TLR4 expression and functionality are downregulated in glioblastoma cells and in tumor-associated macrophages: A new mechanism of immune evasion?

Glioblastoma (GB) is the most common and aggressive form of primary brain tumor, in which the presence of an inflammatory environment, composed mainly by tumor-associated macrophages (TAMs), is related to its progression and development of chemoresistance. Toll-Like Receptors (TLRs) are key components of the innate immune system and their expression in both tumor and immune-associated cells may impact the cell communication in the tumor microenvironment (TME), further modeling cancer growth and response to therapy. Here, we investigated the participation of TLR4-mediated signaling as a mechanism of induced-immune escape in GB. Initially, bioinformatics analysis of public datasets revealed that TLR4 expression is lower in GB tumors when compared to astrocytomas (AST), and in a subset of TAMs. Further, we confirmed that TLR4 expression is downregulated in chemoresistant GB, as well as in macrophages co-cultured with GB cells. Additionally, TLR4 function is impaired in those cells even following stimulation with LPS, an agonist of TLR4. Finally, experiments performed in a cohort of clinical primary and metastatic brain tumors indicated that the immunostaining of TLR4 and CD45 are inversely proportional, and confirmed the low TLR4 expression in GBs. Interestingly, the cytoplasmic/nuclear pattern of TLR4 staining in cancer tissues suggests additional roles of this receptor in carcinogenesis. Overall, our data suggest the downregulation of TLR4 expression and activity as a strategy for GB-associated immune escape. Additional studies are necessary to better understand TLR4 signaling in TME in order to improve the benefits of immunotherapy based on TLR signaling.

Isolation of human mesenchymal stem cells from amnion, chorion, placental decidua and umbilical cord: comparison of four enzymatic protocols.

OBJECTIVE: To compare four enzymatic protocols for mesenchymal stem cells (MSCs) isolation from amniotic (A-MSC) and chorionic (C-MSC) membranes, umbilical cord (UC-MSC) and placental decidua (D-MSC) in order to define a robust, practical and low-cost protocol for each tissue. RESULTS: A-MSCs and UC-MSCs could be isolated from all samples using trypsin/collagenase-based protocols; C-MSCs could be isolated from all samples with collagenase- and trypsin/collagenase-based protocols; D-MSCs were isolated from all samples exclusively with a collagenase-based protocol. CONCLUSIONS: The trypsin-only protocol was least efficient; the collagenase-only protocol was best for C-MSCs and D-MSCs; the combination of trypsin and collagenase was best for UC-MSCs and none of tested protocols was adequate for A-MSCs isolation.

Autophagy analysis in oral carcinogenesis.

OBJECTIVE: The aim of this study was to evaluate the levels of autophagy in oral leukoplakia and squamous cell carcinoma and to correlate with clinical pathological features, as well as, the evolution of these lesions. METHODOLOGY: 7 Normal oral mucosa, 51 oral leukoplakias, and 120 oral squamous cell carcinomas (OSCC) were included in the study. Histological sections of the mucosa and leukoplakias were evaluated throughout their length, while the carcinomas were evaluated using Tissue Microarray. After the immunohistochemical technique, LC3-II positive cells were quantified in the different epithelial layers of the mucosa and leukoplakias and in the microarrays of the squamous cell carcinomas. The correlation between positive cells with the different clinical-pathological variables and with the evolution of the lesions was tested using the t test, ANOVA, and Kaplan-Meier survival analysis. RESULTS: We observed increased levels of autophagy in the oral squamous cell carcinomas (p<0.001) in relation to the other groups, but without any association with poorer evolution or survival of these patients. Among the leukoplakias, we observed a higher percentage of positive cells in the intermediate layer of the dysplastic leukoplakias (p=0.0319) and in the basal layer of lesions with poorer evolution (p=0.0133). CONCLUSION: The levels of autophagy increased during the process of oral carcinogenesis and are correlated with poorer behavior of the leukoplakias.

Treatment of dilated cardiomyopathy in rabbits with mesenchymal stem cell transplantation and platelet-rich plasma.

Dilated cardiomyopathy (DCM) is a major cause of cardiovascular mortality and morbidity, and there is evidence to suggest that stem cell transplantation may be a viable treatment option for this condition. Therefore, the goal of the present study was to assess myocardial regeneration in rabbits with doxorubicin-induced DCM treated with adipose mesenchymal stem cells (MSC) alone or in combination with platelet-rich plasma (PRP). Twenty New Zealand rabbits received doxorubicin for the induction of DCM and were divided into four groups according to treatment: saline, MSC, PRP and MSC + RP. Treatment agents were injected directly into the left ventricular myocardium following a thoracoscopy. Rabbits were assessed through echocardiographic and electrocardiographic examinations, as well as serum cardiac troponin I measurements at baseline, after the induction of DCM and 15 days after treatment. Animals were euthanased following the last assessment, and hearts were collected for histopathological analyses. The MSC group showed improvements in all parameters assessed, while the PRP group showed significantly impaired heart function. Histopathology of the heart revealed that the MSC group displayed the lowest number of lesions, while rabbits in the MSC + PRP, saline and PRP groups had steadily advancing lesions. These results suggest that MSC transplantation can improve heart function in rabbits with DCM, and underscore the need for further studies of the effects of PRP on the myocardium.

Bone regeneration in mandible defect with autograft bone and cell suspension from bone marrow in rabbits / Regeneração óssea em defeitos mandibulares com auto-enxerto ósseo e suspensão celular da medula óssea em coelhos

The objective of this study was to investigate the bone regeneration of a "gold standard" (autograft) from iliac crest associated with cellular therapy in rabbits. A bone defect was created with 10x5x5mm in 28 rabbit mandibles. The control group animals (n=14) were repaired with autograft of iliac crest and the experimental group animals (n=14) received iliac crest autograft in association with mononuclear cells from the bone marrow of the femur. Weekly radiographs were taken of the surgery region and histological analyses was performed in seven animals in each group at 15 days and in seven animals of each group at 30 days after the surgery. A gradual increase of bone density was observed and the experimental animals presented the bone bridge in 85.7 percent (6/7) of the cases, while only 42.8 percent (3/7) of the animals in the control group presented this structure 28 days after the surgery. The histopathological parameters analyzed did not show any statistical difference between the control and experimental group in 15 and 30 days of analysis. The results suggest that the mononuclear cells from the marrow bone can better support the autograft regeneration in mandible defects in rabbits.

Avaliou-se a regeneração óssea de auto-enxerto, considerado "padrão ouro" da crista ilíaca associado à terapia celular da medula óssea em coelhos. Foi criado um defeito ósseo de 10x5x5mm na mandíbula de 28 coelhos, distribuídos em grupo-controle com, 14 animais, reparados com auto-enxerto de crista ilíaca, e grupo experimental com, 14 animais, em que o auto-enxerto foi associado a células mononucleares da medula óssea autógena do fêmur. Foram realizadas radiografias semanais da região operada e análise histológica em sete animais de cada grupo aos 15 e em sete de cada grupo aos 30 dias do pós-operatório. Houve aumento gradativo da densidade óssea, e 85,7 por cento (6/7) dos animais do grupo experimental e 42,8 por cento (3/7) do grupo-controle apresentaram formação de ponte óssea 28 dias após a cirurgia. Na análise histopatológica aos 15 dias, os enxertos foram facilmente visualizados e a atividade das células fagocitárias foi intensa. Já aos 30 dias, a visualização foi mais difícil e, quando possível, apenas um resquício foi visualizado. Os resultados sugerem que a adição de células mononucleares da medula óssea favorece a regeneração do auto-enxerto em defeitos mandibulares de coelhos.