Antibacterial activity of Eriobotrya japonica leaf extracts against methicillin-resistant Staphylococcus aureus

Aim: To investigate antimethicillin-resistant Staphylococcus aureus (MRSA) activity of E. japonica leaf and to elucidate its anti-MRSA mechanism. Methodology: Methanolic extract of dried E. japonica leaf was partitioned in n-hexane, dichloromethane, ethyl acetate (EtOAc) and n-butyl alcohol. Antibacterial activity of extracts was determined by disk diffusion assay and minimum inhibitory concentration.Total RNA isolation was performed by treating MRSA culture with EtOAc sub-fractions. All MRSA isolates were tested for the presence of genes by reverse transcription polymerase chain reaction, followed by Western Blot. Results: The highest MRSA activity was observed from EtOAc sub-fraction 03 of methanolic extract. The minimum inhibitory concentration values of EF03 ranged from 32 to 64 µg ml-1 against methicillin-susceptible and -resistant S. aureus. EF03 fractions inhibited the expression of resistant genes in a dose-dependent manner. Moreover, the results of Western Blot assay indicated that the EF03 fractions inhibited the expression levels of resistant protein, PBP2a in a dose-dependent manner. Interpretation: EtOAc soluble fraction of E. japonica leaf evidenced profound antimicrobial activity, and inhibited expression of resistant genes against MRSA.

Heterotopic ossification in appendiceal mucinous neoplasms: clinicopathological characteristics of 3 cases

Heterotopic bone formation is a very rare event in the gastrointestinal tract including in the appendix. Here we report three cases of heterotopic ossification in appendiceal mucinous neoplasms, one occurring in an appendiceal mucinous cystadenoma, another in a low-grade appendiceal mucinous neoplasm, and the third occurring in an appendiceal mucinous adenocarcinoma. The clinicopathologic characteristics of these three present cases and two previously reported cases are discussed in detail. The mechanism of heterotopic ossification in appendiceal mucinous neoplasm is still unclear, but mucin extravasation and subsequent calcification may be predisposing events.

Berberine induces apoptosis via ROS generation in PANC-1 and MIA-PaCa2 pancreatic cell lines

Pancreatic cancer is the fourth leading cause of cancer death. Gemcitabine is widely used as a chemotherapeutic agent for the treatment of pancreatic cancer, but the prognosis is still poor. Berberine, an isoquinoline alkaloid extracted from a variety of natural herbs, possesses a variety of pharmacological properties including anticancer effects. In this study, we investigated the anticancer effects of berberine and compared its use with that of gemcitabine in the pancreatic cancer cell lines PANC-1 and MIA-PaCa2. Berberine inhibited cell growth in a dose-dependent manner by inducing cell cycle arrest and apoptosis. After berberine treatment, the G1 phase of PANC-1 cells increased by 10% compared to control cells, and the G1 phase of MIA-PaCa2 cells was increased by 2%. Whereas gemcitabine exerts antiproliferation effects through S-phase arrest, our results showed that berberine inhibited proliferation by inducing G1-phase arrest. Berberine-induced apoptosis of PANC-1 and MIA-PaCa2 cells increased by 7 and 2% compared to control cells, respectively. Notably, berberine had a greater apoptotic effect in PANC-1 cells than gemcitabine. Upon treatment of PANC-1 and MIA-PaCa2 with berberine at a half-maximal inhibitory concentration (IC50), apoptosis was induced by a mechanism that involved the production of reactive oxygen species (ROS) rather than caspase 3/7 activation. Our findings showed that berberine had anti-cancer effects and may be an effective drug for pancreatic cancer chemotherapy.

Detection of Iga Antibody Against Epstein-Barr Virus Nuclear Antigen 1 in Nasopharyngeal Carcinoma Patients by ELISA

Nasopharyngeal carcinoma (NPC) is a multi-factorial disease caused by genetic, viral (Epstein Barr virus, EBV) and environmental factors. The elevation of IgA antibody titers against EBV viral capsid antigen (VCA) measured by indirect immunofluorescence assay (IFA) had been use as ‘gold standard’ for NPC diagnosis for over thirty years. However, IFA is unsuitable for mass screening among population since it is time-consuming, inconvenient to perform and difficult to standardize. To date, these difficulties of IFA have been solved by using recombinant protein-based enzyme-linked immunosorbent assay (ELISA). The EBV nuclear antigen 1 (EBNA1) is the only latent EBV antigen consistently expressed in NPC tissues. Recently, it has been found that IgA antibody against EBNA1 (IgA/EBNA1) measured by ELISA may be a useful marker for NPC and the early detection of this cancer. The purpose of this study is to evaluate the usefulness of IgA/EBNA1 from a commercial kit in Thai NPC cases. The concentration of serum IgA/EBNA1 was measured in 54 NPC patients and 122 age match healthy controls by using Sinoclone EBV IgA ELISA kit. The normal cut off value (mean+2SD) of serum IgA/EBNA1 showed a relative optical density (rOD) at 1.26 units. Serum IgA/EBNA1 level was positive in 52 (96.30%) out of 54 NPC patients and in 5 (4.10%) out of 122 healthy controls. NPC cases showed significantly higher serum IgA/EBNA1 level than healthy controls (P \< 0.001). In NPC patients, the serum IGA/EBNA1 level was increased with aggressiveness and advance stages of the disease. Detection of IgA/EBNA1 by Sinoclone EBV IgA ELISA kit in serum had a sensitivity, a specificity, positive predictive values and negative predictive values of 96.30, 95.90, 91.23 and 98.32%, respectively, for the diagnosis of NPC. The results of our study suggest that serum IgA/EBNA1 may be a suitable marker for diagnosis and prognosis of NPC in Thailand and that this test may be a useful addition to the panel of tests used for this purpose. Further studies are currently underway to evaluate the effectiveness of this marker as an early detection tool for NPC in Thailand.

Exploratory calcineurin inhibitor-free regimens in living-related kidney transplant recipients

Chronic allograft nephropathy is among the major causes of graft loss even in low-risk kidney transplant recipients and correlates with acute nephrotoxic events during the first year post-transplant. Therefore, calcineurin inhibitor-free regimens may improve patient and graft survival among recipients of living-related kidney transplants. To confirm this hypothesis, we evaluated the efficacy and safety of two calcineurin inhibitor-free regimens in 92 low-risk recipients of one-haplotype living-related kidney transplants. Immunosuppression consisted of tacrolimus, azathioprine and prednisone (group I, GI, N = 38), 2 doses of daclizumab, mycophenolate mofetil (MMF), and prednisone (GII, N = 33) and 2 doses of daclizumab, MMF, sirolimus and prednisone (GIII, N = 21). At 12 months, treatment failure (biopsy-confirmed acute rejection, graft loss or death) was higher in GII compared to GIII and GI (54.5 vs 24.0 vs 13.1 percent, P < 0.01, respectively). In patients of black ethnicity the incidence of acute rejection was 25 vs 83.3 vs 20 percent (P = 0.055), respectively. Patient and graft survival was comparable. There were no differences in mean creatinine or calculated creatinine clearance at 12 months. Overall incidence of post-transplant diabetes mellitus (3.3 percent) and cytomegalovirus disease (4.3 percent) was similar in all groups. Further development of effective calcineurin inhibitor-free regimens should exclude patients of black ethnicity and may need full-induction therapy, perhaps with depleting agents, and concentration-controlled use of sirolimus and MMF.

Impact of initial exposure to calcineurin inhibitors on kidney graft function of patients at high risk to develop delayed graft function

We conducted a retrospective analysis of the influence of full doses of calcineurin inhibitors [8-10 mg kg-1 day-1 cyclosporine (N = 80), or 0.2-0.3 mg kg-1 day-1 tacrolimus (N = 68)] administered from day 1 after transplantation on the transplant outcomes of a high-risk population. Induction therapy was used in 13 percent of the patients. Patients also received azathioprine (2 mg kg-1 day-1, N = 58) or mycophenolate mofetil (2 g/day, N = 90), and prednisone (0.5 mg kg-1 day-1, N = 148). Mean time on dialysis was 79 ± 41 months, 12 percent of the cases were re-transplants, and 21 percent had panel reactive antibodies >10 percent. In 43 percent of donors the cause of death was cerebrovascular disease and 27 percent showed creatinine above 1.5 mg/dL. The incidence of slow graft function (SGF) and delayed graft function (DGF) was 15 and 60 percent, respectively. Mean time to last dialysis and to nadir creatinine were 18 ± 15 and 34 ± 20 days, respectively. Mean creatinine at 1 year after transplantation was 1.48 ± 0.50 mg/dL (DGF 1.68 ± 0.65 vs SGF 1.67 ± 0.66 vs immediate graft function (IGF) 1.41 ± 0.40 mg/dL, P = 0.089). The incidence of biopsy-confirmed acute rejection was 22 percent (DGF 31 percent, SGF 10 percent, IGF 8 percent). One-year patient and graft survival was 92.6 and 78.4 percent, respectively. The incidence of cytomegalovirus disease, post-transplant diabetes mellitus and malignancies was 28, 8.1, and 0 percent, respectively. Compared to previous studies, the use of initial full doses of calcineurin inhibitors without antibody induction in patients with SGF or DGF had no negative impact on patient and graft survival.

Relationship of cyclosporin and sirolimus blood concentrations regarding the incidence and severity of hyperlipidemia after kidney transplantation

The influence of drug concentrations on the development of persistent posttransplant hyperlipidemia was investigated in 82 patients who received cyclosporin A (CsA) and prednisone plus sirolimus (SRL) (52) or azathioprine (AZA) (30) during the first year after transplantation. Blood levels of CsA and SRL, daily doses of AZA and prednisone, and cholesterol, triglyceride, and glucose concentrations were determined during each visit (pretransplant and 30, 60, 90, 120, 180, and 360 days posttransplant). Persistent hyperlipidemia was defined as one-year average steady-state cholesterol (CavCHOL) or triglyceride (CavTG) concentrations above 240 and 200 mg/dL, respectively. Mean cholesterol and triglyceride concentrations increased after transplantation (P < 0.01) and were higher in patients receiving SRL compared to AZA (P < 0.001). Patients receiving SRL showed a significantly higher number of cholesterol (>229 or >274 mg/dL) and triglyceride (>198 or >282 mg/dL) determinations in the upper interquartile ranges. CsA and SRL interquartile ranges correlated with cholesterol concentrations (P = 0.001) whereas only SRL interquartile ranges correlated with triglyceride concentrations (P < 0.0001). Only pretransplant cholesterol concentration >205 mg/dL was independently associated with development of persistent hypercholesterolemia (CavCHOL >240 mg/dL, relative risk (RR) = 20, CI 3.8-104.6, P = 0.0004) whereas pretransplant triglyceride concentration >150 mg/dL (RR = 7.2, CI 1.6-32.4, P = 0.01) or >211 mg/dL (RR = 19.8, CI 3.6-107.9, P = 0.0006) and use of SRL (RR = 3, CI 1.0-8.8, P = 0.0049) were independently associated with development of persistent hypertriglyceridemia (CavTG >200 mg/dL). Persistent hypercholesterolemia was more frequent among patients with higher pretransplant cholesterol concentrations and was dependent on both CsA and SRL concentrations. Persistent hypertriglyceridemia was more frequent among patients with higher pretransplant triglyceride concentrations and was dependent on SRL concentrations.

Standardization of an antihemorrhagic potency test of antivenoms prepared from two different Agkistrodon halys venoms

To establish Korea National Standards for venoms and antivenoms, it is necessary to have standardized assay methods. In this study, we standardized a method to evaluate the antihemorrhagic potency of two horse-derived antivenoms using rabbit intracutaneous injection. We expressed the capability of these antivenoms to neutralize the hemorrhagic activities triggered by the venoms of Agkistrodon halys from Japan and Jiangzhe Agkistrodon halys from China as Minimum Hemorrhagic Dose (MHD). We also performed cross-neutralization tests employing the parallel line assay on different pairings of venoms and antivenoms to check the possibility of using Jiangzhe Agkistrodon halys venom as a substitute for the standard Agkistrodon halys venom in measurements of the antihemorrhagic activity, since A. halys venom is not easily available. Slope function ratio (S.R.) was 0.957 for Agkistrodon halys venom either with Agkistrodon halys antivenom or with Jiangzhe Agkistrodon halys antivenom (p>0.05). Similarly, S.R. was 0.348 for Jiangzhe Agkistrodon halys venom either with Agkistrodon halys antivenom or with Jiangzhe Agkistrodon halys antivenom (p>0.05). Thus, in this study we established antihemorrhagic potency test methods for both Agkistrodon halys and Jiangzhe Agkistrodon halys antivenoms and we could also show it is possible to use Jiangzhe Agkistrodon halys venom as a standard.

Standardization of anti-lethal toxin potency test of antivenoms prepared from two different Agkistrodon halys venoms

In Korea, antivenoms for the treatment of patients bitten by venomous snakes have been imported from Japan or China. Although there is cross-reactivity between these antibodies and venoms from snakes indigenous to Korea (e.g. Agkistrodon genus), protection is not optimal. Antivenoms specifically prepared to neutralize Korean snake venoms could be more effective, with fewer side effects. To this end, we established an infrastructure to develop national standards and created a standardized method to evaluate the efficacy of two horse-derived antivenoms using mouse lethal toxin test. Additionally, we determined the antivenoms neutralizing activity against lethal doses (LD50) of Agkistrodon halys (from Japan) and Jiangzhe Agkistrodon halys (from China) venoms. We also performed cross-neutralization tests using probit analysis on each pairing of venom and antivenom in order to check the possibility of using Jiangzhe A. halys venom as a substitute for A. halys venom, the current standard. Slope of A. halys venom with A. halys antivenom was 10.2 and that of A. halys venom with Jiangzhe A. halys antivenom was 9.6. However, Slope of Jiangzhe A. halys venom with A. halys antivenom was 4.7 while that of Jiangzhe A. halys venom with Jiangzhe A. halys antivenom was 11.5. Therefore, the significant difference in slope patterns suggests that Jiangzhe A. halys venom cannot be used as a substitute for the standard venom to test the anti-lethal toxin activity of antivenoms (p<0.05).(AU)

Pharmacokinetic/pharmacodynamic relationships of FTY720 in kidney transplant recipients

FTY720 is a new and effective immunosuppressive agent, which produces peripheral blood lymphopenia through a lymphocyte homing effect. We investigated the relationship between the dose of FTY720 or blood concentration (pharmacokinetics, PK) and peripheral lymphopenia (pharmacodynamics, PD) in 23 kidney transplant recipients randomized to receive FTY720 (0.25-2.5 mg/day) or mofetil mycophenolate (2 mg/day) in combination with cyclosporine and steroids. FTY720 dose, blood concentrations and lymphocyte counts were determined weekly before and 4 to 12 weeks after transplantation. The effect of PD was calculated as the absolute lymphocyte count or its reductions. PK/PD modeling was used to find the best-fit model. Mean FTY720 concentrations were 0.36 ± 0.05 (0.25 mg), 0.73 ± 0.12 (0.5 mg), 3.26 ± 0.51 (1 mg), and 7.15 ± 1.41 ng/ml (2.5 mg) between 4 and 12 weeks after transplantation. FTY720 PK was linear with dose (r² = 0.98) and showed low inter- and intra-individual variability. FTY720 produced a dose-dependent increase in mean percent reduction of peripheral lymphocyte counts (38 vs 42 vs 56 vs 77, P < 0.01, respectively). The simple Emax model [E = (Emax * C)/(C + EC50)] was the best-fit PK/PD modeling for FTY720 dose (Emax = 87.8 ± 5.3 percent and ED50 = 0.48 ± 0.08 mg, r² = 0.94) or concentration (Emax = 78.3 ± 2.9 percent and EC50 = 0.59 ± 0.09 ng/ml, r² = 0.89) vs effect ( percent reduction in peripheral lymphocytes). FTY720 PK/PD is dose dependent and follows an Emax model (EC50 = 0.5 mg or 0.6 ng/ml). Using lymphopenia as an FTY720 PD surrogate marker, high percent reductions ( about 80 percent) in peripheral lymphocytes are required to achieve best efficacy to prevent acute allograft rejection.

Preservation of graft function in low-risk living kidney transplant recipients treated with a combination of sirolimus and cyclosporine

The use of sirolimus (SRL) in combination with full doses of cyclosporin A (CsA) results in reduced one-year kidney allograft function, which is associated with shorter long-term allograft survival. We determined the effect of reduced CsA exposure on graft function in patients receiving SRL and prednisone. Ninety recipients of living kidney transplants receiving SRL (2 mg/day, po) were compared to 35 recipients receiving azathioprine (AZA, 2 mg kg-1 day-1, po). All patients also received CsA (8-10 mg kg-1 day-1, po) and prednisone (0.5 mg kg-1 day-1). Efficacy end-point was a composite of biopsy-confirmed acute rejection, graft loss, or death at one year. Graft function was measured by creatinine, creatinine clearance, and graft function deterioration between 3 and 12 months (delta1/Cr). CsA concentrations in patients receiving SRL were 26 percent lower. No differences in one-year composite efficacy end-point were observed comparing SRL and AZA groups (18 vs 20 percent) or in the incidence of biopsy-proven acute rejection (14.4 and 14.3 percent). There were no differences in mean ± SD creatinine (1.65 ± 0.46 vs 1.60 ± 0.43 mg/dl, P = 0.48) or calculated creatinine clearances (61 ± 15 vs 62 ± 13 ml/min, P = 0.58) at one year. Mean ± SD delta1/Cr (-11 ± 17 vs -14 ± 15 percent, P = 0.7) or the percentage of patients with >20 percent (26 vs 31 percent, P = 0.6) or >30 percent delta1/Cr (19 vs 17 percent, P = 1) did not differ between the two groups. The use of 2-mg fixed oral doses of SRL and reduced CsA exposure was effective in preventing acute rejection and preserving allograft function.