Temporal morphologic changes in human colorectal carcinomas following xenografting.

The temporal morphologic changes of human colorectal carcinomas following xenografting into immunosuppressed mice were investigated by the use of light and transmission electron microscopy. The results show that colorectal carcinomas undergo a series of morphologic changes during the initial 30-day period following transplantation. During the initial 1-5-day period the majority of tumor cells die, and during the following 5-10-day period the necrotic debris created during the 1-5-day period is removed by host-supplied inflammatory cells. Only small groups of peripherally placed tumor cells survived at the end of the first 10 days. During the 10-20-day period the tumor cell populations of xenografts were reestablished by a morphologically heterogeneous population of tumor cells, and during the 20-30 day period consolidation of this process continued and some xenografts showed macroscopic evidence of growth. The authors hypothesize that human colorectal carcinomas, like the antecedent epithelium, contain subpopulations of undifferentiated cells that give rise to populations of more-differentiated cells.     

内毒素诱导血小板20kD蛋白磷酸化、钙转运及纤维蛋白原受体显露

本文观察了E.Coli大肠杆菌内毒素对人血小板蛋白磷酸化,钙转运及纤维蛋白原受体的影响,实验采用SDS-凝胶电泳及放射自显影技术分离血小板蛋白并测定其磷酸化程度,结果发现内毒素刺激血小板后,内源性20kD蛋白迅速磷酸化,这一作用在细胞外Ca~(2 )存在时明显加强。同时还观察到血小板对~(45)Ca~(2 )摄取加强,胞浆游离Ca~(2 )升高,血小板结合荧光标记纤维蛋白原的阳性细胞百分率明显提高。提示:内毒素可能通过诱导Ca~(2 )动员,蛋白磷酸化,使血小板纤维蛋白原受体显露,导致血小板聚集。     

Lymphokines and platelets promote human monocyte adherence to fibrinogen and fibronectin in vitro

Monocytes must accumulate in areas of tissue injury and inflammation to effect phagocytosis, antigen presentation, and monokine production. Fibrinogen/fibronectin matrices have been demonstrated in healing wounds and in delayed-type hypersensitivity skin reactions. We have developed an in vitro system for investigation of the ability of fibrinogen and fibronectin matrices to serve as substrata for human peripheral blood monocyte adherence. Monocyte adherence was greatest on matrices of both fibrinogen and fibronectin, less to fibrinogen alone, and least to fibronectin alone. Lymphokines increased adherence of monocytes to all three surfaces but not to albumin-coated surfaces. Addition of platelets also caused a dose-dependent increase in monocyte adherence to all three surfaces. This increased adherence was not a simple function of arachidonic acid metabolites, stable platelet products, nor monocyte binding sites on the platelet membrane. The effect of platelets was not additive to the effect of lymphokines. We conclude that fibrinogen and fibronectin provide a framework for monocyte adherence and that factors present in areas of inflammation and wound healing, such as lymphokines and platelets, can augment this adherence. Such adherence facilitates the transformation of monocytes into macrophages in vitro and may also foster such transformation in vivo.     

Immunofluorescent localization of adhesive glycoproteins in resting and thrombin-stimulated platelets.

The distribution and transport of thrombospondin (TSP), fibrinogen (Fbg), fibronectin (Fn), and Factor VIII-related antigen (VIII:RAg) in resting and thrombin-stimulated platelets was investigated by immunofluorescence microscopy. In resting intact cells, little surface staining was seen for these proteins. In permeable resting cells, punctate staining similar to that reported for platelet factor 4 was observed. Double-label immunofluorescence staining for Fbg and either beta-thromboglobulin (beta TG), TSP, or Fn demonstrated co-localization, indicating their presence in the same intracellular structures. VIII:RAg showed general co-localization; however, the staining was finer, suggesting a possible differential intragranular localization. Thrombin stimulation induced the appearance of larger (approximately 0.5 mu) immunofluorescent masses of these proteins. In thrombin-stimulated cells, co-localization of all proteins in these masses was observed by double label immunofluorescence. Thus, TSP, Fbg, Fn, and beta TG are localized in the same structure in resting cells. Thrombin stimulates formation of common larger masses of these proteins prior to their release, suggesting that they reach the cell surface through a common intermediate.     

Immunohistochemical localization of progesterone receptor in human decidua of early pregnancy.

Rat monoclonal antibodies to human progesterone receptor were used for immunolocalization studies in human decidua of early pregnancy. Frozen sections of 42 specimens of decidua were stained by the peroxidase-antiperoxidase method (PAP). Progesterone receptor was localized exclusively in the nuclei of decidual and myometrial cells with no specific staining in the cytoplasm. In the decidualized endometrium, stroma were always positively stained. Smooth muscle, pericyte and endothelial cells of blood vessels were extensively stained. Glandular epithelia showed variation in staining, which was positive in the basal but very weak or negative in the superficial layer of the decidua. No specific staining could be detected in the control sections. Of special interest was the positive staining of the endothelium of decidual blood vessels, a finding which has not been reported previously. The cells of the inner lining of vessels that stained with the antiprogesterone receptor antibodies were also Factor VIII positive, thus confirming the endothelial nature of these cells. It is concluded from these results that endothelial cells from human first trimester decidua express progesterone receptors.     

Proliferative and morphologic changes in rat colon following bypass surgery.

In this study the proliferative and morphologic changes that occur in the colon of normal and dimethylhydrazine-treated rats following surgical bypass of the middle third of the colon are reported. Proliferative changes were measured by estimating accumulated mitotic indexes following vinblastine treatment and morphologic changes were observed with the use of light microscopy and scanning electron microscopy. Data were collected on Days 0, 7, 14, 30, and 72 after surgery. The results show that surgical bypass produces contrasting effects in the segments proximal to and distal to the suture line. In the proximal segment there was morphologic evidence of hyperplasia, although proliferative activity was unchanged except for an increase at 7 days in normal rats. In the distal segment there was a long-lived increase in the mitotic index, although morphologic changes were not seen. The results for DMH-treated rats were similar to those in normal rats. Groups of isolated dysplastic epithelial cells were often seen in the submucosa adjacent to sutures up to 72 days after surgery. Increased lymphoid infiltration was seen in segments proximal to but not distal to the suture line. It is hypothesized that the different responses of the proximal and distal segments may be related to the different embryologic origins of those segments. It is also hypothesized that the seeding of the submucosa with epithelial cells during suturing may be a factor in tumor recurrence.     

Interactions of thermally denatured fibrinogen on polyethylene with plasma proteins and platelets.

During the investigation of fibrin deposition onto hydrophobic polymers in contact with human blood, a model was developed in which fibrinogen was denatured and irreversibly coated onto a polyethylene surface by heating to 70 degrees C for 10 min. The denatured fibrinogen-polyethylene surface is resistant to fluid wall shear rates of up to 550 s-1 and the fibrinogen does not desorb in the presence of plasma proteins. Compared to uncoated polyethylene, little albumin or fibrinogen adsorbs to heat-denatured fibrinogen. Thrombin binds to the denatured fibrinogen-coated polyethylene with low affinity and also acts on the surface-bound denatured fibrinogen and cleaves fibrinopeptide A (FPA) quantitatively. Washed, 51Cr-labeled platelets do not adhere to the thermally denatured fibrinogen at either low or high shear rates compared to surfaces coated with undenatured fibrinogen (p < 0.01). These observations support the role of the D domain of fibrinogen in platelet adhesion because this is the region that is denatured by heating. In contrast, the E domain of fibrinogen is not altered by heating to 70 degrees C and hence remains susceptible to thrombin and/or plasmin cleavage. The characteristics of this surface are such that it can be used to develop fibrin-coated surfaces for use in studies of thrombus formation on artificial surfaces.     

Subcellular localization of the heparin-neutralizing factor in blood platelets.

1. The distribution of the heparin-neutralizing factor (platelet factor 4, PF4) in subcellular organelles of blood platelets of rabbits and man was investigated. 2. In both species the organelles storing 5-hydroxytryptamine (5-HT storage organelles) contained only trivial amounts of PF4. 3. In contrast, the content of PF4 was highest in the subcellular fractions rich in alpha-granules. 4. In conclusion, PF4 is probably localized in the alpha-granules and therefore the platelets contain at least two types of organelles (5-HT organelles and alpha-granules) capable of releasing their contents in response to the same stimuli, such as exposure to collagen, thrombin, etc.     

Platelet adhesiveness, plasma fibrinogen, and fibrinolytic activity in juvenile-onset and maturity-onset diabetes mellitus.

The nature of vascular disease and its complications are different in juvenile-onset (JOD) and maturity-onset (MOD) diabetes mellitus. In order to explore the disturbances in the coagulation-fibrinolytic system, platelet adhesiveness, plasma fibrinogen, and euglobulin lysis time were estimated in 26 cases of MOD. Two groups of age- and sex-matched controls were also studied. Platelet adhesiveness and plasma fibrinogen were essentially normal while euglobulin lysis time was significantly decreased in the JOD group. The MOD group, on the other hand, showed a reversed pattern in the form of enhanced platelet adhesiveness and plasma fibrinogen and no compensatory increase in fibrinolysis.     

In vivo defibrination results in markedly decreased amounts of fibrinogen in rat megakaryocytes and platelets.

Recently evidence was provided for a pathway whereby circulating fibrinogen enters megakaryocyte granules by an endocytic mechanism. Synthesis of fibrinogen by megakaryocytes has been reported. To determine the relationship between plasma fibrinogen and alpha-granule fibrinogen in megakaryocytes and platelets, the fibrinogen content of these cells was studied in rats defibrinated by use of Ancrod, a thrombinlike enzyme purified from the venom of Agkistrodon rhodostoma. Unlike thrombin, Ancrod does not induce platelet secretion. Rats were injected with Ancrod (50 units/kilogram body weight) at 8-hour intervals for 5 days. There were no significant changes in platelet counts. Blood from the treated rats failed to clot, and plasma fibrinogen levels were less than 15 mg/dl. Bone marrow from defibrinated rats and untreated control rats was stained immunohistochemically for fibrinogen and two other alpha-granule proteins, albumin and platelet factor 4 (PF4), in plastic-embedded sections. The presence of these three proteins in platelets was detected by Western blots. Only trace amounts of fibrinogen were detected in megakaryocytes and platelets from defibrinated rats, but fibrinogen in control megakaryocytes and platelets was readily demonstrated. However defibrinated and control rats did not differ in albumin and PF4 content in megakaryocytes and platelets. It is concluded that a major portion of rat platelet fibrinogen is derived from plasma by endocytosis by megakaryocytes.     

Bound fibrinogen distribution on stimulated platelets. Examination by confocal scanning laser microscopy.

Previous studies have suggested that qualitative changes in platelet bound fibrinogen modulate platelet aggregation. The present study used confocal scanning laser microscopy to further evaluate post-ligand binding events over a 60-minute time course. When fluorescein isothiocyanate (FITC)-streptavidin was added to ADP-stimulated platelets 1 minute after biotinylated fibrinogen binding at 22 degrees C, bound fibrinogen was found in variously sized patches on the cell surface. When streptavidin was added 60 minutes later, bound fibrinogen had been cleared from the platelet surface and was observed in clusters penetrating into platelets to various extents. ADP-activated platelets did not stain with a monoclonal antibody against CD62 suggesting that platelets were not permeabilized during the experiment and had not released alpha-granules. Additional studies using either biotinylated fibrinogen that had been prelabeled with FITC-streptavidin or FITC-labeled fibrinogen revealed similar patterns of platelet-associated fibrinogen clearance and redistribution. Pretreatment of platelets with cytochalasin D prevented this redistribution. Dual labeling experiments using biotinylated fibrinogen and FITC-streptavidin as well as a monoclonal anti-GPIIIa antibody labeled with rhodamine-conjugated anti-mouse IgG demonstrated the co-localization of fibrinogen and GPIIIa. Similar observations were made with fibrinogen bound to thrombin-stimulated platelets. In contrast, fibronectin bound to thrombin-activated platelets retained a predominantly surface membrane distribution under identical experimental conditions. Since surface-cleared fibrinogen was accessible to exogenous FITC-streptavidin under conditions that did not lead to platelet permeabilization, the data suggest fibrinogen deposition in compartments that are accessible to the extracellular milieu. This is consistent with the ability of exogenous plasmin to completely remove cleared fibrinogen pools without detectable fibrinogen reexpression on the platelet surface or alpha-granule secretion. The data provide morphological evidence for the selective, GPIIb-IIIa mediated, actin-dependent clearance of bound fibrinogen from the activated platelet surface, suggesting a mechanism for preventing and limiting thrombus development.     

Cellular localization of the Trk neurotrophin receptor family in human non-neuronal tissues.

We investigated the distribution of the high-affinity neurotrophin receptors TrkA, TrkB, and TrkC in a wide range of normal non-neuronal tissues of adult human by immunohistochemical methods. Trk immunoreactivity (IR) was detected at various levels in all tissues examined, except for the heart and liver. The gastric parietal cells showed strong TrkA and TrkC IR and all of the Trks had IR for the putative intestinal neuroendocrine cells. In the pancreas, TrkA and TrkC IR was detected in the sub-intralobular ducts, whereas TrkB IR was found specifically in the alpha-islet cells. The lymph node and spleen exhibited TrkB IR in monocytes/macrophages. The adrenal cortex showed selective TrkA IR with TrkC IR in the medulla. In the reproductive system, TrkA IR was detected in the prostatic epithelial cells, TrkC in the ovarian theca and granulosa cells, TrkA and TrkC in the secretory-phase endometrium, and TrkA in the mammary ducts. The kidney showed strong TrkA and TrkC IR in it tubules, but no Trk receptors were present in the glomeruli. In the skin, TrkA and TrkB/TrkC were present in the basal and granular layers of the epidermis, respectively.     

Immunohistochemical localization of neurotrophin receptor proteins in human skin

The target organs of neurotrophin-dependent sympathetic and sensory neurons, including the skin, synthesize and release neurotrophins, primarily NGF. Neurotrophins undergo retrograde axonal transport, and exert specific function in the perikarya of the responsive neurons. Moreover, evidence exists for an autocrine and/or paracrine function of neurotrophins in the skin. This study analyses the immunohistochemical localization of low (gp75) and high-affinity (gp140 trkA, gp145trkB and gp145trkC) neurotrophin receptor proteins in the human glabrous skin. We consider that the expression of neurotrophin receptors may be indicative of neurotrophin activity. Specific gp75 and gp140trkA-like immunoreactivity (IR) were observed highly co-localized in (1) epidermis, primarily in the basal keratinocytes; 2) sweat glands; (3) blood vessel walls, mainly in the muscular layer; (4) Schwann and perineurial cells of nerve trunks; (5) periaxonic cells forming sensory nerve formations (Meissner's and Pacini's corpuscles); (6) large axons of nerve bundles and of sensory corpuscles; gp145trkB-like and gp145trkC-like were found labelling nerve fibers and sensory nerve formations, as well as blood vessels and sweat glands, but not epidermic cells. The results suggest that, in addition to the well known neurotrophic functions, neurotrophins may also regulate unknown functions in non-nervous cutaneous cells, which are targets for neurotrophin-dependent sympathetic and sensory neurons.     

Unilateral obstructive nephropathy in the rabbit. II. Late morphologic changes

The late histologic and ultrastructural effects of complete unilateral ureteral obstruction in the rabbit kidney were studied from 7 to 32 days postobstruction. Papillary necrosis occurred in all animals by the 7th day. There was progressive widening of the cortical interstitial space with proliferation of fibroblasts. Increased interstitial collagen fibers first became apparent on the 7th day and showed a progressive increase with time. The fibroblasts underwent myofibroblast transformation beginning on the 7th day. All segments of the nephron showed ultrastructural evidence of sublethal cell injury with loss of cell specialization. The dedifferentiation of the tubular cells was associated with a repetitive formation of new lamellae of basement membrane on the luminal side of the original basement membranes beginning on the 16th day of obstruction. These morphologic changes are discussed in terms of the physiologic changes previously reported in this model and the known clinical abnormalities found in obstructive uropathy.     

PTA1在人巨核母细胞系HEL细胞和血小板的表达及分布

应用抗人血小板和Ｔ细胞活化抗原１单克隆抗体间接免疫荧光染色和流式细胞仪分析，以及胶体金免疫电镜技术，检测了人巨核母细胞系ＨＥＬ细胞和血小板膜表面ＰＴＡ１分子的表达水平，并对静止与活化血小板膜表面ＰＴＡ１分子的表达和分布进行了比较。     

Residence-time dependent changes in fibrinogen adsorbed to polymeric biomaterials.

It has generally been accepted that biomaterials adsorbing the least amount of the plasma protein fibrinogen following exposure to blood will support less platelet adhesion and therefore exhibit less thrombogenicity. Several studies suggest, however, that the conformation or orientation of immobilized fibrinogen rather than the total amount adsorbed plays an important role in determining the blood compatibility of biomaterials. The purpose of this study was to investigate time-dependent functional changes in fibrinogen adsorbed to polytetrafluoroethylene (PTFE), polyethylene (PE), and silicone rubber (SR). Fibrinogen was adsorbed to these materials for 1 min and then allowed to 'reside" on the surfaces for up to 2 h prior to assessing its biological activity. Changes in fibrinogen reactivity were determined by measuring the adhesion of 51Cr-labeled platelets, the binding of a monoclonal antibody (mAb) directed against an important functional region of the fibrinogen molecule (the gamma-chain dodecapeptide sequence 400-411), and the ability of blood plasma to displace previously adsorbed fibrinogen. Platelet adhesion differed among the polymeric materials studied, and PTFE and PE samples exhibited a small decrease in adhesion with increasing fibrinogen residence time. Platelet adhesion to SR was the least among all materials studied and showed no variation with residence time. When using PTFE and SR as substrates, mAb recognition of adsorbed fibrinogen did not change with residence time whereas that on PE decreased slightly. The mAb binding was least to fibrinogen adsorbed to SR, which is in agreement with the platelet adhesion results. Finally, the ability of plasma to displace previously adsorbed fibrinogen decreased dramatically with increasing residence time on all materials. These in vitro studies support the hypothesis that fibrinogen undergoes biologically significant conformational changes upon adsorption to polymeric biomaterials, a phenomenon that may contribute to the hemocompatibility of the materials following implantation in the body.