Liver Targeting of Interferon Through Pullulan Conjugation

Purpose. The purpose of this study was to actively target interferon (IFN) to the liver through its chemical conjugation with pullulan, a water-soluble polysaccharide with a high affinity for the liver. Methods. Chemical conjugation of IFN with pullulan was achieved by a cyanuric chloride method. Following intravenous injection of the conjugates to mice, their body distribution and the activity of an IFN-induced enzyme, 2,5-oligoadenylate (2-5A) synthetase in the liver and other organs, were evaluated. Results. The cyanuric chloride method enabled us to prepare an IFN-pullulan conjugate that retained approximately 7–9 % of the biological activity of IFN. Pullulan conjugation enhanced the liver accumulation of IFN and the retention period with the results being reproducible. When injected intravenously to mice, the IFN-pullulan conjugate enhanced the activity of 2-5A synthetase in the liver. The activity could be induced at IFN doses much lower than those of free IFN injection. In addition, the liver 2-5A synthetase induced by conjugate injection was retained for 3 days, whereas it was lost within the first day for the free IFN-injected mice. Conclusions. IFN-pullulan conjugation was promising for IFN targeting to the liver with efficient exertion of its antiviral activity therein.     

Metabolism-based inactivation of neuronal nitric-oxide synthase by components of cigarette and cigarette smoke.

It has been shown that administration of cigarette smoke to rats leads to loss of neuronal nitric-oxide synthase (nNOS) activity and nNOS protein in penile tissue. The exact mechanism for this loss of activity and protein is not known. In the current study, we investigated whether extracts prepared from cigarette smoke or from the cigarette itself could directly inhibit nNOS activity. We discovered that the cigarette smoke extract and the cigarette extract cause a time-, concentration-, and calmodulin-dependent inactivation of nNOS in an in vitro system containing the purified enzyme. L-Arginine, but not D-arginine, protects nNOS from this time-dependent inactivation, suggesting an active site directed event. The kinetics of inactivation are consistent with the metabolism-based or suicide inactivation of nNOS. Based on studies with other metabolism-based inactivators, this cigarette-mediated inactivation may render nNOS more susceptible to proteasomal degradation and thereby may explain the loss of nNOS protein in vivo. The component(s) responsible for nNOS inactivation is not volatile, is not retained by a 3,000 molecular weight cut-off membrane, binds to activated charcoal, and is highly water-soluble under both acidic and basic conditions. The discovery of a direct inactivation of nNOS by an organic, cationic compound(s) present in tobacco and tobacco smoke provides a basis for further study of not only the mechanisms responsible for the biological effects of tobacco but also a search for a potentially novel inactivator of nNOS.     

Expression level of valosin-containing protein is strongly associated with progression and prognosis of gastric carcinoma.

PURPOSE: Valosin-containing protein (VCP; also known as p97) was shown to be associated with antiapoptotic function and metastasis via activation of nuclear factor kappa-B signaling pathway. In this study, association of VCP expression with recurrence of gastric carcinoma (GC), in which lymphatic vessels are the main route of spread, was examined. PATIENTS AND METHODS: VCP expression in 330 patients with GC (242 males and 88 females) with ages ranging from 26 to 81 years (median, 60 years) was analyzed by immunohistochemistry, in which staining intensity in tumor cells was categorized as weaker (level 1) or equal to or stronger (level 2) than that in endothelial cells. RESULTS: Ninety-four (28.7%) patient cases showed level 1 and 233 patient cases (71.3%) showed level 2 VCP expression. Patients with level 2 expression showed higher rates of large tumor size (P <.0001), undifferentiated histologic subtype (P <.05), presence of vascular and lymphatic invasion (P <.0001 for both), presence of lymph node metastasis (P <.0001), deep tumor invasion (P <.0001), and poorer disease-free and overall survivals (P <.0001 for both) compared with those with level 1 VCP expression. Multivariate analysis revealed VCP expression level as an independent prognosticator for disease-free and overall survival. VCP level was an indicator for disease-free and overall survival in the early (pT1; P <.01 and P <.05, respectively) and advanced (pT2-4; P <.05 for both) group of pathologic tumor-node-metastasis system classification. CONCLUSION: The prognostic significance of VCP expression level in GC was demonstrated.     

Inhibition of tumor growth and metastasis by angiogenesis inhibitor TNP-470 on breast cancer cell lines in vitro and in vivo

Antitumor and antimetastatic activity of the angiogenesis inhibitor O-(chloroacetyl-carbamoyl) fumagillol (TNP-470), a semisynthetic analogue offumagillin, was evaluated in breast cancer cell lines. In an in vitro MTTassay, after 72 hrs continuous exposure to TNP-470, growth inhibition wasobserved in all seven cell lines of murine (JYG-A, JYG-B, DD-762, andBALB/c-MC) or human (KPL-1, MDA-MB-231, and MKL-F) origin, in which the50% inhibitory concentrations (IC₅₀) at 72 hrstreatment were 4.6, 4.4, 4.6, 10.1, 35.0, 25.3, and 33.4 µg/ml,respectively. In an in vivo assay using JYG-A, JYG-B, KPL-1, and MDA-MB-231cells by orthotopic (right thoracic mammary fat pad) transplantation infemale nude mice, TNP-470 at 30 or 50 mg/kg body weight was injected s.c.every other day from the day of tumor cell inoculation until the end of theexperiment. The inhibitory effect on primary tumor growth was obtained inall four cell lines in a dose-dependent manner. In the 50 mg/kgTNP-470-treated group, the reductions in tumor weight of the JYG-A, JYG-B,KPL-1, and MDA-MB-231 cells with respect to the controls were 50%,30%, 4%, and 49%, respectively. Metastasis was seen inthe JYG-A, JYG-B, and KPL-1 cells. The numbers of mice bearing pulmonarymetastases of JYG-A and JYG-B cells and regional axillary lymph nodemetastases of KPL-1 cells were reduced, and TNP-470 at the 50 mg/kg dose toKPL-1 cells significantly reduced lymph node metastases compared with thecontrol. Although the weight gain was retarded in the TNP-470-treated mice,weight loss was not seen. TNP-470 was highly effective in the treatment ofbreast cancer cells. These results suggest that the clinical use of TNP-470may be a promising treatment for breast cancer patients.     

In vitro schedule-dependent interaction between paclitaxel and SN-38 (the active metabolite of irinotecan) in human carcinoma cell lines

Paclitaxel and irinotecan are important new anticancer agents. The combination of these two agents has been considered for use against a variety of advanced solid tumors. Since the schedule-dependent effects of this combination may be crucial to its use, we studied the interaction of paclitaxel and SN-38 (the active metabolite of irinotecan) in various schedules in four human cancer cell lines in culture. Cell growth inhibition after 5 days was determined using an MTT assay. The effects of drug combinations at the IC₈₀ level were analyzed by the isobologram method. Simultaneous exposure to paclitaxel and SN-38 for 24 h produced antagonistic (subadditive and protective) effects in the human lung cancer cell line A549, the breast cancer cell line MCF7, and the colon cancer cell line WiDr, and produced additive effects in the ovarian cancer cell line PA1. Sequential exposure to paclitaxel for 24 h followed by SN-38 for 24 h, and the reverse sequence, produced additive effects in all four cell lines. These findings suggest that sequential administration, not simultaneous administration, may be the appropriate schedule for the therapeutic combination of paclitaxel and irinotecan. Continued preclinical and clinical studies should provide further insights and assist in determining the optimal schedule for this combination in clinical use. Received: 25 February 1997 / Accepted: 6 November 1997     

Effects of cellular parameters on the in vitro osteogenic potential of dual-gelling mesenchymal stem cell-laden hydrogels

This work investigated the effects of cellular encapsulation density and differentiation stage on the osteogenic capacity of injectable, dual physically and chemically gelling hydrogels comprised of thermogelling macromers and polyamidoamine crosslinkers. Undifferentiated and osteogenically predifferentiated mesenchymal stem cells (MSCs) were encapsulated within 20 wt% composite hydrogels with gelatin microparticles at densities of six or 15 million cells/mL. We hypothesized that a high encapsulation density and predifferentiation would promote increased cellular interaction and accelerate osteogenesis, leading to enhanced osteogenic potential in vitro. Hydrogels were able to maintain the viability of the encapsulated cells over a period of 28 days, with the high encapsulation density and predifferentiation group possessing the highest DNA content at all time points. Early alkaline phosphatase activity and mineralization were promoted by encapsulation density, whereas this effect by predifferentiation was only observed in the low seeding density groups. Both parameters only demonstrated short-lived effects when examined independently, but jointly led to greater levels of alkaline phosphatase activity and mineralization. The combined effects suggest that there may be optimal encapsulation densities and differentiation periods that need to be investigated to improve MSCs for biomaterial-based therapeutics in bone tissue engineering.     

Acarbose treatments improve arterial stiffness in patients with type 2 diabetes mellitus

Aims/Introduction: Although the improvement of postprandial hyperglycemia by an alpha‐glucosidase inhibitor (α‐GI) has been associated with a risk reduction of cardiovascular events, the relationship between postprandial hyperglycemia and arterial stiffness has not been well understood. We therefore examined whether ameliorating the postprandial state by α‐GI leads to an improvement in arterial stiffness. Materials and Methods: A total of 22 patients with type 2 diabetes mellitus were treated with acarbose. Cardio‐ankle vascular index (CAVI) as the arterial stiffness was measured by using a VaSera CAVI instrument before and 12 months after acarbose treatment. Serum high‐sensitivity C‐reactive protein (hs‐CRP), pentraxin‐3 (PTX3) and matrix metalloproteinase (MMP) ‐2, ‐9 were measured at the same time points. Furthermore, circulating peripheral blood mononuclear cells were examined for the frequencies of CD14 positive cells expressing membrane type‐1 MMP (MT1‐MMP) at the single cell level using flow cytometry. Results: After acarbose treatment, postprandial glucose and glycosylated hemoglobin (HbA_1c) were significantly decreased. Serum levels of hs‐CRP, PTX3, MMP‐2 and MMP‐9 were significantly decreased. CAVI showed a significant reduction, although the changes were not significant in blood pressure and heart rate. MT1‐MMP expression was significantly decreased by acarbose treatment. In multivariate analysis, improvement of blood glucose, decrease of PTX3 levels and MT1‐MMP expression were independent predictors of beneficial change in CAVI. Conclusions: The present study showed that the beneficial effects of acarbose on arterial stiffness are mediated by an improvement of postprandial hyperglycemia and vascular remodeling markers. In conclusion, acarbose treatment might reduce the risk of cardiovascular diseases by altering the arterial stiffness in postprandial hyperglycemic status. (J Diabetes Invest, doi: 10.1111/j.2040‐1124.2010.00079.x , 2010)     

Review of Risk Assessment of Major Anatomical Variations in Clinical Dentistry

Most of the studies of the mandible's anatomical variations have presented the authors' speculations, and only a limited number has provided evidence that demonstrated the actual complications injury to the variant structures caused. To our knowledge, no study has evaluated the risks associated with these variant anatomical structures' injury. We reviewed articles that described clinical cases of the injury to, and anatomical studies of, three anatomical variants of the mandible—the accessory mental, lingual, and retromolar foramina—with which dentists are relatively familiar and that are mentioned often in the context of implant and third molar surgeries, to describe risk assessment methods with which to evaluate potential complications preoperatively. Only a limited number of the clinical reports of injury to the mandible's accessory foramina were available. The potential severe complication of injury of the accessory mental foramen (AMF) is sensory disturbance of the lower lip. Risk of neurosensory disturbance of lower lip can be assessed by AMF/MF ratio and positional relations to the MF. Potential severe complication of injury of the lingual foramen is bleeding and hemorrhage in the oral cavity's floor. Risk of bleeding can be assessed by diameter and positional relation between the mental spine/mylohyoid line. A risk assessment of the retromolar foramen could not be made because of inadequate data. We hope the risk assessments suggested will encourage dentists to predict intraoperative/postoperative complications caused by damaging the mandible's accessory foramina. Clin. Anat. 32:672–677, 2019. © 2019 Wiley Periodicals, Inc.