Effect of a new cross-linked hyaluronan gel on the staple line after sleeve gastrectomy in a rat model

Abstract Purpose: To evaluate the effect of a new cross-linked hyaluronan (NCHA) gel on healing of the staple line in an experimental sleeve gastrectomy. Methods: Eighteen rats were randomly divided into three groups. The control group (n = 6) received no medication. In the saline group (n = 6) and NCHA gel group (n = 6), saline and NCHA gel were respectively administered onto the staple line and intraperitoneally into the abdominal cavity after the standard stapling procedure. Results: The fibroblast activity and collagen deposition were significantly higher in the NCHA gel group than in the control group (p = 0.00, p = 0.017) and saline group (p = 0.004, p = 0.015). The tissue hydroxyproline protein level was significantly higher in the NCHA gel group than in the control group (p = 0.041). Adhesion formation was significantly lower in the NCHA gel group than in the control and saline groups (p = 0.015, p = 0.041). Conclusions: New cross-linked hyaluronan gel could be an effective approach to improve staple line wound healing and prevent potential leakage after sleeve gastrectomy. Moreover, NCHA gel helps to prevent adhesion formation without compromising healing of the staple line.

The effects of riboflavin and ultraviolet light on keratocytes cultured in vitro / Avaliação do efeito da riboflavina e luz ultravioleta sobre os ceratócitos in vitro

ABSTRACT Purpose: To culture quiescent human keratocytes and evaluate the effects of ultraviolet light and riboflavin on human corneal keratocytes in vitro. Methods: Keratocytes were obtained from remaining corneoscleral ring donor corneas previously used in corneal transplant surgeries and cultured in DMEM/F12 with 2% FBS until confluence. Characterization of cultured cells was performed by immunofluorescence analysis for anti-cytokeratin-3, anti-Thy-1, anti-α-smooth muscle actin, and anti-lumican. Immunofluorescence was performed before and after treatment of cultured cells with either ultraviolet light or riboflavin. Corneal stromal cells were covered with collagen (200 µL or 500 µL) and 0.1% riboflavin, and then exposed to ultraviolet light at 370 nm for 30 minutes. After 24 hours, cytotoxicity was determined using MTT colorimetric assays, whereas cell viability was assessed using Hoechst 33342 and propidium iodide. Results: Cell cultures achieved confluence in approximately 20 days. Expression of the lumican was high, whereas no expression of CK3, Thy-1, and α-SMA was observed. After crosslinking, MTT colorimetric assays demonstrated a low toxicity rate, whereas Hoechst 33342/propidium iodide staining demonstrated a low rate of apoptosis and necrosis, respectively, in all collagen-treatment groups. Conclusion: Keratocytes can be successfully cultured in vitro and characterized by immunofluorescence using lumican. MTT colorimetric assays, and Hoechst 33342, and propidium iodide staining demonstrated a higher rate of cell death in cells cultured without collagen, indicating collagen protects keratocytes from the cytotoxic effects of ultraviolet light.

RESUMO Objetivo: Avaliar o efeito da aplicação da luz ultravioleta e riboflavina sobre ceratócitos da córnea humana in vitro. Métodos: Os ceratócitos foram obtidos a partir das rimas corneoesclerais remanescentes da trepanação de córneas previamente utilizadas em cirurgias de transplante de córnea e cultivadas em meio DMEM/F12 com 2% de FBS até atingir confluência. As culturas de células foram caracterizadas por imunofluorescência com os anticorpos K3 (marcador de células epiteliais), Thy-1 (marcador de fibroblasto) SMA (marcador de miofibroblasto) e Lumican (marcador de ceratócitos). Imunofluorescência também foi feita após o tratamento. As células do estroma da córnea foram cobertas com colágeno (200 µL e 500 µL) e 0,1% de riboflavina e exposta a luz UVA a 370 nm por 30 minutos. Após 24 horas, citotoxicidade foi determinada por ensaio de MTT e a viabilidade celular foi feita por Hoechst 33342/Iodeto de propideo. Resultados: As culturas de células atingiram confluência em aproximadamente 20 dias. Imunofluorescência apontou alta expressão para o marcador de ceratócitos (Lumican) e expressão negativa par os marcadores de células epiteliais (K3), fibroblasto (Thy-1) e miofibroblasto (α-SMA). Após o cross linking a análise de MTT mostrou baixa taxa de toxicidade e com a coloração de Hoechst 33342/Iodeto de propideo baixa taxa de apoptose e necrose respectivamente em todos os grupos que continham colágeno. Conclusão: As culturas de ceratócitos foram obtidas e caracterizadas por imunofluorescência através do marcador Lumican com sucesso. O ensaio de MTT e a coloração por Hoechst 33342 e iodeto de propídio, apresentaram maior índice de morte celular nos grupos que não continham colágeno, provando que protege as células contra os efeitos da luz UVA.

Six-month outcomes of corneal crosslinking with dextran-free isotonic riboflavin solution / Resultados após seis meses de crosslinking de córnea com solução isotônica de riboflavina sem dextrano

ABSTRACT Purpose: To analyze the short-term clinical and topographic outcomes in patients with keratoconus after corneal collagen cross-linking treatment (CXL) with dextran-free isotonic riboflavin solution. Methods: In this retrospective case series, 26 eyes from 26 patients with keratoconus were studied. The best corrected visual acuity (BCVA) and refractive and topographic findings were analyzed at a 6-month follow-up. Results: The mean BCVA (Snellen lines) values before and 1, 3, and 6 months after CXL were 0.51 ± 0.2, 0.48 ± 0.2, 0.57 ± 0.2, and 0.64 ± 0.2, respectively, and the difference between the preoperative and 6-month values was statistically significant (p=0.006). The mean spherical equivalent refraction decreased from -5.6 ± 2.4 diopters (D) preoperatively to -5.0 ± 2.1 D, and mean simulated keratometry decreased from 48.5 ± 2.5 D to 47.8 ± 2.6 D at 6 months. (p=0.145 and p=0.001, respectively). In addition, the maximum keratometry decreased progressively and significantly from the preoperative value during follow-up (p=0.003). The central and minimal corneal thicknesses, including those of the epithelium, also decreased from 442.8 ± 25.6 µm and 430.5 ± 23.9 µm preoperatively to 420.7 ± 31.8 µm and 409.3 ± 28.7 µm at the most recent follow-up (p<0.001), respectively. No intraoperative or postoperative complications were observed. Conclusions: CXL with dextran-free isotonic riboflavin solution appears to be a safe treatment alternative for keratoconus and yields sustained short-term improvements in visual acuity, keratometric readings, and corneal thickness. However, long-term results are needed to confirm these outcomes.

RESUMO Objetivo: Analisar os resultados clínicos e topográficos curto prazo após crosslinking (CXL) de córnea com solução isotônica de riboflavina sem dextrano, em pacientes com ceratocone. Método: Estudamos 26 olhos de 26 pacientes com ceratocone, nesta série retrospectiva de casos. Melhor acuidade visual corrigida (BCVA), refração e achados topográficos foram analisados aos 6 meses de acompanhamento. Resultados: BCVA pré-operatória (linhas de Snellen) foi de 0,51 ± 0,2. BCVA após CXL foram de 0,48 ± 0,2, 0,57 ± 0,2 e 0,64 ± 0,2 no 1º, 3º e 6º meses, respectivamente. A diferença entre a BCVA pré-operatória e mais recente foi estatisticamente significativa (p=0,006). O equivalente esférico médio diminuiu de -5,6 ± 2,4 dioptrias (D) no pré-operatório para -5.0 ± 2.1 D e a média da ceratometria simulada diminuiu de 48,5 ± 2,5 D para 47, 8± 2,6 D aos 6 meses. (p=0,145 e p=0,001, respectivamente). A ceratometria máxima diminuiu progressivamente durante o acompanhamento com as mudanças sendo significativamente diferentes do valor pré-operatório (p=0,003). As espessuras corneanas central e mínima, diminuiram de 442,8 ± 25,6 µm e 430,5 ± 23,9 µm para 420,7 ± 31,8 µm e 409,3 ± 28,7 µm, respectivamente, na visita mais recente (p<0,001). Não foram observadas complicações intraoperatórias e pós-operatórias. Conclusões: CXL com solução de riboflavina isotônica sem dextrano parece ser uma opção segura de tratamento para o ceratocone com melhora mantida na acuidade visual, ceratometria e espessura corneana, no curto prazo. Resultados a longo prazo são necessários para confirmar estes resultados.

Factors affecting visual acuity after accelerated crosslinking in patients with progressive keratoconus / Fatores que afetam a acuidade visual após crosslinking acelerado entre pacientes com ceratocone progressivo

ABSTRACT Purpose: The present study aimed to report the outcomes of patients with progressive keratoconus who were treated via accelerated crosslinking (CXL) 6 months earlier and to determine the factors that promoted improved visual acuity after treatment. Methods: This retrospective study included 35 eyes of 34 patients with progressive keratoconus who underwent CXL. Topographical measurements were obtained preoperatively and in the first, third, and sixth months postoperatively using a rotating Scheimpflug camera. The uncorrected visual acuity (UCVA), best-corrected visual acuity (BCVA), flat keratometry (K) value (K1), steep K value (K2), average K value (avgK), topographic cylindrical value (Cyl), apical keratoscopy front (AKf), apical keratoscopy back (AKb), symmetry index front (SIf), symmetry index back (SIb), and thinnest point of the cornea (ThkMin) were recorded. Results: At the 6-month follow-up, the mean UCVA and BCVA values were improved, and the K values remained stable. Statistically significant decreases in AKf (p=0.04) and the thinnest point of the cornea (p=0.001) and a statistically significant increase in AKb (p=0.01) were observed. A correlation analysis revealed that the preoperative BCVA, UCVA, K1, K2, avgK, AKf, and AKb values significantly affected visual acuity at the 6-month follow-up. Conclusions: Accelerated CXL is an effective treatment for the prevention or even reversal of keratoconus progression. The preoperative K values and apexes of the anterior and posterior cornea were found to affect visual acuity at 6 months after accelerated CXL. Both AKb steepening and AKf flattening appear to be important factors in the stabilization of keratometric values and improvement of visual outcomes.

RESUMO Objetivo: O objetivo do estudo é relatar os resultados do sexto mês após o tratamento de crosslinking acelerado (CXL) em pacientes com ceratocone progressivo e determinar os fatores que afetam a melhora da acuidade após o tratamento. Métodos: Neste estudo retrospectivo, foram incluídos 35 olhos de 34 pacientes com ceratocone progressivo que se submeteram CXL. Acuidade visual não corrigida (UCVA) e melhor acuidade visual corrigida (BCVA) foram registradas. Medidas topográficas foram obtidas utilizando uma câmara Scheimpflug rotativa no pré-operatório e no 1º, 3º e 6º meses após a cirurgia. Os valores de ceratometria (K) mais plana (K1), K mais curva (K2), médio de K (avgK), astigmatismo topográfico (Cyl), ápice anterior da ceratoscopia (AKf), ápice posterior da ceratoscopia (AKb), índice anterior de simetria (SIf), índice posterior de simetria (SIb) e ponto mais fino da córnea (ThkMin) foram avaliados. Resultados: A média UCVA e BCVA melhoraram, enquanto valores de K ficaram estáveis 6º mês. Houve uma diminuição estatisticamente significativa na AKf e um aumento estatisticamente significativo na AKb (p=0,04, p=0,01, respectivamente). O ponto mais fino da córnea diminuiu significativamente (p=0,001). Na análise de correlações, além da UCVA e BCVA pré-operatórias; valores K1, K2, avgK, AKf e AKb pré-operatórios influenciaram significativamente a acuidade visual no 6º mês de acompanhamento. Conclusões: CXL acelerado é uma forma eficaz de tratamento na prevenção ou no mesmo inversão da progressão do ceratocone. A acuidade visual no 6º mês após CXL acelerado foi afetada a partir dos valores de K e dos ápice anterior e posterior da córnea. Encurvamento do AKb e aplanamento do AKf parecem ser fatores importantes na estabilização dos valores ceratométricos e na melhora da acuidade visual.

Effects of epicatechin, a crosslinking agent, on human dental pulp cells cultured in collagen scaffolds

ABSTRACT Objective The purpose of this study was to investigate the biological effects of epicatechin (ECN), a crosslinking agent, on human dental pulp cells (hDPCs) cultured in collagen scaffolds. Material and Method To evaluate the effects of ECN on the proliferation of hDPCs, cell counting was performed using optical and fluorescent microscopy. Measurements of alkaline phosphatase (ALP) activity, alizarin red staining, and real-time polymerase chain reactions were performed to assess odontogenic differentiation. The compressive strength and setting time of collagen scaffolds containing ECN were measured. Differential scanning calorimetry was performed to analyze the thermal behavior of collagen in the presence of ECN. Results Epicatechin increased ALP activity, mineralized nodule formation, and the mRNA expression of dentin sialophosphoprotein (DSPP), a specific odontogenic-related marker. Furthermore, ECN upregulated the expression of DSPP in hDPCs cultured in collagen scaffolds. Epicatechin activated the extracellular signal-regulated kinase (ERK) and the treatment with an ERK inhibitor (U0126) blocked the expression of DSPP. The compressive strength was increased and the setting time was shortened in a dose-dependent manner. The number of cells cultured in the ECN-treated collagen scaffolds was significantly increased compared to the cells in the untreated control group. Conclusions Our results revealed that ECN promoted the proliferation and differentiation of hDPCs. Furthermore, the differentiation was regulated by the ERK signaling pathway. Changes in mechanical properties are related to cell fate, including proliferation and differentiation. Therefore, our study suggests the ECN treatment might be desirable for dentin-pulp complex regeneration.

Corneal thickness changes during corneal collagen cross-linking with UV-A irradiation and hypo-osmolar riboflavin in thin corneas / Alterações na espessura da córnea durante "cross-linking" do colágeno com irradiação UV-A e riboflavina hipo-osmolar em córneas finas

PURPOSE: To evaluate the thinnest corneal thickness changes during and after corneal collagen cross-linking treatment with ultraviolet-A irradiation, using hypo-osmolar riboflavin solution in thin corneas. METHODS: Eighteen eyes of 18 patients were included in this study. After epithelium removal, iso-osmolar 0.1% riboflavin solution was instilled to the cornea every 3 minutes for 30 minutes. Hypo-osmolar 0.1% riboflavin solution was then applied every 20 seconds for 5 minutes or until the thinnest corneal thickness reached 400 µm. Ultraviolet-A irradiation was performed for 30 minutes. During irradiation, iso-osmolar 0.1% riboflavin drops were applied every 5 minutes. Ultrasound pachymetry was performed at approximately the thinnest point of the cornea preoperatively, after epithelial removal, after iso-osmolar riboflavin instillation, after hypo-osmolar riboflavin instillation, after ultraviolet-A irradiation, and at 1, 6 and 12 months after treatment. RESULTS: Mean preoperative thinnest corneal thickness was 380 ± 11 µm. After epithelial removal it decreased to 341 ± 11 µm, and after 30 minutes of iso-osmolar 0.1% riboflavin drops, to 330 ± 7.6 µm. After hypo-osmolar 0.1% riboflavin drops, mean thinnest corneal thickness increased to 418 ± 11 µm. After UVA irradiation, it was 384 ± 10 µm. At 1, 6 and 12 months after treatment, it was 372 ± 10 µm, 381 ± 12.7, and 379 ± 15 µm, respectively. No intraoperative, early postoperative, or late postoperative complications were noted. CONCLUSIONS: Hypo-osmolar 0.1% riboflavin solution seems to be effective for swelling thin corneas. The swelling effect is transient and short acting. Corneal thickness should be monitored throughout the procedure. Larger sample sizes and longer follow-up are required in order to make meaningful conclusions regarding safety.

OBJETIVO: Avaliar as alterações da espessura mínima da córnea durante e após o cross-linking do colágeno corneano com radiação ultravioleta A e solução hipo-osmolar de riboflavina em córneas finas. MÉTODOS: Dezoito olhos de 18 pacientes foram incluídos neste estudo. Após a remoção do epitélio, solução iso-osmolar de riboflavina 0,1% foi instilada a cada 3 minutos por 30 minutos. Solução hipo-osmolar de riboflavina 0,1% foi então aplicada a cada 20 segundos por 5 minutos ou até que a espessura mínima da córnea atingisse 400 µm. Irradiação UVA foi feita durante 30 minutos. Durante a irradiação, riboflavina iso-osmolar 0,1% foi aplicada a cada 5 minutos. Paquimetria ultrassônica foi realizada no ponto mais fino da córnea antes da cirurgia, após a remoção do epitélio, após a instilação de riboflavina iso-osmolar, após a instilação de riboflavina hipo-osmolar, após a irradiação com UVA e após 1, 6 e 12 meses do tratamento. RESULTADOS: Antes da cirurgia, a espessura mínima da córnea era de 380 ± 11 µm. Após a remoção do epitélio, este valor foi reduzido para 341 ± 11 µm e após 30 minutos de riboflavina iso-osmolar, caiu para 330 ± 7,6 µm. Após a riboflavina hipo-osmolar, a espessura mínima da córnea aumentou para 418 ± 11 µm. Após a irradiação com UVA, era de 384 ± 10 µm. Após 1, 6 e 12 meses do tratamento este valor era de 372 ± 10, 381 ± 12,7 e 379 ± 15 µm, respectivamente. Não foram observadas complicações no intra ou no pós-operatório precoce ou tardio. CONCLUSÕES: A solução de riboflavina hipo-osmolar 0,1% parece ser eficaz para edemaciar córnea finas. Este efeito é transitório e de curta duração. A espessura da córnea deveria ser monitorada durante todo o procedimento. Maior número de casos e seguimento prolongado são necessários para tirarmos conclusões quanto à segurança.

Efeito da mitomicina-C tópica sobre os depósitos de colágeno total na submucosa das pregas vocais íntegras de suínos / Effect of topical mitomycin-C on total collagen deposits on the submucosa of intact vocal folds in swine

OBJETIVO: Estudar o efeito da aplicação tópica de Mitomicina-C em diferentes concentrações sobre os depósitos de colágeno total na submucosa de pregas vocais íntegras de suínos. MÉTODOS: Os animais foram divididos em três grupos de acordo com o conteúdo da solução tópica aplicada sobre as pregas vocais: solução fisiológica 0,9 por cento (grupo controle); Mitomicina-C tópica 4 mg/mL (grupo 1); e Mitomicina-C 8 mg/mL (Grupo 2). Após 30 dias da aplicação, os animais foram submetidos à eutanásia, coletado amostras das pregas vocais e coradas pela técnica do picrosirius red com polarização para a quantificação computadorizada da deposição do colágeno, através do programa Image Pro plus 4.5Ò. Compararam-se as médias de área do colágeno depositado na submucosa das pregas vocais dos três grupos através do teste não paramétrico de Mann-Whitney. RESULTADOS: As médias das áreas de depósito colágeno na submucosa das pregas vocais foram de 3.110,44 micrômetros quadrados (mm²); 3.115,98 mm² e 3.105,78 mm² nos grupos controle, 1 e 2 respectivamente. Não houve diferença significativa nas áreas de depósitos de colágeno total da submucosa das pregas vocais entre os três grupos estudados (p>0,05). CONCLUSÃO: A mitomicina-C aplicada topicamente em pregas vocais suínas íntegras não alterou significativamente a deposição de colágeno na submucosa.

OBJECTIVE: To compare the effects of topical mitomycin-C at different concentrations on submucosal collagen deposition on the vocal folds of swine. METHODS: The animals were divided into three groups according to the composition of the topical solution to be applied to the vocal folds: 0.9 percent saline solution (control group); 4 mg/ml mitomycin-C (group 1) and 8 mg/ml mitomycin-C (group 2). Thirty days after the application, all animals were sacrificed, their vocal folds were collected and stained by the picrosirius red technique, and submucosal collagen deposition areas were estimated by the Image Pro Plus 4.5® software. Mann-Whitney test was used to compare differences between parameters of each group. RESULTS: The means of the areas of submucosal collagen deposits on vocal folds were 3110.44 square micrometers (mm²), 3115.98 mm² and 3105.78 mm² for groups control, 1 and 2, respectively. There were no statistical differences across the three groups (p>0.05). CONCLUSION: Mitomycin-C topically applied to intact vocal folds of swine did not alter submucosal collagen deposition.

Plasma Factor XIII Activity in Patients with Disseminated Intravascular Coagulation

The objective of this study was to investigate the correlation between factor XIII (FXIII) activity and disseminated intravascular coagulation (DIC) parameters and also to evaluate the clinical usefulness of DIC diagnosis. Citrated plasma from eighty patients with potential DIC was analyzed for FXIII activity. The primary patient conditions (48 male and 32 female, mean age, 51 years) were malignancy (n = 29), infection (n = 25), inflammation (n = 6), heart disease (n= 3), thrombosis (n = 2), injury (n = 2), and other miscellaneous conditions (n = 13). FXIII testing was performed using the CoaLinkTM FXIII Incorporation Assay Kit (PeopleBio Inc.). Among 80 patients who were suspected to have DIC based on clinical analysis, 46 (57.5%) fulfilled the overt DIC criteria (DIC score > = 5) according to the International Society of Thrombosis and Haemostasis. FXIII levels in the plasma were significantly decreased in overt DIC compared to non-overt DIC patients (mean 75.1% and 199.7% respectively, p < 0.0001). Interestingly, we found a significant inverse correlation between DIC scores and FXIII activity. In addition, FXIII activity significantly correlated with other hemostatic markers that included platelet count, prothrombin time, activated partial thromboplastin time, fibrinogen, and D-dimer. FXIII levels were significantly lower in patients with liver or renal dysfunction. In conclusion, FXIII cross-linking activity measurements may have differential diagnostic value as well as predictive value in patients who are suspected to have DIC.

Preparation of arsenic trioxide albumin microspheres and its release characteristics in vitro / 华中科技大学学报（医学）（英德文版）

Arsenic trioxide albumin microspheres (As2O3-BSA-NS) were prepared by using methods of chemical cross-linking. The desirability function (DF), calculated according to the size (0.05). The release experiment in vitro showed that As2O3 in As2O3-BSA-NS was released more slower than pure As2O3. It was concluded that regular As2O3-BSA-NS may be prepared by the methods of chemical cross-linking, which was optimized by orthogonal experimental analysis of different factors, and the microspheres can release As2O3 slowly.

Regulation of activity of the yeast TATA-binding protein through intra-molecular interactions.

Dimerization is proposed to be a regulatory mechanism for TATA-binding protein (TBP) activity both in vitro and in vivo. The reversible dimer-monomer transition of TBP is influenced by the buffer conditions in vitro. Using in vitro chemical cross-linking, we found yeast TBP (yTBP) to be largely monomeric in the presence of the divalent cation Mg2+, even at high salt concentrations. Apparent molecular mass of yTBP at high salt with Mg2+, run through a gel filtration column, was close to that of monomeric yTBP. Lowering the monovalent ionic concentration in the absence of Mg2+, resulted in dimerization of TBP. Effect of Mg2+ was seen at two different levels: at higher TBP concentrations, it suppressed the TBP dimerization and at lower TBP levels, it helped keep TBP monomers in active conformation (competent for binding TATA box), resulting in enhanced TBP-TATA complex formation in the presence of increasing Mg2+. At both the levels, activity of the full-length TBP in the presence of Mg2+ was like that reported for the truncated C-terminal domain of TBP from which the N-terminus is removed. Therefore for full-length TBP, intra-molecular interactions can regulate its activity via a similar mechanism.

The transcriptional activator GAL4-VP16 regulates the intra-molecular interactions of the TATA-binding protein.

Binding characteristics of yeast TATA-binding protein (yTBP) over five oligomers having different TATA variants and lacking a UASGAL, showed that TATA-binding protein (TBP)-TATA complex gets stabilized in the presence of the acidic activator GAL4-VP16. Activator also greatly suppressed the non-specific TBP-DNA complex formation. The effects were more pronounced over weaker TATA boxes. Activator also reduced the TBP dimer levels both in vitro and in vivo, suggesting the dimer may be a direct target of transcriptional activators. The transcriptional activator facilitated the dimer to monomer transition and activated monomers further to help TBP bind even the weaker TATA boxes stably. The overall stimulatory effect of the GAL4-VP16 on the TBP-TATA complex formation resembles the known effects of removal of the N-terminus of TBP on its activity, suggesting that the activator directly targets the N-terminus of TBP and facilitates its binding to the TATA box.

Differential effects of Fas cross-linking on phospholipase D activation and related lipid metabolism in Fas-resistant A20 cells

A20 murine lymphoma cells undergoing Fas-mediated apoptosis showed increase in the activity of phospholipase D (PLD), which is involved in proliferative or mitogenic cellular responses. Using A20 cell lines that were resistant to Fas-induced apoptosis, we investigated the differential effects of Fas cross-linking on PLD activity and sphingolipid metabolism. The basal PLD activities in all of the selected three Fas-resistant clones (#5, #8, and #11) were about 2~4 folds higher than that of wild type A20 cells. Among the PLD isoforms, PLD2 expression was increased in all of the selected Fas-resistant clones. The Fas downstream signaling events triggered by Fas cross-linking, including the activations of PLD, phosphatidy-lcholine-specific phospholipase C (PC-PLC), sphingomyelinase (SMase), the increase in diacylglycerol (DAG) and protein phosphorylation levels, and the translocation of protein kinase C to membrane were not changed in both of Fas-resistant clone #5 and #8. In contrast, Fas cross-linking stimulated the activity of PLD, PC-PLC, and SMase, translocation of PKC, and protein phosphorylation in Fas-resistant clone #11, similar to that of wild type cells. We also found that clone #11 had a different Fas sequence encoding Fas B which has been known to inhibit Fas-induced apoptosis. These findings suggest that increased PLD2 expression resulting in increased basal PLD activity and the blockade of Fas downstream signaling cascades may be involved to limit apoptosis induced by Fas cross-linking.