OBJECTIVES: To describe the clinical course and treatment response of a case of thrombotic thrombocytopenic purpura (TTP) after a month of therapeutic plasma exchange (TPE) and immunosuppressive agents and to review related published literature regarding TTP and its response to TPE and immunosuppressive agents.
CASE AND DISCUSSION: A 64-year-old female presented with fever, bicytopenia, change in sensorium, and seizure of one day duration. Metabolic panel showed normal electrolytes with normal brain imaging. Hematologic work-up showed anemia, thrombocytopenia and leukocytosis with normal differential count. Reticulocyte was elevated. Peripheral smear showed significant schistocytes with nucleated red blood cells and marked thrombocytopenia. Thrombotic thrombocytopenic purpura was the initial consideration with the fulfilment of four out of the pentad of TTP, namely: microangiopathichaemolytic anemia, thrombocytopenia, fever and neurologic symptoms. Therapeutic plasma exchange was immediately initiated with daily platelet count and Lactate dehydrogenase (LDH) determination to assess response. However, despite daily plasma exchanges and continuous plasma infusion, there was inadequate response. In this light, immunosuppressive agents were started in the following order: high dose methylprednisolone, weekly rituximab and intravenous cyclophosphamide. On the 31st hospital day, after daily TPE and combined immunosuppression, she achieved complete response with a platelet count of 170 X 109/L to 250 X 109/L (baseline-14 X 109/L) and LDH 227U/L (baseline-1,191 U/L).
CONCLUSION: This study presented a challenging case of TTP which was successfully treated with the standard of care together with the available adjunctive treatment options.

Human ; Female ; Middle Aged ; Plasma Exchange ; Purpura, Thrombotic Thrombocytopenic ; Rituximab ; Thymidine 5'-triphosphate ; Lactate Dehydrogenase 1 ; Isoenzymes ; L-lactate Dehydrogenase ; Anemia

PURPOSE: To evaluate the response and cellular changes of cultured human corneal epithelium according to the concentration of topical cyclosporine. METHODS: Human corneal epithelial cells were exposed to cyclosporine A concentrations of 1 ug/ml (0.0001%), 10 ug/ml (0.001%), 100 ug/ml (0.01%), and 500 ug/ml (0.05%) for 5 and 10 minutes. An MTT-based colorimetric assay was performed to assess the metabolic activity of cellular proliferation and a lactate dehydrogenase (LDH) leakage assay was used to determine cellular toxicity. The modulations of extracellular matrix proteins such as PIP and laminin were evaluated. The levels of pro-inflammatory cytokine, TNF-alpha, and IL-1 were evaluated by ELISA kits. RESULTS: The inhibitory effects of human corneal epithelial cellular proliferation did not show a concentration- or exposure time-dependent response. Activity of LDH did not show a statistically significant difference for the different concentrations and exposure times. The inhibitory effects of extracellular matrix proteins such as PIP or laminin synthesized from human corneal epithelial cells were compared with those in the control group and showed a statistically significant difference at a cyclosporine concentration greater than 0.01%. Released TNF-alpha and IL-1 from human corneal epithelial cells did not show any difference according to concentration or cyclosporine exposure time. CONCLUSIONS: To modulate human corneal epithelial cellular proliferation and the levels of extracellular matrix proteins such as PIP and laminin, a concentration of cyclosporine A greater than 0.01% for longer than 5 minutes of exposure is needed. There were little effects of cyclosporine A on pro-inflammatory cytokine secreted from corneal epithelial cells.
Cell Proliferation ; Cyclosporine ; Enzyme-Linked Immunosorbent Assay ; Epithelial Cells ; Epithelium, Corneal ; Extracellular Matrix Proteins ; Humans ; Interleukin-1 ; L-Lactate Dehydrogenase ; Laminin ; Tumor Necrosis Factor-alpha

OBJECTIVETo explore whether coal tar pitch smoke extract (CTP) induced pyroptosis in human bronchial epithelial cells (BEAS-2B).

METHODSBEAS-2B cells were treated with different concentrations of CTP (1, 3 µg/ml) for 8h and 24 h, respectively. Lactic dehydrogenase (LDH) activity and interleukin-1 beta (IL-1β) levels in the supernatants of cell culture media were measured with LDH activity or human IL-1β ELISA kit, respectively. The activity of Caspase-1 was measured with Caspase-1 colorimetric assay kit.

RESULTSThe activity of caspase-1 in 1 and 3 µg/ml CTP groups were (9.29 ± 0.30) and (8.67 ± 0.59) µmol/ml respectively which were both significantly increased compared to that (7.42 ± 0.59) µmol/ml in the control group (P < 0.05) after 8 h exposure, but there was no significant difference in the activity of LDH and levels of IL-1β in the cell culture media among the CTP and control groups. 24 h after exposure, the activity of LDH in the CTP (1, 3 µg/ml) groups were (1323.03 ± 28.53) and (1148.45 ± 16.42) U/dl respectively which were significantly higher than that (1091.93 ± 26.64) U/dl in the control group (P < 0.05), and the levels of IL-1β in the CTP (1 and 3 µg/ml) groups were (125.37 ± 25.00) pg/ml and (92.04 ± 19.09) pg/ml respectively which were significantly higher than that (46.20 ± 14.43) pg/ml in the control group (P < 0.05), but there was no significant difference in the activity of Caspase-1 among CTP and control groups (P < 0.05).

CONCLUSIONCTP treatment induced early increase in caspase-1 activity followed by the increase in LDH activity and IL-1 levels, indicative of pyroptosis in human bronchial epithelial cells.

Apoptosis ; Bronchi ; cytology ; Caspase 1 ; metabolism ; Cell Line ; Coal Tar ; adverse effects ; Epithelial Cells ; cytology ; Humans ; Interleukin-1beta ; metabolism ; L-Lactate Dehydrogenase ; metabolism ; Smoke ; adverse effects

OBJECTIVETo investigate whether the cardioprotective effect of hemin against ischemia/reperfusion (I/R) injury is through the inhibition of calpain activity, and to explore its underlying mechanism.

METHODSSixty-four SD rats were randomly divided into eight groups (n = 8): sham, I/R, MDL+ I/R, MDL, hemin + I/R, hemin, and ZnPP + hemin+ I/R, ZnPP. Iangendorff isolated rat heart perfusion model was used. The rat hearts were suffered from 40 min of ischemia followed by 30 min of reperfusion. After that, left ventricular developed pressure (LVDP) was recorded. Infarct size and release of lactate dehydrogenase (LDH) were measured. Calpain, heme oxygenase (HO), and caspase 3 activities were evaluated. Expression of calpastatin protein was detected by Western blot.

RESULTS(1) After suffered from ischemia/reperfusion, the calpain activity and caspase 3 activity increased. MDL28170, an inhibitor of calpain, prevented ischemia/reperfusion induced increases in LDH and infarct size, improved the LVDP recovery. (2) Compared with ischema/reperfusion rat hearts, pretreatment of hemin enhanced the HO-1 activity, decreased the calpain and caspase 3 activities, declined LDH release and infarct size, and improved LVDP recovery. (3) Ischemia/reperfusion reduced the expression of calpastatin protein in rat hearts, which was inhibited by hemin pretreatment. And HO-1 inhibitor could abolish the cardioprotection of hemin.

CONCLUSIONCardioprotective effect of hemin against ischemia/reperfusion injury is through the inhibition of calpain activity, the mechanism might be involved in the increase in calpastatin protein expression.

Animals ; Calpain ; metabolism ; Cardiotonic Agents ; pharmacology ; Caspase 3 ; metabolism ; Heme Oxygenase-1 ; metabolism ; Hemin ; pharmacology ; L-Lactate Dehydrogenase ; metabolism ; Myocardial Reperfusion Injury ; drug therapy ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the influence of hypoxia induction factor-1alpha (HIF-1alpha) on glycosis of rat myocardial cell under hypoxic condition.

METHODSThe myocardial cells of the rats were routinely isolated and cultured. The cells were divided into single hypoxia (H) and HIF-1alpha inhibiting (I) groups. The cells in H group were cultured in glucose-free medium with mixed low-oxygen gas [1% O2, 94% N2 and 5% CO2 (v/v)]. While the cells in I group were cultured with low-oxygen gas after the cell model of low expression of HIF-1alpha protein constructed by RNAi technique. The cells in both groups were all observed before hypoxia (routine culture) and at the time points of 1, 3, 6, 12 and 24 hours of hypoxia. The LA (lactate acid ) content in the supernatant of the culture and the activity of the key enzymes in glycolysis such as hexokinase (HK), phosphofructokinase (PFK) and lactate dehydrogenase (LDH) of both groups of cells were determined at all the time points.

RESULTS(1) After hypoxia, the HK and PFK activities of the rat myocardial cells in H and I groups were obviously increased at the beginning and decreased thereafter when compared with that before hypoxia. While the activities of HK and PFK in H group at 1, 3 and 6 hours after hypoxia were evidently higher than those in I group (P <0.05 or 0.01), and the peak activity of them in H and I groups was 159 +/- 13 U/g vs 133 +/- 55 U/g, and 298 +/- 44 U/g vs 188 +/- 55 U/g, respectively. (2) Compared with normal control (92 +/- 12 U/g), the LDH activity of the cells in H group after hypoxia increased significantly, reaching the peak at 6 hours after hypoxia (2 568 +/- 125 U/g, P < 0. 01), and it decreased thereafter, while that in I group peaked at 3 hours after hypoxia (2125 +/- 126 U/g, P <0.01). The LA content in the culture supernatant in H group increased significantly after hypoxia with the passage of time, while that in I group increased in smaller magnitude (P <0.01).

CONCLUSIONHigh expression of HIF-1alpha in the rat myocardial cells after hypoxia could directly cause continuous enhancement of cell glycolysis, which was beneficial to the protection of myocardial cells under hypoxic condition.

Animals ; Cell Hypoxia ; Cells, Cultured ; Glycolysis ; Hexokinase ; metabolism ; Hypoxia-Inducible Factor 1, alpha Subunit ; metabolism ; L-Lactate Dehydrogenase ; metabolism ; Myocytes, Cardiac ; metabolism ; Phosphofructokinase-1 ; metabolism ; RNA Interference ; Rats ; Rats, Sprague-Dawley

BACKGROUND: Growing tumors adapt to a hypoxic environment and increase anaerobic glycolysis. This metabolic switch is related to aggressive behavior. We investigated the relationship between glycolytic metabolism biomarkers associated with hypoxia-inducible factor (HIF)-1alpha and prognosis. METHODS: We performed immunohistochemical staining of HIF-1alpha, pyruvate dehydrogenase kinase (PDK) 1 and lactate dehydrogenase (LDH) 5 in 74 patients with oral squamous cell carcinoma (SCC) who had received curative radical resection. RESULTS: High reactivity of HIF-1alpha, PDK 1 and LDH 5 was observed in 29 (39.2%), 32 (43.2%) and 54 (73.0%) patients, respectively. Expression levels of the three biomarkers were significantly correlated. All three markers were highly expressed in 16 (21.6%) patients. Elevated expression of the three markers was associated with increased invasiveness (p = 0.043) and recurrence (p = 0.017) of tumors. In survival analysis, upregulation of the three markers was additionally associated with shorter disease free survival (DFS, p = 0.001) and overall survival (OS, p = 0.002). High expression of all three markers was a strong independent prognostic factor for DFS (p = 0.030) and OS (p = 0.026). CONCLUSIONS: Oral SCC with altered glycolytic metabolism exhibits a more invasive and aggressive phenotype. Our results indicate that glycolytic metabolism biomarkers related to HIF-1alpha may be independent prognostic factors in patients with oral SCC.
Biomarkers ; Carcinoma, Squamous Cell ; Disease-Free Survival ; Glycolysis ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; Isoenzymes ; L-Lactate Dehydrogenase ; Oxidoreductases ; Phenotype ; Phosphotransferases ; Prognosis ; Protein-Serine-Threonine Kinases ; Pyruvic Acid ; Recurrence ; Up-Regulation

OBJECTIVETo investigate nephroprotective effects of a mixture of 8 L-amino acids on renal ischemia-reperfusion injury and its effects on renal endothelin-1 (ET-1).

METHODSThe mixture of 8 L-amino acids includes glycine, alanine, threonine, serine, valine, leucine, isoleucine and proline. Acute ischemic renal injury was induced by clamping renal pedicle for 45 minutes in rats. Sixty male Sprague-Dawley rats were randomly divided into 3 groups: a sham-operated group (Group A, n=8), a control group (Group B, n=26) and an amino acid-treated group (Group C, n=26). Amino acids were infused at a rate of 1 ml x 100g(-1) x h(-1) I hour before ischemia and during 3 hours of the whole reperfusion. The serum creatinine values, BUN levels, creatinine clearance, urine sodium and potassium excretion, urine lactate dehydrogenase (LDH), the rate of urine flow and histological examination were measured. Renal ET-1 levels were assayed with radioimmunological assay (RIA) RESULTS: The creatinine clearance was 471.0 microl/min+/-121.5 microl/min in Group C and 227.0 microl/min+/-27.0 microl/min in Group B 3 hours after reperfusion, P<0.01). The urine flow rate was 63.6 microl/min+/-15.2 microl/min in Group C and 24.3 microl/min+/-7.7 microl/minin Group B, P<0.01) 1.5 hours after reperfusion. The serum creatinine was 85.0 microl/min+/-7.7 micromol/L and BUN concentration 11.4 mmol/L+/-3.9 mmol/L in Group C and 112.7 micromol/L+/-19.5 micromol/L and 20.7 mmol/L+/-6.6 mmol/L respectively in Group B after 24 hours of reperfusion (P<0.05). The mean histological score by standards of Paller in kidneys was 108.7+/-15.7 in Group C, and 168.8+/-14.8in Group B (P<0.01). The renal ET-1 levels 15 minute and 3 hours after reperfusion were 7.2 pg/mg+/-0.8 pg/mg and 9.6 pg/ml+/-1.0 pg/ml in Group C, and 10.1 pg/ml+/-2.8 pg/ml and 13.0 pg/ml+/-2.7pg/ml in Group B (P<0.01).

CONCLUSIONSThe mixture of 8 L-amino acids can provide remarkable protection against renal ischemia-reperfusion injury in rats. This may associate with attenuation of renal ET-1 disorder.

Amino Acids ; pharmacology ; Animals ; Antineoplastic Combined Chemotherapy Protocols ; Blood Urea Nitrogen ; Creatinine ; urine ; Endothelin-1 ; analysis ; Glomerular Filtration Rate ; Kidney ; blood supply ; L-Lactate Dehydrogenase ; urine ; Male ; Radioimmunoassay ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; prevention & control

OBJECTIVETo investigate the effects of resveratrol (Res) on neonatal rat cardiomyocyte lesion induced by hypoxia.

METHODThe cardiomyocyte of neonatal rats were cultured in vitro and the model of cardiomyocyte hypoxia was established. The cardiomyocyte vitalities were determined by MTT assay, the HIF-1alpha expression levels in myocardial cells was detected by immunohistochemical, the activities of peroxidase (GSH-Px) and lactate dehydrogenase (LDH) were measured as well.

RESULTAfter the administration of hypoxia for 24 hours, the HIF-1alpha expression in myocardial cells was significantly increased. The LDH level in the culture medium was increased from (93.07 +/- 15.84) U x L(-1) to (750.77 +/- 181.51) U x L(-1) (P < 0.01). The intracellular GSH-Px activity was decreased from (46.96 +/- 8.36) U x mL(-1) to (27.13 +/- 4.76) U x mL(-1) (P < 0.05). Res 25, 50 and 75 micromol x L(-1) could dose-dependently inhibit the raising of the HIF-1alpha expression in myocardial cells induced by hypoxia. The LDH activities were decreased dose-dependently to (486.17 +/- 69.97), (189.43 +/- 32.07), (155.34 +/- 29.57) U x L(-1), respectively (P <0.05 or P <0.01). The GSH-Px activities were increased dose-dependently (33.55 +/- 6.34), (37.67 +/- 6.73), (41.44 +/- 7.91) U x mL(-1) (P < 0.05 or P < 0.01).

CONCLUSIONRes has a protective effect on neonatal rat cardiomyocyte lesion induced by hypoxia.

Animals ; Animals, Newborn ; Cell Hypoxia ; physiology ; Cells, Cultured ; Dose-Response Relationship, Drug ; Hypoxia-Inducible Factor 1, alpha Subunit ; metabolism ; L-Lactate Dehydrogenase ; metabolism ; Myocytes, Cardiac ; drug effects ; metabolism ; Rats ; Rats, Sprague-Dawley ; Stilbenes ; pharmacology

OBJECTIVETo investigate the influence of microtubule intervention drugs on glycolytic key enzymes in myocardial cells after hypoxia.

METHODSThe primary passage of cultured myocardial cells from neonatal rats were divided into A group (with hypoxia), B group (with hypoxia and administration of l0 micromol/L colchicine), C group (with hypoxia and administration of 5 micromol/L taxol), D group (with hypoxia and administration of 10 micromol/L taxol), E group (with hypoxia and administration of 15 micromol/L taxol). The morphology of microtubule was observed with laser scanning microscope (LSM). The cell vitality was assayed by cell counting kit (CCK). The activities of hexokinase (HK), pyruvate kinase (PK), phosphofructokinase (PFK) and lactate dehydrogenase (LDH) were assayed with colorimetry.

RESULTSIn group B and E, the microtubule structure was damaged heavily, and the cell vitality was decreased significantly [The cell vitality was (89.99 +/- 3.47)% in B group and (84.56 +/- 6.61)% in E group, respectively, at 1.0 post hypoxia hour (PHH), and hoth values were obviously lower than that in A group (97.44 +/- 1.76)%, P < 0.01]. The HK, PK and PFK activities decreased obviously. The activities of HK, PK and PFK in group C were similar to those of the A group. Compared with that in other groups, the degree of damage of microtubule structure in D group was milden. The activities of HK, PK and PFK in D group during 0.5 - 6.0 PHH were significantly higher than those in A group. The activity of LDH in each group was increased after hypoxia.

CONCLUSIONProper concentration of microtubule-stabilizing drugs can alleviate the damages to microtubule structure, and enhance the activity of glycolytic key enzymes of myocardial cells at early stage of hypoxia.

Animals ; Cell Hypoxia ; Cells, Cultured ; Glycolysis ; drug effects ; Hexokinase ; metabolism ; L-Lactate Dehydrogenase ; metabolism ; Microtubules ; drug effects ; metabolism ; Myocytes, Cardiac ; enzymology ; metabolism ; Phosphofructokinase-1 ; metabolism ; Pyruvate Kinase ; metabolism ; Rats ; Rats, Sprague-Dawley

OBJECTIVE: To explore the mechanisms of large-conductance calcium-activated potassium channel (BKCa) involved in inflammatory response in sepsis. METHODS: The serum levels of BKCa were measured by enzyme-linked immunosorbent assay (ELISA) in patients with sepsis (28 cases), patients with common infection (25 cases) and healthy people (25 cases). The relationship between levels of BKCa and acute physiology and chronic health evaluation II (APACHE II) were analyzed. Cultured RAW 264.7 cells were stimulated by lipopolysaccharide (LPS). In some experiments, a cell model of sepsis was constructed using Nigericin as the second stimulus signal. The mRNA and protein expressions of BKCa in RAW 264.7 cells stimulated with LPS (0, 50, 100, 1 000 μg/L) were measured by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) and Western blotting. RAW 264.7 cells were transfected with small interfering RNA of BKCa (siRNA-BKCa), and the levels of caspase-1 precursor (pro-caspase-1), interleukin-1β precursor (pro-IL-1β) in cell, and the levels of caspase-1 p20, IL-1β p17 of cell culture medium, and NOD-like receptor protein 3 (NLRP3), nuclear factor-κB (NF-κB) were measured by Western blotting. The apoptosis were detected by staining with propidium iodide (PI), the release rate of lactate dehydrogenase (LDH) were measured, and the expression of apoptotic protein Gasdermin D (GSDMD) was measured by Western blotting to evaluate the effect of silencing BKCa on cell pyrosis. RESULTS: The level of serum BKCa in patients with sepsis was significantly higher than that in patients with common infection and health peoples (ng/L: 165.2±25.9 vs. 102.5±25.9, 98.8±20.0, both P < 0.05). In addition, the level of serum BKCa in patients with sepsis was significantly positively correlated with APACHE II score (r = 0.453, P = 0.013). LPS could construct a sepsis cell model by which LPS could promote BKCa expression in mRNA and protein with a concentration-dependent manner. The mRNA and protein expressions of BKCa in the cells stimulated by 1 000 μg/L LPS were significantly higher than that in the blank group (0 μg/L) [BKCa mRNA (2^-ΔΔCt): 3.00±0.36 vs. 1.00±0.16, BKCa/β-actin: 1.30±0.16 vs. 0.37±0.09, both P < 0.05]. Compared with the control group, the ratios of caspase-1 p20/pro-caspase-1 and IL-1β p17/pro-IL-1β in the model group were significantly increased (caspase-1 p20/pro-caspase-1: 0.83±0.12 vs. 0.27±0.05, IL-1β p17/pro-IL-1β: 0.77±0.12 vs. 0.23±0.12, both P < 0.05), however, transfection of siRNA-BKCa induced the decrease both of them (caspase-1 p20/pro-capase-1: 0.23±0.12 vs. 0.83±0.12, IL-1β p17/pro-IL-1β: 0.13±0.05 vs. 0.77±0.12, both P < 0.05). Compared with the control group, the number of apoptotic cells, LDH release rate and GSDMD expression in the model group were significantly increased [LDH release rate: (30.60±8.40)% vs. (15.20±7.10)%, GSDMD-N/GSDMD-FL: 2.10±0.16 vs. 1.00±0.16, both P < 0.05], however, transfection of siRNA-BKCa induced the decrease both of them [LDH release rate: (15.60±7.30)% vs. (30.60±8.40)%, GSDMD-N/GSDMD-FL: 1.13±0.17 vs. 2.10±0.16, both P < 0.05]. The mRNA and protein expressions of NLRP3 in sepsis cells were significantly higher than those in the control group [NLRP3 mRNA (2^-ΔΔCt): 2.06±0.17 vs. 1.00±0.24, NLRP3/GAPDH: 0.46±0.05 vs. 0.15±0.04, both P < 0.05]. However, the expression of NLRP3 after siRNA-BKCa transfection was significantly lower than that in model group [NLRP3 mRNA (2^-ΔΔCt): 1.57±0.09 vs. 2.06±0.17, NLRP3/GAPDH: 0.19±0.02 vs. 0.46±0.05, both P < 0.05]. Compared with the control group, the NF-κB p65 nuclear transfer of sepsis cell were significantly increased (NF-κB p65/Histone: 0.73±0.12 vs. 0.23±0.09, P < 0.05). However, the NF-κB p65 expression in the nucleus were decreased after siRNA-BKCa transfection (NF-κB p65/Histone: 0.20±0.03 vs. 0.73±0.12, P < 0.05). CONCLUSIONS BKCa is involved in the pathogenesis of sepsis, and its possible mechanism is to activate NF-κB/NLRP3/caspase-1 signaling pathway to induce inflammatory factor production and cell death.
Humans ; Histones ; Caspase 1 ; Large-Conductance Calcium-Activated Potassium Channels ; Lipopolysaccharides ; NF-kappa B ; NLR Family, Pyrin Domain-Containing 3 Protein ; L-Lactate Dehydrogenase ; Sepsis ; RNA, Small Interfering ; Caspases