Transformation of LMTK- cells with purified HLA class I gene. VI. Serological characterization of HLA-B7 and AW24 molecules

Serological characterization of HLA-B7 and HLA-AW24 class I molecules following transfection of murine LMTK- cells with purified HLA class I genes was performed using human alloantisera. Induction by murine alpha interferon of the expression of class I molecules was required to obtain unambiguous identification of these molecules which appear serologically identical to the HLA-B7 and HLA-AW24 molecules expressed at the surface of human peripheral blood lymphocytes of 20 unrelated individuals. Analysis of the transformed cells with 8 different anti-HLA class I monoclonal antibodies results in the definition of 3 separate clusters of antigenic determinants shared by all HLA class I molecules. These studies further suggest the existence of locus-specific serological reactivities associated either with the HLA-A or with the HLA-B and C gene products.     

综合康复对膝关节功能障碍的影响

目的：探讨综合康复对膝关节功能活动障碍的影响.方法：共选取膝关节功能活动障碍患者138例,随机分为治疗组与对照组,康复组给予关节CPM治疗、关节松动、关节功能锻炼与蜡疗与中频电疗;对照组仅给予未经指导的自我训练.结果：患者经四周治疗后,两组患者膝关节活动范围及膝关节功能均较治疗前明显改善,但治疗组明显优于对照组,（P〈0.05）.结论：综合康复治疗可明显改善膝关节功能障碍患者ROM,促进关节功能进一步恢复.随着交通、建筑施工中意外事故的增加,经常发生外伤所致膝关节内骨折或靠近膝关节部位的骨折,而由此造成的膝关节活动受限甚至关节强直比较常见.若膝关节ROM（range of motio）〈60°,将对患者的日常生活、工作造成较大影响.如何最大程度改善膝关节骨折后功能障碍,提高患者的生活质量,本研究就此进行探讨.     

股骨髁支撑钢板侧方内固定结合CPM治疗股骨远端C型骨折

目的 探讨解剖型股骨髁支撑钢板内固定结合CPM治疗股骨远端C型骨折临床效果。方法 采用解剖型股骨髁支撑钢板侧方内固定治疗股骨远端C型骨折23例，术后结合CPM锻炼。结果 随访3个月-3年，平均15个月。骨折全部愈合。膝关节功能恢复良好，按Merchan等标准，优5例，良13例，优良率为18／23。结论 解剖型股骨髁支撑钢板贴合股骨远端外形，固定稳定可靠，术后3d进行CPM训练，安全有效，并发症少，是治疗股骨远端C型骨折的较好选择。     

Activation requirements and responses to TLR ligands in human CD4 T cells: Comparison of two T cell isolation techniques

Direct regulation of T cell function by microbial ligands through Toll-like receptors (TLR) is an emerging area of T cell biology. Currently either immunomagnetic cell sorting (IMACS) or fluorescence-activated cell sorting (FACS), are utilized to isolate T-cell subsets for such studies. However, it is unknown to what extent differences in T cell purity between these isolation techniques influence T cell functional assays. We compared the purity, response to mitogen, activation requirements, and response to TLR ligands between human CD4⁺ T cells isolated either by IMACS (IMACS-CD4⁺) or by IMACS followed by FACS (IMACS/FACS-CD4⁺). As expected, IMACS-CD4⁺ were less pure than IMACS/FACS-CD4⁺ (92.5% ± 1.4% versus 99.7% ± 0.2%, respectively). Consequently, IMACS-CD4⁺ proliferated and produced cytokines in response to mitogen alone and had lower activation requirements compared to IMACS/FACS-CD4⁺. In addition IMACS-CD4⁺ but not IMACS/FACS-CD4⁺ responses were upregulated by the TLR-4 ligand lipopolysaccharide (LPS). On the other hand, TLR-2 and TLR-5 engagement induced costimulation in both IMACS-CD4⁺ and highly purified IMACS-/FACS-CD4⁺. Altogether these results indicate that small differences in cell purity can significantly alter T cell responses to TLR ligands. This study stresses the importance of a stringent purification method when investigating the role of microbial ligands in T cell function.     

人工膝关节置换术后下肢被动关节活动器的应用和康复护理

人工膝关节置换术(TKA)病人的下肢被动关节活动器(CPM)应用在整个治疗过程中,起着举足轻重的作用,它直接关系到患者肢体及关节功能的恢复。TKA的主要目的是膝关节获得良好的关节功能,减轻患者膝的疼痛,提高患者的生活质量。为此,CPM的正确应用是TKA后所必须的,康复训练直接影响膝关节术后的功能恢复。在进行CPM治疗过程中,做好患者的康复训练,是治疗计划顺利完成的保证。我们对11例TKA患者行CPM治疗和康复训练进行分析和总结。1一般资料2004年5月~2006年12月,行人工膝关节置换11例,男2例,女9例,年龄55~68岁,平均61.5岁。本组类风…     

下肢CPM训练器的研制

基于CPM理论，研制出适用于膝，髋，踝关节功能锻炼的下肢CPM训练器，介绍了该机的基本结构。主要功能，工作原理及临床机理和作用，具体给出了该机的主要技术参数和控制系统的设计方法。     

"Y"形钢板内固定结合CPM治疗肱骨髁部粉碎性骨折

目的 探讨Y形钢板内固定结合CPM功能锻炼治疗肱骨髁部粉碎性骨折的疗效。方法 19例骨折按AO／ASIF分类法：A3型7例，C1型7例，C2型4例，C3型1例。均采用肘后正中纵形切口，骨折复位后用Y形钢板内固定，术后结合上肢CPM进行功能锻炼。结果 平均随访13个月，按Aitken和Rorabeek标准进行肘关节功能评定，优14例，良4例，可1例，无差病例。总优良率为94．7％。无骨不连、骨折畸形愈合、骨化性肌炎、迟发性尺神经炎、内固定失败等发生。结论 采用Y形钢板内固定结合CPM功能锻炼治疗肱骨髁部粉碎性骨折具有固定牢靠、操作简单、肘关节功能恢复满意等优点。     

Diffusion-weighted MR findings of central pontine and extrapontine myelinolysis

Diffusion-weighted MR (DWI) can detect changes in water diffusion associated with cellular dysfunction, which enables the differentiation of cytotoxic edema from vasogenic edema. In this study on DWI findings in central pontine (CPM) and extrapontine myelinolysis (EPM), DWI showed high signal intensities in the bilateral pons, midbrain, and genu of the corpus callosum. The corresponding apparent diffusion coefficient values were rather low. This suggests that cytotoxic edema does in fact exist in CPM and EPM and that DWI can be useful in the rapid diagnosis and prediction of the various types of edema occurring in active demyelinating diseases.     

卷曲霉素不良反应的临床观察

目的　了解卷曲霉素 (CPM)的不良反应。方法 调查61例用卷曲霉素治疗的复治结核病人出现的不良反应。结果 61例中出现14例,发生率22.9%。共出现5种不良反应,其频率依次为肾功能损害、低血钾、第八对颅神经损害、低血钙、局部硬结,出现时间多在给药1个月左右。结论CPM引起的不良反应常见,以肾功能损害及电解质紊乱较重,应引起临床工作者高度重视。     

In situ selectivity profiling and crystal structure of SML-8-73-1, an active site inhibitor of oncogenic K-Ras G12C

Directly targeting oncogenic V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (K-Ras) with small-molecule inhibitors has historically been considered prohibitively challenging. Recent reports of compounds that bind directly to the K-Ras G12C mutant suggest avenues to overcome key obstacles that stand in the way of developing such compounds. We aim to target the guanine nucleotide (GN)-binding pocket because the natural contents of this pocket dictate the signaling state of K-Ras. Here, we characterize the irreversible inhibitor SML-8-73-1 (SML), which targets the GN-binding pocket of K-Ras G12C. We report a high-resolution X-ray crystal structure of G12C K-Ras bound to SML, revealing that the compound binds in a manner similar to GDP, forming a covalent linkage with Cys-12. The resulting conformation renders K-Ras in the open, inactive conformation, which is not predicted to associate productively with or activate downstream effectors. Conservation analysis of the Ras family GN-binding pocket reveals variability in the side chains surrounding the active site and adjacent regions, especially in the switch I region. This variability may enable building specificity into new iterations of Ras and other GTPase inhibitors. High-resolution in situ chemical proteomic profiling of SML confirms that SML effectively discriminates between K-Ras G12C and other cellular GTP-binding proteins. A biochemical assay provides additional evidence that SML is able to compete with millimolar concentrations of GTP and GDP for the GN-binding site.Multiple lines of evidence suggest that if achievable, inhibiting V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (K-Ras) signaling may have therapeutic advantages in cancer. Approximately 30% of all human cancers contain activating Ras mutations, making them one of the most common identifiable molecular drivers of cancer (1, 2). K-Ras mutation–positive tumors tend to be less responsive to current therapy than other biological subtypes, and patients with these tumors have worse cancer outcomes (3, 4). We aim to develop and evaluate direct-acting inhibitors of K-Ras as a therapeutic strategy.Ras proteins are GTPase enzymes that transduce extracellular signals when growth factors bind to extracellular receptors, resulting in cellular responses such as proliferation, apoptosis, and differentiation (5). Activating signals are transmitted when Ras is bound to GTP and cease once the bound GTP is hydrolyzed to GDP. Two structurally dynamic loops, so-called “switch I” (residues 25–40 in K-Ras) and “switch II” (residues 57–75 in K-Ras) form a key portion of the binding interface between K-Ras and a number of regulators and downstream effectors, including Raf kinases, PI3 kinases, and RalGDS (6–8). We hypothesize that to be successful, direct-acting K-Ras inhibitors must alter the conformation of switch I and/or switch II such that it becomes incapable of transmitting activating signals. Because the guanine nucleotide (GN)-binding pocket dictates the switch conformation, we postulate that developing compounds binding to this region will have a high likelihood of modulating K-Ras signaling.Development of GN competitive inhibitors of K-Ras is challenging because GTP and GDP bind with subnanomolar affinity and intracellular concentrations of GTP and GDP are high. Recently, our group (9) and Shokat and coworkers (10) independently developed and reported two classes of compounds that have a direct impact on productive nucleotide binding to the GN-binding site. Both target a common activating mutation, G12C, to achieve irreversible binding. K-Ras G12C is present in an estimated 10–20% of Ras-driven cancers and in roughly 50% of Ras-mutated lung adenocarcinomas (11–13). For lung cancer alone, this means that therapeutics targeting the G12C mutation could treat roughly 25,000 people per year in the United States (14). Mutations at codon 12, along with the other most common cancer-causing Ras mutations at codons 13 and 61, decrease intrinsic GTPase activity to some extent and impair interactions with GTPase-activating proteins that modulate (GTP) hydrolysis. Codon 12 is adjacent to the active site, such that the mutation places a solvent-accessible cysteine near the GN terminal phosphate.We previously reported development of SML-8-73-1 (SML), a GDP analog containing an electrophilic warhead extending from the beta-phosphate that undergoes a Michael reaction addition to Cys-12, forming a stable thioether linkage (9). Even in the presence of large excesses of GDP and GTP, quantitative complete irreversible binding was observed by MS. In biochemical assays, SML prevents K-Ras association with the Ras-binding domain of the downstream effector BRaf. Preliminary cellular tests using a cell-permeable caged prodrug version, SML-10-70-1, demonstrated that treatment of a G12C mutant K-Ras cancer cell line with the SML class of compounds, albeit at a high concentration (100 μM), leads to inactivation of K-Ras and down-regulation of Akt and Erk signaling pathways, demonstrating as a proof of concept that the GN-binding pocket is a viable target for inhibitors of Ras signaling.Here, we provide three lines of evidence to support further the concept of targeting the GN-binding pocket of K-Ras. First, we report high-resolution X-ray crystal structures of K-Ras, including a structure containing the irreversible inhibitor, SML, bound to K-Ras G12C. The models are analyzed with the aim of understanding implications for K-Ras interactions with downstream effectors. Second, we address compound selectivity with MS-based in situ profiling demonstrating that SML preferentially interacts with K-Ras G12C over most other cellular GTP-binding proteins. Finally, we provide additional evidence that in a purified system, SML is able to compete for the GN-binding site of K-Ras G12C in the presence of millimolar concentrations of GTP and GDP, similar to those found in a living cell. The prospects for developing GTPase inhibitors are also broadly considered.