Poly(ethylene glycol) hydrogels formed by thiol-ene photopolymerization for enzyme-responsive protein delivery

Degradable hydrogels have been extensively used in biomedical applications such as drug delivery, and recent interest has grown in hydrogels that degrade in recognition of a cellular response. This contribution describes a poly(ethylene glycol) (PEG) hydrogel platform with human neutrophil elastase (HNE) sensitive peptide cross-links formed using thiol-ene photopolymerization rendering the gel degradable at sites of inflammation. Further, protein therapeutics can be physically entrapped within the network and selectively released upon exposure to HNE. HNE-responsive hydrogels exhibited surface erosion where the degradation kinetics was influenced by changes in peptide k_cat, concentration of HNE, and concentration of peptide within the gel. Using this platform, we were able to achieve controlled, zero-order release of bovine serum albumin (BSA) in the presence of HNE, and release was arrested in the absence of HNE. To further exploit the advantages of surface eroding delivery systems, a smaller protein (carbonic anhydrase) was delivered at the same rate as BSA and only dependent on gel formulation and environmental conditions. Also, protein release was predicted from a 3-layered hydrogel device using mass loss data. Lastly, the bioactivity of lysozyme was maintained above 90% following the exposure to thiol-ene photopolymerization conditions.     

Reduction in inflammation-induced sensitization of dorsal horn neurons by transcutaneous electrical nerve stimulation in anesthetized rats

Transcutaneous electrical nerve stimulation (TENS) is utilized to treat a variety of painful conditions. Inflamed animals present with an increased response to noxious stimuli, i.e., hyperalgesia, at the site of injury (primary hyperalgesia) and outside the site of injury (secondary hyperalgesia). Further, following acute inflammation, dorsal horn neurons show an increased responsiveness to peripherally applied stimuli, which has been termed sensitization. Previous studies demonstrate a reduction in dorsal horn neuron activity following TENS treatment in normal animals and a reduction in primary and secondary hyperalgesia in acutely inflamed animals. The purpose of this study was to examine the effects of TENS on dorsal horn neurons sensitized by acute inflammation. Extracellular recordings from wide dynamic range (WDR), high threshold (HT) and low threshold (LT) dorsal horn neurons in anesthetized rats were assessed for spontaneous activity, responses to innocuous and noxious mechanical stimulation and receptive field size. Responses were measured before and 3 h after induction of inflammation, and immediately and 1 h after application of either high (100 Hz) or low (4 Hz) frequency TENS (motor intensity, pulse duration = 100 microseconds). TENS was applied to the inflamed paw to encompass the receptive field of the neuron for 20 min. WDR and HT dorsal horn neurons sensitized to mechanical stimulation after induction of inflammation. Application of either high or low frequency TENS to the inflamed paw reduced both innocuous and noxious evoked responses of WDR and HT dorsal horn neurons immediately and 1 h after treatment with TENS. Comparison of responses after TENS with baseline responses showed that the evoked responses in the majority of WDR and HT cells returned to or fell below baseline responses. TENS had no effect on responses of LT neurons. In summary, central neuron sensitization is reduced by TENS and may underlie the reduction in hyperalgesia observed after treatment with TENS.     

帕夫林联用甲氨蝶呤治疗类风湿关节炎的临床观察

目的:探讨治疗类风湿关节炎经济、有效、安全的治疗方案。方法:回顾性总结2005年3月至2006年3月我院门诊及住院115例类风湿关节炎患者的临床疗效及肝肾功能,通过统计学分析比较单用甲氨蝶呤,甲氨蝶呤联合柳氮磺胺吡啶,帕夫林联用甲氨蝶呤三组患者治疗1y的临床疗效、实验室指标,比较三组间肝肾功异常的发生率。结果:甲氨蝶呤组(单联组)、甲氨蝶呤+柳氮磺吡啶对照组(对照组)、甲氨蝶呤+帕夫林组(治疗组)三组病例用药前一般资料无统计学差异(P0.05),对照组及治疗组的临床疗效明显优于单联组(P0.01),而治疗组肝肾功异常发生率明显低于单联组和对照组(P0.01)。结论:帕夫林联用甲氨蝶呤治疗类风湿关节炎1y疗效优于单用甲氨蝶呤,而肝肾功异常的发生率小于单用甲氨蝶呤和甲氨蝶呤联用柳氮磺胺吡啶。     

Ⅱ型胶原诱导的类风湿关节炎模型的制作

Ⅱ型胶原诱导的类风湿关节炎模型CIA模型是目前最为理想的类风湿关节炎动物模型。受诸多因素影响，模型制作难度较大，成功率不高。结合参考国内外大量文献，本室多年研究经验，从动物品系的选择，胶原的配制和准备，注射的部位和方法，评价的指标等方面为CIA模型的制作提供了参考。     

Immunomodulating therapy of rheumatoid arthritis by high-dose intravenous immunoglobulin

Summary 11 patients with rheumatoid arthritis were treated with intravenous immunoglobulin (IVGG). In 6 patients clinical results were impressive, although lasting responses could be achieved in 3 patients only. This treatment was immunomodulating, since the immunoregulatory T-cell ratio (CD4/CD8) decreased following therapy by reducing CD4-positive cells in-vivo. By use of anti--antibodies as a B-cell specific mitogen, IVGG-treatment was seen to suppress early processes of B cell activation. In parallel to these cellular effects, IVGG led to a reduction in the levels of polyethyleneglycol-precipitated circulating immune complexes as measured by lasernephelometry.Abkürzungsverzeichnis IVGG Intravenous Immunoglobulin - ITP Idiopathic Thrombocytopenic purpura - RA Rheumatoid Arthritis - ARA American Rheumatism Association - AI Activity-Index - CIC Circulating Immune complexes - TCU Tetanus complex-Units - PBMC Peripheral Blood mononuclear cells - SI Stimulation-Index - PWM Pokeweed mitogen - IG Immunoglobulin     

Animal models of rheumatoid arthritis

Animal models have been used extensively in studies of rheumatoid arthritis pathogenesis. Despite the inherent limitations of all animal models, several rodent models have significantly progressed our understanding of the fundamental mechanisms underpinning rheumatoid arthritis and contributed to several current major advances in treatment. These models include the induced arthritis models such as collagen‐induced arthritis, collagen‐antibody‐induced arthritis, zymosan‐induced arthritis, and the methylated BSA model, and the genetically manipulated or spontaneous arthritis models such as the TNF‐α‐transgenic mouse, K/BxN mouse, and the Skg mouse. Here, we describe these animal models and discuss their advantages and limitations. Note added after publication: For an update on this topic, please see the latest review     

Efficacy of low-dose methotrexate in rheumatoid arthritis

Twenty-eight patients with refractory rheumatoid arthritis completed a randomized 24-week double-blind crossover trial comparing oral methotrexate (2.5 to 5 mg every 12 hours for three doses weekly) with placebo. The methotrexate group had significant reductions (P less than 0.01 as compared with the placebo group) in the number of tender or painful joints, the duration of morning stiffness, and disease activity according to physician and patient assessments at the 12-week crossover visit; reductions in the number of swollen joints (P less than 0.05) and 15-m walking time (P less than 0.03) also occurred. These variables, as well as the grip strength and erythrocyte sedimentation rate, showed significant (P less than 0.01) improvement at 24 weeks in the population crossed over to methotrexate. A significantly increased frequency (P less than 0.03) of the HLA-DR2 haplotype occurred in the eight patients with the most substantial response to methotrexate. Adverse reactions during methotrexate therapy included transaminase elevation (21 per cent), nausea (18 per cent), and diarrhea (12 per cent); one patient was withdrawn from the trial because of diarrhea. One patient died while receiving the placebo. Methotrexate did not affect measures of humoral or cellular immunity. We conclude that this trial provides evidence of the short-term efficacy of methotrexate in rheumatoid arthritis, but the mechanism of action is unknown. Longer trials will be required to determine the ultimate safety and effectiveness of this drug.     

Anti-inflammatory effects of intravenous methotrexate associated with lipid nanoemulsions on antigen-induced arthritis

OBJECTIVE:To test the hypothesis that intravenous use of methotrexate associated with lipid nanoemulsions can achieve superior anti-inflammatory effects in the joints of rabbits with antigen-induced arthritis compared with commercial methotrexate.METHODS:Arthritis was induced in New Zealand rabbits sensitized with methylated bovine serum albumin and subsequently intra-articularly injected with the antigen. A nanoemulsion of methotrexate labeled with ³H-cholesteryl ether (4 mg/kg methotrexate) was then intravenously injected into four rabbits to determine the plasma decaying curves and the biodistribution of the methotrexate nanoemulsion by radioactive counting. Additionally, the pharmacokinetics of the methotrexate nanoemulsion were determined by high-pressure liquid chromatography. Twenty-four hours after arthritis induction, the animals were allocated into three groups, with intravenous injection with saline solution (n=9), methotrexate nanoemulsion (0.5 µmol/kg methotrexate, n=7), or commercial methotrexate (0.5 µmol/kg, n=4). The rabbits were sacrificed 24 h afterward. Synovial fluid was then collected for protein leakage and cell content analyses and synovial membranes were collected for histopathological analysis.RESULTS:The methotrexate nanoemulsion was taken up mainly by the liver and the uptake by arthritic joints was two-fold greater than that by control joints. The methotrexate nanoemulsion treatment reduced leukocyte influx into the synovial fluid by nearly 65%; in particular, mononuclear and polymorphonuclear cells were reduced by 47 and 72%, respectively. In contrast, cell influx was unaffected following treatment with commercial methotrexate. Protein leakage into the arthritic knees of the rabbits was also more limited following methotrexate nanoemulsion treatment than following commercial methotrexate treatment.CONCLUSIONS:The intravenous methotrexate nanoemulsion showed anti-inflammatory effects on the synovia of arthritic joints that were clearly superior to the effects of a commercial methotrexate preparation. This result is conceivably due to greater methotrexate uptake by the joints when the drug is associated with a nanoemulsion.     

Diatom silica microparticles for sustained release and permeation enhancement following oral delivery of prednisone and mesalamine

Diatoms are porous silica-based materials obtained from single cell photosynthetic algae. Despite low cost, easy purification process, environmentally safe properties, and rapidly increasing potentials for medical applications, the cytotoxicity of diatoms and the effect on drug permeation of oral formulations have not been studied so far. Herein, we have evaluated the potential of diatom silica microparticles (DSMs) for the delivery of mesalamine and prednisone, which are two commonly prescribed drugs for gastrointestinal (GI) diseases. Transmission electron microscopy analysis of the morphological surface changes of Caco-2/HT-29 monolayers and the cell viability data in colon cancer cells (Caco-2, HT-29 and HCT-116) showed very low toxicity of diatoms at concentrations up to 1000 μg/mL. The mesalamine and prednisone release under simulated GI conditions indicated prolonged release of both drugs from the diatoms. Furthermore, drug permeation across Caco-2/HT-29 co-culture monolayers demonstrated that diatoms are capable to enhance the drug permeability. Overall, this study evaluated DSMs' cytotoxicity in colon cancer cells and the effect of DSMs on drug permeability across Caco-2/HT-29 monolayers. Our results demonstrate that DSMs can be considered as a non-cytotoxic biomaterial with high potential to improve the mesalamine and prednisone bioavailability by sustaining the drug release and enhancing drug permeability.     

ERK信号转导通路与类风湿关节炎

丝裂原活化蛋白激酶(mitogen-activated protein kinases,MAPKs)信号转导通路是细胞外信号引起细胞核内反应的重要通路,而细胞外信号调节激酶(extracellular signal-regulated kinase, ERK)通路是MAPKs家族中的重要成员,其异常活化与类风湿关节炎(rheumatoid arthritis,RA)关节破坏的病理过程密切相关.     

Gold nanoparticles with a monolayer of doxorubicin-conjugated amphiphilic block copolymer for tumor-targeted drug delivery

Gold (Au) nanoparticles (NPs) stabilized with a monolayer of folate-conjugated poly(l-aspartate-doxorubicin)-b-poly(ethylene glycol) copolymer (Au-P(LA-DOX)-b-PEG-OH/FA) was synthesized as a tumor-targeted drug delivery carrier. The Au-P(LA-DOX)-b-PEG-OH/FA NPs consist of an Au core, a hydrophobic poly(l-aspartate-doxorubicin) (P(LA-DOX)) inner shell, and a hydrophilic poly(ethylene glycol) and folate-conjugated poly(ethylene glycol) outer shell (PEG-OH/FA). The anticancer drug, doxorubicin (DOX), was covalently conjugated onto the hydrophobic inner shell by acid-cleavable hydrazone linkage. The DOX loading level was determined to be 17 wt%. The Au-P(LA-DOX)-b-PEG-OH/FA NPs formed stable unimolecular micelles in aqueous solution. The size of the Au-P(LA-DOX)-b-PEG-OH/FA micelles were determined as 24–52 and 10–25 nm by dynamic light scattering (DLS) and transmission electron microscopy (TEM), respectively. The conjugated DOX was released from the Au-P(LA-DOX)-b-PEG-OH/FA micelles much more rapidly at pH 5.3 and 6.6 than at pH 7.4, which is a desirable characteristic for tumor-targeted drug delivery. Cellular uptake of the Au-P(LA-DOX)-b-PEG-OH/FA micelles facilitated by the folate-receptor-mediated endocytosis process was higher than that of the micelles without folate. This was consistent with the higher cytotoxicity observed with the Au-P(LA-DOX)-b-PEG-OH/FA micelles against the 4T1 mouse mammary carcinoma cell line. These results suggest that Au-P(LA-DOX)-b-PEG-OH/FA NPs could be used as a carrier with pH-triggered drug releasing properties for tumor-targeted drug delivery.     

Mice transgenic for intracellular interleukin-1 receptor antagonist type 1 are protected from collagen-induced arthritis

Interleukin-1 receptor antagonist (IL-1Ra) is a natural IL-1 inhibitor, which competitively inhibits binding of IL-1 to its receptors. IL-1Ra is produced as four different isoforms, one secreted (sIL-1Ra) and three intracellular (icIL-1Ra1, 2, 3), derived from the same gene. We previously observed increased production of icIL-1Ra1 in the joints of mice with collagen-induced arthritis (CIA). However, due to its intracellular localization, the biological role of icIL-1Ra1 remains unknown. The aim of the present study was to examine the effect of the icIL-1Ra1 isoform, as compared to that of sIL-1Ra, in the CIA model by comparing transgenic (tg) mice overexpressing icIL-1Ra1 or sIL-1Ra to their wild-type littermates. Serum levels of tg human IL-1Ra were elevated in sIL-1Ra and, to a lesser extent, also in icIL-1Ra1 mice. Clinical scoring indicated that none of the icIL-1Ra1 or siL-1Ra tg mice developed CIA, whereas arthritis was present in, respectively, 60% and 100% of their wild-type littermates. Histological and radiological analyses confirmed the absence of arthritis in icIL-1Ra1 and sIL-1Ra tg mice. Accordingly, circulating levels of the acute-phase protein serum amyloid A tended to be lower in icIL-1Ra1 tg mice than in their wild-type littermates and were significantly lower in sIL-1Ra tg mice than in controls. In contrast, no difference was observed between the groups regarding serum levels of anti-type II collagen antibodies and ex vivo spleen cell proliferative response to collagen. In conclusion, icIL-1Ra1, which is released into the extracellular space when produced in high amounts, has a similar anti-arthritic effect as sIL-1Ra.     

Amphiphilic multi-arm-block copolymer conjugated with doxorubicin via pH-sensitive hydrazone bond for tumor-targeted drug delivery

Folate-conjugated unimolecular micelles based on amphiphilic hyperbranched block copolymer, Boltorn^® H40-poly(l-aspartate-doxorubicin)-b-poly(ethylene glycol)/FA-conjugated poly(ethylene glycol) (H40-P(LA-DOX)-b-PEG-OH/FA), were synthesized as a carrier for tumor-targeted drug delivery. The anticancer drug DOX was covalently conjugated onto the hydrophobic segments of the amphiphilic block copolymer arms by pH-sensitive hydrazone linkage. The size of the unimolecular micelles was determined as 17–36 and 10–20 nm by dynamic light scattering (DLS) and transmission electron microscopy (TEM), respectively. The release profiles of the DOX from the H40-P(LA-DOX)-b-PEG-OH/FA micelles showed a strong dependence on the environmental pH values. The DOX release rate increased in the acidic medium due to the acid-cleavable hydrazone linkage between the DOX and micelles. Cellular uptake of the H40-P(LA-DOX)-b-PEG-OH/FA micelles was found to be higher than that of the H40-P(LA-DOX)-b-PEG-OH micelles because of the folate-receptor-mediated endocytosis, thereby providing higher cytotoxicity against the 4T1 mouse mammary carcinoma cell line. Degradation studies showed that the H40-P(LA-DOX)-b-PEG-OH/FA copolymer hydrolytically degraded into polymer fragments within six weeks. These results suggest that H40-P(LA-DOX)-b-PEG-OH/FA micelles could be a promising nanocarrier with excellent in vivo stability for targeting the drugs to cancer cells and releasing the drug molecules inside the cells by sensing the acidic environment of the endosomal compartments.     

Prediction of response to methotrexate in rheumatoid arthritis

Introduction: Methotrexate (MTX) is the first-line disease-modifying drug of choice in controlling active inflammation of the synovium that characterises rheumatoid arthritis, a chronic autoimmune inflammatory condition. However, many patients do not respond to treatment with MTX or cannot tolerate the medication. Pre-treatment characteristics that predict response to MTX are, therefore, of particular interest and potential clinical utility.

Areas covered: This narrative review seeks to cover various genotypic and phenotypic characteristics that have been investigated as predictors of treatment response to MTX in RA. Ovid Medline searches (1946 to January 2018) were carried out for ‘methotrexate’ and ‘rheumatoid arthritis’, in combination with relevant terms. All papers identified were English language, with abstracts. Relevant references were also reviewed.

Expert commentary: Despite the introduction of biologic medication and targeted therapies, MTX is likely to remain the mainstay of RA treatment, largely due to its much cheaper cost. Development of a multifactorial predictive algorithm for response to MTX may be of clinical utility, as well as routine MTX drug level testing to improve medication adherence and persistence.     

The use of nanolipoprotein particles to enhance the immunostimulatory properties of innate immune agonists against lethal influenza challenge

Recent studies have demonstrated that therapies targeting the innate immune system have the potential to provide transient, non-specific protection from a variety of infectious organisms; however, the potential of enhancing the efficacy of such treatments using nano-scale delivery platforms requires more in depth evaluation. As such, we employed a nanolipoprotein (NLP) platform to enhance the efficacy of innate immune agonists. Here, we demonstrate that the synthetic Toll-like receptor (TLR) agonists monophosphoryl lipid A (MPLA) and CpG oligodeoxynucleotides (CpG) can be readily incorporated into NLPs. Conjugation of MPLA and CpG to NLPs (MPLA:NLP and CpG:NLP, respectively) significantly enhanced their immunostimulatory profiles both in vitro and in vivo compared to administration of agonists alone, as evidenced by significant increases in cytokine production, cell surface expression of activation markers, and upregulation of immunoregulatory genes. Importantly, enhancement of cytokine production by agonist conjugation to NLPs was also observed in primary human dendritic cells. Furthermore, BALB/c mice pretreated with CpG:NLP constructs survived a lethal influenza challenge whereas pretreatment with CpG alone had no effect on survival.     

壳聚糖水凝胶用于局部药物控释的研究进展

<正>大量的活性化合物具有作为药物治疗的潜力,但在临床应用上很少取得满意的效果,主要是因为它们在体内的生物利用度低[1]。这些复合物的生物活性主要取决于药物的输入方式和器官的生理代谢特征[2]。利用高分子化合物的疏水和     

Muscle involvement in rheumatoid arthritis: an ultrastructural study

An electron microscopic investigation has been carried out on muscle bioptic samples from patients affected by rheumatoid arthritis (RA). This study was undertaken to seek further ultrastructural alterations affecting striated muscles in RA pathology. Bioptic samples were collected on a total of 30 surgical interventions of hip (10), knee(8), and foot (12). This yielded three muscle types: gluteus maximus, vastus lateralis, and extensor digitorum communis. Muscle samples from 12 patients with no RA stigmata, selected to match RA patients by age and gender, constituted the control group. Tissue samples were prepared both for conventional histochemical methods and according to conventional electron microscopic procedures, including morphometric analysis. Although to a different extent in each sample, in muscles from RA vs. controls the authors observed the simultaneous presence of discrete muscular alterations such as wider separation of myofibrils, myelin figures, dilated sarcotubular system, pleomorphic mitochondria, myofibril flaking, and lipofuscin deposition in the subsarcolemmal region. In addition to a progressive atrophy, the above findings are suggestive of rheumatoid myositis and lend further support to the still poorly documented presence of an idiopathic inflammatory myopathy and inclusion body myositis associated with RA.     

影响类风湿关节炎X线平片进展的临床多因素分析

Objective To investigate the clinical factors related to radiological progression in rheumatoid arthritis(RA) and the role of radiology in the assessment of RA outcomes. Methods A total of 178 cases of active rheumatic arthritis were enrolled in a 2-year trial given radiological assessment at baseline and during follow up. These cases were classified into three groups, namely groups 1, 2 and 3 for mild,moderate and severe based on baseline X-ray images of joint damage. Radiological progression, clinical remission and Health Assessment Questionnaire (HAQ) were used in the two-year follow-up and analyzed for the correlation between each other. Results At the end point, groups 1 and 2 did not differ in radiological progression (P＞0.05). Significant difference was found between group 1 (15.4%, 12/78)and group 2 (30.0%,9/30) (P＜0.05) , as well as between group 3 (11.9% , 7/59) and group 3 (P＜0.05) , suggesting that radiological damages may deteriorate more in patients with higher level of bone erosion at baseline. After 2years of medications, the rate of clinical remission in all the cases was 85.0% (142/167) and was not different among the three groups (P＞0.05), which indicated that clinical remission had no relevance with radiological progression. Conclusion Female gender, positive rheumatoid factor, high HAQ scores and peri- articular lesions are risk factors for radiological progression in RA. Early use of disease-modifying antirheumatic drugs (within less than 6 months) is beneficial for patients with radiological progression. X-ray is sufficiently effective to confirm, re-evaluate and determine the articular erosion. Clinical remission may not necessarily indicate suspension of radiological progression and routine X-ray is required in the whole follow up process of RA in order to modify medications based on progression of articular lesions.     

Flexible microfluidic devices supported by biodegradable insertion scaffolds for convection-enhanced neural drug delivery

Convection enhanced delivery (CED) can improve the spatial distribution of drugs delivered directly to the brain. In CED, drugs are infused locally into tissue through a needle or catheter inserted into brain parenchyma. Transport of the infused material is dominated by convection, which enhances drug penetration into tissue compared with diffusion mediated delivery. We have fabricated and characterized an implantable microfluidic device for chronic convection enhanced delivery protocols. The device consists of a flexible parylene-C microfluidic channel that is supported during its insertion into tissue by a biodegradable poly(DL-lactide-co-glycolide) scaffold. The scaffold is designed to enable tissue penetration and then erode over time, leaving only the flexible channel implanted in the tissue. The device was able to reproducibly inject fluid into neural tissue in acute experiments with final infusate distributions that closely approximate delivery from an ideal point source. This system shows promise as a tool for chronic CED protocols.     

Macrophage activation syndrome in a child with systemic juvenile rheumatoid arthritis

Macrophage activation syndrome (MAS) is a rare and potentially fatal complication of rheumatic disorders in children. We describe a 13-month-old boy in whom MAS developed as a complication of systemic juvenile rheumatoid arthritis (S-JRA). He suffered from fever and generalized rash followed by multiple joints swelling for four months before admission. Physical examination revealed cervical lymphadenopathy and hepatosplenomegaly. Laboratory findings were: abnormal liver enzymes, increased triglyceride and ferritin levels, coagulopathies resembling disseminated intravascular coagulation, anemia and thrombocytopenia. Hyperplasia of hemophagocytic macrophages was remarkable in his bone marrow. Methylprednisolone and cyclosporin therapy resulted in clinical and laboratory improvements. This is the third case of MAS associated with S-JRA in Koreans, and the first one, in which hemophagocytic macrophages were proven in bone marrow.