In vitro drug release of natamycin from β-cyclodextrin and 2-hydroxypropyl-β-cyclodextrin-functionalized contact lens materials

Purpose: The antifungal agent natamycin can effectively form inclusion complexes with beta-cyclodextrin (β-CD) and 2-hydroxypropyl-β-cyclodextrin (HP-βCD) to improve the water solubility of natamycin by 16-fold and 152-fold, respectively (Koontz, J. Agric. Food. Chem. 2003). The purpose of this study was to develop contact lens materials functionalized with methacrylated β-CD (MβCD) and methacrylated HP-βCD (MHP-βCD), and to evaluate their ability to deliver natamycin in vitro. Methods: Model conventional hydrogel (CH) materials were synthesized by adding varying amounts of MβCD and MHP-βCD (0, 0.22, 0.44, 0.65, 0.87, 1.08% of total monomer weight) to a monomer solution containing 2-hydroxyethyl methacrylate (HEMA). Model silicone hydrogel (SH) materials were synthesized by adding similar concentrations of MβCD and MHP-βCD to N,N-dimethylacrylamide (DMAA)/10% 3-methacryloxypropyltris(trimethylsiloxy)silane (TRIS). The gels were cured with UV light, washed with ethanol and then, hydrated for 24 h (h). The model materials were then incubated with 2 mL of 100 μg/mL of natamycin in phosphate buffered saline (PBS) pH 7.4 for 48 h at room temperature. The release of natamycin from these materials in 2 mL of PBS, pH 7.4 at 32 ± 2 °C was monitored using UV–vis spectrophotometry at 304 nm over 24 h. Results: For both CH and SH materials, functionalization with MβCD and MHP-βCD improved the total amount of drugs released up to a threshold loading concentration, after which further addition of methacrylated CDs decreased the amount of drugs released (p < 0.05). The addition of CDs did not extend the drug release duration; the release of natamycin by all model materials reached a plateau after 12 h (p < 0.05). Overall, DMAA/10% TRIS materials released significantly more drug than HEMA materials (p < 0.05). The addition of MHP-βCD had a higher improvement in drug release than MβCD for both HEMA and DMAA/10% TRIS gels (p < 0.05). Conclusions: A high loading concentration of methacrylated CDs decreases overall drug delivery efficiency, which likely results from an unfavorable arrangement of the CDs within the polymer network leading to reduced binding of natamycin to the CDs. HEMA and DMAA/10% TRIS materials functionalized with MHP-βCD are more effective than those functionalized with MβCD to deliver natamycin.     

Can local Erythropoietin administration enhance bone regeneration in osteonecrosis of femoral head?

Osteonecrosis of femoral head (ONFH) is a challenging disease. Regardless of underlying causes, the ultimate result in all cases is disruption of femoral head blood supply. Once the disease starts, it is progressive in 80% of cases. Since the majority of the affected individuals are young, every effort should be focused on preserving the patients own femoral head. These years, the role of angiogenic growth factors has been investigated with promising results in animal models of ONFH. Erythropoietin (EPO) is a well known hormone that has been used in treatment of chronic anemia for many years with few side effects. Considering the angiogenic properties of EPO, we hypothesize that local delivery of recombinant human EPO during core decompression will enhance bone regeneration in ONFH. In this way we also can avoid systemic side effects of EPO.     

A luminescence study of the interaction of sulfobutylether-β-cyclodextrin with rutin

The luminol/sulfobutylether-β-cyclodextrin (SBE(7M)-β-CD) chemiluminescence (CL) system and the interaction of SBE(7M)-β-CD/rutin were first described by flow injection (FI) CL method. It was found that SBE(7M)-β-CD with luminol could form 1:1 complex online, which could accelerate the electrons transferring rate of excited 3-aminophthalate, giving the enhanced CL intensity of luminol. The enhancement of CL intensity was proportional to the concentrations of SBE(7M)- β-CD with a linear range from 25 to 1750 μmol L?1. It was also found that rutin could inhibit the CL intensity from luminol/SBE(7M)-β-CD system, and the decrement of CL intensity was logarithm over the concentrations of rutin ranging from 0.1 to 100.0 nmol L?1, giving the regression equation ΔI = 32.90lgC(rutin) + 16.26 (R2 = 0.9952) with a detection limit of 0.03 nmol L?1 (3σ). According to the proposed CL model, the binding constant (K(CD-R)) and the stoichiometric ratio of SBE(7M)-β-CD/rutin complex were obtained as 1.6 × 10? L2 mol?2 and 2:1. The possible mechanism of luminol/SBE(7M)-β- CD/rutin interaction was also discussed. The method was successfully applied to monitor rutin in human urine samples after ingesting SBE(7M)-β-CD/rutin complex, with a total excretion of 68.8% within 8.0 h.     

Combination of BMP-2-releasing gelatin/β-TCP sponges with autologous bone marrow for bone regeneration of X-ray-irradiated rabbit ulnar defects

The objective of this study is to evaluate the feasibility of gelatin sponges incorporating β-tricalcium phosphate (β-TCP) granules (gelatin/β-TCP sponges) to enhance bone regeneration at a segmental ulnar defect of rabbits with X-ray irradiation. After X-ray irradiation of the ulnar bone, segmental critical-sized defects of 20-mm length were created, and bone morphogenetic protein-2 (BMP-2)-releasing gelatin/β-TCP sponges with or without autologous bone marrow were applied to the defects to evaluate bone regeneration. Both gelatin/β-TCP sponges containing autologous bone marrow and BMP-2-releasing sponges enhanced bone regeneration at the ulna defect to a significantly greater extent than the empty sponges (control). However, in the X-ray-irradiated bone, the bone regeneration either by autologous bone marrow or BMP-2 was inhibited. When combined with autologous bone marrow, the BMP-2 exhibited significantly high osteoinductivity, irrespective of the X-ray irradiation. The bone mineral content at the ulna defect was similar to that of the intact bone. It is concluded that the combination of bone marrow with the BMP-2-releasing gelatin/β-TCP sponge is a promising technique to induce bone regeneration at segmental bone defects after X-ray irradiation.     

Health correlates and mode of administration of hormones—Are there any differences between parenteral and oral estrogen preparations?

Objectives

To investigate use-associated differences between parental and oral hormone therapy (HT) users in reference to HT non-users regarding self-rated general health status, quality of life, health service utilization, and selected chronic diseases.

Methods

All cases of last-week medicine use were recorded among 2248 women aged 40–79 who participated in the German Health Interview and Examination Survey 1997–1999. 89 current parenteral HT users and 322 oral HT users were identified. Health correlates were compared between the two groups in reference to HT non-users.

Results

Oral HT users had a poorer current health status as well as an impaired health status compared to the year before, were less satisfied with their health and life in general, and showed a lower quality of life regarding ‘body pain’ and ‘vitality’ in comparison with hormone non-users (all p < .05). Parenteral HT users showed no significant difference compared with HT non-users and oral HT users, respectively, in these health correlates except for a less satisfaction with health found in comparison with HT non-users (p = .002). Prevalences of cerebral-cardiovascular diseases were not different among women using parenteral or oral HT use. Parenteral HT users visited the offices of general practitioner and gynecologists more frequently than oral HT users as well as hormone non-users (all p < .05).

Conclusions

Oral HT use is associated with a negative assessment for health well-being whereas parenteral HT use shows largely a neutral effect. Further designated studies could clarify whether the mode of hormone administration consistently affects health-related quality of life and whether the mode of hormone treatment influences the choice of outpatient facilities for surveillance of therapy.     

Age-dependent loss of naïve T cells in TCR transgenic bone marrow chimeras

Study of immune senescence is complicated by low numbers of antigen-specific lymphocytes, particularly na?ve T (Tn)cells which disappear with aging. Although T cell receptor (TCR) transgenic mice facilitate aging research, their large number of Ag-specific T cells renders their T cell pool abnormal, precluding normal in vivo immunity. To create a physiologically relevant model with measurable numbers of TCR transgenic CD4+ T cells in the context of normal lymphocytes, mixed (DO11.10+BALB/c) bone marrow (BM) chimeras were constructed. As found in normal mice, the total number of transgenic T cells and the Tn:memory T cell ratio declined with the aging of the BM chimeras. Although these shifts in T cell subsets were evident in both the lymph nodes and the spleen (SP), they were more pronounced in the SP. Unlike DO11.10 mice, transgenic T cells in the chimera acquired an effector/memory phenotype upon antigen challenge. These results reveal a pliable model to study the impact of the constriction of the Tn cell repertoire upon optimal vaccine responses in the elderly.     

Downregulation of Wnt/β-catenin signaling causes degeneration of hippocampal neurons in vivo

The possibility that the degeneration of hippocampal neurons can be caused by mis-regulation of Wnt/β-catenin signaling was tested. Downregulation of Wnt signaling by the inducible expression of Axin, ICAT, and dnTcf4E causes degeneration of hippocampal neurons, while upregulation of Wnt signaling by the inducible expression of Dvl and β-catenin has a negligible effect. Treatment with ICG-001, a small molecule known to inhibit Wnt signaling, causes degeneration of hippocampal neurons, while the treatment with a JNK specific inhibitor does not show any effect. The results from LDH and TUNEL assays suggest that degeneration occurs via apoptotic processes. Inhibition of Wnt signaling reduced IGF-1 expression and the addition of IGF-1 blocked degeneration, which suggests that downregulation of IGF-1/Akt signaling is partially responsible for the degeneration. Inducible expression of Axin in the hippocampal neurons isolated from Axin2P-rtTA/pBI-EGFP-Axin double transgenic mice also causes degeneration. Consistent with the findings, these mice had more neuronal cell death in hippocampus and had differences in contextual conditioning upon the inducible expression of Axin. In summary, our data strongly support the idea that downregulation of Wnt/β-catenin signaling causes degeneration of hippocampal neurons in vivo and may be a cause of neurodegenerative diseases related to an anxiety related response.     

Repair of rabbit radial bone defects using bone morphogenetic protein-2 combined with 3D porous silk fibroin/β-tricalcium phosphate hybrid scaffolds

Our study aimed to investigate the effect of bone morphogenetic protein-2 (BMP-2) bound to silk fibroin and β-tricalcium phosphate (SF/β-TCP) hybrid on the healing of critical-size radial defects in rabbits. A 15-mm critical-size defect was induced at mid-diaphysis in the left radius of 20 New Zealand white rabbits (average age, 3.5 months; weight, 2.5–3.0 kg). The animals were randomized into Group 1 (SF/β-TCP combined with BMP-2), Group 2 (SF/β-TCP alone), and Group 3 (nothing implanted). Radiographs were obtained every 2 weeks and euthanasia was performed after 8 weeks for visual, radiological, micro-computed tomography (micro-CT), and histological studies. Eight weeks after implantation (SF/β-TCP combined with BMP-2), radiographs showed that new bone formed on the surface of the implant and had bridged the defect in Group 1. Micro-CT imaging also confirmed the formation of new bone around the implant, and the newly formed bone was quantified. Histological examination revealed newly formed bone in the implanted area. Meanwhile, there was no formation of new bone in Group 3. Among the groups, most active formation of new bones was found in Group 1, while there was no difference between Group 2 and Group 3. Based on these results, we concluded that BMP-2-SF/β-TCP showed significant improvement in healing of critical-size defects. Therefore, the combination of BMP-2 and SF/β-TCP would be useful in the field of bone tissue engineering.     

Effect of long-term parenteral administration of empty andl-Dopa-loaded liposomes on the turnover of dopamine and its metabolites in the striatum of mice with experimental Parkinson’s syndrome

It is found that the dose ofl-Dopa required for correction of dopamine metabolism in experimental Parkinson’s syndrome in C57B1/6 mice can be reduced 10-fold when the preparation is incorporated into small unilamellar liposomes (60 nm) composed from egg yolk phosphatidylcholine and cholesterol (7:3). High-performance liquid chromatography shows that a 14-day treatment with bothl-Dopa incorporated into liposomes (5 mg/kg) and freel-Dopa (50 mg/kg) equally increases the content of dopamine and homovanillic acid and their ratio in mouse striatum. The content of dihydroxyphenylacetic acid markedly increases after administration of freel-Dopa. Translated fromByulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 123, No. 2, pp. 150–153, February, 1997     

Are the antioxidative effects of 17β-estradiol modified by concomitant administration of a progestin?

Objective: Estrogens are known to exhibit antioxidative effects. At present little information exists on the influence of co-administered progestins upon this effect. Therefore we investigated the influence of levonorgestrel, a potent antiestrogenic progestin, on the inhibition of the low-density lipoprotein (LDL) oxidation by 17β-estradiol or 17β-estradiol valerate in vitro and ex vivo. Methods: After isolation from blood, the in vitro oxidation of LDL was induced by copper ions and measured continuously by monitoring the formation of conjugated dienes. In 21 female ovariectomized White New Zealand rabbits the antioxidative action of 17β-estradiol alone or in combination with levonorgestrel after subcutaneous infusion for 3 days was determined using the copper-induced LDL-oxidation as an endpoint. Eleven postmenopausal women were exposed to sequential estrogen-progestin replacement therapy (day 1–21: 2 mg estradiol valerate/day, day 10–21: 0.15 mg levonorgestrel/day). Blood samples were collected at three times: on day 1, on day 10, on day 22 (after the combination phase). The lag time of ex vivo oxidation of LDL, the plasma estradiol and estrone levels were estimated. Results: In the chosen cell-free system, 17β-estradiol increased the lag time of the LDL-oxidation in a dose-dependent manner. Levonorgestrel showed neither pro-oxidative nor antioxidative effects when administered alone in different concentrations. Co-administration of different doses of levonorgestrel did not modify the antioxidative action of estrogen either. The two ex-vivo models confirmed these results. In rabbits the co-administered 19-nortestosterone derivative levonorgestrel did not impair or reverse the estradiol-dependent effect. In postmenopausal women the daily oral administration of levonorgestrel in conjunction with 17β-estradiol valerate did not diminish the antioxidative action of this estrogen given for the first 9 days. Conclusion: The antioxidative potential of estradiol and estradiol valerate is maintained in the presence of levonorgestrel.     

Long-term estradiol-17β administration reduces population of neurons in the sympathetic chain ganglia supplying the ovary in adult gilts

Elevated levels of endogenous estrogens occurring in the course of pathological states of ovaries (follicular cysts, tumors) as well as xenoestrogens may result in hyperestrogenism. In rat, a close relationship between estrogens and sympathetic and sensory neurons supplying the genito-urinary system was reported. Recently, we have shown that long-term estradiol-17β (E₂) administration affected morphological and immunochemical organization of the sympathetic ovarian neurons in the caudal mesenteric ganglion of adult gilts. In this study, the influence of E₂ overdose on the number and distribution of neurons in the sympathetic chain ganglia (SChG) projecting to the ovary of adult pigs was investigated. The numbers of ovarian dopamine-β-hydroxylase (DβH-), neuropeptide Y (NPY-), somatostatin (SOM-), galanin (GAL-) and estrogen receptors (ERs-) immunoreactive perikarya as well as the density of the intraganglionic nerve fibers containing DβH and/or NPY, SOM, GAL were also determined. On day 3 of the estrous cycle the ovaries of both the control and experimental gilts were injected with retrograde neuronal tracer Fast Blue, to identify the neurons innervating gonads. From day 4 of the estrous cycle to the expected day 20 of the second studied cycle, the experimental gilts were injected with E₂, while the control gilts were receiving oil. After the last E₂/oil injection, the SChG Th16–S2 were collected and processed for double-labeling immunofluorescence. Injections of E₂: (1) increased the E₂ level in the peripheral blood ~ 4–5 fold, (2) reduced the total number of Fast Blue-positive postganglionic neurons in the ganglia under investigation, (3) decreased the number of perikarya in the L2–L4 ganglia, (4) reduced the number of perikarya in the ventral, dorsal and central regions of the SChG, (5) decreased the numbers of DβH⁺/NPY⁺ and DβH⁺/GAL⁺ perikarya and the numbers of DβH⁺ but NPY⁻, SOM⁻ and GAL⁻ perikarya in the SChG, (6) decreased the number of perikarya expressing ERs subtype α and β, and (7) decreased the total number of the intraganglionic nerve fibers containing DβH and/or NPY. These results show that long-term E₂ treatment of adult gilts down-regulates the population of both noradrenergic and ERs expressing the SChG ovary supplying neurons. Our findings suggest also that elevated E₂ levels that occur during pathological states may regulate gonadal function(s) by affecting ovary supplying neurons.     

Lower concentrations of methyl-β-cyclodextrin combined with interleukin-2 can preferentially induce activation and proliferation of natural killer cells in human peripheral blood

Previous studies have demonstrated that high concentrations of methyl-β-cyclodextrin (MβCD, 10-15 mM) can interfere with the formation of lipid rafts and inhibit activation of lymphocytes. In this report, we determined that lower concentrations of MβCD (1-4 mM) could accelerate the proliferation of lymphocytes in human peripheral blood mononuclear cells (PBMCs). In the expanded cells, CD3(-)CD56(+) natural killer (NK) cells were the dominant subpopulation, and a significant dose-effect relationship existed between the proportion of NK cells and the concentration of MβCD. In the groups treated with 3-4 mM MβCD, the proportions of NK cells reached a level of more than 60%. When PBMCs were treated with MβCD, CD69 was more preferentially expressed on CD3(-)CD56(+) cells than on CD3(+) cells at 48 and 72 hours. The expression of CD25 had no distinct difference at 48 hours, but when recombinant human interleukin-2 (IL-2) was added for a further 24 hours, it was also preferentially expressed on NK cells. MβCD and IL-2 synergistically could also induce interferon-γ (IFN-γ) production in CD56(+) human PBMCs. Mechanistic studies revealed that IFN-γ production in response to MβCD plus IL-2 was IL-12 independent but depended on endogenous IL-18 and IL-1β, and CD56(+)CD14(+) dendritic cell-like cells and B cells might mediate the ability of MβCD to activate NK cells. The MβCD-activated NK cells also had high cytotoxicity against the natural killer cell-sensitive K562 cells or lymphokine-activated killer cell-sensitive DAUDI cells in vitro. These studies indicated that lower concentrations of MβCD combined with IL-2 can preferentially induce activation and proliferation of NK cells in PBMCs.     

NOD2 and Crohn's disease: loss or gain of function?

The human nucleotide binding and oligomerization domain (NOD)-containing protein family consists of some 25 members related to the Apaf-1/Ced-4 family of apoptosis regulators and certain plant disease-resistance genes (Inohara et al., 2004). One member, NOD2 (CARD15), has gained recent prominence through its association with increased susceptibility to several clinically important human inflammatory diseases, especially Crohn's disease (CD). NOD2 is a cytoplasmic molecule involved in sensing microbial cell wall components and regulating inflammatory processes and apoptosis. This review focuses on recent insights into the functions of normal and variant NOD2 proteins and the pathogenetic mechanisms underlying NOD2-associated inflammatory diseases.     

Effect of rhTGF-β1 combined with bone grafts on human periodontal cell differentiation

Various techniques and materials have been proposed for the treatment of periodontal defects. In periodontal regeneration, periodontal ligament (PDL) cell differentiation as well as certain growth factors and their delivery system applied are critical. The purpose of this study was to evaluate the in vitro effect of recombinant human transforming growth factor-beta 1 (rhTGF-β1) combined with two different bone grafts on human PDL (hPDL) cell differentiation. The hPDL cells were treated with TGF-β1 alone or in combination with a calcified freeze-dried bone allograft (FDBA) and a porous biphasic calcium phosphate (BC) bone graft. Cell differentiation effect was estimated by measuring alkaline phosphatase (ALPase) activity and osteocalcin secretion. Results demonstrated that rhTGF-β1 alone or in combination with FDBA and BC provoked a significant (p < 0.05) increase in ALPase activity as compared with controls. The findings of this study confirmed the beneficial role of rhTGF-β1 combined with FDBA and BC as carriers in periodontal regeneration.     

Effect of rhTGF-β1 combined with bone grafts on human periodontal cell differentiation

Various techniques and materials have been proposed for the treatment of periodontal defects. In periodontal regeneration, periodontal ligament (PDL) cell differentiation as well as certain growth factors and their delivery system applied are critical. The purpose of this study was to evaluate the in vitro effect of recombinant human transforming growth factor-beta 1 (rhTGF-β1) combined with two different bone grafts on human PDL (hPDL) cell differentiation. The hPDL cells were treated with TGF-β1 alone or in combination with a calcified freeze-dried bone allograft (FDBA) and a porous biphasic calcium phosphate (BC) bone graft. Cell differentiation effect was estimated by measuring alkaline phosphatase (ALPase) activity and osteocalcin secretion. Results demonstrated that rhTGF-β1 alone or in combination with FDBA and BC provoked a significant (p<0.05) increase in ALPase activity as compared with controls. The findings of this study confirmed the beneficial role of rhTGF-β1 combined with FDBA and BC as carriers in periodontal regeneration.     

Combined administration of NAN-190 and 17β-estradiol abolishes the amnesic effect of scopolamine in middle-aged ovariectomized rats

Chronic combined administration of 5-HT_1A receptor antagonist NAN-190 and 17β-estradiol improved conditioned passive avoidance performance in middle-aged ovariectomized rats with scopolamine-induced amnesia. Our results suggest that the ovarian hormonal, cholinergic, and serotoninergic systems play a role in age-related cognitive disturbances under conditions of hypoestrogenic syndrome. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 142, No. 7, pp. 90–93, July, 2006     

Enhanced responsiveness to T-cell help causes loss of B-lymphocyte tolerance to a β-cell neo-self-antigen in type 1 diabetes prone NOD mice

Self-reactive B lymphocytes contribute to type 1 diabetes pathogenesis as APC and auto-Ab producers in NOD mice and humans. To shed light on the mechanisms responsible for the breakdown in B-lymphocyte self-tolerance to β-cell Ag, we utilised a model whereby hen-egg lysozyme (HEL)-specific Ig Tg (IgHEL-Tg)-Tg B lymphocytes were allowed to develop in or were transferred into mice expressing the HEL Tg under an insulin promoter (insHEL-Tg). IgHEL-Tg B lymphocytes enhanced type 1 diabetes susceptibility of insHEL-Tg NOD mice. A comparison of the tolerogenic activity of IgHEL-Tg B lymphocytes with NOD and non-autoimmune-prone C57BL/6 genetic backgrounds showed that both were rendered anergic in the presence of insHEL when competing with polyclonal B lymphocytes. Nevertheless, NOD IgHEL-Tg B lymphocytes transferred into insHEL-Tg mice were more readily susceptible to rescue from anergy than their C57BL/6 counterparts, following provision of in vivo T-cell help. The different tolerogenic outcomes were an intrinsic property of B lymphocytes rather than being related to the quality of T-cell help, with the defective response being at least partially controlled by genes mapping to insulin-dependent diabetes (Idd) susceptibility loci on Chromosome 1 (Idd5) and 4 (Idd9/11).     

Long-term follow-up and prognosis of chronic granulomatous disease in Yugoslavia: is there a role for early bone marrow transplantation?

We report the long-term follow-up of 12 pediatric-aged patients with chronic granulomatous disease (CGD). The mean age at the onset of infections was 5 months with a median delay in diagnosis of 2.5 years. Bacille Calmette–Guérin lymphadenitis was the most common presenting infection (6) followed by suppurative lymphadenitis (4), liver abscess (1), or Salmonella sepsis (1). Prophylaxis with cotrimoxazole was recommended to all patients. During the mean follow-up of 10 years (range, 4–23 years) pneumonitis was the most prevalent infection (91%) followed by lymphadenitis (83%), aphtous stomatitis (58%), and liver abscesses (25%). Seven (58%) patients developed chronic lung disease due to grossly delayed diagnosis (3) or poor compliance with antimicrobial prophylaxis (4). Five (41%) patients died during the second decade of life of aspergillosis (3) or chronic lung disease (2). Probability of survival into the third decade of life was estimated to be only 19%. We argue that HLA-identical bone marrow transplantation (BMT), if possible, should be attempted at early age because of significant morbidity and mortality in adolescence. BMT also should be considered in patients who suffer severe infections despite antimicrobial prophylaxis or patients with evidence of chronic lung disease. Possibility of elective BMT from unrelated donors remains to be carefully evaluated.