge to young students; sensitize and guide them to pursue academic-scientific careers; reduce stereotypes about scientific work and scientists; and help students understand the social, political, and ethical roles of science within society. All activities were led by postgraduate students and professors from our postgraduate program, physicians, nurses, physiotherapists, biomedicals, and veterinarians from Hospital Israelita Albert Einstein, as well as medical students from Faculdade Israelita de Ciências da Saúde Albert Einstein . Activities in the short training included lectures on cinema and science, strategies to combat fake news, non-violent communication, innovation, design-thinking framework, and developing a scientific project. During the long training period, discussions were focused on nanotechnology, animal research, big data, bioinformatics, meditation, blood and bone marrow donation, telemedicine, sex and sexually-transmitted infections, rehabilitation, career opportunities, and scientific integrity. In addition, practical activities were further expanded using optical and confocal microscopy, cytometry, and basic concepts regarding the structure and function of living cells. The program also included the launching of the open-air outreach Education E-natureza activity, which turned students into ambassadors of nature. In conclusion, the Scientists of Tomorrow Program was innovative and enabled young students to learn that science is a collective activity that can enhance public health. In Brief Rangel et al. enumerated the Scientists of Tomorrow/Cientistas do Amanhã program, an immersive science initiative conducted in collaboration with a public school. The program, which involved 15 students, aimed to promote science, share knowledge, inspire academic paths, and underscore societal impacts. Led by postgraduates, professors, and healthcare experts, the program included diverse lectures and practical laboratory activities. Highlights Every research endeavor commences with a fundamental question. Sharing of findings by researchers and students contributes toward the expansion of knowledge. Teaching scientific methodology is a pivotal step in nurturing critical thinking skills. Science permeates our daily lives and plays a crucial role in addressing societal issues.

AKT inhibition interferes with the expression of immune checkpoint proteins and increases NK-induced killing of HL60-AML cells.

OBJECTIVE: To determine the role of the AKT pathway in the regulating of natural Killer-induced apoptosis of acute myeloid leukemia cells and to characterize the associated molecular mechanisms. METHODS: BALB/c nude mice were injected with HL60 cells to induce a xenogenic model of subcutaneous leukemic tumors. Mice were treated with perifosine, and their spleens were analyzed using biometry, histopathology, and immunohistochemistry. Gene expression analysis in leukemia cells was performed by real-time PCR. Protein analysis of leukemia and natural Killer cells was performed by flow cytometry. AKT inhibition in HL60 cells, followed by co-culture with natural Killer cells was performed to assess cytotoxicity. Apoptosis rate was quantified using flow cytometry. RESULTS: Perifosine treatment caused a reduction in leukemic infiltration in the spleens of BALB/c nude mice. In vitro , AKT inhibition reduced HL60 resistance to natural Killer-induced apoptosis. AKT inhibition suppressed the immune checkpoint proteins PD-L1, galectin-9, and CD122 in HL60 cells, but did not change the expression of their co-receptors PD1, Tim3, and CD96 on the natural Killer cell surface. In addition, the death receptors DR4, TNFR1, and FAS were overexpressed by AKT inhibition, thus increasing the susceptibility of HL60 cells to the extrinsic pathway of apoptosis. CONCLUSION: The AKT pathway is involved in resistance to natural Killer-induced apoptosis in HL60 cells by regulating the expression of immune suppressor receptors. These findings highlight the importance of AKT in contributing to immune evasion mechanisms in acute myeloid leukemia and suggests the potential of AKT inhibition as an adjunct to immunotherapy.

Scientists of Tomorrow/ Cientistas do Amanhã : a project to inspire, stimulate scientific thinking, and introduce scientific methodology for young students

ABSTRACT The Scientists of Tomorrow/ Cientistas do Amanhã project is an immersive science training program developed by the Program of Post-Graduation in Health Sciences at Hospital Israelita Albert Einstein. This program was conducted in partnership with Volunteering and Escola Municipal de Ensino Fundamental Professor Paulo Freire in Paraisópolis, São Paulo, Brazil. The Scientists of Tomorrow Program comprised a short training period conducted in May 2022 involving 37 students, and a long training period from August to December 2022, which included 15 students. It aimed to popularize science through practical activities; transfer knowledge to young students; sensitize and guide them to pursue academic-scientific careers; reduce stereotypes about scientific work and scientists; and help students understand the social, political, and ethical roles of science within society. All activities were led by postgraduate students and professors from our postgraduate program, physicians, nurses, physiotherapists, biomedicals, and veterinarians from Hospital Israelita Albert Einstein, as well as medical students from Faculdade Israelita de Ciências da Saúde Albert Einstein . Activities in the short training included lectures on cinema and science, strategies to combat fake news, non-violent communication, innovation, design-thinking framework, and developing a scientific project. During the long training period, discussions were focused on nanotechnology, animal research, big data, bioinformatics, meditation, blood and bone marrow donation, telemedicine, sex and sexually-transmitted infections, rehabilitation, career opportunities, and scientific integrity. In addition, practical activities were further expanded using optical and confocal microscopy, cytometry, and basic concepts regarding the structure and function of living cells. The program also included the launching of the open-air outreach Education E-natureza activity, which turned students into ambassadors of nature. In conclusion, the Scientists of Tomorrow Program was innovative and enabled young students to learn that science is a collective activity that can enhance public health.

AKT inhibition interferes with the expression of immune checkpoint proteins and increases NK-induced killing of HL60-AML cells

ABSTRACT Objective To determine the role of the AKT pathway in the regulating of natural Killer-induced apoptosis of acute myeloid leukemia cells and to characterize the associated molecular mechanisms. Methods BALB/c nude mice were injected with HL60 cells to induce a xenogenic model of subcutaneous leukemic tumors. Mice were treated with perifosine, and their spleens were analyzed using biometry, histopathology, and immunohistochemistry. Gene expression analysis in leukemia cells was performed by real-time PCR. Protein analysis of leukemia and natural Killer cells was performed by flow cytometry. AKT inhibition in HL60 cells, followed by co-culture with natural Killer cells was performed to assess cytotoxicity. Apoptosis rate was quantified using flow cytometry. Results Perifosine treatment caused a reduction in leukemic infiltration in the spleens of BALB/c nude mice. In vitro , AKT inhibition reduced HL60 resistance to natural Killer-induced apoptosis. AKT inhibition suppressed the immune checkpoint proteins PD-L1, galectin-9, and CD122 in HL60 cells, but did not change the expression of their co-receptors PD1, Tim3, and CD96 on the natural Killer cell surface. In addition, the death receptors DR4, TNFR1, and FAS were overexpressed by AKT inhibition, thus increasing the susceptibility of HL60 cells to the extrinsic pathway of apoptosis. Conclusion The AKT pathway is involved in resistance to natural Killer-induced apoptosis in HL60 cells by regulating the expression of immune suppressor receptors. These findings highlight the importance of AKT in contributing to immune evasion mechanisms in acute myeloid leukemia and suggests the potential of AKT inhibition as an adjunct to immunotherapy.

Evaluation of Atypical Chemokine Receptor Expression in T Cell Subsets.

Chemokines are molecules that pertain to a family of small cytokines and can generate cell chemotaxis through the interaction with their receptors. Chemokines can trigger signaling via conventional G-protein-coupled receptors or through atypical chemokine receptors. Currently, four atypical chemokine receptors have been are described (ACKR1, ACKR2, ACKR3 and ACKR4). ACKRs are expressed in various cells and tissues, including T lymphocytes. These receptors' main function is related to the internalization and degradation of chemokines, as well as to the inflammation control. However, the expression of these receptors in human T lymphocytes is unclear in the literature. The objective of this study was to evaluate the expression of ACKRs in different subpopulations of T lymphocytes. For this, peripheral blood from healthy donors was used to analyze the expression of ACKR2, ACKR3 and ACKR4 by immunophenotyping CD4, CD8 T lymphocytes and, in their subsets, naive, transition and memory. Results obtained in this study demonstrated that ACKR2, ACKR3 and ACKR4 receptors were expressed by T lymphocytes subsets in different proportions. These receptors are highly expressed in the cytoplasmic milieu of all subsets of T lymphocytes, therefore suggesting that their expression in plasma membrane is regulated after transcription, and it must be dependent on a stimulus, which was not identified in our study. Thus, regarding ACKRs function as scavenger receptors, at least for the ACKR3, this function does not impair the chemotaxis exert for their ligand compared to the typical counterpart receptor.

Biologic Agents in Crohn's Patients Reduce CD4+ T Cells Activation and Are Inversely Related to Treg Cells.

Crohn's disease (CD) is a chronic inflammatory disease with a complex interface of broad factors. There are two main treatments for Chron's disease: biological therapy and nonbiological therapy. Biological agent therapy (e.g., anti-TNF) is the most frequently prescribed treatment; however, it is not universally accessible. In fact, in Brazil, many patients are only given the option of receiving nonbiological therapy. This approach prolongs the subsequent clinical relapse; however, this procedure could be implicated in the immune response and enhance disease severity. Our purpose was to assess the effects of different treatments on CD4+ T cells in a cohort of patients with Crohn's disease compared with healthy individuals. To examine the immune status in a Brazilian cohort, we analyzed CD4+ T cells, activation status, cytokine production, and Treg cells in blood of Crohn's patients. Patients that underwent biological therapy can recover the percentage of CD4+CD73+ T cells, decrease the CD4+ T cell activation/effector functions, and maintain the peripheral percentage of regulatory T cells. These results show that anti-TNF agents can improve CD4+ T cell subsets, thereby inducing Crohn's patients to relapse and remission rates.

Invariant Natural Killer T-cells and their subtypes may play a role in the pathogenesis of endometriosis.

OBJECTIVE: To evaluate the frequencies of iNKT cells and their subsets in patients with deep endometriosis. METHODS: A case-control study was conducted between 2013 and 2015, with 73 patients distributed into two groups: 47 women with a histological diagnosis of endometriosis and 26 controls. Peripheral blood, endometriosis lesions, and healthy peritoneal samples were collected on the day of surgery to determine the frequencies of iNKT cells and subtypes via flow cytometry analysis. RESULTS: The authors observed a lower number of iNKT (p = 0.01) and Double-Negative (DN) iNKT cells (p = 0.02) in the blood of patients with endometriosis than in the control group. The number of DN iNKT IL-17+ cells in the secretory phase was lower in the endometriosis group (p = 0.049). There was an increase in the secretion of IL-17 by CD4+ iNKT cells in the blood of patients with endometriosis and severe dysmenorrhea (p = 0.038), and severe acyclic pelvic pain (p = 0.048). Patients with severe dysmenorrhea also had a decreased number of CD4+ CCR7+ cells (p = 0.022). CONCLUSION: The decreased number of total iNKT and DN iNKT cells in patients with endometriosis suggests that iNKT cells play a role in the pathogenesis of endometriosis and can be used to develop new diagnostic and therapeutic agents.

Site-Specific Regulation of Sulfatase and Aromatase Pathways for Estrogen Production in Endometriosis.

Endometriosis is a highly prevalent gynecological disease characterized by lesions in different sites. Regulation of specific estrogen pathways may favor the formation of distinct microenvironments and the progression of endometriosis. However, no study has simultaneously evaluated the gene and protein regulation of the main estrogen-synthesizing enzymes in endometriosis. Thus, our goals were to study the relationship between gene and protein expression of aromatase (CYP19A1 or ARO), steroid sulfatase (STS), and hydroxysteroid 17-beta dehydrogenase (HSD17B1) in superficial (SUP), ovarian (OMA), and deep infiltrating (DIE) endometriotic lesion sites as well as in the eutopic endometrium of patients with (EE) and without (control) endometriosis in the same and large cohort of patients. The site-specific expression of these enzymes within different cells (glandular and stromal components) was also explored. The study included 108 patients surgically diagnosed with endometriosis who provided biopsies of EE and endometriotic lesions and 16 disease-free patients who collected normal endometrium tissue. Our results showed that CYP19A1 was detected in all endometriosis tissues and was in higher levels than in control. Unique patterns of the STS and HSD17B1 levels showed that they were most closely regulated in all tissues, with manifestation at greater levels in DIE compared to the other endometriotic lesion sites, OMA and SUP. Gene and protein expression of ARO, STS, and HSD17B1 occurred at different rates in endometriotic sites or EE. The distinctive levels of these estrogen-synthesizing enzymes in each endometriotic site support the hypothesis of a tissue microenvironment that can both influence and be influenced by the expression of different estrogenic pathways, locally affecting the availability of estrogen needed for maintenance and progression of endometriotic lesions.

Invariant Natural Killer T-cells and their subtypes may play a role in the pathogenesis of endometriosis

Abstract Objective To evaluate the frequencies of iNKT cells and their subsets in patients with deep endometriosis. Methods A case-control study was conducted between 2013 and 2015, with 73 patients distributed into two groups: 47 women with a histological diagnosis of endometriosis and 26 controls. Peripheral blood, endometriosis lesions, and healthy peritoneal samples were collected on the day of surgery to determine the frequencies of iNKT cells and subtypes via flow cytometry analysis. Results The authors observed a lower number of iNKT (p= 0.01) and Double-Negative (DN) iNKT cells (p= 0.02) in the blood of patients with endometriosis than in the control group. The number of DN iNKT IL-17+ cells in the secretory phase was lower in the endometriosis group (p= 0.049). There was an increase in the secretion of IL-17 by CD4+ iNKT cells in the blood of patients with endometriosis and severe dysmenorrhea (p= 0.038), and severe acyclic pelvic pain (p= 0.048). Patients with severe dysmenorrhea also had a decreased number of CD4+ CCR7+ cells (p= 0.022). Conclusion The decreased number of total iNKT and DN iNKT cells in patients with endometriosis suggests that iNKT cells play a role in the pathogenesis of endometriosis and can be used to develop new diagnostic and therapeutic agents.

Effect of unilateral renal ischemia on the contralateral kidney assessed by Caspase 3 expression.

BACKGROUND: Studies have demonstrated with histological analysis and Doppler flow measurement analysis that unilateral renal ischemia, which is performed in some surgeries, interfered with the contralateral kidney, identifying the phenomenon of kidney-kidney crosstalk. OBJECTIVES: To identify the effects on the ischemic and contralateral kidney of renal ischemia induced by two types of clamping technique by analyzing the volume of kidney cells positive for Caspase 3. METHODS: Sixteen pigs were divided into 2 groups, as follows: A (n = 8) - clamping of left renal artery only and AV (n = 8) - clamping of left renal artery and vein. Immunohistochemical analyses (anti Caspase 3) were conducted with biopsy specimens collected from the ischemic and contralateral kidney at 0, 30, 60, and 90 minutes of ischemia and morphometric analysis was performed, taking the mean to represent the volume of the Caspase 3 positive area (%). RESULTS: Morphometric analysis of specimens collected at 30, 60, and 90 minutes of ischemia showed that the mean area marked for Caspase 3 was statistically larger in the contralateral kidney than the ischemic kidney in both groups: clamped renal artery (A) and clamped renal artery and vein (AV). Comparing the ischemic and contralateral kidney, there was no statistically significant difference in the area marked for Caspase 3 between the two types of clamping. CONCLUSIONS: In the experimental model of unilateral renal ischemia, the non-ischemic kidney exhibited cell damage, demonstrated by Caspase 3 expression. The type of hilum clamping does not appear to influence the area marked for Caspase 3.

Efeito da isquemia renal unilateral no rim contralateral avaliada pela expressão de Caspase 3 / Effect of unilateral renal ischemia on the contralateral kidney assessed by Caspase 3 expression

Resumo Contexto Estudos demonstraram, por análise histológica e Dopplerfluxométrica, a interferência da isquemia renal unilateral, realizada em algumas cirurgias, sobre o rim contralateral, identificando o fenômeno de kidney-kidney crosstalk. Objetivos Identificar o efeito da isquemia de duas estratégias de oclusão da vasculatura renal esquerda sobre o rim contralateral através do volume de células renais positivas para Caspase 3. Métodos Suínos foram divididos em 2 grupos: A (n = 8), artéria renal esquerda clampeada, e AV (n = 8), artéria e veia renais esquerdas clampeadas. Foi realizado o estudo imuno-histoquímico (anti-Caspase 3), com o material de biópsias coletadas do rim isquêmico e contralateral em 0, 30, 60 e 90 minutos de isquemia, e análise morfométrica, sendo que a média representou o volume de área de Caspase 3 positiva (%). Resultados A análise morfométrica do rim contralateral nos tempos 30, 60 e 90 minutos de isquemia mostrou que a média da área marcada por Caspase 3 foi estatisticamente superior à média do rim isquêmico nos dois grupos: artéria renal clampeada (A) e artéria e veia renais clampeadas (AV). Comparando o rim isquêmico e contralateral nos dois tipos de clampeamento, não houve diferença estatisticamente significante da área marcada por Caspase 3. Conclusões No modelo experimental de isquemia renal unilateral, o rim não isquêmico apresentou dano celular, demonstrado pela expressão da Caspase 3 de forma aguda em decorrência da isquemia contralateral. O tipo de clampeamento do hilo não parece ter influência sobre o volume de área marcada por Caspase 3.

Abstract Background Studies have demonstrated with histological analysis and Doppler flow measurement analysis that unilateral renal ischemia, which is performed in some surgeries, interfered with the contralateral kidney, identifying the phenomenon of kidney-kidney crosstalk. Objectives To identify the effects on the ischemic and contralateral kidney of renal ischemia induced by two types of clamping technique by analyzing the volume of kidney cells positive for Caspase 3. Methods Sixteen pigs were divided into 2 groups, as follows: A (n = 8) - clamping of left renal artery only and AV (n = 8) - clamping of left renal artery and vein. Immunohistochemical analyses (anti Caspase 3) were conducted with biopsy specimens collected from the ischemic and contralateral kidney at 0, 30, 60, and 90 minutes of ischemia and morphometric analysis was performed, taking the mean to represent the volume of the Caspase 3 positive area (%). Results Morphometric analysis of specimens collected at 30, 60, and 90 minutes of ischemia showed that the mean area marked for Caspase 3 was statistically larger in the contralateral kidney than the ischemic kidney in both groups: clamped renal artery (A) and clamped renal artery and vein (AV). Comparing the ischemic and contralateral kidney, there was no statistically significant difference in the area marked for Caspase 3 between the two types of clamping. Conclusions In the experimental model of unilateral renal ischemia, the non-ischemic kidney exhibited cell damage, demonstrated by Caspase 3 expression. The type of hilum clamping does not appear to influence the area marked for Caspase 3.

Comparison of senescence progression in mesenchymal cells from human umbilical cord walls measured by immunofluorescence and flow cytometry of p16 and p21.

OBJECTIVE: To follow the expansion of mesenchymal stem cells from umbilical cords by two classic senescence markers, p16 (INK4A) and p21 (CDKN1A), using practical, fast, and less expensive methods than the gold standard Western blotting technique, to evaluate its applicability in the laboratory. METHODS: Mesenchymal stem cells from umbilical cords were isolated from Wharton's jelly and, after quality control, morphological and immunophenotypic characterization by flow cytometry, were expanded in culture until coming close to cell cycle arrest (replicative senescence). RESULTS: A comparison was made between young cells, at passage 5, and pre-senescent cells, at passage 10, evaluating the protein expression of the classic cell senescence markers p16 and p21, comparing the results obtained by Western blotting with those obtained by flow cytometry and indirect immunofluorescence. CONCLUSION: Follow-up of cell cultures, through indirect p16 immunofluorescence, allows the identification of mesenchymal stem cells from umbilical cord cultures at risk of reaching replicative senescence.

Burdock (Arctium lappa L.) root attenuates preneoplastic lesion development in a diet and thioacetamide-induced model of steatohepatitis-associated hepatocarcinogenesis.

Nonalcoholic steatohepatitis (NASH) is considered growing risk factor for hepatocellular carcinoma development in high-income countries. Diet- and chemically induced rodent models have been applied for the translational study of NASH-associated hepatocarcinogenesis due to their morphological and molecular similarities to the corresponding human disease. Arctium lappa L. (burdock) root tea has been extensively consumed in Traditional Chinese Medicine due to its potential therapeutic properties. Indeed, the bioactive compounds of A. lappa root, as the polyphenols, have already showed antioxidant and anti-inflammatory properties in different in vivo and in vitro bioassays. In this study, we investigated whether burdock root ethanolic extract (BRE) administration attenuates NASH-associated hepatocarcinogenesis. Eight-week-old male Wistar rats received choline-deficient high-fat diet for 8 weeks and multiple thioacetamide doses for 4 weeks in order to induce NASH and preneoplastic glutathione-S-transferase pi (GST-P)+ preneoplastic foci. Subsequently, rats were treated with BRE (100 or 200 mg/kg body weight) or vehicle by oral gavage for 2 weeks. BRE displayed high levels of chlorogenic and caffeic acids and BRE administration reduced total fatty acid and lipid hydroperoxide levels, while increasing the activities of antioxidant superoxide dismutase and catalase enzymes in the liver. Furthermore, burdock intervention diminished the size of GST-P+ remodeling preneoplastic lesions (PNLs) and displayed a trend on reducing hepatocyte proliferation (Ki-67) inside them. These findings suggest that short-term exposure to BRE alleviated remodeling PNL development in NASH-associated hepatocarcinogenesis.

Comparison of senescence progression in mesenchymal cells from human umbilical cord walls measured by immunofluorescence and flow cytometry of p16 and p21 / Comparação da progressão da senescência em células mesenquimais de parede de cordão umbilical humano medida por imunofluorescência e citometria de fluxo de p16 e p21

ABSTRACT Objective To follow the expansion of mesenchymal stem cells from umbilical cords by two classic senescence markers, p16 (INK4A) and p21 (CDKN1A), using practical, fast, and less expensive methods than the gold standard Western blotting technique, to evaluate its applicability in the laboratory. Methods Mesenchymal stem cells from umbilical cords were isolated from Wharton's jelly and, after quality control, morphological and immunophenotypic characterization by flow cytometry, were expanded in culture until coming close to cell cycle arrest (replicative senescence). Results A comparison was made between young cells, at passage 5, and pre-senescent cells, at passage 10, evaluating the protein expression of the classic cell senescence markers p16 and p21, comparing the results obtained by Western blotting with those obtained by flow cytometry and indirect immunofluorescence. Conclusion Follow-up of cell cultures, through indirect p16 immunofluorescence, allows the identification of mesenchymal stem cells from umbilical cord cultures at risk of reaching replicative senescence.

RESUMO Objetivo Acompanhar a expansão de células-tronco mesenquimais de cordão umbilical por dois marcadores clássicos de senescência, p16 (INK4A) e p21 (CDKN1A), usando métodos práticos, rápidos e com custo menor do que a técnica padrão-ouro de Western blotting, para avaliar sua aplicabilidade em laboratório. Métodos Células-tronco mesenquimais de cordão umbilical foram isoladas da geleia de Wharton e, após controle de qualidade e caracterização morfológica e imunofenotípica por citometria de fluxo, foram expandidas em cultura, até chegarem próximas à parada do ciclo celular (senescência replicativa). Resultados Foi feita a comparação entre células jovens, na passagem 5, e células pré-senescentes, na passagem 10, avaliando a expressão proteica dos marcadores clássicos de senescência celular p16 e p21, comparando os resultados obtidos por Western blotting com os obtidos por citometria de fluxo e imunofluorescência indireta. Conclusão O seguimento de culturas celulares, por meio da imunofluorescência indireta de p16, permite identificar as culturas de células-tronco mesenquimais de cordão umbilical em risco de atingirem a senescência replicativa.

Validation of Laser Capture Microdissection Protocol in Endometriosis Studies.

Background and Objectives: The presence of endometrial-like tissue outside the uterine cavity is a key feature of endometriosis. Although endometriotic lesions appear to be histologically quite similar to the eutopic endometrium, detailed studies comparing both tissues are required because their inner and surrounding cellular arrangement is distinct. Thus, comparison between tissues might require methods, such as laser capture microdissection (LCM), that allow for precise selection of an area and its specific cell populations. However, it is known that the efficient use of LCM depends on the type of studied tissue and on the choice of an adequate protocol. Recent studies have reported the use of LCM in endometriosis studies. The main objective of the present study is to establish a standardized protocol to obtain good-quality microdissected material from eutopic or ectopic endometrium. Materials and Methods: The main methodological steps involved in the processing of the lesion samples for LCM were standardized to yield material of good quality to be further used in molecular techniques. Results: We obtained satisfactory results regarding the yields and integrity of RNA and protein obtained from LCM-processed endometriosis tissues. Conclusion: LCM can provide more precise analysis of endometriosis biopsies, provided that key steps of the methodology are followed.

Expansive Vascular Remodeling and Increased Vascular Calcification Response to Cholecalciferol in a Murine Model of Obesity and Insulin Resistance.

Objective- We hypothesized that ob/ob mice develop expansive vascular remodeling associated with calcification. Approach and Results- We quantified and investigated mechanisms of vascular remodeling and vascular calcification in ob/ob mice after vitamin D3(VD) stimulation or PBS (control), compared with C57BL/6 mice. Both ob/ob (OBVD [VD-treated ob/ob mice]) and C57BL/6 (C57VD [VD-treated C57BL/6 mice]) received 8×103 IU/day of intraperitoneal VD for 14 days. Control ob/ob (OBCT [PBS-treated ob/ob mice]) and C57BL/6 (C57CT [PBS-treated C57BL/6 mice]) received intraperitoneal PBS for 14 days. Hypervitaminosis D increased the external and internal elastic length in aortae from OBVD, resulting in increased total vascular area and lumen vascular area, respectively, which characterizes expansive vascular remodeling. OBVD decreased the aortic wall thickness, resulting in hypotrophic vascular remodeling. We demonstrated increased collagen deposition, elastolysis, and calcification in aortae from OBVD. Our results showed a positive correlation between expansive vascular remodeling and vascular calcification in OBVD. We demonstrated increased serum calcium levels, augmented Bmp (bone morphogenetic protein)-2 and osteochondrogenic proteins expression in OBVD aortae. Furthermore, aortae from OBVD increased oxidative stress, coincidently with augmented in situ MMP (matrix metalloproteinase) activity and exhibited no VDR (VD receptor) inhibition after VD. Conclusions- Our data provide evidence that obese and insulin-resistant mice (ob/ob) developed expansive hypotrophic vascular remodeling correlated directly with increased vascular calcification after chronic VD stimulation. Positive hypotrophic vascular remodeling and vascular calcification in this mouse model is possibly mediated by the convergence of absence VDR downregulation after VD stimulation, increased reactive oxygen species generation, and MMP activation.

Recovery of the Cholangiocytes After Ischemia and Reperfusion Injury: Ultra-Structural, Hystological and Molecular Assessment in Rats.

INTRODUCTION: Ischemia-reperfusion (I/R) injury of the liver is a common area of interest to transplant and hepatic surgery. Nevertheless, most of the current knowledge of I/R of the liver derives from the hepatocyte and little is known of what happens to the cholangiocytes. Herein, we assess the sequence of early events involved in the I/R injury of the cholangiocytes. METHODS: Sixty Wistar rats were randomized in a SHAM group and I/R group. Serum biochemistry, histopathology, immunohistochemistry, transmission electron microscopy (TEM) and laser capture microdissection (LCM) were used for group comparison. RESULTS: There was peak of alkaline phosphatase 24 h after IR injury, and an increase of aspartate aminotransferase and alanine aminotransferase after 6 h of reperfusion, followed by a return to normal levels 24 h after injury. The I/R group presented the liver parenchyma with hepatocellular degeneration up to 6 h, followed by hepatocellular necrosis at 24 h. TEM showed cholangiocyte injury, including a progressive nuclear degeneration and cell membrane rupture, beginning at 6 h and peaking at 24 h after reperfusion. Cytokeratin-18 and caspase-3-positive areas were observed in the I/R group, peaking at 24-h reperfusion. Anti-apoptotic genes Bcl-2 and Bcl-xl activity were expressed from 6 through 24 h after reperfusion. BAX expression showed an increase for 24 h. CONCLUSIONS: I/R injury to the cholangiocyte occurs from 6 through 24 h after reperfusion and a combination of TEM, immunohistochemistry and LCM allows a better isolation of the cholangiocyte and a proper investigation of the events related to the I/R injury. Apoptosis is certainly involved in the I/R process, particularly mediated by BAX.

Human Lymph Node-Derived Fibroblastic and Double-Negative Reticular Cells Alter Their Chemokines and Cytokines Expression Profile Following Inflammatory Stimuli.

Lymph node (LN) is a secondary lymphoid organ with highly organized and compartmentalized structure. LNs harbor B, T, and other cells among fibroblastic reticular cells (FRCs). FRCs are characterized by both podoplanin (PDPN/gp38) expression and by the lack of CD31 expression. FRCs are involved in several immune response processes but mechanisms underlying their function are still under investigation. Double-negative cells (DNCs), another cell population within LNs, are even less understood. They do not express PDPN or CD31, their localization within the LN is unknown, and their phenotype and function remain to be elucidated. This study evaluates the gene expression and cytokines and chemokines profile of human LN-derived FRCs and DNCs during homeostasis and following inflammatory stimuli. Cytokines and chemokines secreted by human FRCs and DNCs partially diverged from those identified in murine models that used similar stimulation. Cytokine and chemokine secretion and their receptors expression levels differed between stimulated DNCs and FRCs, with FRCs expressing a broader range of chemokines. Additionally, dendritic cells demonstrated increased migration toward FRCs, possibly due to chemokine-induced chemotaxis since migration was significantly decreased upon neutralization of secreted CCL2 and CCL20. Our study contributes to the understanding of the biology and functions of FRCs and DNCs and, accordingly, of the mechanisms involving them in immune cells activation and migration.

Connexin32 deficiency exacerbates carbon tetrachloride-induced hepatocellular injury and liver fibrosis in mice.

OBJECTIVE: Liver fibrosis results from the perpetuation of the normal wound healing response to several types of injury. Despite the wealth of knowledge regarding the involvement of intracellular and extracellular signaling pathways in liver fibrogenesis, information about the role of intercellular communication mediated by gap junctions is scarce. METHODS: In this study, liver fibrosis was chemically induced by carbon tetrachloride in mice lacking connexin32, the major liver gap junction constituent. The manifestation of liver fibrosis was evaluated based on a series of read-outs, including collagen morphometric and mRNA analysis, oxidative stress, apoptotic, proliferative and inflammatory markers. RESULTS: More pronounced liver damage and enhanced collagen deposition were observed in connexin32 knockout mice compared to wild-type animals in experimentally triggered induced liver fibrosis. No differences between both groups were noticed in apoptotic signaling nor in inflammation markers. However, connexin32 deficient mice displayed decreased catalase activity and increased malondialdehyde levels. CONCLUSION: These findings could suggest that connexin32-based signaling mediates tissue resistance against liver damage by the modulation of the antioxidant capacity. In turn, this could point to a role for connexin32 signaling as a therapeutic target in the treatment of liver fibrosis.