Prediction of serum M30 and M65 levels for the short-term prognosis of patients with hepatitis B virus related acute-on-chronic liver failure during artificial liver support system therapy / 中华传染病杂志

Objective@#To analyze the dynamic changes of serum M30 and M65 levels in patients with hepatitis B virus related acute-on-chronic liver failure (HBV-ACLF) during artificial liver support system(ALSS) therapy, and to explore their predictive efficiency and clinical values for short-term prognosis of HBV-ACLF.@*Methods@#Seventy-six patients with HBV-ACLF who underwent ALSS therapy for the first time from May 2016 to May 2019 in the First Hospital of Jiaxing were selected.The patients were divided into improvement group (38 cases) and non-recovered group (38 cases)according to their prognosis, and 38 healthy persons were selected as control group during the same period.The serum levels of M30 and M65 were detected by enzyme-linked immunosorbent assay(ELISA). The predictive values of M30 and M65 levels for short-term prognosis in patients receiving ALSS were calculated by receiver operating characteristic analysis curve (ROC). M30 and M65 levels before and after ALSS were compared by two-way repeated measures analysis of variance.@*Results@#The levels of M30 and M65 in the improvement group, non-recovered group and control group were significantly different before the first ALSS therapy (F=109.36 and 90.42, respectively, both P<0.01). The levels of M30 and M65 were not significantly different between improvement group and non-recovered group before treatment (t=0.836 and 0.286, respectively, both P>0.05). However, after twice ALSS therapy, the levels of M30 and M65 in non-recovered group were significantly higher than those in improvement group (t=30.699 and 64.777, respectively, both P<0.01). Moreover, after the second ALSS therapy, the levels of M30 and M65 were both significantly lower compared to those after the first-time therapy in the improvement group (t=3.350 and 5.932, respectively, both P<0.01). The areas under curve (AUC) of M30, M65 and the combination of M30 and M65 for prognosis prediction were 0.796, 0.844 and 0.906, respectively. The AUC of combination of M30 and M65 was significantly higher than M30 or M65 alone (Z=2.163 and 2.141, respectively, P=0.031 and 0.032, respectively). The cut-off values of M30 and M65 were 591.91 and 924.50 U/L, respectively. The sensitivity and specificity of combined M30 and M65 were 94.7% and 82.5%, respectively.@*Conclusions@#Serum M30 and M65 levels can predict the short-term prognosis of HBV-ACLF patients after ALSS therapy.The combination of M30 and M65 is of better diagnostic value.

Prediction of serum M30 and M65 levels for the short-term prognosis of patients with hepatith B virus related acute-on-chronic liver failure during artificial liver support system therapy / 中华传染病杂志

The levels of M30 and M65 in the improvement group,non-recovered group and control group were significantly different before the first ALSS therapy(F=109.36 and 90.42,respectively,both P＜0.01).The levels of M30 and M65 were not significantly different between improvement group and non-recovered group before treatment(t=0.836 and 0.286,respectively,both P＞0.05).However,after twice ALSS therapy,the levels of M30 and M65 in non-recovered group were significantly higher than those in improvement group(t=30.699 and 64.777,respectively,both P＜0.01).Moreover,after the second ALSS therapy,the levels of M30 and M65 were both significantly lower compared to those after the first-time therapy in the improvement group(t=3.350 and 5.932,respectively,both P＜0.01).The areas under Curve(AUC)of M30,M65 and the combination of M30 and M65 for prognosis prediction were 0.796,0.844 and 0.906,respectively.The AUC of combination of M30 and M65 was significantly higher than M30 or M65 alone(Z=2.163 and 2.141,respectively,P=0.031 and 0.032,respectively).The cut-off values of M30 and M65 were 591.91 and 924.50 U/L,respectively.The sensitivity and specificity of combined M30 and M65 were 94.7％and 82.5％,respectively.Conclusions Serum M30 and M65 levels can predict the short-term prognosis of HBV-ACLF patients after ALSS therapy.The combination of M30 and M65 is of better diagnostic value.

Clinical value of serum markers M30 and M65 for primary biliary cholangitis / 临床检验杂志

Objective@#To quantify the serum levels of apoptosis-related molecules M30 and M65 in the patients with primary biliary cholangitis (PBC) and analyze their clinical significance for PBC. @*Methods@#A total of 79 patients with PBC and 40 healthy individuals were involved in this study from January 2017 to April 2019. The levels of serum M30 and M65 were measured by enzyme-linked immunosorbent assay (ELISA) and the ratio of M30/M65 was calculated. The parameters of liver function were tested by automatic biochemical analyzer. ALT, AST and GGT were determined by rate method, ALP was assayed by the method of NPP substrate-AMP buffer and T-Bil was determined by oxidation method. Autoantibodies including AMA-M2, anti-GP210 and anti-SP100 were detected by automated multiplexed bead-based and immunoblotting assays. The correlation of M30, M65 and M30/M65 ratio with liver function items were analyzed by spearman assay. The levels of M30, M65 and M30/M65 ratio were analyzed by ROC curve to evaluate their values in PBC screening. @*Results@#M65 level in PBC group was significantly higher than that in control group (P＜0.001) with statistically significant difference and M30/M65 ratio was significantly lower than that in control group with statistically significant difference (P＜0.001). There was no significant difference for the level of M30 between the two groups (P=0.641). Among the PBC patients, 18 were anti-GP210-positive and 17 were anti-SP100-positive. In anti-GP210 positive group the levels of M30 and M65 were significantly higher than those in the negative group (P values were 0.002 and 0.001 respectively). In anti-SP100 positive group the level of M65 was significantly higher than that in the negative group (P=0.027) and M30/M65 ratio was significantly lower than that in negative group with statistically significant difference (P=0.005). Spearman correlation analysis showed that the levels of M30 and M65 were positively correlated with ALT, AST and T-Bil (P＜0.05). The cut off values of M30 and M65 were 63.62 U/L and 155.37 U/L and M30/M65 was 0.32, respectively. The screening sensitivities were 63.30%, 89.10% and 93.30%, and the specificities were 51.60%, 90.00% and 75.00%, respectively. @*Conclusion@#The level of M65 in the serum of PBC patients significantly increased and M30/M65 ratio significantly decreased, which could be used as a promising serum marker of laboratory for PBC screening.

Expression of M30 and M65 in celiac disease. Analytical cross-sectional study

ABSTRACT BACKGROUND: The role of villous atrophy in apoptosis, a distinctive feature of celiac disease, is a matter of controversy. The aim of this study was to determine the apoptosis rate through immunohistochemical staining for M30 and M65 in celiac disease cases. DESIGN AND SETTING: Analytical cross-sectional study in a tertiary-level center. METHODS: Duodenal biopsies from 28 treatment-naive patients with celiac disease, 16 patients with potential celiac disease, 10 patients with a gluten-free diet and 8 controls were subjected to immunohistochemical staining for the end-apoptotic marker M30 and the total cell death marker M65. H-scores were compared. Several laboratory parameters were recorded concomitantly, and at the one-year follow-up for celiac disease and potential celiac disease patients. RESULTS: There was a significant difference in H-score for M30 expression between the celiac disease, potential celiac disease and gluten-free diet groups (P = 0.009). There was no significant difference in H-score for M65 expression. There was a positive correlation between the H-score for M30 expression and the anti-tissue transglutaminase immunoglobulin A (anti-tTgIgA) and anti-tissue transglutaminase immunoglobulin G (anti-tTgIgG) levels (R = 0.285, P = 0.036; and R = 0.307, P = 0.024, respectively); and between the H-score for M65 expression and the anti-tTgIgA and anti-tTgIgG levels (R = 0.265, P = 0.053; and R=0.314, P = 0.021, respectively). There was no difference between celiac disease and potential celiac disease patients regarding the laboratory parameters selected. CONCLUSION: The rates of apoptosis and nutritional deficiencies in patients with potential celiac disease were similar to those in patients with celiac disease.

Clinical value of Serum CK-18 M30 levels in patients with nonalcoholic steatohepatitis / 实用医学杂志

Objective To detect the level of serum fragmented cytokeratin 18 (CK-18 M30) in patients with nonalcoholic steatohepatitis (NASH), to explore the relationship between the expression of CK-18 M30 and NASH. Methods 33 healthy people as control group, 24 nonalcoholic simple fatty liver (NAFL) patients, and 21 NASH patients were included in this study. CK-18 M30, ALT, AST and GGT were detected in all patients’ vein blood. NAFLD activity points (NAS) was examined in biopsy specimens of NAFL patients and NASH patients. Pearson correlation was applied to analyze the correlations between serum CK-18 M30, ALT, AST, GGT and the NAS of liver tissue in NASH group. Results Serum CK-18 M30 level of healthy control, NAFL and NASH group were (96.557 2 ± 41.226 8)U/L, (104.321 7 ± 45.167 3)U/L, (263.125 5 ± 61.578 1)U/L respectively. Serum CK-18 M30 level in NASH patients positively correlated with both NAS of liver tissue and serum ALT, which correlation coefficient r values were 0.601 5 and 0.420 6. Conclusion The concentration of serum CK-18 M30 could be used as a marker in the diagnosis of NASH.

Propofol or sevoflurane anaesthesia does not affect hepatic integrity as assessed by the M30 & M65 cell death markers & liver enzymes.

Background & objectives: General anaesthetics may induce apoptosis. The pro-apoptotic/necrotic markers M30 (caspase-cleaved cytokeratin-18) and M65 (intact cytokeratin-18) have been used to identify early apoptosis in liver disease. The aim of this study was to detect the effect of propofol and sevoflurane anaesthesia on these markers and blood transaminase levels in female patients undergoing elective surgery. Methods: Sixty-seven women undergoing mastectomy or thyroidectomy under general anaesthesia were randomly allocated to the propofol or sevoflurane groups. Venous blood samples for measuring the apoptotic and necrotic markers M30 and M65 as well as for measuring the alanine aminotransferase (ALT) and the aspartate aminotransferase (AST) liver enzymes were collected before induction of anaesthesia, immediately after completion of surgery, and 24 and 48 h postoperatively. Results. The M30 values preoperatively and 0, 24 and 48 h postoperatively were 280±229, 300±244, 267±198 and 254±189 U/l in the propofol group and 237±95, 242±109, 231±94 and 234±127 U/l in the sevoflurane group, respectively. The M30 values did not differ within or between the groups. The M65 levels at the same time intervals were 470±262, 478±271, 456±339 and 485±273 in the propofol group and 427±226, 481±227, 389±158 and 404±144 U/l in the sevoflurane group, respectively. No significant changes were found in the M65 either within or between the propofol and the sevoflurane groups. The ALT and AST levels did not change at these time intervals. Interpretation & conclusions: Under the present study design propofol or sevoflurane anaesthesia did not induce apoptosis or affected the liver function as assessed by the M30, M65 markers and liver enzymes in patients undergoing mastectomy or thyroidectomy under general anaesthesia.

Various methods available for detection of apoptotic cells- A review.

Apoptosis is a process of programmed cell death occurring in multicellular organisms in whom development, maintenance and sculpturing organs and tissues. Taken together, apoptotic processes are of widespread biological significance; being involved in e.g. development, differentiation, proliferation/homoeostasis, regulation and function of the immune system and in the removal of defected harmful cells. Dys regulation of apoptosis can play a primary or secondary role leading to cancer whereas excessive apoptosis contributes to neuro degeneration, autoimmunity, AIDS, and ischemia. Gaining insight into the techniques for detecting apoptotic cells will allow the development of more effective, higher specific and therefore better-tolerable therapeutic approaches. The goal of this review article is to provide a general overview of current knowledge, on the various technical approaches for detecting apoptotic cells.

Effects of different doses of UVA1 irradiation on the expressions of CD34 and M30 in skin homogenate from a mouse model of scleroderma / 中华皮肤科杂志

Objective To observe the expression changes of CD34 and M30 in skin homogenate from a mouse model of scleroderma after irradiation with different doses of UVA1, and to investigate the effect of UVA1 phototherapy on vascular endothelial cell function in scleroderma. Methods The experimental mouse models of scleroderma were established by the injection with bleomycin and randomly divided into model control group (n = 10), UVA1 irradiation group (n = 30) and unirradiated group (n = 10). The UVA1 irradiation group was further equally divided into 3 groups, HD-UVA1 group irradiated with UVA1 at 100 J/cm2, MD-UVA1group with UVA1 at 60 J/cm2, and LD-UVA1 group with UVA1 at 20 J/cm2; phototherapy was performed thrice weekly for 10 weeks followed by the sacrifice of mice. The mice in model control group were killed immediately after the establishment of models, and the mice in unirradiated group received no irradiation after the establishment of models and were maintained till the killing of mice in UVA1 irradiation groups. Skin specimens were obtained from the bleomycin-induced scleroderma lesions of mice and separated into two parts, one was subjected to histopathological examination, and the other one was used to prepare skin homogenate for the detection of CD34 and M30 content with ELISA assay. Results After 30 sessions of treatment with UVA1,the softening and thinning of sclerotic skin were seen by the naked eye, with the most obvious changes in HDUVA1 group; pathological examination revealed a reduction in dermal thickness and the presence of hair follicular structures in subcutaneous fat tissue with no obvious proliferation of collagen in these mice. Compared with the mice in model control group and unirradiated group, there was an increase in CD34 and decrease in M30 content in skin homogenate from UVA 1-irradiated mice, with the most marked changes in mice irradiated with UVA1 at 100 J/cm2. The concentration of CD34 and M30 in skin homogenate from unirradiated group and model control group was significantly different from that in HD-UVA1 group (22.25 ± 8.91 μg/L and 31.97 ±17.97 μg/L vs. 72.39 ± 13.04 μg/L, 162.41 ± 58.00 U/L and 195.71 ± 71.09 U/L vs. 38.06 ± 19.89 U/L, all P ＜ 0.01 ). Additionally, significant differences were observed between the three UVA1 groups in the concentration of CD34 and M30 (F = 21.23, 15.32, respectively, both P ＜ 0.01 ). Conclusions UVA1 phototherapy could up-regulate the expression of CD34 but down-regulate that of M30 in skin homogenate from the mouse model of scleroderma, and the effect is correlated with the intensity and cumulative dose of irradiation.

Measurement of apoptosis using M30 in culture media of cell lines treated with anti-cancer agents / 대한산부인과학회지

OBJECTIVE: We investigated a possible use of the induced apoptosis as a biomarker in the cells and their media treated with commonly used anti-cancer agents in gynecologic malignancies. METHODS: After treatments with low and high concentrations of paclitaxel, cisplatin, and camptothecin in HeLa and OVCAR-3 cells, the levels of M30 antigen were detected in the cells and their media by immunofluorescence staining and ELISA methods, respectively. RESULTS: The percentages of M30-fluoresein isothiocyanate (FITC) positive cells in HeLa and OVCAR-3 cells treated with paclitaxel, cisplatin, and camptothecin were 4.3% vs 18.1% vs 34.87% and 4.07% vs 18.6% vs 32.63%, 4.3% vs 17.87% vs 32.38% and 4.07% vs 16.83% vs 32%, and 4.3% vs 16.75% vs 31.3% and 4.07% vs 15.18% vs 29.9% in control, low dose, and hight dose groups, respectively (P<0.001). M30 antigen levels (U/L) measured in culture media of HeLa and OVCAR-3 cells treated with paclitaxel, cisplatin, and camptothecin were 53.03 vs 101.53 vs 355.59 and 86 vs 114.41 vs 412.04, 53.03 vs 79.84 vs 327.64 and 86 vs 125.44 vs 385.09, and 53.03 vs 88.41 vs 295.005 and 86 vs 108.42 vs 263.1 in control, low dose, and hight dose groups, respectively (P<0.001). CONCLUSION: Our results obtained in this preclinical study suggests that measurement of the levels of M30 antigen may help to predict the clinical responses and to select the effective anti-cancer agents in clinical settings, rapidly and quantitatively.

Detection of chemosensitivity using K18-Asp(396) (M30) antibody in HeLa and OVCAR-3 cell lines treated with anticancer agents / 대한산부인과학회지

OBJECTIVE: The aim of this study was to detect the levels of M30-antigens as a biomarker of apoptosis in cells and their culture media after treatments with anticancer drugs as a preclinical study. METHODS: After HeLa and OVCAR-3 cells were treated respectively with paclitaxel, cisplatin, and camptothecin, the harvested cells were stained sequentially with M30 monoclonal antibodies and propidium iodide (PI). Afterwards, they were analyzed using a FACScan flow cytometer and observed under an immunofluorescence microscope for M30-FITC immunofluorescences. Levels of M30 antigens were also detected in their culture media using M30-Apoptosense ELISA kit. RESULTS: The levels of M30-FITC immunofluorescences were elevated in both cell lines after each drug treatments compared with those of control cells. The levels of M30 antigens detected by ELISA in media culturing each cell line treated with each of drugs were elevated compared with those of control cells. CONCLUSION: This study suggests that M30-antigens representing chemotherapy induced apoptosis may be a useful biomarker for predicting and monitoring the response of neoadjuvant chemotherapy in patients with gynecologic cancers.

Role of apoptosis in erosive and reticular oral lichen planus exhibiting variable epithelial thickness

Oral lichen planus (OLP) is a chronic inflammatory disease with different clinical types. Reticular and erosive forms are the most common. Although the cause of OLP remains speculative, many findings suggest auto-immune involvement, mediated by T lymphocytes against the basal keratinocytes. Inflammation, mechanical trauma or toxic agents can affect the epithelial homeostasia. Increased apoptosis may cause a decrease in epithelial thickness reflecting in the activity of the lesion. The objective of this study was to evaluate the occurrence of apoptosis and epithelial thickness in reticular and erosive forms of OLP. 15 samples of OLP each type (reticular and erosive) plus 10 of healthy mucosa were collected and processed. After morphometry, the apoptotic index and epitelial thickness were obtained. TUNEL and M30 CytoDEATH immunohistochemical assay were used to validate the morphologic criteria used. Apoptosis in the erosive OLP was significantly more intense than in the reticular type and both forms of OLP presented more apoptosis than the healthy oral mucosa. Healthy oral mucosa was thicker than both OLP forms and thicker in OLP reticular form than in the erosive one. The clinical differences between reticular and erosive forms of OLP are related to variations in epithelial thickness and in intensity of apoptosis.

O líquen plano oral (LPO) é uma doença inflamatória crônica com diferentes tipos clínicos. As mais comuns são as formas reticular e erosiva. Embora a causa do LPO permaneça no campo especulativo, muitos achados sugerem tratar-se de uma doença auto-imune, mediada por linfócitos T que têm como alvo os ceratinócitos basais. Inflamação, trauma mecânico ou agentes tóxicos podem afetar a homeostasia epitelial. O aumento da apoptose pode levar a uma diminuição da espessura epitelial e isto refletir na atividade da doença. O objetivo deste estudo foi avaliar a ocorrência de apoptose e a espessura epitelial nas formas reticular e erosiva de LPO. 15 amostras de LPO de cada tipo reticular e erosivo, além de 10 amostras de mucosa saudável foram coletadas e processadas. Depois da morfometria, o índice apoptótico (IA) e a espessura do epitélio foram obtidas. Reação de TUNEL e imunohistoquímica do M30 CytoDeath foram usadas para validação dos critérios morfológicos. A apoptose no LPO erosivo foi significativamente maior que no tipo reticular e ambas as formas de LPO apresentaram mais apoptose que a mucosa oral normal. A mucosa oral normal foi mais espessa que ambas as formas de LPO, sendo que, a forma reticular foi mais espessa que o tipo erosivo. As diferenças clínicas entre as formas reticular e erosiva de LPO têm relação com as variações na espessura epitelial e na intensidade da apoptose.

Assessment of Apoptosis by M30 Immunoreactivity and the Relationship with the MSI status and the Clinicopathological Characteristics of Colorectal Carcinomas

BACKGROUND: The monoclonal antibody M30 recognizes a neoepitope of cytokeratin 18 that's produced during the process of apoptosis, and it is reactive in formalin-fixed, paraffin-embedded tissue. The detailed nature of apoptosis in colorectal cancer is unclear, especially in regard to the MSI status and the clinicopathologic factors. The purpose of this study was to elucidate the apoptosis assessed by M30 immunoreactivity in colorectal cancer and its relationship with the MSI status and the various clinicopathologic factors of colorectal cancers. METHODS: 101 colorectal cancers were classified according to levels of MSI as 12 MSI-H, 4 MSI-L and 85 MSS. Apoptosis was quantified by immunohistochemistry with using M30 CytoDEATH anti-body. RESULTS: The apoptotic index assessed by M30 was significantly increased in the MSI-H and MSI-L colorectal cancer compared to that in the MSS colorectal cancer. Right sided colon cancer showed a significant higher apoptotic index than did the left sided colon cancer. There was also a tendency for decreased apoptosis in metastatic colorectal cancers (Duke's stage D). There was somewhat of an increase of apoptosis in colorectal cancers with mucinous carcinoma and medullary carcinoma, and also in the colorectal cancers with an increased TIL count, but this was not statistically significant. CONCLUSION: M30 immunoreactivity is a valuable method to detect apoptosis in formalin-fixed, paraffin-embedded tissue and it might explain that MSI-H colorectal cancer shows better clinical behavior than MSS colorectal cancer in regard to the increased apoptosis.

Increased Apoptotic Activity of Human Trophoblasts in Pregnancy-Induced Hypertension: Assessment by the Caspase-related M30 CytoDeath Antibody / 대한해부학회지

Pregnancy-induced hypertension (PIH) is a multi-system disorder unique to human pregnancy. Although the etiology of PIH is still unknown, there is a large evidence suggesting that abnormal trophoblast invasion is occurring in early pregnancy and that this may contribute to relative placental hypoxia and oxidative stress that may further exacerbate placental pathology. This study was undertaken to determine placental Cu/Zn superoxide dismutase (SOD) and Mn SOD activities and evaluate the use of M30 CytoDeath antibody to assess trophoblasts apoptotic activity in normal and PIH pregnancies. We also compared apoptotic rates as detected by M30 and TdT-mediated dUTP nickend labelling (TUNEL). Placental expression of Cu/Zn SOD and Mn SOD were reduced in PIH as compared to normal pregnancies. M30 immunoreactivity occurred predominantly in the syncytiotrophoblasts, and a significantly higher number of M30 positive cells in the preeclamptic placentas were found when compared with normal placentas. The number of M30 positive cells correlated with another apoptotic index previously detected by TUNEL method.