[Characterization of Supersulfide in Serum Albumin and Its Therapeutic Application].

Recent studies have shown that proteins already possess supersulfides during the translation. However, the distribution and the role of supersulfides are not fully understood. In this review, we focus on supersulfides in biological fluids, especially in serum. Various methods for measuring supersulfides have been developed, and these methods have elucidated the presence of supersulfides in serum proteins including serum albumin. Since the levels of supersulfides in serum and serum albumin of patients with chronic kidney disease were lower than those in healthy subjects and recovered by hemodialysis, the levels of supersulfides in serum would be an indicator reflecting oxidative stress. In addition, it has long been known that serum albumin is responsible for sulfur transference. We have applied this phenomenon to the synthesis of sulfur-added albumin (Sn-HSA) by the reaction of serum albumin with sodium polysulfide (Na2Sn). Sn-HSA suppressed the melanin production via scavenging oxidative stress. As described above, studies on the characterization of supersulfides in serum albumin may contribute to the monitoring of redox balance and prevention of oxidative stress-related diseases.

8-Prenylnaringenin tissue distribution and pharmacokinetics in mice and its binding to human serum albumin and cellular uptake in human embryonic kidney cells.

8-Prenylnaringenin (8-PN), a hop flavonoid, is a promising food substance with health benefits. Compared with nonprenylated naringenin, 8-PN exhibits stronger estrogenic activity and prevents muscle atrophy. Moreover, 8-PN prevents hot flushes and bone loss. Considering that prenylation reportedly improves the bioavailability of flavonoids, we compared the parameters related to the bioavailability [pharmacokinetics and tissue distribution in C57/BL6 mice, binding affinity to human serum albumin (HSA), and cellular uptake in HEK293 cells] of 8-PN and its mother (non-prenylated) compound naringenin. C57/BL6 mice were fed an 8-PN or naringenin mixed diet for 22 days. The amount of 8-PN (nmol/g tissue) in the kidneys (16.8 ± 9.20), liver (14.8 ± 2.58), muscles (3.33 ± 0.60), lungs (2.07 ± 0.68), pancreas (1.80 ± 0.38), heart (1.71 ± 0.27), spleen (1.36 ± 0.29), and brain (0.31 ± 0.09) was higher than that of naringenin. A pharmacokinetic study in mice demonstrated that the C max of 8-PN (50 mg/kg body weight) was lower than that of naringenin; however, the plasma concentration of 8-PN 8 h after ingestion was higher than that of naringenin. The binding affinity of 8-PN to HSA and cellular uptake in HEK293 cells were higher than those of naringenin. 8-PN bioavailability features assessed in mouse or human model experiments were obviously different from those of naringenin.

TERT/BMI1-transgenic human dermal papilla cells enhance murine hair follicle formation in vivo.

BACKGROUND: Dermal papilla cells (DPCs) are one type of mesenchymal cells; they play a key role on hair follicle induction. Their hair inductivity and proliferation abilities are rapidly lost during the 2-dimensional culture. Cell senescence is induced by inadequate culture conditions and telomere shortening. We previously reported that overexpression of TERT coding telomerase reverse transcriptase and BMI1 coding human B-cell-specific Moloney murine leukemia virus insertion region 1 (BMI1) avoided senescence of murine DPC and restored hair inductive activity. OBJECTIVE: To evaluate the function of TERT and BMI1 in the human DPCs (hDPCs). METHODS: Cultured hDPCs obtained from human scalp hair were transduced with TERT alone (hDP-T), BMI1 alone (hDP-B), both TERT and BMI1 (hDP-TB) and empty vector (hDP-E). The hair inductive activity of those cells was assessed by chamber assay in vivo. Gene expressions were analyzed by quantitative PCR (q-PCR). RESULTS: hDP-TB proliferated more than hDP-T and hDP-B in vitro and only hDP-TB showed hair inductivity in vivo. Moreover, the expressions of VCAN, CTNNB1, LEF1, FGF7 and VEGFA in hDP-TB were elevated compared to those in hDP-E. CONCLUSION: Overexpression of both TERT and BMI1 extends the life span of cultured hDPCs and ameliorates their hair inducing ability on mouse hair follicles.

Vaginal palpation versus transabdominal ultrasound in the comprehension of pelvic floor muscle contraction after vaginal delivery: a randomised controlled trial.

BACKGROUND: Pelvic floor muscles support the pelvic organs and control voiding. The first choice in the repair of pelvic floor function that is damaged during pregnancy and delivery is pelvic floor muscle training, which involves repeated muscle relaxation and contraction. However, as muscle contractions cannot be visualised, it is difficult to assess whether patients understand how to contract them. Therefore, we assessed patients' comprehension of pelvic floor muscle contraction by comparing two teaching methods, vaginal palpation and transabdominal ultrasound, following vaginal delivery. We hypothesised that vaginal palpation is better than transabdominal ultrasound in this regard. METHODS: This randomised controlled trial conducted in facilities in Tokyo, Japan between July 2018 and January 2019 included women aged ≥ 20 years at 4-6 weeks after vaginal delivery. The randomisation involved website-based centralised allocation. The primary outcome was a change in bladder base displacement during pelvic floor muscle contraction before and after training, which was measured using transabdominal ultrasound. Participants performed three contractions for 3 s, and the mean value was used for statistical analysis. The secondary outcome was a change in understanding the contraction before and after training, which was measured using a five-point Likert scale questionnaire. Outcomes were analysed using Welch's t-test. RESULTS: Sixty-five participants were randomly allocated to the vaginal palpation group (n = 32) and transabdominal ultrasound group (n = 33). Baseline characteristics were similar between the groups. Changes in bladder base displacement were not significantly different between the groups (p = 0.181). Within-group analyses showed that bladder base displacement was large in both groups after the respective intervention. There were no significant differences in any of the outcomes between the two groups before and after the intervention. CONCLUSIONS: Vaginal palpation and transabdominal ultrasound might be useful for comprehending pelvic floor muscle contraction after vaginal delivery. TRIAL REGISTRATION: UMIN 000032304. Registered 18 April 2018, https://upload.umin.ac.jp/cgi-open-bin/icdr_e/ctr_view.cgi?recptno=R000036820 .

Thioredoxin-albumin fusion protein prevents urban aerosol-induced lung injury via suppressing oxidative stress-related neutrophil extracellular trap formation.

The number of deaths from air pollution worldwide is estimated at 8.8 million per year, more than the number of deaths from smoking. Air pollutants, such as PM2.5, are known to induce respiratory and cardiovascular diseases by inducing oxidative stress. Thioredoxin (Trx) is a 12-kDa endogenous protein that exerts antioxidant activity by promoting dithiol disulfide exchange reactions. We previously synthesized human serum albumin-fused thioredoxin (HSA-Trx), which has a longer half-life in plasma compared with Trx, and demonstrated its efficacy against various diseases including respiratory diseases. Here, we examined the effect of HSA-Trx on urban aerosol-induced lung injury in mice. Urban aerosols induced lung injury and inflammatory responses in ICR mice, but intravenous administration of HSA-Trx markedly inhibited these responses. We next analyzed reactive oxygen species (ROS) production in murine lungs using an in vivo imaging system. The results show that intratracheal administration of urban aerosols induced ROS production that was inhibited by intravenously administered HSA-Trx. Finally, we found that HSA-Trx inhibited the urban aerosol-induced increase in levels of neutrophilic extracellular trap (NET) indicators (i.e., double-stranded DNA, citrullinated histone H3, and neutrophil elastase) in bronchoalveolar lavage fluid (BALF). Together, these findings suggest that HSA-Trx prevents urban aerosol-induced acute lung injury by suppressing ROS production and neutrophilic inflammation. Thus, HSA-Trx may be a potential candidate drug for preventing the onset or exacerbation of lung injury caused by air pollutants.

Formulation Stability of Amphiphilic Poly(γ-Glutamic Acid) Nanoparticle and Evaluation of Cardiotoxicity of NPs With Human iPSC-Derived 3D-Cardiomyocyte Tissues.

We conducted a stability study of biodegradable and amphiphilic nanoparticles (NPs) consisting of phenylalanine-attached poly(γ-glutamic acid) for drug delivery to find the optimal formulation and define the optimal storage conditions using novel quantitative analytical methods. The stability of NP suspension and lyophilized NP powder manufactured by a dimethyl sulfoxide-based and an ethanol-based process was assessed under 5°C, 25°C/60% relative humidity and 40°C/75% relative humidity. The content of phenylalanine-attached poly(γ-glutamic acid), impurities, absolute molecular weight, appearance, clarity of solution, particle size, zeta potential, particle matter, osmolality, water content, and pH were evaluated as parameters of NP stability. Lyophilized NPs with trehalose showed better stability. The lyophilized NP formulation could therefore provide a stable and high-quality product for clinical studies and shows promise as an effective drug delivery system carrier. The cardiotoxicity of prospective impurities contained in NPs and reagents used in the manufacturing process with human-induced pluripotent stem cell-derived 3-dimensional cardiomyocyte tissues by centrifugation layer-by-layer technique was also evaluated. As a result, cardiotoxicity for NPs and reagents was not observed, and it was clarified that the potential risk to human safety from NPs is low. The applicability of the cardiotoxicity evaluation approaches with human-induced pluripotent stem cell-derived 3-dimensional cardiomyocyte tissues will be evaluated by centrifugation layer-by-layer technique.

Distribution of Polysulfide in Human Biological Fluids and Their Association with Amylase and Sperm Activities.

Intracellular polysulfide could regulate the redox balance via its anti-oxidant activity. However, the existence of polysulfide in biological fluids still remains unknown. Recently, we developed a quantitative analytical method for polysulfide and discovered that polysulfide exists in plasma and responds to oxidative stress. In this study, we confirmed the presence of polysulfide in other biological fluids, such as semen and nasal discharge. The levels of polysulfide in these biological fluids from healthy volunteers (n = 9) with identical characteristics were compared. Additionally, the circadian rhythm of plasma polysulfide was also investigated. The polysulfide levels detected from nasal discharge and seminal fluid were approximately 400 and 600 µM, respectively. No correlation could be found between plasma polysulfide and the polysulfide levels of tear, saliva, and nasal discharge. On the other hand, seminal polysulfide was positively correlated with plasma polysulfide, and almost all polysulfide contained in semen was found in seminal fluid. Intriguingly, saliva and seminal polysulfide strongly correlated with salivary amylase and sperm activities, respectively. These results provide a foundation for scientific breakthroughs in various research areas like infertility and the digestive system process.

Involvement of SAPK/JNK Signaling Pathway in Copper Enhanced Zinc-Induced Neuronal Cell Death.

Zinc (Zn) plays an important role in many organisms in various physiological functions such as cell division, immune mechanisms and protein synthesis. However, excessive Zn release is induced in pathological situations and causes neuronal cell death. Previously, we reported that Cu ions (Cu2+) markedly exacerbates Zn2+-induced neuronal cell death by potentiating oxidative stress and the endoplasmic reticulum stress response. In contrast, the stress-activated protein kinase/c-Jun amino-terminal kinase (SAPK/JNK) signaling pathway is important in neuronal cell death. Thus, in this study, we focused on the SAPK/JNK signaling pathway and examined its involvement in Cu2+/Zn2+-induced neurotoxicity. Initially, we examined expression of factors involved in the SAPK/JNK signaling pathway. Accordingly, we found that phosphorylated (ie, active) forms of SAPK/JNK (p46 and p54) are increased by CuCl2 and ZnCl2 co-treatment in hypothalamic neuronal mouse cells (GT1-7 cells). Downstream factors of SAPK/JNK, phospho-c-Jun, and phospho-activating transcription factor 2 are also induced by CuCl2 and ZnCl2 co-treatment. Moreover, an inhibitor of the SAPK/JNK signaling pathway, SP600125, significantly suppressed neuronal cell death and activation of the SAPK/JNK signaling pathway induced by CuCl2 and ZnCl2 cotreatment. Finally, we examined involvement of oxidative stress in activation of the SAPK/JNK signaling pathway, and found that human serum albumin-thioredoxin fusion protein, an antioxidative protein, suppresses activation of the SAPK/JNK signaling pathway. On the basis of these results, our findings suggest that activation of ZnCl2-dependent SAPK/JNK signaling pathway is important in neuronal cell death, and CuCl2-induced oxidative stress triggers the activation of this pathway.

Development of analytical methods for evaluating the quality of dissociated and associated amphiphilic poly(γ-glutamic acid) nanoparticles.

A quantitative method of analyzing nanoparticles (NPs) for drug delivery is urgently required by researchers and industry. Therefore, we developed new quantitative analytical methods for biodegradable and amphiphilic NPs consisting of polymeric γ-PGA-Phe [phenylalanine attached to poly(γ-glutamic acid)] molecules. These γ-PGA-Phe NPs were completely dissociated into separate γ-PGA-Phe molecules by adding sodium dodecyl sulfate (SDS). The dissociated NPs were chromatographically separated to analyze parameters such as the γ-PGA-Phe content in the NPs, the impurities present [using reverse-phase (RP) HPLC with an ultraviolet (UV) detector], and the absolute MW [using size-exclusion chromatography (SEC) with refractive index detection (RI) and multiangle light scattering (MALS) detection, i.e., SEC-RI/MALS]. The chromatographic patterns of the NPs were equivalent to those of the component polymer (γ-PGA-Phe), and excellent chromatographic separation for the quantitative evaluation of NPs was achieved. To the best of our knowledge, this is the first report of the quantitative evaluation of NPs in the field of NP-based delivery systems. Furthermore, these methods were applied to optimize and evaluate the NP manufacturing process. The results showed that impurities were effectively removed from the γ-PGA-Phe during the manufacturing process, so the purity of the final γ-PGA-Phe NPs was enhanced. In addition, the appearance, clarity of solution, particle size, zeta potential, particle matter, osmolarity, and pH of the product were evaluated to ensure that the NPs were of the required quality. Our approach should prove useful for product and process characterization and quality control in the manufacture of NPs. γ-PGA-Phe NPs are known to be a powerful vaccine adjuvant, so they are expected to undergo clinical development into a practical drug-delivery system. The analytical methods established in this paper should facilitate the reliable and practical quality testing of NP products, thus aiding the clinical development of γ-PGA-Phe-based drug-delivery systems. Moreover, since these analytical methods employ commonly used reagents and chromatographic systems, the methods are expected to be applicable to other NP-based drug-delivery products too. Graphical abstract NPs were completely dissociated into separate γ-PGA-Phe polymeric molecules, which yielded a similar chromatogram to that seen for the NPs.

Characterization and analytical development for amphiphilic poly(γ-glutamic acid) as raw material of nanoparticle adjuvants.

Amphiphilic graft copolymer consisting of poly(γ-glutamic acid) (γ-PGA) as the hydrophilic backbone and L-phenylalanine ethyl ester (Phe) as the hydrophobic side chain is an important biodegradable polymer with great potential in medical applications. In this research, we established analytical methods for the characterization and quality control of γ-PGA-graft-Phe (γ-PGA-Phe), which forms nanoparticles in aqueous solution, as a deployment platform in practical applications for vaccine adjuvants. The SEC-RI/MALS system, which uses size exclusion chromatography (SEC) coupled with a multi_angle light scattering (MALS) detector and refractive index (RI) detector, was developed to evaluate the characteristics of various types of polymers. By this method, it was indicated that absolute molecular weight (MW) should be used to measure the branch polymer. A gradient reversed phase HPLC (RP-HPLC) method was developed for the content of γ-PGA-Phe and the impurity levels to control product quality and safety. This quantitative approach could become key elements for identifying and characterizing γ-PGA-Phe. In addition, the degradation mechanism of γ-PGA-Phe was also identified as cleavage of main-chain of γ-PGA-Phe based on the stability study of γ-PGA-Phe in buffer solution with various pH values. The analytical developments described above will be important for use in both characterization and formulation design of biopolymers. Nanoparticles (NPs) composed of well-characterized biodegradable γ-PGA-Phe are expected to have a variety of potential clinical applications such as their use as drug and vaccine carriers.

Dual Therapeutic Effects of an Albumin-Based Nitric Oxide Donor on 2 Experimental Models of Chronic Kidney Disease.

Chronic kidney disease (CKD) is accompanied by a variety of complications, typically renal anemia and kidney fibrosis. Accordingly, it is desirable to develop the novel therapeutics that can treat these CKD conditions. Since nitric oxide (NO) has multiple functions including hypoxia inducible factor stabilizing, anti-inflammatory, anti-oxidative, and anti-apoptoic activities, the use of NO for the CKD therapy has attracted considerable interest. Here, we evaluate the therapeutic impacts of S-nitrosated human serum albumin (SNO-HSA), a long-lasting NO donor, on 2 animal models of CKD. SNO-HSA increased the expression of erythropoietin (EPO), VEGF, and eNOS by stabilizing hypoxia inducible factor-1α in HepG2 and HK-2 cells. SNO-HSA increased hematopoiesis in both healthy and renal anemia rats, suggesting the promotion of EPO production. In unilateral ureteral obstruction-treated mice, SNO-HSA ameliorated kidney fibrosis by suppressing the accumulation of renal extracellular matrix. SNO-HSA also inhibited unilateral ureteral obstruction-induced α-smooth muscle actin increase and E-cadherin decrease, suggesting that SNO-HSA might suppress the accumulation of myofibroblasts, an important factor of fibrosis. SNO-HSA also inhibited the elevations of fibrosis factors, such as transforming growth factor-ß, interleukin-6, and oxidative stress, while it increased EPO production, an anti-fibrosis factor. In conclusion, SNO-HSA has the potential to function as a dual therapeutics for renal anemia and kidney fibrosis.

A novel S-sulfhydrated human serum albumin preparation suppresses melanin synthesis.

Products of ultraviolet (UV) irradiation such as reactive oxygen species (ROS) and nitric oxide (NO) stimulate melanin synthesis. Reactive sulfur species (RSS) have been shown to have strong ROS and NO scavenging effects. However, the instability and low retention of RSS limit their use as inhibitors of melanin synthesis. The free thiol at Cys34 on human serum albumin (HSA) is highly stable, has a long retention and possess a high reactivity for RSS. We report herein on the development of an HSA based RSS delivery system. Sulfane sulfur derivatives released from sodium polysulfides (Na2Sn) react readily with HSA. An assay for estimating the elimination of sulfide from polysulfide showed that almost all of the sulfur released from Na2Sn bound to HSA. The Na2Sn-treated HSA was found to efficiently scavenge ROS and NO produced from chemical reagents. The Na2Sn-treated HSA was also found to inhibit melanin synthesis in B16 melanoma cells and this inhibition was independent of the number of added sulfur atoms. In B16 melanoma cells, the Na2Sn-treated HSA also inhibited the levels of ROS and NO induced by UV radiation. Finally, the Na2Sn-treated HSA inhibited melanin synthesis from L-DOPA and mushroom tyrosinase and suppressed the extent of aggregation of melanin pigments. These data suggest that Na2Sn-treated HSA inhibits tyrosinase activity for melanin synthesis via two pathways; by directly inhibiting ROS signaling and by scavenging NO. These findings indicate that Na2Sn-treated HSA has potential to be an attractive and effective candidate for use as a skin whitening agent.

Quantitative determination of polysulfide in albumins, plasma proteins and biological fluid samples using a novel combined assays approach.

Hydrogen sulfide (H2S) signaling involves polysulfide (RSSnSR') formation on various proteins. However, the current lack of sensitive polysulfide detection assays poses methodological challenges for understanding sulfane sulfur homeostasis and signaling. We developed a novel combined assay by modifying Sulfide Antioxidant Buffer (SAOB) to produce an "Elimination Method of Sulfide from Polysulfide" (EMSP) treatment solution that liberates sulfide, followed with methylene blue (MB) sulfide detection assay. The combined EMSP-MB sulfide detection assay performed on low molecular weight sulfur species showed that sulfide was produced from trisulfide compounds such as glutathione trisulfide and diallyl trisulfide, but not from the thiol compounds such as cysteine, cystine and glutathione. In the case of plasma proteins, this novel combined detection assay revealed that approximately 14.7, 1.7, 3.9, 3.7 sulfide mol/mol released from human serum albumin, α1-anti-trypsin, α1-acid glycoprotein and ovalbumin, respectively, suggesting that serum albumin is a major pool of polysulfide in human blood circulation. Taken together with the results of albumins of different species, the liberated sulfide has a good correlation with cysteine instead of methionine, indicating the site of incorporation of polysulfide is cysteine. With this novel sulfide detention assay, approximately 8,000, 120 and 1100 µM of polysulfide concentrations was quantitated in human healthy plasma, saliva and tear, respectively. Our promising polysulfide specific detection assay can be a very important tool because quantitative determination of polysulfide sheds light on the functional consequence of protein-bound cysteine polysulfide and expands the research area of reactive oxygen to reactive polysulfide species.

Apoptosis induction of poly-S-nitrosated human serum albumin in resistant solid tumor under hypoxia can be restored by phosphodiesterase 5 inhibition.

Poly-S-nitrosated human serum albumin (Poly-SNO-HSA) delivered and accumulated nitric oxide (NO) in tumors and induces apoptosis. Tumor hypoxia is strongly associated with malignant progression and tumor resistance to therapy. In this study, we examined the cytotoxic effect of Poly-SNO-HSA under hypoxia on the murine colon 26 adenocarcinoma (C26) cells in vitro and in vivo. Under hypoxia, at about 4 times LD50 dose of Poly-SNO-HSA in vitro, the reactive oxygen species production was hindered but apoptotic cells were induced via cGMP pathway as the effect was suppressed by a soluble guanylate cyclase inhibitor, NS2028. The apoptosis induction effect of low dose Poly-SNO-HSA on C26 cells in vitro under hypoxia can be restored by a phosphodiesterase 5 (PDE5) inhibitor, vardenafil. In C26-bearing mice, Poly-SNO-HSA/vardenafil combination treatment significantly suppressed the tumor volume compared with Poly-SNO-HSA or vardenafil treatment alone. Furthermore, the core tumor tissues showed increased expression of caspase-3 than the non-core tissue. The expression of caspase-3 appeared to overlap with the hypoxic zone of tumor tissues. Similar results were also obtained when the experiments were repeated using Epimedium extract, a natural herbal supplement with PDE5 inhibition activity. In conclusion, Poly-SNO-HSA/PDE5 inhibitors combination therapy is a promising approach for enhancing the anticancer therapeutic effects of Poly-SNO-HSA against not only anti-cancer drug resistance but also hypoxic stress related solid tumor resistance.

Human serum albumin hydropersulfide is a potent reactive oxygen species scavenger in oxidative stress conditions such as chronic kidney disease.

Recently, hydropersulfide (RSSH) was found to exist in mammalian tissues and fluids. Cysteine hydropersulfide can be found in free cysteine residues as well as in proteins, and it has potent antioxidative activity. Human serum albumin (HSA) is the most abundant protein in mammalian serum. HSA possesses a free thiol group in Cys-34 that could be a site for hydropersulfide formation. HSA hydropersulfide of high purity as a positive control was prepared by treatment of HSA with Na2S. The presence of HSA hydropersulfide was confirmed by spectroscopy and ESI-TOFMS analysis where molecular weights of HSA hydropersulfide by increments of approximately 32 Da (Sulfur atom) were detected. The fluorescent probe results showed that Alexa Fluor 680 conjugated maleimide (Red-Mal) was a suitable assay and bromotrimethylammoniumbimane bromide appeared to be a selective reagent for hydropersulfide. The effect of oxidative stress related disease on the existence of albumin hydropersulfides was examined in rat 5/6 nephrectomy model of chronic kidney disease (CKD). Interestingly, the level of hydropersulfides in rat 5/6 nephrectomy model serum was decreased by a uremic toxin that increases oxidative stress in rat 5/6 nephrectomy model. Furthermore, we demonstrated that the levels of HSA hydropersulfide in human subjects were reduced in CKD but restored by hemodialysis using Red-Mal assay. We conclude that HSA hydropersulfide could potentially play an important role in biological anti-oxidative defense, and it is a promising diagnostic and therapeutic marker of oxidative diseases.

S-Nitrosated human serum albumin dimer as novel nano-EPR enhancer applied to macromolecular anti-tumor drugs such as micelles and liposomes.

The enhanced permeability and retention (EPR) effect is a unique phenomenon of solid tumors, and it can serve as a basis for the development of macromolecular anticancer therapy. We have previously found that recombinant human serum albumin dimer, and especially its S-nitrosated form (SNO-HSA-Dimer), is an enhancer of the EPR effect. In this study, we investigated the influence of SNO-HSA-Dimer on the anti-tumor effect of two types of macromolecular anti-tumor drugs, namely N-(2-hydroxypropyl)methacrylamide polymer conjugated with zinc protoporphyrin, which forms micelles and can be used for fluorescence studies. The other was PEGylated liposomal doxorubicin (Doxil), a typical example of a stealth liposome approved for medical usage. In mice having C26 tumors with highly permeable vasculature, SNO-HSA-Dimer increases tumor accumulation of the drugs by a factor 3-4 and thereby their anti-tumor effects. Experiments with Evans blue revealed increased EPR effect in all parts of the tumor. Furthermore, SNO-HSA-Dimer improves the anti-metastatic effects of Doxil and reduces its minor uptake in non-tumorous organs such as liver and kidney. Tumor accumulation of Doxil in B16 tumors, which are characterized by a low permeable vasculature, increased even more (6-fold) in the presence of SNO-HSA-Dimer, and the improved accumulation lead to decreased tumor volume and increased survival of the animals. The administration of SNO-HSA-Dimer itself is safe, because it has no effect on blood pressure, heart rate or on several biochemical parameters. The present findings indicate that SNO-HSA-Dimer is promising for enhancing the EPR effect and consequently the specific, therapeutic effects of macromolecular anticancer drugs.

Poly-S-nitrosated human albumin enhances the antitumor and antimetastasis effect of bevacizumab, partly by inhibiting autophagy through the generation of nitric oxide.

Autophagy is one of the major causes of drug resistance. For example, the angiogenesis inhibitor bevacizumab shows only transient and short-term therapeutic effects, whereas long-term therapeutic benefits are rarely observed, probably due to hypoxia-induced autophagy. Nitric oxide (NO) is an important molecule with multiple functions, and it has recently been reported to function as a regulator of autophagy. Therefore, a reasonable therapeutic strategy for overcoming drug resistance by NO would involve it being directly delivered to the tumor. Here, we investigated the inhibitory effect of NO on autophagy by using a macromolecular NO donor S-nitrosated human serum albumin (SNO-HSA) with a high degree of NO loading and tumor targeting potential. In colon 26 (C26) cells, SNO-HSA significantly suppressed hypoxia-induced autophagy by inhibiting the phosphorylation of JNK1 and the expression of its downstream molecule Beclin1. The effect of SNO-HSA was also confirmed in vivo by combining it with Bev. In C26-bearing mice, significant suppression of tumor growth as well as lung metastasis was achieved in the combination group compared to the SNO-HSA or bevacizumab alone group. Similar to the in vitro experiments, the immunostaining of tumor tissues clearly showed that SNO-HSA inhibited the autophagy of tumor cells induced by bevacizumab treatment. In addition to other known antitumor effects of SNO-HSA, that is, the induction of apoptosis and the inhibition of multidrug efflux pumps, these data may open alternate strategies for cancer chemotherapy by taking advantage of the ability of SNO-HSA to suppress autophagy-mediated drug resistance and enhance the efficacy of chemotherapy.

RR interval variability during galvanic vestibular stimulation correlates with arterial pressure upon head-up tilt.

RR interval variability (RRIV) in the supine position without and with galvanic vestibular stimulation (GVS (off) and GVS (on), respectively), changes in mean arterial pressure (MAP) at the onset of 60° head-up tilt (HUT) during GVS (off), and their relationship were analyzed in 25 healthy young subjects. MAP decreased by less than 5mmHg or increased upon HUT in 12 subjects (UP), but MAP decreased by more than 5mmHg in 13 subjects (DOWN). Applying sinusoidal GVS of 2mA at a random frequency of 0.2 to 10.0Hz did not change the RR intervals or MAP. However, the high frequency component (HF) of RRIV increased in both UP and DOWN subjects. The increase in DOWN subjects was larger than that in UP subjects. The ratio of the low frequency component to HF (L/H) increased in UP subjects during GVS (on), but did not reach a significant level in DOWN subjects. The changes in the HF were significantly correlated with changes in MAP at the onset of HUT; i.e., the subjects with larger increases in the HF during GVS (on) showed larger decreases in MAP. Thus, GVS or vestibular input during HUT possibly activates the vagal nerves, and the dominance of excitation in sympathetic or vagal nerves during vestibular stimulation is important for controlling MAP at the onset of HUT.

Effective elimination of contaminants after oral care in elderly institutionalized individuals.

After mechanical cleaning in oral care, eliminating residual oral contaminants has an important role in preventing their aspiration, especially in individuals with weak airway protection. We examined the effectiveness of wiping the oral cavity after oral care on eliminating contaminants in 31 patients who were hospitalized in our neurology inpatient unit. The amount of bacteria on the tongue, palate, and buccal vestibule was counted before and just after oral care, after eliminating contaminants either by rinsing with water and suction or by wiping with mouth wipes, and 1 h after oral care. Oral bacteria amounts were decreased significantly by both elimination procedures after oral care. These findings suggest that wiping with mouth wipes is as effective as mouth rinsing to decrease bacteria following oral care. With a lower risk of contaminant aspiration, wiping may be a suitable alternative to rinsing, especially in dysphagic individuals.

[Pathological complete response in a case of advanced esophageal cancer invading aorta treated by preoperative chemotherapy with docetaxel and cisplatin plus 5-FU].

The patient was a 64-year-old man, diagnosed as cStage IVa esophageal cancer invading the aorta with lymph node metastasis. He received combination chemotherapy with docetaxel/cisplatin/5-FU(DFP therapy). After one course, CT and endoscopic examination showed remarkable reduction of the primary lesion and lymph node metastasis. We performed subtotal esophagectomy and gastric tube reconstruction by the retroposterior mediastinum route. The pathological specimen evidenced fibrosis and infiltration of inflammatory cells on almost all layers, but showed no viable malignant cells in the middle thoracic esophagus. Therefore, the pathological effect was judged as Grade 3(pCR). This case suggested that DFP combination chemotherapy may prove to be a useful treatment for advanced esophageal cancer with invasion to other organs.