Innate immune response of bovine mammary epithelial cells in Mycoplasma bovis mastitis using an in vitro model of bovine mammary gland infection.

Mycoplasma bovis mastitisis highly contagious and disrupts lactation, posing a significant threat to the dairy industry. While the mammary gland's defence mechanism involves epithelial cells and mononuclear cells (MNC), their interaction with M. bovis remains incompletely understood. In this study, we assessed the immunological reactivity of bovine mammary epithelial cells (bMEC) to M. bovis through co-culture with MNC. Upon co-culture with MNC, the mRNA expression levels of interleukin (IL)-1ß,IL-6, IL-8 and tumor necrosis factor (TNF)-α in bMEC stimulated by M. bovis showed a significant increase compared to monoculture. Additionally, when stimulated with M. bovis, the culture supernatant exhibited significantly higher concentrations of IL-6 and interferon (IFN)-γ, while IL-1ß concentration tended to be higher in co-culture with MNC than in monoculture. Furthermore, the mRNA expression levels of toll-like receptor (TLR) 2 in bMEC stimulated with M. bovis tended to increase, and TLR4 significantly increased when co-cultured with MNC compared to monocultures. However, the surface expression levels in bMEC did not exhibit significant changes between co-culture and monoculture. Overall, our research indicates that the inflammatory response of bMEC is increased during co-culture with MNC, suggesting that the interaction between bMEC and MNC in the mammary gland amplifies the immune response to M. bovis in cows affected by M. bovis mastitis.

Possible Involvement of Protein Binding Inhibition in Changes in Dexmedetomidine Concentration in Extracorporeal Circuits during Midazolam Use.

It was recently reported that the dexmedetomidine concentration within the extracorporeal circuit decreases with co-administration of midazolam. In this study, we investigated whether displacement of dexmedetomidine by midazolam from the binding site of major plasma proteins, human serum albumin (HSA) and α1-acid glycoprotein (AAG), would increase levels of free dexmedetomidine that could be adsorbed to the circuit. Equilibrium dialysis experiments indicated that dexmedetomidine binds to a single site on both HSA and AAG with four times greater affinity than midazolam. Midazolam-mediated inhibition of the binding of dexmedetomidine to HSA and AAG was also examined. The binding of dexmedetomidine to these proteins decreased in the presence of midazolam. Competitive binding experiments suggested that the inhibition of binding by midazolam was due to competitive displacement at site II of HSA and due to non-competitive displacement at the site of AAG. Thus, our present data indicate that free dexmedetomidine displaced by midazolam from site II of HSA or from AAG is adsorbed onto extracorporeal circuits, resulting in a change in the dexmedetomidine concentration within the circuit.

Preparation and in Vitro Characterization of Fatty-Acid Modified Pirarubicin Nanosuspensions Stabilized by Albumin.

Pirarubicin (THP) shows more rapid intracellular uptake, more effective antitumor activity, and less cardiac toxicity, compared to doxorubicin. However, THP is distributed to both tumor and normal tissues indiscriminately. This study aimed to develop a nanosuspension to deliver THP to tumor tissues more efficiently. Fatty-acid-modified THPs (FA-THPs; octanoic acid, dodecanoic acid, palmitic acid-THPs) were synthesized to increase the hydrophobicity of THP. Nanosuspensions of these FA-THPs were then prepared using an antisolvent precipitation technique. Among the FA-THPs, the most efficiently drug-loaded nanosuspension was obtained from palmitic acid-THP (pal-THP) using an aqueous antisolvent containing bovine serum albumin as a stabilizer. The pal-THP nanoparticles in the nanosuspension were confirmed to be of optimal size (100-125 nm) for delivery to tumor tissues using dynamic light scattering and transmission electron microscopy. The pal-THP nanosuspension showed cytotoxicity in colon 26 cells. The nanosuspension was shown to disintegrate in the presence of surfactants such as lecithin, liberating pal-THP, which was converted to free THP in acidic media. It is therefore proposed that pal-THP nanoparticles that reach tumor cells after intravenous administration would exert antitumor effect by liberating pal-THP (i.e., disintegration of nanoparticles by the interaction with cell membrane), followed by the release of free THP in the acidic milieu of tumor cells. These findings indicate that FA-THP nanosuspensions, particularly pal-THP nanosuspension, hold promise as a candidate for cancer treatment. However, further in vivo studies are necessary.

Development of α-Cyclodextrin-Based Orally Disintegrating Tablets for 4-Phenylbutyrate.

Despite major improvements brought about by the introduction of taste-masked formulations of 4-phenylbutyrate (PB), poor compliance remains a significant drawback to treatment for some pediatric and dysphagic patients with urea cycle disorders (UCDs). This study reports on the development of a cyclodextrin (CD)-based orally disintegrating tablet (ODT) formulation for PB as an alternative to existing formulations. This is based on previous reports of the PB taste-masking potential of CDs and the suitability of ODTs for improving compliance in pediatric and dysphagic populations. In preliminary studies, the interactions of PB with α and ßCD in the solid state were characterized using X-ray diffraction, scanning electron microscopy, dissolution, and accelerated stability studies. Based on these studies, lyophilized PB-CD solid systems were formulated into ODTs after wet granulation. Evaluation of the ODTs showed that they had adequate physical characteristics, including hardness and friability and good storage stability. Notably, the developed αCD-based ODT for PB had a disintegration time of 28 s and achieved a slightly acidic and agreeable pH (≈5.5) in solution, which is suitable for effective PB-CD complexation and taste masking. The developed formulation could be helpful as an alternative to existing PB formulations, especially for pediatric and dysphagic UCD patients.

Study of the Structural Chemistry of the Inclusion Complexation of 4-Phenylbutyrate and Related Compounds with Cyclodextrins in Solution: Differences in Inclusion Mode with Cavity Size Dependency.

4-phenylbutyrate (PB) and structurally related compounds hold promise for treating many diseases, including cancers. However, pharmaceutical limitations, such as an unpleasant taste or poor aqueous solubility, impede their evaluation and clinical use. This study explores cyclodextrin (CD) complexation as a strategy to address these limitations. The structural chemistry of the CD complexes of these compounds was analyzed using phase solubility, nuclear magnetic resonance (NMR) spectroscopic techniques, and molecular modeling to inform the choice of CD for such application. The study revealed that PB and its shorter-chain derivative form 1:1 αCD complexes, while the longer-chain derivatives form 1:2 (guest:host) complexes. αCD includes the alkyl chain of the shorter-chain compounds, depositing the phenyl ring around its secondary rim, whereas two αCD molecules sandwich the phenyl ring in a secondary-to-secondary rim orientation for the longer-chain derivatives. ßCD includes each compound to form 1:1 complexes, with their alkyl chains bent to varying degrees within the CD cavity. γCD includes two molecules of each compound to form 2:1 complexes, with both parallel and antiparallel orientations plausible. The study found that αCD is more suitable for overcoming the pharmaceutical drawbacks of PB and its shorter-chain derivative, while ßCD is better for the longer-chain derivatives.

Encapsulation of an Antioxidant in Redox-Sensitive Self-Assembled Albumin Nanoparticles for the Treatment of Hepatitis.

Hepatitis is an inflammation of the liver caused by the inadequate elimination of reactive oxygen species (ROS) derived from Kupffer cells. Edaravone is clinically used as an antioxidant but shows poor liver distribution. Herein, we report on the design of a Kupffer cell-oriented nanoantioxidant based on a disulfide cross-linked albumin nanoparticle containing encapsulated edaravone (EeNA) as a therapeutic for the treatment of hepatitis. Since the edaravone is bound to albumin, this results in a soluble and stable form of edaravone in water. Exchanging the intramolecular disulfide bonds to intermolecular disulfide bridges of albumin molecules allowed the preparation of a redox responsive albumin nanoparticle that is stable in the blood circulation but can release drugs into cells. Consequently, EeNA was fabricated by the nanoscale self-assembly of edaravone and albumin nanoparticles without the additives that are contained in commercially available edaravone preparations. EeNA retained its nanostructure under serum conditions, but the encapsulated edaravone was released efficiently under intracellular reducing conditions in macrophages. The EeNA was largely distributed in the liver and subsequently internalized into Kupffer cells within 60 min after injection in a concanavalin-A-induced hepatitis mouse. The survival rate of the hepatitis mice was significantly improved by EeNA due to the suppression of liver necrosis and oxidative stress by scavenging excessive ROS. Moreover, even through the postadministration, EeNA showed an excellent hepatoprotective action as well. In conclusion, EeNA has the potential for use as a nanotherapeutic against various types of hepatitis because of its Kupffer cell targeting ability and redox characteristics.

Characteristics of Mycoplasma bovis, Mycoplasma arginini, and Mycoplasma californicum on immunological response of bovine synovial cells.

Mycoplasma arthritis in calves caused by M. bovis exhibits joint swelling, lameness, and immobility. In contrast to M. bovis, M. arginini, and M. californicum which were similarly isolated from the affected joints, only induced mild inflammation. The changes in pathogenesis that depended on species, however, remained unknown. This investigation aims to examine the characteristics of immune responses to M. bovis, M. arginini, and M. californicum in synovial cells. Intracellular M. bovis was detected by gentamicin assay, but M. arginini and M. californicum were not detected. M. bovis-infected synovial cells were encouraged to proliferate and had their apoptosis suppressed. We suggest that M. bovis invaded and inhibited apoptosis of synovial to evade host immunity, which led to long term survival in joints. M. bovis infection significantly increased IL-6 mRNA expression compared to control, although M. arginini and M. californicum infection were comparable to control. We suggest that M. arginini and M. californicum have low abilities to induce inflammation in joints and therefore do not cause severe pathology. Our findings are the first to show the variations in synovial cell immune responses to M. bovis, M. arginini, and M. californicum, which are thought to be related to the pathogenicity of arthritis.

[Elucidation of Drug Transport Mechanism by Serum Protein and Development for Pancreatic Cancer Treatment].

Human serum albumin (HSA) and α1-acid glycoprotein (AGP) are the major drug-binding proteins in the blood and regulate the tissue transfer of bound drugs. We succeeded in clarifying the three-dimensional structure of AGP for the first time in the world from X-ray crystal structure analysis. Using a site-directed mutagenesis method by constructing yeast expression systems as well as the three-dimensional structure, we elucidated the properties of drug binding sites of AGP. We also found that structural change due to the interaction between AGP and cell membranes causes the release of bound drugs and reported an "AGP-mediated drug transport process." Pancreatic cancer has an extremely low response rate to anticancer drugs compared to other cancers and is resistant to starvation of nutrients including fatty acids. We clarified that glutamine metabolism is involved in this tolerance. Furthermore, aiming at efficient drug delivery and effective treatment for pancreatic cancer, we focused on nitric oxide (NO) which increases pancreatic blood flow and has a cell-killing effect on tumors and surrounding stromal tissues. We successfully synthesized nitrated phenylbutyrate (NPB), which binds to HSA and has an antitumor effect in vitro and vivo. The binding of NPB to HSA is considered to be useful for delivery to tumors through the enhanced permeability and retention (EPR) effect and HSA receptors.

Structural Basis of the Change in the Interaction Between Mycophenolic Acid and Subdomain IIA of Human Serum Albumin During Renal Failure.

Mycophenolic acid (MP) is an active metabolite of mycophenolate mofetil, a widely used immunosuppressive drug. MP normally exhibits high plasma protein binding (97-99%), but its binding rate is decreased in patients with renal insufficiency. This decreased protein binding is thought to be associated with leukopenia, a side effect of MP. In this study, we characterized the change in protein binding of MP in renal failure patients. Our findings indicate that MP binds strongly to subdomain IIA of human serum albumin. X-ray crystallographic data indicated that the isobenzofuran group of MP forms a stacking interaction with Trp214, and the carboxyl group of MP is located at a position that allows the formation of hydrogen bonds with Tyr150, His242, or Arg257. Due to the specific binding of MP to subdomain IIA, MP is thought to be displaced by uremic toxin (3-carboxy-4-methyl-5-propyl-2-furan-propionic acid) and fatty acids (oleate or myristate) that can bind to subdomain IIA, resulting in the decreased plasma protein binding of MP in renal failure.

Study of the inclusion complexes formed between 4-phenylbutyrate and α-, ß- and γ-cyclodextrin in solution and evaluation on their taste-masking properties.

OBJECTIVES: 4-Phenylbutyrate (PB), which is used in the management of urea cycle disorders, has an unpleasant taste leading to poor patient compliance. Existing PB formulations though helpful, have some limitations in their use. This study reports on attempts to mask this unpleasant taste by complexing PB with cyclodextrins (CDs) to improve patient compliance. METHODS: α, ß and γCD were used as CDs. Phase solubility studies, circular dichroism, 1H-NMR spectroscopy, including ROESY, and molecular modelling were used to investigate and characterize the PB-CD interactions in solution. The taste-masking effect of the CDs was evaluated using in vitro taste sensor measurements. KEY FINDINGS: PB interacts with α, ß and γCD in solution to form 1:1, 1:1 and 1:2 CD: PB inclusion complexes, respectively, with stability constants in the order αCD > ßCD > γCD. Taste evaluation revealed that the CDs significantly mask the taste of PB through the formation of the inclusion complexes. Notably, αCD masked the bitter taste of PB to 30% of the initial taste at a 1:1 molar ratio. CONCLUSION: αCD significantly masks the unpleasant taste of PB in solution and can be used to formulate PB to address the limitations of existing formulations and improve patient compliance and quality of life.

Chlorine Atoms of an Aripiprazole Molecule Control the Geometry and Motion of Aripiprazole and Deschloro-aripiprazole in Subdomain IIIA of Human Serum Albumin.

Aripiprazole (ARP), an antipsychotic drug, binds more strongly to human serum albumin (HSA) than the other ARP derivatives. In addition, the signs for the extrinsic Cotton effects for HSA complexed with ARP or deschloro-ARP are reversed. In this study, we report on a structural-chemical approach using circular dichroism (CD) spectroscopic analysis, X-ray crystallographic analysis, and molecular dynamics simulations. The objective was to examine the relationship between the induced CD spectra and the structural features of the HSA complexes with ARP or deschloro-ARP. The intensity of the induced CD spectra of the HSA complexes with ARP or deschloro-ARP was reduced with increasing temperature. We determined the crystal structure of the HSA complexed with deschloro-ARP in this study and compared it to HSA complexed with ARP that we reported previously. The comparison of these structures revealed that both ARP and deschloro-ARP were bound at the site II pocket in HSA and that the orientation of the molecules was nearly identical. Molecular dynamics simulations indicated that the molecular motions of ARP and deschloro-ARP within the site II pocket were different from one another and the proportion of stacking interaction formations of Tyr411 with the dihydroquinoline rings of ARP and deschloro-ARP was also different. These findings indicate that the induced CD spectra are related to the molecular motions and dynamic interactions of ARP and deschloro-ARP in HSA and may help to understand the molecular recognition and motion that occurs within the binding site for the other HSA ligands more clearly.

Novel nitric oxide donor, nitrated phenylbutyrate, induces cell death of human pancreatic cancer cells and suppresses tumor growth of cancer xenografts.

Pancreatic cancer has a low response rate to chemotherapy due to the low drug transferability caused by the low blood flow around the tumor. In the present study, focusing on nitric oxide (NO) for its vasodilatory and antitumor effects, a novel NO donor, a nitrated form of phenylbutyrate (NPB) was synthesized and the antitumor effect on human pancreatic cancer cells (AsPC1 and BxPC3) and xenografts was examined. Using Annexin V, NPB was confirmed to induce cell death against AsPC1 and BxPC3 in a time­ and concentration­dependent manner. In NPB­exposed cells, DAF­FM DA (a probe to detect intracellular NO) derived fluorescence was observed. Release of nitrite and nitrate from NPB in aqueous solution was very gradual until even 72 h after dissolution. Phenylbutyrate (PB) and hydroxy PB in which the nitro group of NPB was replaced with a hydroxyl group did not have the cell death­inducing effect as observed in NPB. These results suggest that the effect of NPB was dependent on NO release form NPB. Apoptosis inhibitor, Z­VAD FMK, had no effect on the cell death­inducing effect of NPB, and NPB did not show significant activation of caspase­3/7. In addition, NPB significantly decreased cellular ATP levels, suggesting that necrosis is involved in the effect of NPB. NPB also accumulated cells specifically at the S phase of the cell cycle. A single dose of NPB (10 mg/kg) into mice with established BxPC3 xenografts significantly suppressed tumor growth for at least 7 weeks without apparent toxicity. The findings of the present study indicate that NPB has potential as a novel therapeutic agent for NO­based therapy of pancreatic cancer.

Effect of Fatty Acids and Uremic Toxins on the Binding of Nateglinide, an Insulin Secretagogue, to Site II on Human Serum Albumin.

Nateglinide (NAT) is used to treat diabetes, stimulating pancreatic islet ß-cells with residual insulin secretory capacity to increase insulin secretion. NAT has been reported to bind to human serum albumin (HSA), but the detail is still unclear. In the current study, we investigated the location and the affinity for the binding of NAT to HSA. Quantitative analysis data from the ultrafiltration experiment indicated that NAT binds strongly to a primary site on HSA with a high affinity. The presence of diazepam (DZP) or ibuprofen (IB), the specific site II ligands of HSA, decreased the binding constants of NAT respectively, without the significant changes in the number of binding sites. Whereas warfarin (WF), a site I specific ligand, did not affect the binding of NAT. Fluorescent replacement experiment showed that NAT replaced dansylsarcosine (DNSS), a site II probe of HSA, but not WF. An increasing level of myristate and uremic toxins, indoxyl sulphate (IS), indoxyl acetate (IA) and p-cresyl sulphate (PCS), during renal disease significantly increased the concentration of unbound NAT. These findings suggest that NAT specifically binds to site II of HSA and the binding capacity and pharmacokinetics of NAT change in renal diseases.

Effects of Myristate on the Induced Circular Dichroism Spectra of Aripiprazole Bound to Human Serum Albumin: A Structural-Chemical Investigation.

The effects of myristate on the induced circular dichroism spectra of aripiprazole (ARP) bound to human serum albumin (HSA) were investigated. High concentrations of myristate reversed the Cotton effects induced in the ARP-HSA system. The observed ellipticities increased with increasing drug concentration up to an ARP-to-HSA molar ratio of 1:1 and then decreased, indicating that the extrinsic Cotton effects were generated by the binding of ARP molecules to the high- and low-affinity sites in HSA. The data for the concentration of free ARP show that myristate displaces ARP molecules from HSA. Moreover, the free fractions of ARP in the ARP-HSA-myristate system increased significantly when adding fusidic acid, a subdomain IB ligand. In the crystal structure of the ARP-HSA-myristate ternary complex, one ARP molecule is bound to subdomain IB, and the interaction between the carbonyl group of ARP and the aromatic ring of Tyr138 in subdomain IB is essential for binding to occur. Meanwhile, the ARP molecule in the ARP-HSA binary complex structure is bound only to subdomain IIIA. Consequently, the inversion in the extrinsic Cotton effects in the ARP-HSA system can be attributed to the modification of the geometry within the binding pocket, in addition to the transfer of ARP from subdomain IIIA to subdomain IB through the displacement as a result of the binding of myristate to subdomain IIIA.

The Nitrated Form of Nateglinide Induces Apoptosis in Human Pancreatic Cancer Cells Through a Caspase-dependent Mechanism.

BACKGROUND/AIM: Nitric oxide (NO) has antitumor activity against various solid tumor cell types in addition to its vasodilatory effect. In the current study, we investigated the effect and mechanism of the synthetic nitrated form (NO2-NAT) of nateglinide, a hypoglycemic agent, on the induction of cell death in human pancreatic cancer cells. MATERIALS AND METHODS: NO production was evaluated by measuring nitrite (NO2) and nitrate (NO3) (NOx). Apoptotic cell numbers were determined using annexin V. RESULTS: NO2-NAT released nitrate and nitrite ions immediately upon dissolving in aqueous solution. NO2-NAT caused significant extracellular leakage of lactate dehydrogenase (LDH) in the human pancreatic cancer cell lines AsPC1 and BxPC3, and increased annexin-positive cells in a time- and concentration-dependent manner. NO2-NAT also significantly increased the activity of caspases 3 and 7. Exposure to Z-VAD-FMK, a caspase inhibitor, significantly suppressed NO2-NAT-induced cell death. CONCLUSION: These results indicated that NO2-NAT induces apoptosis in human pancreatic cancer cells and may serve as a new NO-based chemotherapeutic agent for the treatment of pancreatic cancer.

In Vitro and In Vivo Assessment of Atemoya Fruit (Annona atemoya) for Food-Drug Interactions.

BACKGROUND AND OBJECTIVES: Atemoya (Annona atemoya) is increasingly being consumed worldwide because of its pleasant taste. However, only limited information is available concerning possible atemoya-drug interactions. In the present study, the issue of whether atemoya shows food-drug interactions with substrate drugs of the major drug-metabolizing cytochrome P450s (i.e., CYP1A2, CYP2C9, and CYP3A) is addressed. METHODS: The ability of atemoya juice to inhibit the activities of phenacetin O-deethylase (CYP1A2), diclofenac 4'-hydroxylase (CYP2C9), and midazolam 1'-hydroxylase (CYP3A) was examined in vitro using human and rat liver microsomes. The in vivo pharmacokinetics of phenacetin and metabolites derived from it in rats when atemoya juice or fluvoxamine (a CYP1A2 inhibitor) was preadministered were also investigated. RESULTS: Atemoya juice significantly inhibited CYP1A2 activity in human liver microsomes, but not the activities of CYP2C9 and CYP3A. In spite of this inhibition, preadministration of atemoya had no effect on the pharmacokinetics of phenacetin, a CYP1A2 substrate, in rats. Meanwhile, preadministration of fluvoxamine significantly extended the time needed for the elimination of phenacetin, possibly due to the inhibition of CYP1A2. This suggests that the intake of an excess amount of atemoya juice is necessary to cause a change in the pharmacokinetics of phenacetin when the IC50 values for CYP1A2 inhibition by atemoya and fluvoxamine are taken into account. CONCLUSION: The results indicate that a daily intake of atemoya would not change the pharmacokinetics of CYP1A2 substrates such as phenacetin as well as CYP2C9- and CYP3A-substrate drugs.

Effects of intra-articular inoculation with Mycoplasma bovis on immunological responses in calf joints.

Mycoplasma arthritis that caused by Mycoplasma bovis exhibit severe lameness. This disease is difficult to cure with antibiotics, but the detailed pathological mechanisms have not been fully clarified. In this study, we examined the effects of intra-articular inoculation with M. bovis on immunological responses in calf joints. We inoculated three calves each with M. bovis or phosphate buffer saline (control) into the right stifle joint and dissected them at 15 days postinoculation. Mycoplasma bovis-inoculated calves exhibited swelling of the stifle joint, increases in synovial fluid, fibrin deposition, and cartilage thinning. Intracellular M. bovis was detected in synovial tissues analyzed by immunohistochemistry and transmission electron microscopy. Messenger RNA expressions of interleukin (IL)-1ß, IL-6, IL-8, IL-12p40, and IL-17A in synovial fluid cells and synovial tissues from M. bovis-inoculated calves were significantly higher than those from control calves. Protein levels of these cytokines in synovial fluid from M. bovis-inoculated calves were markedly higher than those from control calves. Our study clarified that inoculation with M. bovis into the stifle joint induced the production of inflammatory cytokines by synovial fluid cells and synovial tissues, causing a severe inflammatory response in joints. Additionally, M. bovis could invade cells in synovial tissues, which may have aided it in evading antibiotics and host immune surveillance.

An in-vitro comparative study of the binding of caspofungin and micafungin to plasma proteins.

OBJECTIVES: Echinocandins are widely used for the treatment of invasive fungal diseases. While they bind strongly to plasma proteins, our knowledge of this process is not sufficient to permit their pharmacokinetics and pharmacodynamics targets to be discussed. In this study, we characterized the binding of two echinocandins, caspofungin and micafungin, to plasma proteins, human serum albumin (HSA) and human α 1-acid glycoprotein (AAG). METHODS: The binding parameters, number of binding sites (n) and association constant (K) for caspofungin and micafungin to HSA and AAG were determined by equilibrium dialysis. The binding site on HSA for these echinocandins was identified by conducting inhibition experiments. KEY FINDINGS: Caspofungin was found to bind strongly to a single site on HSA (n = 1.26, K = 0.45 × 106 M-1) and AAG (n = 0.99, K = 0.29 × 106 M-1). Micafungin was found to bind more strongly to HSA (n = 1.35, K = 1.44 × 106 M-1) and AAG (n = 1.32, K = 1.16 × 106 M-1). The binding site for these drugs on HSA appears to be within subdomain IA. CONCLUSIONS: Free fraction of caspofungin and micafungin in patients may not be substantially affected due to the contribution of AAG to the overall protein binding and the binding to subdomain IA on HSA, which is different from the major drug-binding sites within subdomains IB, IIA and IIIA.

The Binding of Aripiprazole to Plasma Proteins in Chronic Renal Failure Patients.

The binding of drugs to plasma protein is frequently altered in certain types of renal diseases. We recently reported on the effects of oxidation and uremic toxins on the binding of aripiprazole (ARP) to human serum albumin. In our continuing investigations, we examined the binding of ARP to plasma pooled from patients with chronic renal dysfunction. We examined the issue of the molecular basis for which factors affect the changes in drug binding that accompany renal failure. The study was based on the statistical relationships between ARP albumin binding and biochemical parameters such as the concentrations of oxidized albumin and uremic toxins. The binding of ARP to plasma from chronic renal patients was significantly lower than healthy volunteers. A rational relationship between the ARP binding rate and the concentration of toxins, including indoxyl sulphate (IS) and p-cresyl sulphate (PCS), was found, particularly for IS. Moreover, multiple regression analyses that involved taking other parameters such as PCS or oxidized albumin ratio to IS into account supports the above hypothesis. In conclusion, the limited data reported in this present study indicates that monitoring IS in the blood is a very important determinant in the dosage plan for the administration of site II drugs such as ARP, if the efficacy of the drug in renal disease is to be considered.

Innate immune response in bovine neutrophils stimulated with Mycoplasma bovis.

Mycoplasma bovis (M. bovis) is a significant worldwide pathogen of cattle. Neutrophils have an important role in the innate immune response during infection with M. bovis. However, even though neutrophils accumulate in M. bovis infection, the interaction of M. bovis and neutrophils has not been fully elucidated. We attempted to elucidate the innate immune response of neutrophils stimulated with M. bovis and evaluate the transcriptome and functional analysis of bovine neutrophils stimulated with M. bovis. Proinflammatory cytokines, such as inducible nitric oxide (iNOS), which was the most increased gene in transcriptome analysis, were increased in quantitative polymerase chain reaction analysis of bovine neutrophils stimulated with live or heat-killed M. bovis. Nitric oxide and intracellular reactive oxygen species production of neutrophils stimulated with M. bovis was significantly increased. Neutrophils stimulated with M. bovis showed an increased ratio of nonapoptotic cell death compared to unstimulated controls. We demonstrated that neutrophil extracellular traps (NETs) formation was not recognized in neutrophils stimulated with live M. bovis. However, heat-killed M. bovis induced NETs formation. We also showed the interaction with M. bovis and bovine neutrophils regarding proinflammatory cytokine gene expression and functional expression related to NETs formation. Live and killed M. bovis induced innate immune responses in neutrophils and had the potential to induce NETs formation, but live M. bovis escaped NETs.