BACKGROUND: Intracellular ionized calcium plays a central role in the transduction of external stimuli as a critical second messenger. The spectral properties of fluo-3 allows the analysis of intracellular ionized calcium level by flow cytometers. The aim of this study is to assess the performance of flow cytometer for measuring intracellular ionized calcium level using fluo-3 and to define the reference interval of intracellular ionized calcium level of lymphocytes from healthy people, and to find out the clinical implications according to various disorders. METHODS: For the analytical performance of flow cytometer on determining the concentration of intracellular ionized calcium, precision study, lowest limit of detection, analytical range, and the loading stability of fluo-3 were per foamed. Fifty-four cases of healthy people, 52 cases of renal transplant patients, and 20 cases of diabetes mellitus patients were included in this study. RESULTS: Loading effect of fluo-3 at room temperature was stable upto 5 hours. Lowest limit of detection of ionized calcium concentration was 4.34 nM at in-situ calibration procedure. Within-run and among-day intraindividual CVs of in-situ calibration procedure were 6.67% and 13.99% respectively, and of optical calibration procedure were 13.86% and 16.12% respectively. The reference interval of cytosolic free calcium level for healthy people ranged 73.54 - 155.09 nM without sexual differences. The level of intracellular ionized calcium was lowered by 36.9% on renal transplant group in comparison with healthy control group. But, level of cytosolic free calcium was Increased upto 276.0% on acute rejection group and 159.1% on diabetes mellitus group compared to control group. CONCLUSIONS: These results reveal that in-situ calibration method for intra cellular ionized calcium using flow cytometry with flue-3 can be regarded as an accurate and standardized method. Quantitation of intracellular ionized calcium level might be used as the monitoring test for early detection of acute rejection after renal transplantation.
Calcium* ; Calibration ; Cytosol ; Diabetes Mellitus ; Flow Cytometry* ; Humans* ; Kidney Transplantation ; Limit of Detection ; Lymphocytes* ; Second Messenger Systems

BACKGROUND: Reactive oxygen species (ROS) induce lipid peroxidation and tissue damage in the isolated rabbit thoracic aorta. The aim of this study was to explore the influence of the propofol and midazolam against ROS in the isolated rabbit thoracic aortic endothelium. METHODS: Eighteen white male rabbits (weighing 2.0-2.5 kg) were used. The thoracic aorta was dissected free and cut into rings (3-4 mm) and then suspended in a organ bath filled with 10 ml Krebs solution bubbled with 5% CO2 95% O2 at 37 degrees C. Aortic rings were then equilibrated for 90 min, and a resting tension of 1.5 g was applied. The Krebs solution was changed every 15 min. Isometric tension was recorded with transducer coupled to a data acqusition system (Biopac Inc. USA) on a PC. After precontraction with norepinephrine (NE, 10(-6)M), changes in tension were measured following the cumulative administration of acetylcholine (ACh 3x10(-7), 10(-6) and 3x10(-6)M) and nitroglycerin (NTG, 10(-5)M). Data are expressed as percentage of the 10 5 M NTG-induced relaxation (ACh/NTG). The ACh/NTG, before and after electrolysis were defined as the control and the experimental groups. The aortic rings were pretreated with propofol (3x10(-5), 10(-4), 3x10(-4) and 5.7x10(-4) M, n = 8, 10, 15, 13), midazolam (10(-4)M, n = 7), catalase (1,000 U/ml, n = 12), mannitol (3x10(-4)M, n = 5) or not pretreated group (Free, n = 6). After 30 minutes, the aortic rings were exposed to ROS generated by electrolysis (DC 9 V, 20 mA, aortic rings 1 cm away from electrode) in Krebs solution for 2 minutes, which was then changed for physiologic buffered salt solution. The aortic rings were precontracted with NE and vasorelaxation was induced with ACh and NTG at the above mentioned concentrations. RESULTS: Propofol produced vasorelaxation of NE-precontracted thoracic aorta in a dose-dependent fashion in all groups of propofol (3x10(-5), 10(-4), 3x10(-4) and 5.7x10(-4)M) even after ROS attack (P < 0.05 vs control value). Catalase produced vasorelaxation after ROS attack (P < 0.05 vs control value).On the other hand, ACh-induced significant endothelium-dependent vasorelaxation were not observed in the midazolam or mannitol pretreated group or the non-pretreated group (P <0.05 vs control group). CONCLUSIONS: These findings suggest that propofol and catalase preserve ACh induced endothelium-dependent vasorelaxation and that propofol has a concentration dependent ROS scavenging effect like catalase.
Acetylcholine ; Aorta, Thoracic ; Baths ; Catalase ; Electrolysis ; Endothelium ; Hand ; Humans ; Lipid Peroxidation ; Male ; Mannitol ; Midazolam ; Nitroglycerin ; Norepinephrine ; Propofol* ; Rabbits ; Reactive Oxygen Species* ; Relaxation ; Transducers ; Vasodilation

BACKGROUND: Morphine has a direct action on morphine receptors in the brain and spinal cord. Intrathecally administered L-NAME, a nitric oxide synthase inhibitor, is known to have an antinociceptive effect on formalin-induced pain in animal studies. Efficacy of intrathecally administered ketorolac, a cyclooxygenase inhibitor, is somewhat controversial. The interactions of intrathecally administered morphine, ketorolac and L-NAME on formalin-induced nociception was studied. METHODS: Male Sprague-Dawley rats were implanted with chronic lumbar intrathecal catheters and were tested for paw flinch by a formalin injection. Drugs were intrathecally administered 15 min before the formalin injection, and biphasic painful behaviors were observed. We obtained the ED50 for each agent (ketorolac, L-NAME and morphine). ED50 fractions (1, 1/2 and 1/4) of drug combinations of L-NAME-ketorolac, morphine-L-NAME and ketorolac-morphine were administered. The ED50 of each combined drug was established and isobolographic analysis of the drug interactions was carried out. RESULTS: Intrathecal administration of ketorolac, L-NAME and morphine produced a dose-dependent suppression of pain behaviors in phase 2. ED50 values were 297.04micro gram for ketorolac, 207.46micro gram for L-NAME and 0.17micro gram for morphine in phase 2. Isobolographic analysis showed that the combination of intrathecal morphine and L-NAME synergistically reduced pain behaviors in phase 2. CONCLUSIONS: Intrathecally administered morphine, L-NAME and ketorolac produced a dose-dependent decrease in the number of paw flinches in both phase 1 and phase 2 on the formalin test. Morphine with L-NAME showed synergistic analgesic effects on formalin-induced pain in phase 2.
Animals ; Brain ; Catheters ; Drug Combinations ; Drug Interactions ; Formaldehyde ; Humans ; Ketorolac* ; Male ; Morphine* ; NG-Nitroarginine Methyl Ester* ; Nitric Oxide Synthase ; Nociception ; Pain Measurement ; Prostaglandin-Endoperoxide Synthases ; Rats* ; Rats, Sprague-Dawley ; Receptors, Opioid, mu ; Spinal Cord

OBJECTIVE: A number of disease-modifying anti-rheumatic drugs (DMARDs) have been shown to be more effective than placebo in the management of rheumatoid arthritis (RA). However, most course of DMARDs, except methotrexate, are discontinued after 2 or 3 years, because of toxicity, lack of efficacy or escape from control. The multi-drug resistance (MDR) is a phenomenon in which cells develop cross-resistance to many agents such as anthracyclin, vinca alkaloids and colchicine. In our hypothesis, MDR phenomenon could be implicated in acquired resistance to DMARDs in RA. We have established a mdr1 cell line and tested whether DMARDs are substrate for P-glycoprotein (P-gp). METHODS: The mdr1-cDNA was cloned into retroviral vector, and the recombinant retroviral vector was transfected into PA317 cells. The target cells, NIH3T3, were infected with recombinant retroviruses. A colony most resistant to vinblastin was selected for the following experiments; expression of mdr1 gene in NIH3T3 cells was confirmed by RT-PCR, and biological function of mdr1 gene product, P-gp, was tested using Rhodamine-123 (Rh123) efflux assay. Resistance of the target cells expression P-gp which can survive against hydroxychloroquine (HCQ) and methotrxate (MTX) were measured by MTT assay. RESULTS: RT-PCR for mdr1 gene showed successful transfer of the gene into the NIH3T3 cells. Rh123 assay revealed expression of P-gp on the selected cells as follows; Rh123 efflux activity of uninfected NIH3T3 cells was 6%, that of PLXSN was 0.2%, and that of selected cells was 44%. The 50% proliferation inhibitory capacity of the selected cells were twice for HCQ but there was no difference of that for MTX. CONCLUSION: We established a mdr1 cell line and using the cell line, HCQ was a substrate of MDR, but MTX was not related to MDR.
Antirheumatic Agents* ; Arthritis, Rheumatoid ; Cell Line ; Clone Cells ; Colchicine ; Drug Resistance, Multiple ; Hydroxychloroquine ; Methotrexate ; P-Glycoprotein ; Retroviridae ; United Nations ; Vinca Alkaloids ; Zidovudine

Purpose: This study aimed to describe the desperate situation where the clinician should make decisions to further manage patients having experienced adverse drug reaction (ADR) to lamotrigine that is indicated to not easily controlled neuropsychiatric diseases. Methods: A descriptive analysis was done by thoroughly reviewing medical records of patients who were reported to have ADR to lamotrigine in a regional drug-safety center between 2010 and 2018. Results: Eighty-four cases of lamotrigine-related ADRs occurred in 80 patients. Skin lesions were most commonly observed in 70 cases (83.3%) and 14 cases (16.7%) had severe ADRs. Sixty-three subjects (78.8%) discontinued lamotrigine, while 17 (21.3%) continued it.At the time of discontinuation, 30.0% were prescribed aromatic antiepileptic drugs. Among 4 subjects who were eventually prescribed lamotrigine again after a period of discontinuation, 3 (75.0%) experienced its recurrence. Among patients who had taken alternative medications, the incidence of ADRs was higher in those being prescribed aromatic antiepileptic drugs than in the others being prescribed other than aromatic antiepileptic drugs (P = 0.013). Regarding the control of underlying diseases, as many as 65 (86.7%) and 68 (90.7%) failed to reach maintaining the resolved state from 6 months and 12 months after the substitution, respectively. Conclusion Patients can be easily trapped between the recurrence of ADRs and the treatment failure to a certain drug like lamotrigine, in which we can hardly find a reasonable alternative to manage them.

OBJECTIVES: To evaluate the effects of progesterone/progestin and in combination with estrogen in relaxation of rat aorta. METHODS: Eight weeks after bilateral oophorectomy, the descending aorta of Spague-Dawley rats (n=10) were quickly removed and placed in organ bath containing Krebs solution. Each aorta ring in 2-3 mm length was connected to an isometric force transducer (FT 03, Grass, USA) and the changes in tension were recorded with an AD converter system (MP 100, Biopac Inc, USA) in a personal computer. After precontraction of the rings with norepinephrine (1 umol/L) or KCl (40 mmol/L), estradiol, progesterone, medroxyprogesterone acetate (MPA), norethisterone acetate (NETA) (10-5-10-8 M/L in each) were added to each ring and they were incubated for 15 minutes. The relaxation was expressed as a percentage of the tonic contraction. RESULTS: Estrogen relax the aorta in all concentrations. The degree of relaxation was dose dependent (P<0.001). All of the progesterone, MPA, NETA relax the aorta and the effects was different according to the concentration of steroids (P<0.0001). The degree of relaxation was not different between estrogen and those of progesterone, MPA, NETA except MPA 10-8 M, NETA 10-5 M. Addition of progesterone, MPA and NETA to the estrogen showed similar vascular effects compared to those of estrogen alone. CONCLUSION: Not only estrogen but also progesterone, MPA, and NETA acutely relax aorta. Progesterone/progestin have not been found attenuate the action of estrogen in our animal in vitro study.
Animals ; Aorta ; Aorta, Thoracic ; Arteries ; Baths ; Estradiol ; Estrogens ; Female ; Medroxyprogesterone Acetate ; Microcomputers ; Norepinephrine ; Norethindrone ; Ovariectomy ; Poaceae ; Progesterone ; Rats ; Relaxation ; Steroids ; Transducers

Leprosy is a granulomatous disease primarily affecting the peripheral nerves. The pathogenesis would be related to the cell-mediated response to mycobacterial antigens, metabolic and biochemical change of Schwann cell, circulating demyelinating factors and other autoimmune process. A specific nerve tissue protein, S-100 protein, has been demonstrated in normal nerves and nerve complexes. The stains of S-100 protein in dermal nerves of leprosy patients have been suggested in assessing the presence of nerve damage. We have estimated the concentration of S-100 protein in the sera of 64 leprosy patients(38 lepromatous leprosy, 26 tuberculoid leprosy) and that of 20 normal controls without neurologic disorders by ELISA. The results obtained were as follows: 1. The mean S-100 protein concentration was 0.0042ng/ml in a total of 64 leprosy patients, with 0.0062ng/ml in lepromatous type and 0.018ng/ml in tuberculoid type. The controls showed 0.0017ng/ml. 2. The analysis of age and serum S-100 protein concentration in both types showed lower value in the fifties of tuberculoid type(p<0.05). With the increase of age, mean S-100 protein concentration in both types tended to increase, but there was no significant correlation(p>0.05). 3. The analysis of duration of illness and serum S-100 protein concentration in both types showed higher value in the forties and fifties in lepromatous type(p<0.05). With the increase of duration of illness, mean S-100 protein concentration tended to increase in lepromatous type and slightly decreased in tuberculoid type, but there was no significant correlation(p>0.05). 4. The mean S-100 protein concentration of patients with neurologic symptoms was 0.0577ng/ml, in contrast with 0.0016ng/ml in patients without neurologic symptoms (p<0.05). In conclusion, the measurement of serum S-100 protein would play a potential role of a useful marker of assessing nerve damage in leprosy patients, esp, with neurologic symptoms.
Coloring Agents ; Enzyme-Linked Immunosorbent Assay ; Humans ; Leprosy* ; Leprosy, Lepromatous ; Nerve Tissue ; Nervous System Diseases ; Neurologic Manifestations ; Peripheral Nerves ; S100 Proteins*

PURPOSE: Tenosynovial giant cell tumors (TSGCT) can be classified into localized and diffuse types. To identify reliable diagnostic markers for these tumors, we compared clinicopathologic and immunohistochemical features in localized and diffuse type TSGCT. MATERIALS AND METHODS: Clinicopathologic and immunohistochemical studies were perfomed. Thirty cases which had been histologically diagnosed as TSGCT after surgery, at our hospital from 2000 to 2012, were analyzed. RESULTS: There was no statistically significant difference between the groups for gender, age, site, recurrence, symptom (p>0.05). Macrophage colony-stimulating factor (MCSF), CD68, and Ki67 expression was identified in localized and diffuse type TSGCT. But there was no statistically significant difference between the groups for MCSF, CD68, and Ki67 expression (p>0.05). CONCLUSION: This study shows that although the markers MCSF, CD68, and Ki67 are expressed by localized and diffuse type TSGCT, their lack of specificity limits their use as a subsidiary immunohistochemical marker in the differential diagnosis of localized and diffuse type TSGCTs.
Diagnosis, Differential ; Giant Cell Tumors* ; Giant Cells* ; Macrophage Colony-Stimulating Factor ; Recurrence ; Sensitivity and Specificity

BACKGROUND: T lymphocytes that bear CD3 but lack CD4 and CD8 (Double negative T cells, DN T cells) are normally present early in ontogeny in the fetal thymus, but constitute only a small proportion of adult thymocytes and peripheral blood. Since DN T TCR alpha beta+ cells have been found to accumulate in the lymphoid organs of lpr and gld mice and to be expanded in patients with autoimmune diseases, their functional properties are now of considerable interest. METHOD: Sixty four rheumatoid arthritis (RA) patients and 24 healthy volunteers were studied from Jan 1997 to Feb 1998. The whole blood from the patients and controls were analyzed by flow cytometry (Elite ESP, Coulter, USA) and XL-II software after the cells were stained with trifluorochrome monoclonal antibodies (anti-CD4-FITC/anti-CD8-PE/anti-CD3-PE-Cy5, anti-CD3-FITC/anti-CD19-PE/anti-CD45-PE-Cy5, anti-CD3-FITC/ CD16+56-PE) (Immunotech, Coulter, USA). We reviewed patient records to find out the inflammatory parameters and Ritchie index. RESULTS: We confirmed the presence of DN T cells in peripheral blood of healthy volunteers and RA patients. DN T cells were lower in RA patients (mean+/-SD; 6.44%+/-4.46), when compared to healthy volunteers (mean+/-SD; 9.97%+/-4.50) (p=0.001). There was no clinical correlations between DN T cells and inflammatory parameters. CONCLUSION: Our study showed that the DN T cells in normal control were about 10% of CD3 positive T cells and the cells were significantly lower in RA patients. Although we did not identify whether these cells have either TCR alpha beta or TCR gamma delta, we could conclude that these cells are not expanded in RA patients. We would like to continue this study further 1) to identify TCR the DN T cells have and 2) to monitor the changes after treatment.
Adult ; Animals ; Antibodies, Monoclonal ; Arthritis, Rheumatoid* ; Autoimmune Diseases ; Flow Cytometry ; Healthy Volunteers ; Humans ; Mice ; Receptors, Antigen, T-Cell, alpha-beta ; Receptors, Antigen, T-Cell, gamma-delta ; T-Lymphocytes* ; Thymocytes ; Thymus Gland

Ketamine hydrochloride is a phencyclidine derivatives and dissociative anesthetics. Ketamine induce the pulmonary vasoconetrietion in vivo. This study was designed to deter- mine the direct effect of the ketamine on the rabbit pulmonary artery in vitro. Isolated pulmonary artery was precontracted with norepinephrine (NE) 10-7M in the 20 ml organ bath. Concentration of ketamine was gradually increased 10-5M, 10 4M and 10-3M at 10 minutes intervals. I divided forty three experimental speeimens into 5 groups : pulmonary artery with and without endothelium, pretreated with indomethacin, nitrow-L-arginine methyl ester(L-NAME) and methylene blue. The results were as follows : 1. Norepinephrine precontracted pulmonary arterial tone wss significantly decreased by ketamine(10-3M), and the relaxing percent were 81.0 19.3, 60.6 55.4 (Mean S.D.) in unrubbed and rubbed endothelium, respectively (p<0.05). 2. The changes of vascular tone in denuding and intact groups were not significantly different. 3. Vasorelaxation induced by ketamine was not related with nitric oxide(NO) synthase, cyclooxygenase and soluble guanylate cyclase. Ketamine induce relaxation of the rabbit pulmonary artery, especially at 10-3 M concentra- tion. The relaxing effect was not related with endothelium presence, nitric oxide synthase, cyclooxygenase and soluble guanylate cyclase pathways. This data suggest that the relaxing effect of ketamine was not associated with endothelium, Nitric oxide, prostacyclin and cyclic guanosinemonophosphate.
Anesthetics, Dissociative ; Arteries ; Baths ; Endothelium ; Epoprostenol ; Guanylate Cyclase ; Indomethacin ; Ketamine* ; Methylene Blue ; NG-Nitroarginine Methyl Ester ; Nitric Oxide ; Nitric Oxide Synthase ; Norepinephrine ; Phencyclidine ; Prostaglandin-Endoperoxide Synthases ; Pulmonary Artery ; Relaxation ; Vasodilation