一种构建改形单域抗体的方法

为验证一种构建改形单域抗体的实用新方法,与以往方法不同的是,该方法不需要对抗体进行空间结构模拟,以确定人源抗体的FRs接受序列及在人源FRs接受序列中哪些氨基酸残基需要突变,并且该方法将抗体的改形与亲和力成熟于同一过程完成,利用该方法构建了改形抗CD28重链单域抗体,根据一种鼠源抗CD28重链单域抗体的氨基酸序列,于GenBank中查得两条与之最同源的人源抗体序列,利用其中一条的FRs作为改形抗体的主框架进行改形构建,将鼠源抗体的CDR区插入到人源FR区后,对人源FR区的一些氨基酸残基进行替换突变,替换的氨基酸残基数及替换原则主要是根据对查到的人源抗体序列,鼠源抗体序列,以及这些序列与Kabat分类中的种属序列进行的比较,为了增加改形抗体基因的多样性,对要被替换的氨基酸残基在基因合成中采用简并的方式,使要被替换的氨基酸残基和替换的氨基酸残基都有机会出现,二者出现的几率各为50%,同时,在将大小不同的合成核苷酸片段采用重叠PCR扩增以获得完整改形抗体基因时,采用高Mg^2 浓度下和使用TaqDNA聚合酶,以进一步随机引入突变,利用重叠PCR产物构建了一个噬菌体抗体库,经过3轮淘选后,获得了几个具有较高免疫学活性的改形抗体,对其中的两个抗体进行了进一步研究,将两个抗体的基因在大肠杆菌BL21（DE3）中表达,复性后的表达蛋白仍具有较高的免疫学活性,结果表明该方法是有效可行的。     

Thumb reconstruction with a sensory flap from the dorsum of the index finger.

The dorsal skin of the index ray is very useful (1) for a one-staged thumb lengthening procedure after amputation, (2) for covering the stump of an avulsed thumb with sensory skin, and (3) for expanding the first web space. The flap may be transferred as a rotation flap, or the dorsal vasculature and nerve supply to the index may be carefully dissected free as a pedicle to permit its use as a neurovascular island flap. We believe that considerably more sensory skin can be transferred by this flap than by the ring finger neurovascular island flap, and that the technical risks and surgical time are less with the index finger flap.     

Reversible Femtosecond Laser-Assisted Myopia Correction: A Non-Human Primate Study of Lenticule Re-Implantation after Refractive Lenticule Extraction

LASIK (laser-assisted in situ keratomileusis) is a common laser refractive procedure for myopia and astigmatism, involving permanent removal of anterior corneal stromal tissue by excimer ablation beneath a hinged flap. Correction of refractive error is achieved by the resulting change in the curvature of the cornea and is limited by central corneal thickness, as a thin residual stromal bed may result in biomechanical instability of the cornea. A recently developed alternative to LASIK called Refractive Lenticule Extraction (ReLEx) utilizes solely a femtosecond laser (FSL) to incise an intrastromal refractive lenticule (RL), which results in reshaping the corneal curvature and correcting the myopia and/or astigmatism. As the RL is extracted intact in the ReLEx, we hypothesized that it could be cryopreserved and re-implanted at a later date to restore corneal stromal volume, in the event of keratectasia, making ReLEx a potentially reversible procedure, unlike LASIK. In this study, we re-implanted cryopreserved RLs in a non-human primate model of ReLEx. Mild intrastromal haze, noted during the first 2 weeks after re-implantation, subsided after 8 weeks. Refractive parameters including corneal thickness, anterior curvature and refractive error indices were restored to near pre-operative values after the re-implantation. Immunohistochemistry revealed no myofibroblast formation or abnormal collagen type I expression after 8 weeks, and a significant attenuation of fibronectin and tenascin expression from week 8 to 16 after re-implantation. In addition, keratocyte re-population could be found along the implanted RL interfaces. Our findings suggest that RL cryopreservation and re-implantation after ReLEx appears feasible, suggesting the possibility of potential reversibility of the procedure, and possible future uses of RLs in treating other corneal disorders and refractive errors.     

Correction of type II breast deformities following conservative cancer surgery.

Breast deformities following conservative cancer surgery are seen with increasing frequency and often represent difficult reconstructive problems. Type II deformities are characterized by localized tissue insufficiency, which can be due to skin insufficiency (type IIa), subcutaneous tissue insufficiency (type IIb), or both (type IIab). Correction of a locally damaged breast is a surgical challenge that can result in a fully restored breast if selection of the surgical procedure is properly carried out. A series of 37 patients who underwent correction of type II deformities from 1980 to 1989 was reviewed. Results obtained with different surgical procedures, including simple submuscular placement of traditional or expandable implants, breast reshaping, transposition of a latissimus dorsi muscle or musculocutaneous flap, TRAM flap, and reverse abdominoplasty, were evaluated. Aesthetic outcome was judged to be good or excellent in 78 percent of patients. Guidelines for selection of the most appropriate surgical procedure according to the defect's etiology, morphology, and location and to the breast's size and shape are presented.     

Expanded applications for transconjunctival lower lid blepharoplasty.

There has been a recent upsurge in interest in the transconjunctival approach for lower lid blepharoplasty. Initial reports have focused on the young patient with isolated fat prominence. We describe our experience with transconjunctival lower lid blepharoplasty in 104 patients over the past 2 years. There have been no instances of prolonged lower lid retraction problems, presumably related to leaving the skin, orbicularis, and orbital septum intact. Our experience with expanding the indications for the transconjunctival approach to include patients with fine skin wrinkling as well as frank skin excess has been extremely favorable. We conclude that the skin excess is often more apparent than real, with the skin being necessary to recontour the lower eyelid after fat excision. Although skin excision may be required during the initial procedure or at a later stage, patients with apparent skin excess need not be excluded from consideration for transconjunctival lower lid blepharoplasty.     

Radial diffusion assay of tissue collagenase and its application in evaluation of collagenase inhibitors.

A radial diffusion assay for tissue collagenase (EC 3.4.24.3) has been devised which is simple, sensitive and capable of application to large numbers of samples. The assay employs an agarose matrix containing solubilized lathyritic rat skin collagen as substrate. Fibril formation is induced for 2 h at 37 degrees C subsequent to 41 h digestion at 28 degrees C. The procedure results in sharply defined zones of lysis which may be measured directly or after photography. The characteristics of the procedure are otherwise similar to those reported for other radial diffusion assays. The new method was used to examine the action of 10 compounds which were known or potential inhibitors of tadpole collagenase. The concentration of inhibitor required to produce 50% inhibition is reported for the following compounds: alpha2-macroglobulin, 142 microng/ml; N-acetylcysteine, greater than or equal to 100 mM; cysteine, 8.7 mM; EDTA, 0.46 mM; histidine, greater than or equal to 100 mM; 2,3-dimercaptopropanol, 0.5 mM and mercaptoacetic acid, 70 mM. The procedure also has potential for clinical determinations (e.g. tears, synovial fluid) since assay dishes may be prepared in advance and only 15 micronl of sample is required.     

Assessing the mechanical energy costs of various tissue reshaping mechanisms

Early-stage embryos must reshape the tissues of which they are made into organs and other life-sustaining structures; and if non-mammalian embryos fail to complete these tasks before the energy provided by their yolk runs out, they die. The aim of this study is to use a cell-level computational model to investigate the energetic cost of a variety of mechanisms that can drive an in-plane reshaping pattern known as convergent extension—a motif in which a tissue narrows in one in-plane direction and expands in another. Mechanisms considered include oriented lamellipodia, directed mitosis, stress fibers, and anisotropic external tension. Both isolated patches of tissue and actively contracting tissues that deform adjacent passive areas are considered. The cell-level finite element model used here assumes that the cell membrane and its associated proteins generate a net tension γ along each cell–cell interface and that the cytoplasm and its embedded networks and structures have an effective viscosity?μ. Work costs are based exclusively on mechanical considerations such as edge lengths and tensions, and because a traditional mechanical efficiency cannot be calculated, mechanisms are compared on the basis of the work they must do to the tissue to cause a specified rate of in-plane reshaping. Although the model contains a number of simplifications compared to real embryonic tissues, it is able to show that the work requirements for tissue reshaping by mitoses and by lamellipodia are of the same order. Lamellipodia are energetically most effective when their tensions are approximately twice as large as the interfacial tensions in the surrounding cells. The model also shows that stress fibers or other direct stretch or compression mechanisms are at least five times more efficient for tissue reshaping than are mitoses or lamellipodia and that the work needed to deform a typical cellular tissue is more than thirty times greater than if it did not contain cell boundaries. Collectively, these findings indicate that common tissue reshaping mechanisms have mechanical efficiencies of less than one percent and that mechanical efficiency is not the primary determinant of which mechanism(s) an embryo uses to reshape its tissues.     

大中型鸟类标本修复翻新方法探索

本文报道一种修复与翻新大中型鸟类标本的方法,包括以下步骤:1.拆标本;2.皮张回软、清洗;3.皮张修复缝合;4.涂抹防腐剂;5.支撑架制作与固定;6.填充与缝合;7.整形与标本固定.皮张回软时,利用药液浸泡可使皮张保持弹性,保证翻新后皮张仍保持完好形态.珍珠棉水果网作为填充物,既环保又可免招虫蛀,亦能使做出的标本形态更加逼真.     

Effects of fronto-occipital cranial reshaping on mandibular form.

Cultural reshaping (artificial deformation or modification) of the neurocranial vault provides an artificially increased range of morphological variability within which the relationship between the growing neurocranium and face can be investigated. We analyze crania which have been fronto-occipitally compressed to ascertain possible morphological effects on the mandible. We collected measures of mandibular breadth, length, and height from 82 modified (N = 48) and unmodified (N = 34) crania from a Peruvian Ancon series. Angle classification was also scored in order to investigate whether or not occlusal relationships were affected by neurocranial reshaping. Only intercondylar distance (posterior mandibular breadth) exhibited significant differences between unmodified and modified groups, though this difference was relatively small compared with vault deformation. The modified crania had a higher frequency of normal occlusion (Class I) than the unmodified crania. Increased intercondylar breadth in modified skulls is due to a cascade of effects which begin with a direct effect of the fronto-occipital deforming device on neurocranial shape (increased neurocranial width). The increase in mandibular breadth may be a compensatory response to increased cranial base breadth and maintains articulation between the cranial base and mandible. The increased posterior breadth, coupled with a slight decrease in mandibular depth, may contribute to the change in occlusal relationships suggested for this sample.     

A new approach for rapidly reshaping single-chain antibody in vitro by combining DNA shuffling with ribosome display

Antibody reshaping is an effective way to reduce the immunogenicity while maintaining or improving the affinity of murine antibodies. This paper describe a new in vitro approach for rapidly reshaping murine antibodies by combining DNA shuffling with ribosome display. With the new method, a reshaping anti-4-1BB single-chain antibody (scFv), Re-4B4-1 scFv, which bound to its antigen (4-1BB) specifically and strongly, was selected from a reshaping library. These results proved definitely the feasibility of the new designed approach for antibody reshaping.     

间伐改形对陇东旱塬密闭苹果园树体冠层结构和发育后期叶片质量的影响

为研究间伐改形对红富士成龄乔化密植果园树体冠层特征、生育后期叶片生理特性、养分积累分配规律、光合生产力和土壤水分时空分布动态的影响,以18年生‘红富士’苹果密闭园为试材,对苹果树单株结构参数、枝量、枝类组成、覆盖率、叶片光合速率等参数进行测定.结果表明: 密闭果园叶片光合作用受非气孔因素限制,导致PSII最大光化学效率、PSII光合潜能、光合性能指数下降了1.2%、11.5%、13.9%.间伐改形后,叶面积指数、树冠覆盖率有所降低,使得冠层直射光透过系数增加了79.0%,树形结构有所改善.苹果园总枝数降低到约1100400条·hm^-2,单株枝量增加了5.0%,短枝比例提高至73.0%.由于冠层光照条件的改善,叶面积、比叶质量、百叶重、叶绿素含量有不同程度的提高.叶片光合速率的提高促进了光合产物的积累,淀粉、蛋白质含量为密闭果园的143.5%、107.8%.叶片的发育质量与其所处的光照辐射环境有着密切联系,密闭果园经间伐、改形后,果园群体结构和冠层光照得以改善,促进了叶片生长发育,提高了叶片光合效能,降低了果园土壤水分的无效消耗,是陇东旱塬苹果产区密闭果园适宜的调整、优化方案.     

The location of silver in frog epidermis after treatment by ranvier's method,and possible implication of the flask cells in transport

Summary Frog skin, bladder wall, and sciatic nerve were treated by Ranvier's silver nitrate method and subsequently fixed and sectioned for electron microscopy. In the epidermis of the skin, more silver is found deposited in the flask cells than elsewhere, especially as a sub-apical plaque in the neck of the flask, which appears after the skin has been flooded with silver nitrate for 5 minutes. Mitochondria rich cells in the bladder also accumulate more silver than the surrounding epithelial cells, but do not show such a distinct sub-apical plaque. In myelinated nerve fibres treated similarly, silver accumulates in the paranodal regions of the axon, and outside the axon at the node. It is suggested that silver may accumulate near a site of ion transport, due to structural specialisations not visible by standard electron microscope techniques, and that the flask cells may therefore be implicated in transport in the frog skin.     

Effects of fronto-occipital artificial cranial vault modification on the cranial base and face.

Artificial reshaping of the cranial vault has been practiced by many human groups and provides a natural experiment in which the relationships of neurocranial, cranial base, and facial growth can be investigated. We test the hypothesis that fronto-occipital artificial reshaping of the neurocranial vault results in specific changes in the cranial base and face. Fronto-occipital reshaping results from the application of pads or a cradle board which constrains cranial vault growth, limiting growth between the frontal and occipital and allowing compensatory growth of the parietals in a mediolateral direction. Two skeletal series including both normal and artificially modified crania are analyzed, a prehistoric Peruvian Ancon sample (47 normal, 64 modified crania) and a Songish Indian sample from British Columbia (6 normal, 4 modified). Three-dimensional coordinates of 53 landmarks were measured with a diagraph and used to form 9 finite elements as a prelude to finite element scaling analysis. Finite element scaling was used to compare average normal and modified crania and the results were evaluated for statistical significance using a bootstrap test. Fronto-occipitally reshaped Ancon crania are significantly different from normal in the vault, cranial base, and face. The vault is compressed along an anterior-superior to posterior-inferior axis and expanded along a mediolateral axis in modified individuals. The cranial base is wider and shallower in the modified crania and the face is foreshortened and wider with the anterior orbital rim moving inferior and posterior towards the cranial base. The Songish crania display a different modification of the vault and face, indicating that important differences may exist in the morphological effects of fronto-occipital reshaping from one group to another.     

DNA-vaccination via tattooing induces stronger humoral and cellular immune responses than intramuscular delivery supported by molecular adjuvants

Tattooing is one of a number of DNA delivery methods which results in an efficient expression of an introduced gene in the epidermal and dermal layers of the skin. The tattoo procedure causes many minor mechanical injuries followed by hemorrhage, necrosis, inflammation and regeneration of the skin and thus non-specifically stimulates the immune system. DNA vaccines delivered by tattooing have been shown to induce higher specific humoral and cellular immune responses than intramuscularly injected DNA. In this study, we focused on the comparison of DNA immunization protocols using different routes of administrations of DNA (intradermal tattoo versus intramuscular injection) and molecular adjuvants (cardiotoxin pre-treatment or GM-CSF DNA co-delivery). For this comparison we used the major capsid protein L1 of human papillomavirus type 16 as a model antigen. L1-specific immune responses were detected after three and four immunizations with 50 μg plasmid DNA. Cardiotoxin pretreatment or GM-CSF DNA co-delivery substantially enhanced the efficacy of DNA vaccine delivered intramuscularly by needle injection but had virtually no effect on the intradermal tattoo vaccination. The promoting effect of both adjuvants was more pronounced after three rather than four immunizations. However, three DNA tattoo immunizations without any adjuvant induced significantly higher L1-specific humoral immune responses than three or even four intramuscular DNA injections supported by molecular adjuvants. Tattooing also elicited significantly higher L1-specific cellular immune responses than intramuscularly delivered DNA in combination with adjuvants. In addition, the lymphocytes of mice treated with the tattoo device proliferated more strongly after mitogen stimulation suggesting the presence of inflammatory responses after tattooing. The tattoo delivery of DNA is a cost-effective method that may be used in laboratory conditions when more rapid and more robust immune responses are required.     

Collagen modifications after deep cutaneous burns in rats]

Acid extracted collegen is prepared from rat skin 48 hours after burning ; the material obtained is purified by salt precipitation and fractionnated by carboxymethylcellulose chromatography as described by Piez (3). Control samples are studied by a similar procedure. It can be concluded that after burning, rat skin collagen becomes insoluble ; the result seems to be consistent with intra and intermolecular crosslinks formation.     

Costimulation blockade, busulfan, and bone marrow promote titratable macrochimerism, induce transplantation tolerance, and correct genetic hemoglobinopathies with minimal myelosuppression.

Mixed hemopoietic chimerism has the potential to correct genetic hemological diseases (sickle cell anemia, thalassemia) and eliminate chronic immunosuppressive therapy following organ transplantation. To date, most strategies require either recipient conditioning (gamma-irradiation, depletion of the peripheral immune system) or administration of "mega" doses of bone marrow to facilitate reliable engraftment. Although encouraging, many issues remain that may restrict or prevent clinical application of such strategies. We describe an alternative, nonirradiation based strategy using a single dose of busulfan, costimulation blockade, and T cell-depleted donor bone marrow, which promotes titratable macrochimerism and a reshaping of the T cell repertoire. Chimeras exhibit robust donor-specific tolerance, evidenced by acceptance of fully allogeneic skin grafts and failure to generate donor-specific proliferative responses in an in vivo graft-versus-host disease model of alloreactivity. In this model, donor cell infusion and costimulation blockade without busulfan were insufficient for tolerance induction as donor-specific IFN-gamma-producing T cells re-emerged and skin grafts were rejected at approximately 100 days. When applied to a murine beta-thalassemia model, this approach allows for the normalization of hemologic parameters and replacement of the diseased red cell compartment. Such a protocol may allow for clinical application of mixed chimerism strategies in patients with end-stage organ disease or hemoglobinopathies.     

Advancement of the orbits and the midface in one piece, combined with frontal repositioning, for the correction of Crouzon's deformities

We describe the use of radical craniofacial osteotomies, to improve the correction of exorbitism and to obtain better esthetic results in children with Crouzon's syndrome. We suggest some minor modifications to improve the fixation and the mechanical stability of the mobilized segments of the skull. Our procedure consists, essentially, of advancement of both orbits and the midface in one piece, plus advancement and reshaping of the frontal area. The results obtained by this technique, in children with a Crouzon's deformity without open bite, have been most satisfactory. We believe the satisfactory resultant appearance will be maintained during and after growth of the face, although these children have not been followed long enough yet to ascertain this with certainty.     

Pinch skin grafting or porcine dermis in venous ulcers: a randomised clinical trial.

Chronic venous ulcers are common, and even with effective compression or elevation large ulcers may take months to heal. Pinch skin grafting may allow healing from epithelial islands throughout the surface area of the ulcer, and a prospective randomised trial was therefore conducted comparing this treatment with porcine dermis dressings. Most patients were treated as outpatients, 25 ulcers being randomised to treatment with pinch skin grafts and 28 to treatment with porcine dermis. Though the groups were well matched, the mean healing rate in the first week was 15 cm2 for pinch skin grafts compared with 3.5 cm2 with porcine dermis (p less than 0.02). By life table analysis 64% of ulcers treated by pinch grafts were healed at six weeks and 74% by 12 weeks compared with 29% and 46% of ulcers, respectively, treated with porcine dermis dressings (chi2 = 4.1; p less than 0.05). All ulcers that failed to heal within 12 weeks included an area posterior to the medial malleolus, where local compression may have been inadequate. Pinch skin grafting improves the rate of healing in large venous ulcers and is a simple technique that may be performed as an outpatient procedure under local anaesthesia.     

Fabrication, quality assurance, and assessment of cultured skin substitutes for treatment of skin wounds

Advances in treatment of skin wounds depend on demonstration of reduced morbidity or mortality either during or after hospitalization. Tissue engineering of skin grafts from cultured cells and biopolymers permits greater amounts of grafts from less donor tissue than conventional procedures. Autologous keratinocytes and fibroblasts isolated from epidermis and dermis of skin may be combined with collagen-based substrates to generate cultured skin substitutes (CSS) with epidermal and dermal components. By regulation of culture conditions, CSS form epidermal barrier and basement membrane, and release angiogenic factors that stimulate vascularization. Prototypes of CSS may be tested for safety and efficacy by grafting to athymic mice which do not reject human tissues. Clinical application of CSS requires establishment of quality assurance assessments, such as, epidermal barrier by measurement of surface hydration, and anatomy by standard histology. Medical benefits of tissue engineered skin for treatment of burns are evaluated quantitatively by the ratio of healed skin to donor skin, and qualitatively by the Vancouver Scar Scale. These benefits may also be extended to other medical conditions including chronic wounds and reconstructive surgery.