Biochemical characterization of I-Ak proteins from mutant antigen-presenting B-cell--B-lymphoma hybridomas

Chemically induced mutants of an I-Ak,d expressing antigen-presenting B-cell--B-lymphoma hybridoma have recently been generated by immunoselection in vitro and were found to possess alterations in some of their serologically and functionally defined I-Ak region dependent functions. In order to identify at the structural level the origin of the differences in serological and functional properties of these mutants, I-Ak molecules from several of these mutant hybridomas were compared biochemically to wild-type I-Ak polypeptides by two-dimensional gel electrophoresis and high-pressure liquid chromatographic tryptic peptide analyses. Two-dimensional gel electrophoresis indicated that no major structural alterations, resulting in changes in mol. wt or charge, had occurred in the Ak alpha or Ak beta polypeptides from the mutant cells. Likewise, Ak alpha peptide maps of the mutants were indistinguishable from the normal Ak alpha peptide maps. However, two of the three mutants studied did exhibit one additional peptide in their Ak beta peptide maps. These results suggest that the major deficiencies in T-cell-activating functions of these mutants are a result of a limited alteration in the Ak beta polypeptide primary structure.     

Human placentae membrane receptor for IgG—i. Studies on properties and solubilization of the receptor

Characterization of the human placental membrane receptor for human ¹²⁵I-IgG is described. The receptor bound specifically both monomers and aggregates of human IgG. Human colostral IgA, bovine, sheep, pig, and horse IgG were not bound. No effect of pH in the range 6.6–7.4, ionic strength in the range 0.1–0.5, and temperature between 4 and 45°C on the binding was found. A water-soluble fraction containing the active receptor (glycoprotein fraction-PGP) was obtained from the placental membranes using lithium diiodosalicylate. The solubilized receptor interacted with IgG better at 4°C than at 20°C or 37°C. The results on replacement of monomeric IgG by aggregated IgG, and vice versa, suggest that both monomers and aggregates of human IgG, were bound to the same receptor sites. The apparent association constant for monomeric human IgG was 0.86 ± 0.2 × 10⁷ mole^?1, and 2.0 ± 0.16 × 10¹⁵ IgG molecules were bound per l mg of the membrane protein. Formaldehyde (0.1%), 2-mercaptoethanol (50 mM), and periodate (4 mM) showed no effect on the binding properties of the membrane-bound and on the solubilized receptor, as well. Higher concentrations of periodate (10 mM or 20 mM) decreased the binding of IgG to membranes but showed no effect on the water-soluble receptor. Both the membrane-bound and the solubilized receptor were sensitive to papain. Pronose abolished the receptor activity after prolonged proteolysis only. Neuraminidase did not affect the activity of the receptor. The decrease of the binding activity of the membrane-bound receptor by trypsin and phospholipase C was due to a release of a material containing an active receptor. No effect of trypsin or phospholipase C on the activity of solubilized receptor was observed. The results obtained suggest a protein character of the placental Fc receptor. After electrophoresis of ¹²⁵I-labeled solubilized receptor in polyacrylamide gel in the presence of SDS, 2 major protein peaks with molecular weights of 74,000 and 104,000 and 3 minor peaks with molecular weights of 56,000, 144,000, and 163,000 were found.     

Catecholaminergic neurites in senile plaques in prefrontal cortex of aged nonhuman primates

Immunocytochemical studies, using a polyclonal antibody directed against tyrosine hydroxylase, identified catecholaminergic axons in prefrontal cortex of young and aged nonhuman primates. Aged monkeys, who showed cortical senile plaques in silver stains, had swollen tyrosine hydroxylase-immunoreactive axons in neocortex. Some of these abnormal processes were associated with deposits of amyloid (visualized by thioflavin-T fluorescence) and were similar in appearance to neurites demonstrated by silver impregnation methods. This study provides evidence for structural abnormalities in catecholaminergic axons/nerve terminals in the neocortices of aged primates.     

Characterization of major histocompatibility antigens on trinitrophenyl-modified cells.

Cell membrane proteins encoded for by the major histocompatibility complex (MHC)¹ are associated with the antigenic determinant(s) recognized on trinitrophenyl (TNP)-modified cells by syngeneic murine cytotoxic T lymphocytes and by hapten-reactive guinea pig T cells. To investigate the relationship of the TNP moiety on TNP-modified cells to these major histocompatibility antigens, peritoneal exudate cells or splenocytes from two inbred guinea pig strains and one inbred murine strain were TNP-modified, radioiodinated and lysed in detergent. TNP-derivatized proteins were then isolated using an anti-TNP immunoabsorbent, and the presence on TNP-derivatized histocompatibility antigens in the eluted proteins was determined by immunoprecipitation experiments and SDS-polyacrylamide gel electrophoretic analysis. Whereas most of the various histocompatibility antigens examined were found to be TNP-derivatized in amounts proportional to the degree of membrane protein derivation as a whole, only small amounts of TNP-modified strain 2 guinea pig Ia antigens were found, and no hapten-modified strain 13 guinea pig Ia antigens were detected. It is concluded that, in contrast to most MHC gene products, strain 13 Ia antigens are not derivatized on TNP-modified cells and, thus, represent an important exception demonstrating that histocompatibility antigens need not be directly TNP-derivatized for T cell recognition and activation.     

超薄细胞检测及TBS分类法在宫颈癌筛查中的应用

[目的]对超薄细胞检测系统(thinprep pap test,TPTP)及Bethesda(TBS)细胞学分类法在宫颈癌筛查中的应用价值进行综合评价。[方法]对进行TPT检查及TBS细胞学分类的1209例临床资料进行回顾性分析。[结果]标本满意率93．38％；鳞状上皮异常96例中，未明确诊断意义的鳞状细胞(ASCUS)14例；低度鳞状上皮内瘤变(LSIL)55例，活检证实CINⅠ-Ⅱ级46例(83．64％)，CINⅢ级2例(3．64％)；高度鳞状上皮内瘤变(HSIL)10例，活检证实CINⅢ级及原位癌8例(80％)，CINⅠ-Ⅱ级1例(10％)，鳞癌早浸1例(10％)；TBs诊断为鳞癌者17例，均经病理证实，准确率100％。超薄涂片找到腺癌4例，经活检证实宫颈腺癌2例，子宫内膜癌1例．卵巢癌宫颈转移1例，未明确诊断意义的腺细胞(AGCUS)2例病理均阴性；不典型腺上皮3例，活检及诊刮结果为子宫内膜癌1例，宫颈腺癌1例，阴性1例。[结论]TPT技术应用于宫颈癌筛查能明显提高涂片满意率及宫颈异常细胞检出率。TBS报告方法内容直观、易懂、具体，便于细胞学医生与临床医生之间的沟通．增加了标本的可信度。     

液基薄层细胞检测及TBS分类在宫颈癌筛查中的应用

目的 :对液基薄层细胞检测系统 Autocyte Prep L iquid based Cytology Test (L CT)及 the Bethesda sytem(TBS)细胞学分类法在宫颈癌筛查中的应用价值进行综合评价。方法 :采用液基薄层细胞检测系统检测宫颈细胞并进行 TBS细胞学分类诊断 ,将诊断意义不明的不典型鳞状细胞 (ASCUS)以上病变均列为阳性病例 ,且在阴道镜下行活检 ,将细胞学检测结果与活检结果作对比分析。结果 :液基薄层细胞检测法标本满意率 95 % ,180 0例涂片中阳性病例 76例 ,阳性率 4 .2 2 % (76 / 180 0 ) ,对 76例异常者行阴道镜下活检与组织病理学诊断总符合率 81.5 % (6 2 / 76 )。结论 :L CT技术应用于宫颈细胞涂片筛查 ,明显提高了涂片的满意率及宫颈异常细胞检出率。 TBS报告方式内容直观、易懂、具体。 L CT检查异常的病例配合阴道镜病理检查进行最后诊断 ,能及早发现宫内早期病变 ,是防治宫颈癌发生的关键     

新柏氏液基细胞学检查在宫颈病变中的应用

目的探讨TCT技术和TBS方案应用于宫颈病变筛查的可靠性。方法收集我院妇科门诊4629例宫颈液基细胞学标本，采用TBS方案诊断，对170例细胞学阳性片行阴道镜及活检诊断，并对其发病年龄进行分析。结果TCT检出100％（4／4）的鳞状细胞癌（SCC），检出94％（47／50）的鳞状上皮内高度病变（HSIL），78．8％（41／52）的鳞状上皮内低度病变（LSIL）。经X^2检验，液基细胞学对HSII．与阴道镜活检，病理组织学诊断的符合率显著高于LSII，（P＜0．05）。细胞学阳性患者年龄分布主要发生于25-50岁年龄段，特别是35-40岁组发生率最高，达30．59％。结论TCT技术和TBS方案对宫颈病变的筛查具有良好的实用性，对宫颈上皮内瘤变，特别是对HSIL及以上的病变敏感性高。25～50岁年龄段妇女应定期做TCT筛查，对35～40岁组应做重点筛查。     

Probiotics inhibit nuclear factor-kappaB and induce heat shock proteins in colonic epithelial cells through proteasome inhibition

BACKGROUND AND AIMS: The extent and severity of mucosal injury in inflammatory bowel diseases are determined by the disequilibrium between 2 opposing processes: reparative and cytoprotective mechanisms vs. inflammation-induced injury. Probiotics may provide clinical benefit by ameliorating colitis; however, their mechanisms of action remain largely unknown. Our objective was to investigate microbial-epithelial interactions that could explain the beneficial therapeutic effects of probiotics. METHODS: The effect of VSL#3-conditioned media on the nuclear factor-kappaB pathway in young adult mouse colonic epithelial cells was assessed by using monocyte chemoattractant protein-1 enzyme-linked immunosorbent assays; IkappaBalpha, IkappaBbeta, and p105 immunoblot analysis; and nuclear factor-kappaB luciferase reporter gene and proteasome assays. Effects on heat shock proteins were determined by electrophoretic mobility shift assay and immunoblot for heat shock proteins 25 and 72 in young adult mouse colonic cells. Cytoprotection against oxidant injury was determined by chromium 51 release and filamentous and globular actin assays. RESULTS: VSL#3 produces soluble factors that inhibit the chymotrypsin-like activity of the proteasome in gut epithelial cells. Proteasome inhibition is an early event that begins almost immediately after exposure of the epithelial cells to the probiotic-conditioned media. In addition, these bacteria inhibit the proinflammatory nuclear factor-kappaB pathway through a mechanism different from the type III secretory mechanisms described for other nonpathogenic enteric flora. They also induce the expression of cytoprotective heat shock proteins in intestinal epithelial cells. CONCLUSIONS: The resulting inhibition of nuclear factor-kappaB and increased expression of heat shock proteins may account for the anti-inflammatory and cytoprotective effects reported for probiotics and may be a novel mechanism of microbial-epithelial interaction. These effects seem to be mediated through the common unifying mechanism of proteasome inhibition.     

Biosynthesis of collagen I,II, RUNX2 and lubricin at different time points of chondrogenic differentiation in a 3D in vitro model of human mesenchymal stem cells derived from adipose tissue

The first aim of the study was to identify the most appropriate time for differentiation of adipose tissue derived mesenchymal stem cells (MSCs) to chondrocytes, through the self-assembly process. For this purpose, the expression of some chondrocyte markers, such as collagen type I, collagen type II, RUNX2 and lubricin was investigated at different times (7, 14, 21 and 28 days) of chondrogenic differentiation of MSCs, by using immunohistochemistry and Western blot analysis. The second aim of the study was to demonstrate that the expression of lubricin, such as the expression of collagen type II, could be a possible biomarker for the detection of chondrocytes well-being and viability in the natural self-assembling constructs, called ‘cell pellets’. Histology (hematoxylin and eosin) and histochemistry (alcian blue staining) methods were used to assess the chondrogenic differentiation of MSCs. The results showed that after 21 days the differentiated chondrocytes, when compared with MSCs cultured without chondrogenic medium (CD44, CD90 and CD105 positive; CD45, CD14 and CD34 negative), were able to produce significant quantities of collagen type I, collagen type II, and lubricin, suggesting hyaline cartilage formation. During the differentiation phase, the cells showed a reduced expression of RUNX2, a protein expressed by osteoblasts. Our studies demonstrated that 21 days is the optimum time for the implantation of chondrocytes differentiated from adipose tissue-derived MSCs. This information could be useful for the future development of cell-based repair therapies for degenerative diseases of articular cartilage.