Comparison of hemostatic efficacy and cytotoxicity of three ferric subsulfate- and chitosan-based styptics in different formulations using a rat tail bleeding model / 대한수의학회지

This study was conducted to compare the hemostatic efficacy of three ferric subsulfate- and chitosan-based styptics as a powder and a gel containing ferric subsulfate and chitosan (FSC-PO and FSC-G, respectively) and a soaked pad containing ferric subsulfate and lidocaine (FSL-SP) using a rat tail bleeding model. The cytotoxicity of the styptics against L-929 mouse fibroblasts was also evaluated using a cell counting kit-8 assay. Four groups of 10 rats each were assigned to the three different styptics and a non-treated control groups. Rat tail tips were transected, after which styptics were applied with pressure. The wounds were observed for hemostasis for 3 min, then irrigated with saline to check for recurrent hemorrhage. L-929 mouse fibroblasts were exposed to extracts of the styptics (100 mg/mL) and their dilutions (1:10, 1:100, and 1:1,000). FSC-PO and FSC-G more effectively controlled initial hemorrhage than FSL-SP (p = 0.033). Additionally, FSC-PO and FSC-G more effectively maintained hemostasis than the control group (p = 0.02 and p < 0.01, respectively). However, all styptics showed enhanced cytotoxicity against L-929 cells in a dose-dependent manner. Therefore, although FSC-PO and FSC-G would be recommended to control hemorrhage, the benefits of styptics must be balanced against the clinical significance of their cytotoxicity.

Resposta imune e mecanismos de evasão na carcinogênese de lábio: um estudo imunoistoquímico / Immune response and evasion mechanisms in lip carcinogenesis: an immunohistochemical study

A vigilância imunológica, principalmente mediada por linfócitos T CD8+ , reconhece e destrói células malignas ou alteradas. Contudo, através de estratégias imunossupressoras, como as vias de sinalização do ligante de morte celular programada-1 (PD-L1) e do antígeno leucocitário humano-G (HLA-G), estas células mutadas conseguem escapar da resposta imune antitumoral. Este estudo investigou a imunoexpressão de PD-L1, HLA-G, CD8 e granzima B (GrB) no microambiente de carcinomas de células escamosas (CCEs) de lábio (n = 40), de queilites actínicas (QAs; n = 55) e de mucosa labial saudável (MLS; n = 10). As amostras foram submetidas à técnica da imunoistoquímica e as análises das imunomarcações seguiram métodos semi-quantitativos (PD-L1 e HLA-G) e quantitativos (CD8 e GrB). A expressão das proteínas foi comparada entre os três grupos de amostras, bem como com parâmetros clinicopatológicos das lesões e sobrevida global dos pacientes com CCE de lábio. A correlação entre as proteínas e o tipo do microambiente tumoral de acordo com a presença de PD-L1 e CD8 também foram avaliados. Os testes estatísticos incluíram o exato de Fisher, Mann-Whitney, Kruskal-Wallis, correlação de Spearman e log-rank para comparação das curvas de sobrevida global construídas pelo método Kaplan-Meier. O nível de significância foi estabelecido em 5%. Os números de células CD8+ e GrB+ aumentaram progressivamente de MLS para CCEs de lábio, com QAs exibindo números intermediários (p < 0,01). A menor expressão dessas proteínas foi associada à metástase para linfonodos e tumores pobremente diferenciados (p < 0,05). A expressão de PD-L1 e HLA-G em células neoplásicas/ceratinócitos e estroma/tecido conjuntivo foi significativamente maior em CCEs de lábio e QAs, em comparação com MLSs (p < 0,05). PDL1 não foi significativamente associado aos aspectos clinicopatológicos das lesões. A maioria dos CCEs de lábio mostrou coexistência de células PD-L1+ e CD8+ (72,5%) no microambiente tumoral. A expressão de PD-L1 foi diretamente correlacionada à infiltração linfocítica CD8+ e GrB+ em CCEs de lábio (p < 0,05). A expressão das proteínas não foi associada com a sobrevida global dos pacientes com CCEs de lábio (p > 0,05). Nossos achados sugerem que as moléculas imunossupressoras PD-L1 e HLA-G estão consistentemente expressas desde QAs e se mantém até fases avançadas dos CCE de lábio. A correlação entre a expressão de PD-L1 e a expressão de CD8 e GrB nos carcinomas sugere que PD-L1 pode surgir como um mecanismo de escape frente a uma resposta antitumoral ativa (AU).

Immune surveillance, mainly mediated by CD8 + T lymphocytes, recognize and destroy malignant or altered cells. However, through immunosuppressive strategies, such as the signaling pathways of the programmed cell death ligand-1 (PD-L1) and human leukocyte antigen-G (HLA-G), these mutated cells often escape the antitumor immune response. The aim of this study was to investigate and compare the immunoexpression of PD-L1, HLA-G, CD8 and granzyme B (GrB) in the microenvironment of lip squamous cell carcinomas (LSCCs; n = 40), actinic cheilitis (ACs; n = 55), and healthy lip mucosa (HLM; n = 10). The samples were submitted to immunohistochemistry and the analysis followed a semi-quantitative (PD-L1 and HLA-G) and quantitative methods (CD8 and GrB). Protein expression was compared between the three groups of samples, as well as with the lesion's clinicopathologic parameters and overall survival of patients with LSCC. Correlation between proteins and the type of tumor microenvironment according to a presence of PD-L1 and CD8 were also evaluated. Statistical tests included Fisher's exact, Mann-Whitney, Kruskal-Wallis, Spearman's correlation, as well as the log-rank for comparison of the overall survival built through Kaplan-Meier method. Significance was set at p < 0.05. The CD8+ and GrB+ cell numbers progressively increased from HLMs to LSCCs, with ACs exhibiting intermediate numbers (p < 0.01). Lower expression of these proteins was associated with lymph node metastasis and poor tumor differentiation (p < 0.05). PD-L1 and HLA-G expression in neoplastic cells/keratinocytes and stroma/connective tissue was significantly higher in LSCCs and ACs, compared to HLMs (p < 0.05). PD-L1 was not significantly associated with clinicopathological aspects of the lesions. Most LSCCs showed coexistence of PD-L1+ and CD8+ cells (72.5%) in the tumor microenvironment. PDL1 was directly correlated to CD8+ and GrB+ lymphocytic infiltration in LSCCs (p < 0.05). Proteins expression was not associated with overall survival of LSCCs patients (p > 0.05). Our findings suggest that immunosuppressive molecules PD-L1 and HLA-G are consistently expressed from ACs and are maintained until advanced stages of LSCCs. The correlation between PD-L1 expression and the expression of CD8 and GrB in carcinomas suggests that that PD-L1 may appear as an escape mechanism against an active antitumor response (AU).

Analysis of platelet eluate for the elucidation of sensitization to HLA in kidney transplant candidate / Análise do eluato de plaqueta para a elucidação da sensibilização ao HLA em um candidato ao transplante de rim

While a 42-year-old male patient was being prepared for deceased-donor renal transplantation, anti-HLA-A2 antibodies were detected in the serum by enzyme-linked immunosorbent assay (ELISA) method. The patient denied any transfusion history and previous transplant. Crossmatch by complement dependent cytotoxicity (CDC) and CDC with anti -human globulin (CDC-AHG) proved negative with a four-cell panel with positive typing for HLA-A2. Adsorption of antibodies with platelets and analysis of eluate were suggested to elucidate discrepancies in results by ELISA and by CDC-AHG. ELISA showed that adsorbed serum with platelets did not reveal antibodies for HLA-A2 specificity and suggested that they were removed by their specific binding with HLA-A2 antigens on the platelet surface. Eluate analysis by ELISA showed antibodies for HLA-A2 specificity. No antibodies for HLA-A2 specificity in the non-adsorbed serum were detected by CDC-AHG method. Revision of patient's data showed that a previous transfusion had occurred, which may have been the source of HLA sensitization. The suggested method may be a contribution towards the evaluation of sensitivity between CDC-AHG and ELISA methods for characterizing antibodies in the patient's serum.

Enquanto um paciente do sexo masculino de 42 anos de idade estava sendo preparado para o transplante renal de doador falecido, anticorpos anti-HLA-A2 foram detectados no soro pelo método de ensaio imunoenzimático (ELISA). O paciente negava história de transfusão e transplante anterior. Prova-cruzada por citotoxicidade dependente de complemento (CDC) e CDC com antiglobulina humana (CDC-AGH) foram negativos com um painel de quatro células com tipagem positiva para HLA-A2. O método de adsorção de anticorpos com plaquetas e análise do eluato foi sugerido para explicar as discrepâncias dos resultados de ELISA e CDC-AGH. O método de ELISA mostrou que o soro adsorvido com plaquetas não revelou anticorpos para especificidade HLA-A2, sugerindo que eles foram removidos por meio de sua ligação específica com os antígenos HLA-A2 na superfície das plaquetas. A análise do eluato por ELISA mostrou anticorpos para especificidade HLA-A2. Nenhum anticorpo para especificidade HLA-A2 foi detectada no soro não adsorvido pelo método de CDC-AGH. Revisão dos dados do paciente mostrou que houve transfusão anterior, podendo ter sido a fonte de sensibilização HLA. O método sugerido é uma contribuição para avaliação da sensibilidade entre os métodos de CDC-AGH e ELISA em caracterizar anticorpos no soro do paciente.

Evaluation of cytolytic activity and phenotypic changes of circulating blood immune cells in patients with colorectal cancer by a simple preparation of peripheral blood mononuclear cells / 대한외과학회지

PURPOSE: This study aimed to assess the cytolytic activity and the phenotype of circulating blood immune cells in cancer patients by using a simple preparation of peripheral blood mononuclear cells (PBMCs). METHODS: Peripheral blood was obtained from 94 diagnosed colorectal cancer (CRC) patients and 112 healthy donors. PBMCs were cocultured with K562 cells for 2 hours and lactate dehydrogenase released from the dead K562 cells was measured by using a spectrophotometer. Meanwhile, PBMCs were stained with fluorescence conjugated monoclonal antibodies (mAbs) and analyzed by flow cytometry. RESULTS: The cytolytic activity of PBMCs were significantly different between CRC patient and healthy groups (8.82% +/- 3.84% vs. 17.51% +/- 8.57%; P < 0.001). However, no significant difference in the cytolytic activity was observed after surgery in the CRC patient group (before surgery, 8.82% +/- 3.84% vs. after surgery, 9.95% +/- 4.94%; P = 0.326). In addition, the percentage of peripheral blood natural killer cells was significantly higher in the preoperative patient group than in the healthy group (19.97% +/- 11.51% vs. 15.60% +/- 5.77%, P = 0.041). In contrast, the percentage of peripheral blood lymphocytes was lower in the preoperative patient group than in the healthy group (28.41% +/- 8.31% vs. 36.4% +/- 8.6%, P < 0.001). CONCLUSION: These results demonstrate that circulating blood immune cells of CRC patients are functionally impaired and undergo an immunophenotypic perturbation, and show that a simple preparation of PBMCs can be useful to evaluate cellular immunity in cancer.

Avaliação in vitro da citotoxicidade de monômeros, plastificante e produtos de degradação liberados a partir de resinas para reembasamento imediato / In vitro cytotoxicity of monomers, plasticizer and degradation by-products released from hard chair side reline resin

O presente estudo teve como objetivo avaliar o efeito citotóxico dos monômeros isobutil metacrilato (IBMA) e 1,6 – Hexanediol dimetacrilato (1,6 – HDMA), do plastificante di-n-butil ftalato (DBP), e dos produtos de degradação ácido metacrílico (AM) e ácido benzóico (AB) sobre células L929. Esses compostos foram testados em faixas de concentrações liberadas, em um período de 30 dias, por materiais reembasadores rígidos, previamente quantificadas em estudos anteriores. O efeito citotóxico foi verificado por meio dos testes de MTT e 3H-timidina, após as células terem sido expostas às substâncias testadas nas concentrações estabelecidas. A classificação da citotoxicidade foi baseada na viabilidade celular em relação ao controle (células expostas ao meio sem as substâncias testadas). A atividade de síntese de DNA foi inibida por todos os compostos. Os resultados do presente estudo demonstraram que o teste de 3H-timidina foi mais sensível que o teste de MTT e que os compostos avaliados mostraram diferentes níveis de citotoxicidade in vitro. A atividade da desidrogenase mitocondrial diminuiu nas células tratadas com os monômeros, o plastificante e o produto de degradação AM; porém, para o AB, a maioria das concentrações testadas não apresentou efeito citotóxico.

The aim of this study was to evaluate the cytotoxic effect of the monomers 1,6 – hexanediol dimethacrylate (1,6 - HDMA) and isobutyl methacrylate (IBMA), the plasticizer di-n-butyl phthalate (DBP), and the degradation by-products methacrylic acid (MA) and benzoic acid (BA) on L929 cells. These compounds were tested in the range of concentrations released in 30 days from hard chairside reline resins that were quantified in previous investigations. Cytotoxic effects were assessed by using MTT and 3H - thymidine assays after the cells had been exposed to the test compounds at the given concentrations. Cytotoxicity was rated based on cell viability relative to controls (cells exposed to medium without test substances). The results presented in this study demonstrated that the 3H-thymidine assay was more sensitive than the MTT assay, and all compounds tested showed varying degrees of cytotoxic effect in vitro. DNA synthesis activity was inhibited by all compounds. Mitochondrial dehydrogenase activity decreased in cells treated with monomers, plasticizer and MA by-product, whereas no cytotoxic effect was observed on contact with BA at the majority of concentrations tested.

Alterations in NK Cell Cytotoxicity Induced by Peripheral Nerve Injury in Mice / 대한마취과학회지

BACKGROUND: Chronic pain is often associated with changes in the immune responses, which highlights the need for the aggressive pain control to obtain a better prognosis. This study examined splenic NK cell cytotoxicity in an attempt to assess the possible changes in the immune function under chronic neuropathic pain after a partial transsection of the sciatic nerve. METHODS: After confirming tactile allodynia in response to the von Frey filament, a modified lactate dehydrogenase (LDH) release assay was used to determine the cytotoxic activity of splenic NK cells on the YAC-1 cell line in C3H/HeN (H-2k) mice (n = 6). NK cells as effector cells were mixed with YAC-1 cells as target cells (1 x 10(4)/100microliter), resulting in an effector-target ratio of 1 : 25, 1 : 50, 1 : 100 in the culture medium. RESULTS: At 1 and 2 weeks after the nerve injury, all the subjects showed significant mechanical sensitivity compared with those observed before surgery. The percentage of NK cell cytotoxicity of the neuropathic mice increased significantly 1 week after the nerve injury but decreased within 2 weeks compared with the normal mice. CONCLUSIONS: In terms of the altered NK cell cytotoxicity, neuropathic pain can cause changes in the normal performance of the immune function.

Antibody dependent cell-mediated cytotoxicity on hepatocytes during immune response in halothane induced hepatitis / 第二军医大学学报

Objective: To study antibody dependent cell-mediated cytotoxicity (ADCC) on hepatocytes during immune response in halothane hepatitis of guinea pigs. Methods: The model of halothane hepatitis of guinea pigs was used. Mixed lymphocyte hepatocytes were cultured and supernatants containing specific antigens and cytokines were added. The changes of hepatic function in immune response of halothane hepatitis were analyzed. Results: The cytotoxicity on hepatocytes induced by halothane 3 times included ADCC mediated by specific antigens. ADCC on hepatocytes induced by halothane once required the second induction. There was no cytotoxicity on hepatocytes of specific antigens and cytokines produced by proliferative lymphocytes induced by Kupffer cells. Conclusion: Humoral immunity is mainly ADCC. Specific antibodies and cytokines can not separately kill hepatocytes. The extent of immune response is related to induction by halothane.