[Performances of breast magnetic resonance imaging in the context of neoadjuvant chemotherapy for breast cancer to predict pathological complete response]. / Performances de l'imagerie par résonance magnétique au cours des chimiothérapies néoadjuvantes du cancer du sein pour prédire la réponse pathologique complète.

PURPOSE: The objective of this work was to estimate the reliability of MRI after neoadjuvant chemotherapy (NAC) for breast cancer to detect a residual tumour by comparing the tumoral size measured by MRI with the histological size. We also estimated the concordance of diagnosis of complete pathological response between histological examination and MRI. MATERIALS AND METHODS: We included all the patients who received a neoadjuvant chemotherapy for breast cancer in the university hospital of Tours from January, 2008 to December 31st, 2012 and in the comprehensive cancer centre of Rennes from January, 2008 till May 31st 201. We considered that the pathological response was complete (pCR) when there was no residual invasive tumour in the mammary surgical specimen. RESULTS: Two hundred and fifty-one women who received NAC for a non-metastatic breast cancer were included in the study: 103 in Tours and 148 in Rennes. Two women (0.8%) refused breast surgery whatever the type. One hundred and twenty-three (49%) women had a breast conservative surgery. One hundred and fifteen (45.8%) had a mastectomy and 11 (4.4%) had breast conservative surgery followed by mastectomy for positive margins. A complete pathological response was present in 54 cases (21.5%). We did not found any significant difference between characteristics of patients with pCR or not. CONCLUSION: Breast MRI remains the most performing examination to evaluate the initial tumoral size and the residual tumour after NAC, but does not add any value at mid or at the end of treatment for the patients to whom a mastectomy is decided at presentation. The correlation between the breast MRI and the histology size is not perfect, but at the moment, MRI stills of the most performing examination to predict the pCR.

Brainstem neurons survive the identical ischemic stress that kills higher neurons: insight to the persistent vegetative state.

Global ischemia caused by heart attack, pulmonary failure, near-drowning or traumatic brain injury often damages the higher brain but not the brainstem, leading to a 'persistent vegetative state' where the patient is awake but not aware. Approximately 30,000 U.S. patients are held captive in this condition but not a single research study has addressed how the lower brain is preferentially protected in these people. In the higher brain, ischemia elicits a profound anoxic depolarization (AD) causing neuronal dysfunction and vasoconstriction within minutes. Might brainstem nuclei generate less damaging AD and so be more resilient? Here we compared resistance to acute injury induced from simulated ischemia by 'higher' hippocampal and striatal neurons versus brainstem neurons in live slices from rat and mouse. Light transmittance (LT) imaging in response to 10 minutes of oxygen/glucose deprivation (OGD) revealed immediate and acutely damaging AD propagating through gray matter of neocortex, hippocampus, striatum, thalamus and cerebellar cortex. In adjacent brainstem nuclei, OGD-evoked AD caused little tissue injury. Whole-cell patch recordings from hippocampal and striatal neurons under OGD revealed sudden membrane potential loss that did not recover. In contrast brainstem neurons from locus ceruleus and mesencephalic nucleus as well as from sensory and motor nuclei only slowly depolarized and then repolarized post-OGD. Two-photon microscopy confirmed non-recoverable swelling and dendritic beading of hippocampal neurons during OGD, while mesencephalic neurons in midbrain appeared uninjured. All of the above responses were mimicked by bath exposure to 100 µM ouabain which inhibits the Na+/K+ pump or to 1-10 nM palytoxin which converts the pump into an open cationic channel. Therefore during ischemia the Na+/K+ pump of higher neurons fails quickly and extensively compared to naturally resilient hypothalamic and brainstem neurons. The selective survival of lower brain regions that maintain vital functions will support the persistent vegetative state.

Histological features and expression of enzymes implicated in melatonin synthesis in pineal parenchymal tumours and in cultured tumoural pineal cells.

Pineal parenchymal tumours (PPT) are rare neoplasms and there have been few in vitro studies. Their capacity for synthesizing and secreting melatonin has been only partially examined. We investigated the presence of messenger RNA (mRNA) encoding tryptophan hydroxylase (TPH), arylalkylamine N-acetyltransferase (AANAT), hydroxyindol-O-methyltransferase (HIOMT), three enzymes involved in melatonin synthesis, and c-myc, a tumoural marker, in 10 PPT, one papillary tumour of the pineal region (PTPR), cell cultures derived from four PPTs and from three other tumours of the pineal region, and in normal pineal gland. Moreover, protein expression of TPH was investigated in three PPT and PTPR. Quantitative real-time reverse transcriptase-polymerase chain reaction and immunohistochemistry were used and the melatonin production by tumoural cells in vitro was analysed by radioimmunoassay. We showed that all the tumoural tissues and cells contained c-myc mRNA. mRNAs encoding TPH, AANAT and HIOMT were detected in all PPT, suggesting that tumour cells can synthesize melatonin. Only PPT expressed TPH protein. Cultured cells lost expression of transcripts throughout passages even if ultrastructural study revealed the presence of characteristic organelles in these tumoural cells. Nevertheless, the basal secretion of melatonin observed in one PPT culture is in favour of a maintained melatonin production and secretion by tumoural pinealocytes, but melatonin production was not stimulated by a beta noradrenergic agonist. Moreover, PTPR never expressed mRNA encoding TPH, AANAT and HIOMT. Our results may contribute to a better understanding of the biology of PTT and PTPR and may help to the diagnosis of these rare tumours.

[Prospective epidemiological study on cutaneous melanomas in the Vendée area]. / Etude épidémiologique prospective des mélanomes cutanés en Vendée.

INTRODUCTION: The aim of this prospective study was to evaluate the incidence of melanoma in the Vendée area, coastal region of Eastern France, in 1997. PATIENTS AND METHODS: The 16 dermatologists in the Vendée area in 1997, grouped together in a local association, completed a sheet for each patient in whom a new melanoma had been diagnosed. During the same period, the hospital and private pathologists of the Vendée and surrounding areas reported their new cases of melanoma in patients residing at least six months of the year in the area. The data concerning the local population was supplied by the INSEE (French national institute of economic and statistical information) and the CPAM (French health authority) supplied data on the practitioners' activity. The raw and standardized incidence was calculated by the epidemiologist and the statistician of the local Vendée Cancer registry. RESULTS: One hundred fifteen new melanomas were diagnosed in Vendée in 1997, 97 by a dermatologist and 18 declared by a pathologist. There were 36 in situ and 79 invasive melanomas discovered in 68 women and 47 men (sex ratio: 1.46). The standardized rate of incidence according to the world model was of 98/100,000 for women and 7.8/100,000 for men. The frequency curve revealed two peaks: the first at 40-50 years of age and the second at 70-75. The mean Breslow index of the 79 invasive cancers was of 1.62 mm (range: 0.10-12.5 mm). The only statistically significant difference between the melanomas of the men and the women concerned the topography; there were more melanomas on the trunk in men and more melanomas on the lower limbs in the women. Forty-seven Dubreuilh melanomas were reported: 28 in situ and 19 invasive. DISCUSSION: This prospective study determined the incidence of melanoma in Vendée in 1997. The figures have been compared with those of the Vendée cancer registry, founded in April 1997, and with those of various published studies. The comparison was difficult because of the difference in study methodology. The only figures obtained in the same conditions are those of the cancer registry and we compared them with those of the Haut-Rhin, area of Eastern France: the incidence was similar for the women in the two areas and greater for the men in the Haut-Rhin with regard to the invasive melanomas. Conversely, 44 in situ melanomas were declared in the Haut-Rhin over a 3 year period versus 37 in Vendée over a period of 1 year. The increase in the incidence of melanoma in Vendée in the years to come should permit the evaluation of the impact of the various preventive campaigns.

[Prospective clinical study on new cutaneous melanomas in the Vendée area]. / Etude clinique prospective des nouveaux mélanomes en Vendée.

INTRODUCTION: In parallel to an epidemiological study on the new cases of melanoma reported in the Vendée area in 1997, private dermatologists conducted a clinical analysis of the characteristics of melanomas (including Dubreuilh's melanoma) and of the population concerned. PATIENTS AND METHODS: For each patient in whom a melanoma had been diagnosed, the dermatologists completed a standardized sheet that included the identification, phototype (comparing the distribution with that of the local population, studied in all the consultants during a randomly selected week), the personal and family history of melanoma, the existence of severe sunburn and exposure to artificial ultra-violet rays, the screening method, the time lapse before treatment, the impact of information campaigns, the number of nevi, atypical or not, and the characteristics of the tumor. The exeresis method and the histological characteristics (type, Clark's score and Breslow's index) were also noted. RESULTS: In 1997, the 16 local dermatologists diagnosed 97 melanomas in 55 women and 42 men with a mean age of 64 years. There was no statistically significant difference between the melanomas of the men and those of the women, other than their localization. Photoype II was significantly more frequent in the population exhibiting a melanoma. Sun exposure was moderate or intensive for three thirds of the patients. Past history of severe sunburn was twofold more frequent in the patients exhibiting a melanoma on the trunk. The association of many nevi was noted in a little more than half of the patients, and with atypical nevi in 5.2 p.cent. The elements that alerted the patients were recorded. The time lapse before diagnosis was calculated in months for nodular and in years for superficial melanomas. Clark's score and Breslow's index are presented in a table. DISCUSSION: Our study underlines several interesting features: the fairly high mean age on discovery, a high rate of invasive melanomas, and patients less well informed than in the Ile de France area. These results should open the debate on the best way to educate the population concerned so as to reduce the incidence of this malignant tumor.

Establishment of human tumoral ependymal cell lines and coculture with tubular-like human endothelial cells.

Ependymomas, rare neoplasms of the central nervous system, occur predominantly in children. They are highly vascularized, and histological findings show many perivascular rosettes of tumoral cells radially organized around capillaries. Treatment of ependymomas relies on surgery combined with radio- or chemotherapy, but the efficiency of chemotherapy is limited, probably because of their multidrug resistance (MDR) phenotype. Progress in the therapy of these neoplasms is dramatically limited by the absence of cell line models. We established conditions for the long-term culture of human tumoral ependymocytes and their 3D coculture in Matrigel with endothelial cells. Histological, immunological, and ultrastructural studies showed that the morphological features (microvilli, cilia, and caveolae) of these cultured cells were similar to those of the tumor in vivo. The cells expressed potential oncological markers related to the immature state of tumoral cells (nestin and Notch-1), their tumorigenicity [caveolae and epidermal growth factor-receptor (EGF-R)], or the MDR phenotype [P-glycoprotein (P-gp)]. The expression of P-gp, EGF-R, and caveolin-1 by these tumoral ependymocytes could be useful in studies on new drugs. This coculture model might represent a new powerful tool to study new therapeutic delivery strategies in tumoral cells.

Differential expression of somatostatin receptors in medulloblastoma.

OBJECT: Somatostatin receptors have been found on a variety of tumours like neuroendocrine breast or brain tumours. Their detection opens new diagnostic and therapeutic paths. The aim of this work was to investigate their expression in medulloblastomas. METHODS: Using both techniques, reverse transcriptase-polymerase chain reaction and immunohistochemistry, we analysed mRNA of different subtypes of somatostatin receptors in 15 medulloblastomas and the localisation of the subtype SSTR2 receptor at the cellular level in 13 medulloblastomas. All five subtypes mRNA were variably expressed in each medulloblastoma. The signal obtained after Southern blotting for SSTR2 receptor amplification was the highest as compared to the signal obtained for the other receptor subtypes. Immunostaining for SSTR2A receptor was present in every tumour specimen and was specifically located to the cellular membrane of neoplastic cells. No staining was identified at the level of peritumoral veins. CONCLUSION: The evidence of predominant expression of SSTR2 receptors in medulloblastomas opens interesting prospects for their diagnosis and therapy.

Astrocytic alterations induced by HTLV type 1-infected T lymphocytes: a role for Tax-1 and tumor necrosis factor alpha.

In the neurological disease associated with HTLV-1 infected T lymphocytes infiltrated within the CNS are suspected of playing a prominent role in pathogenesis via inflammatory cytokines and the viral protein Tax-1. We hypothesized that T lymphocytes initiate functional perturbation in astrocytes, resulting in neuronal alteration as glial cells have a crucial role in CNS homeostasis. In particular, astrocytes manage the steady state level of glutamate and continuously provide metabolite precursors to neurons and oligodendrocytes. Using a model system of HTLV-1-infected T cells-astrocytes interaction, we show that after contact with T cells, astrocyte acquire a phenotype typical of gliosis: secretion of proinflammatory cytokines (TNF-alpha, IL-1alpha, IL-6) and matrix metalloproteinases (MMP-9, MMP-3). The concomitant increase in the expression of MMPs and of their endogenous inhibitors (TIMP-1 and TIMP-3) suggests a perturbation in MMP/TIMP balance. This may alter the extracellular matrix and, in turn, the cell environment. At a functional level, glutamate transport and catabolism are impaired in astrocytes. A decrease in glutamate uptake is associated with downregulated expression of glutamate transporters GLAST and GLT1. The expression of astrocytic enzyme of glutamate metabolism is modified with up-regulation of glutamine synthetase and down-regulation of glutamate dehydrogenase. The involvement of Tax-1 in these alterations, directly or indirectly via TNF-alpha, is shown. Altered glutamate uptake and catabolism associated with impairment in cell connectivity via MMP/TIMP imbalance could compromise the functional integrity of the CNS in general and that of neurons and oligodendrocytes in particular.

Long-term effects of HTLV-1 on brain astrocytes: sustained expression of Tax-1 associated with synthesis of inflammatory mediators.

HTLV-1 is the causative agent of a chronic neurological disease, TSP/HAM. The persistently activated CTL response to the viral protein Tax-1 suggests the existence of persistent viral replication with continuous expression of Tax-1. Although CD4+ T-cell is the main target for HTLV-1, previous observations have indicated that the astrocyte, the major neural cell in close contact with blood vessel and thus with HTLV-1-infected T-cells infiltrating the CNS, may also be infected. We tested in vitro the hypothesis of persistent/restricted infection in human and rat astrocytes after transient contact with an infectious T-cell line (C91PL). Long-term analysis showed prolonged expression of Tax-1 in astrocytes, associated with secretion of inflammatory mediators (TNFalpha, IL1alpha, MMP-2, and MMP-9). These data suggest a possible contribution of Tax-1-expressing astrocytes to TSP/HAM pathogenesis.

Psychosocial factors at work, smoking, sedentary behavior, and body mass index: a prevalence study among 6995 white collar workers.

This cross-sectional study examined whether psychosocial factors at work were associated with smoking, sedentary behavior, and body mass index. The study population was composed of 3531 men and 3464 women employed as white collar workers in 21 organizations. Data were collected at worksites. Psychological demands and decision latitude at work were measured with the Karasek 18-item questionnaire. Smoking, sedentary behavior, and mean body mass index were compared by quartiles of decision latitude and psychological demands and by job strain categories. Prevalence of smoking, mean number of cigarettes smoked per day, prevalence of sedentary behavior, and mean body mass index were not consistently associated with decision latitude, psychological demands, or high job strain. However, prevalence of smoking was elevated in women belonging to the highest quartile of psychological demands (odds ratio [OR], 1.2; 95% confidence interval [CI], 1.0 to 1.6) and in the active job strain groups in both men (OR, 1.6; 95% CI, 1.2 to 2.1) and women (OR, 1.4; 95% CI, 1.0 to 2.0). Prevalence of sedentary behavior was elevated in men in the lowest quartile of decision latitude (OR, 1.3; 95% CI, 1.0 to 1.7), in the passive group (OR, 1.3; 95% CI, 1.0 to 1.5), and in the high strain group (OR, 1.2; 95% CI, 1.0 to 1.6). In women, this prevalence was elevated in the third quartile of psychological demand (OR, 1.3; 95% CI, 1.1 to 1.6). These results provide only partial support for an association between some psychosocial factors at work and the prevalence of smoking and sedentary behavior.

Work problems after breast cancer: an exploratory qualitative study.

People treated for cancer have reported a variety of problems at work. However, there is little data on work experience after breast cancer, particularly for women treated in recent years. This exploratory qualitative study was conducted among 13 breast cancer survivors who had paid employment at diagnosis, returned to work afterwards, and mentioned work-related problems to a clinic nurse or physician. Unstructured, thematic interviews were undertaken. Qualitative thematic content analysis was conducted to identify and group themes which emerged from participants' discourse. Women in various types of jobs reported experiencing job loss, demotion, unwanted changes in tasks, problems with the employer and co-workers, personal changes in attitudes to work and diminished physical capacity. These work problems also preoccupied people treated for cancer more than two decades ago. New areas of concern also emerged: possible positive and negative effects of learning (implicitly or explicitly) about the diagnosis while at work and lack of discussion with health professionals about work and return-to-work issues, suggesting that health professionals' behaviour may influence women's work experience right from diagnosis. The identification of these new problems and confirmation of previously reported ones underlines the pertinence of determining how important and widespread these problems are in women now being treated for breast cancer.

Co-localization of CD9 and GPIIb-IIIa (alpha IIb beta 3 integrin) on activated platelet pseudopods and alpha-granule membranes.

CD9 is a 24-kDa membrane glycoprotein expressed on the surface of human platelets and potentially involved in cellular activation and adhesion functions. This protein belongs to a recently delineated family of cell-surface antigens that span the membrane four times, called tetraspans, and found mainly in leucocytes and tumour cells. As a first approach to clarify the function of CD9, we used immunoelectron microscopy to determine the localization of this antigen in human platelets, and compared its distribution with that of the GPIIb-IIIa integrin, the platelet receptor for fibrinogen. Monoclonal antibodies against CD9 (MAb7) and GPIIb-IIIa (HP1-1D) coupled to colloidal gold of different sizes (5 and 15 nm) were incubated with intact platelets in suspension or on ultrathin sections of platelets embedded in LR white. CD9 was found in association with GPIIb-IIIa on the inner face of alpha-granule membranes. These two antigens also colocalized on pseudopods of activated platelets and in contact regions between adjacent platelets. CD63, another member of the tetraspan family, was absent from alpha-granules but was associated with lysosomal structures. Flow cytometric analysis of platelet CD9 with a series of monoclonal antibodies revealed an increased expression upon thrombin stimulation, confirming the presence of an intracellular granular pool. The observation that CD9 and GPIIb-IIIa are stored in the same intracellular structures and migrate to the same activation zones after platelet stimulation lends support to previous suggestions of a close association between CD9 and GPIIb-IIIa in human platelets and of a possible involvement of CD9 in adhesive functions of platelets.

S-Endo 1, a pan-endothelial monoclonal antibody recognizing a novel human endothelial antigen.

A murine monoclonal antibody (mAb) S-Endo 1 has been produced to detect circulating endothelial cells detached from blood vessels in pathological conditions. We have demonstrated that the associated-antigen (S-Endo 1 Ag) was highly expressed on human vascular structure irrespective of tissue origin or vessel caliber. Its expression was not restricted to endothelium, since it was also detected at low level on smooth muscle cells, stroma cells and follicular dendritic cells. But its absence on hematopoietic cells made S-Endo 1 a helpful reagent to specifically discriminate endothelium from hematopoietic tissues. Biochemical characterization showed that S-Endo 1 recognizes a monomeric structure of approximately 118 kDa on cultured endothelial cells. S-Endo 1 was submitted to the 5th International Workshop (Boston, 1993) and did not cluster in any of the old or new endothelial clusters discussed at the conference, indicating its unique reactivity. Together with the data presented in this paper, this suggested that S-Endo 1 defines a previously undescribed endothelial molecule.

An electron microscopy study into the mechanism of gene transfer with lipopolyamines.

Cationic amphiphiles have been shown to mediate gene transfer to eukaryotic cells, although the nature and fate of the lipid-DNA complexes is still a matter of debate. Negative staining transmission electron microscopy (TEM) of the complexes in physiological medium, as well as thin-section TEM of transfected cells has been used to visualize the particles and the possible pathways leading to transgene expression. Lipopolyamines form a network of tubular micelles into which plasmid DNA is intertwined and condensed; the cationic particles contain hundreds of plasmid molecules and are heterogeneous with respect to size (0.1-0.5 microgram) and shape. Adherent cells (293M, 3T3, MRC5, primary leptomeningeal cells) take them up readily within minutes by spontaneous endocytosis. Among suspension cells, lymphocytes only incidentally show cytoplasmic inclusions and monocytes degrade the particles by phagocytosis. The marked decrease in transfection efficiency generally observed between adherent and nonadherent cells is thus due to reduce cell binding. This suggests that cationic particles bind to membrane components responsible for Ca2+-mediated cell anchoring to the extracellular matrix. Cation/anion-mediated endocytosis leads to endosomes that are entirely filled with the particles. Consequently, two escape mechanisms may operate: disruption of the lamellar envelope in close contact with tubular micelles, and endosome buffering by the lipopolyamine in response to proton entry, leading to osmotic swelling and endosome rupture. Even for moderately transfected MRC5 cells, 10(2)-10(3) particles are found either free or in cytoplasmic vacuoles 24 h after transfection, highlighting a very inefficient nuclear translocation process. Such high numbers are also the clue to the small concentration window between transfection and cytotoxicity that is often observed with nonviral vectors. Nuclear particle inclusions are sometimes seen, yet it is unclear whether plasmid uncoating (before expression) takes place by anion exchange in the cytoplasm or in the nucleus. The still lower efficiency of free plasmid translocation to the nucleus suggests an active role for the cationic lipid during this step. Although the last stages of the transfection mechanism remain unclear, the present work shows that the major barrier which hampers in vitro gene delivery with cationic vectors is nuclear translocation (and cell entry for nonadherent cells), providing precise targets for the design of improved nonviral vectors.

Localization of mRNA coding for the three subtypes of atrial natriuretic factor (ANF) receptors in rat anterior pituitary gland cells.

Atrial Natriuretic Factor (ANF) action is mediated by highly selective and specific receptors. Three subtypes have been characterized and cloned: ANF receptor-A, -B and -C. These subtypes are all expressed in the anterior pituitary of the rat. In the present study, the mRNA for each subtype was detected by in situ hybridization. The amounts of ANFR-A and -B mRNA were found to be similar, and to be twice that of ANFR-C mRNA. At the ultrastructural level, the three types of ANFR mRNA were expressed in three anterior pituitary cell types, namely lactotrophs, corticotrophs, and gonadotrophs, identified by their hormonal content. No signal was revealed in somatotrophs or thyrotrophs. The different forms of mRNA were similar in terms of subcellular localization: in the cytoplasmic matrix and the nuclear euchromatin. These data indicate that the anterior pituitary is an important target tissue for ANF action.

Demonstration of Rickettsia conorii-induced endothelial injury in vivo by measuring circulating endothelial cells, thrombomodulin, and von Willebrand factor in patients with Mediterranean spotted fever.

The endothelial cell (EC) is the primary target for Rickettsia conorii (RC) in Mediterranean spotted fever (MSF). Clinical manifestations such as thrombosis and vasculitis are mediated by pathologic changes localized in blood vessels. To study the in vivo endothelial injury induced by RC, markers of endothelial damage, including circulating EC (CEC), plasmatic thrombomodulin (TM), and von Willebrand factor (vWF), were investigated in 12 patients with MSF. CEC were counted in whole blood by a new immunomagnetic separation assay using a specific anti-EC antibody, S-Endo 1. Plasmatic TM and vWF antigens were measured by enzyme-linked immunosorbent assay. High levels of CEC and cell fragments were found in patients with a severe or malignant form of MSF. Sequential studies of CEC showed a decrease from 162 +/- 454 cells/mL before treatment to 6 +/- 7 cells/mL during treatment and recovery. Mean plasma TM and vWF levels that were also elevated before therapy (TM, 106 +/- 27 ng/mL; vWF, 420% +/- 164%) decreased progressively (TM, 55 +/- 43 ng/mL; vWF, 148% +/- 26%) during treatment. The measurement of cellular and molecular markers of vascular damage such as CEC, plasmatic TM, and vWF contributes to the definition of the Rickettsia-induced endothelial injury in vivo.

Role of cyclic AMP in promoting the thromboresistance of human endothelial cells by enhancing thrombomodulin and decreasing tissue factor activities.

1. The effects of forskolin, prostaglandin E1 (PGE1), dibutyryl cyclic AMP (db cyclic AMP), dibutyryl cyclic GMP (db cyclic GMP) and 3-isobutyl-l-methyl-xanthine (IBMX) were investigated on the expression of tissue factor and thrombomodulin activities on the surface of human saphenous vein endothelial cells (HSVEC) in culture. 2. Forskolin (10(-6) to 10(-4) M), PGE1 (10(-7) to 10(-5) M) and db cyclic AMP (10(-4) to 10(-3) M) caused a concentration-dependent decrease of cytokine-induced tissue factor activity. 3. Similar concentrations of forskolin, PGE1 and db cyclic AMP enhanced significantly constitutive thrombomodulin activity and reversed the decrease of this activity caused by interleukin-1 (IL-1). 4. IBMX (10(-4) M) decreased tissue factor activity and enhanced the effect of forskolin on tissue factor and thrombomodulin activities. 5. Forskolin (10(-4) M) decreased the IL-1-induced tissue factor mRNA and increased the thrombomodulin mRNA level. IL-1 did not change the thrombomodulin mRNA level after 2 h of incubation with HSVEC in culture. 6. Dibutyryl cyclic GMP (10(-4) M to 10(-3) M) did not influence tissue factor or thrombomodulin activity. 7. Our data suggest that elevation of intracellular cyclic AMP levels may participate in the regulation of tissue factor and thrombomodulin expression, thus contributing to promote or restore antithrombotic properties of the endothelium.

Thrombin regulates tissue factor and thrombomodulin mRNA levels and activities in human saphenous vein endothelial cells by distinct mechanisms.

The effects of thrombin, D-phenylalanyl-L-propyl-L-arginine chloromethyl ketone (PPACK)-inhibited thrombin, and thrombin receptor agonist peptide, SFLLRNPNDKYEPF (SFLL, a portion of the receptor unmasked after thrombin cleavage), on the expression of tissue factor (TF) and thrombomodulin by human saphenous vein endothelial cells (HSVECs) in culture were studied. Unstimulated cells contained very low amounts of TF mRNA as measured by the reverse transcriptase-PCR method. Thrombin treatment increased TF mRNA to 8.0 +/- 1.9 (n = 3) times the control level. The increase was detectable within 2 h and declined to near basal level by 6 h. Induction of TF mRNA was not blocked by cycloheximide, treatment with cycloheximide alone also increased TF mRNA levels, and thrombin in combination with cycloheximide further enhanced the accumulation of TF mRNA. Thrombin caused a 14.5 +/- 1.5-fold (n = 5) increase in TF activity on the surface of HSVECs and a 20.5 +/- 1.4-fold (mean +/- S.D., n = 2) increase in the extracellular matrix. The thrombin-induced effects on TF synthesis could be fully reproduced by the thrombin receptor agonist peptide, SFLL, whereas PPACK-inhibited thrombin did not influence TF expression. Thrombin increased thrombomodulin mRNA to 190 +/- 39% (n = 5) of control levels, whereas PPACK-inhibited thrombin or SFLL did not influence thrombomodulin mRNA levels. In contrast, surface-bound thrombomodulin cofactor activity and thrombomodulin antigen in the cell lysates did not change over 24 h of incubation with thrombin. However, thrombin caused a 2-fold increase in thrombomodulin antigen released into the conditioned medium, and immunoelectron microscopy of HSVECs also demonstrated the presence of thrombomodulin vesicles close to the luminal cell surface in thrombin-treated cultures. The Western blot pattern thrombomodulin in the conditioned medium of untreated and thrombin-treated cells was found to be similar, and soluble thrombomodulin occurred mainly as fragments of the cell-associated form. We conclude that the transcriptional control by thrombin causes an increase in both TF and thrombomodulin mRNA. The increase in TF mRNA levels is also paralleled by an increase in surface expression, is dependent on the proteolytic activity of thrombin, and is mediated by the same receptor as the recently cloned thrombin receptor in platelets. Up-regulation of thrombomodulin mRNA levels by thrombin is distinct from this pathway and is associated with unchanged expression on the cell surface.

Quantitative image analysis of angiogenesis in rats implanted with a fibrin gel chamber.

Angiogenesis, a fundamental process in various physiological and pathological events, is generally studied using in vivo models, such as the chick chorioallantoic membrane or the rabbit cornea, which are difficult to quantitate. We developed the quantitation of angiogenesis in an in vivo model previously described by Dvorak et al [6]. Perforated plexiglass chambers filled with human or rat fibrin were implanted into the dorsal subcutaneous space of a Wistar rat. After four days of implantation, a sequentially organized invasion of the fibrin gel by various blood cell types occurred through the holes and neovascularized granulation tissue buds appeared. These buds presented mature neovessels and a new collagen matrix. Three dimensional computer image analysis of the chambers was performed using macroscopic and microscopic parameters: total vascularized area, bud height and equivalent diameter, number of vessels per bud and percentage of central vessels. The time course of the angiogenic response to rat or human fibrin gel was studied and 14 days was found to be the optimum period of implantation. The bud height and number of neovessels were not found to be significantly different when rat or human fibrin gels were used. This quantitative study was a prerequisite for further investigations of the effects of biological and pharmacological agents on angiogenesis.

Health problems of women employed in jobs involving psychological and ergonomic stressors: the case of garment workers in Québec.

Health problems of women whose jobs involved intense time pressure were evaluated in a study of 800 sewing-machine operators employed in Québec between 1976 and 1985. Information on workers' occupational characteristics were obtained from public records. Symptoms of anxiety and depression, use of medication and disability status were determined by interview. Garment workers had an increased prevalence of slight, moderate and severe disability and higher levels of symptoms of anxiety and depression when compared to workers in other occupations. Garment workers paid piecework rates took medication for stomach problems in greater proportion than workers paid an hourly wage. Furthermore, workers who spend 5-9, 10-14, 15-19 and 20 or more years in piecework had an increased prevalence of severe disability compared to the baseline category 0-4 years, with adjusted risk ratios of 2.2 (95% CI = 1.14-4.6), 3.3 (95% CI = 1.5-6.9), 3.6 (95% CI = 1.5-8.4 and 2.3 (95% CI = 0.8-6.6) respectively, independent of age, smoking habits, education, type of task and total length of employment. These findings suggest that short-term, non-disabling conditions associated with time pressure by previous authors may have more important long-term sequelae then had been previously documented.