Course of post-traumatic stress disorder following war in the Balkans: 1-year follow-up study.

BACKGROUND: Prevalence rates of post-traumatic stress disorder (PTSD) following the experience of war have been shown to be high. However, little is known about the course of the disorder in people who remained in the area of conflict and in refugees. Method We studied a representative sample of 522 adults with war-related PTSD in five Balkan countries and 215 compatriot refugees in three Western European countries. They were assessed on average 8 years after the war and reinterviewed 1 year later. We established change in PTSD symptoms, measured on the Impact of Events Scale - Revised (IES-R), and factors associated with more or less favourable outcomes. RESULTS: During the 1-year period, symptoms decreased substantially in both Balkan residents and in refugees. The differences were significant for IES-R total scores and for the three subscales of intrusions, avoidance and hyperarousal. In multivariable regressions adjusting for the level of baseline symptoms, co-morbidity with depression predicted less favourable symptom change in Balkan residents. More pre-war traumatic events and the use of mental health services within the follow-up period were associated with less improvement in refugees. CONCLUSIONS: Several years after the war, people with PTSD reported significant symptom improvement that might indicate a fluctuating course over time. Co-morbid depression may have to be targeted in the treatment of people who remained in the post-conflict regions whereas the use of mental health services seems to be linked to the persistence of symptoms among refugees.

Proteolysis and cell migration: creating a path?

Both serine and metalloproteinases have been implicated in the complex integrated events underlying cell migration but no definitive single mechanism has emerged. Work over the past two years linking both membrane and soluble proteinases with integrins and other adhesion proteins and with intracellular signalling systems could herald the beginnings of a potential expansion of our understanding of the role and regulation of proteolysis in cell migration.

Myelin-specific proteins and glycolipids in rat Schwann cells and oligodendrocytes in culture.

We have used antibodies to identify Schwann cells and oligodendrocytes and to study the expression of myelin-specific glycolipids and proteins in these cells isolated from perinatal rats. Our findings suggest that only Schwann cells which have been induced to myelinate make detectable amounts of galactocerebroside (GC), sulfatide, myelin basic protein (BP), or the major peripheral myelin glycoprotein (P0). When rat Schwann cells were cultured, they stopped making detectable amounts of these myelin molecules, even when the cells were associated with neurites in short-term explant cultures of dorsal root ganglion. In contrast, oligodendrocytes in dissociated cell cultures of neonatal optic nerve, corpus callosum, or cerebellum continued to make GC, sulfatide and BP for many weeks, even in the absence of neurons. These findings suggest that while rat Schwann cells require a continuing signal from appropriate axons to make detectable amounts of myelin-specific glycolipids and proteins, oligodendrocytes do not. Schwann cells and oligodendrocytes also displayed very different morphologies in vitro which appeared to reflect their known differences in myelinating properties in vivo. Since these characteristic morphologies are maintained when Schwann cells and oligodendrocytes were grown together in mixed cultures and in the absence of neurons, we concluded that they are intrinsic properties of these two different myelin-forming cells.

The Schwann cell precursor and its fate: a study of cell death and differentiation during gliogenesis in rat embryonic nerves.

We have characterized a cell, the Schwann cell precursor, that represents a distinct intermediate differentiation stage in the process by which Schwann cells are generated from neural crest cells. The Schwann cell precursor shows radical differences from Schwann cells which include death regulation, antigenic phenotype, pattern of cell-cell interaction, migratory behavior, and morphology. In the nerves of the rat hind limb, Schwann cells are irreversibly generated from these during a brief period, essentially embryonic days 15-17. We also provide evidence that the survival of Schwann cell precursors is regulated by neurons and identify basic fibroblast growth factor as a potential key regulator of apoptosis in Schwann cell precursors and of precursor to Schwann cell conversion. These findings have implications for our understanding of gliogenesis in the peripheral nervous system.

Collagen-mediated dispersion of NBT-II rat bladder carcinoma cells.

During metastatic spread, locomotion mediated by extracellular matrix components of basement membranes and connective tissues has been invoked as a prerequisite to invasion. We studied the interactions of the rat bladder carcinoma cell line NBT-II with fibronectin, laminin, and collagens (types I, III, IV, and V). They all promoted cell attachment and spreading. To analyze their scatter potential, we studied epithelial outgrowth and/or peripheral cell dispersion from tumor aggregates. All matrix components allowed partial collapse of the aggregate and the appearance of a cellular monolayer forming a halo around the aggregate. No peripheral cell dispersion occurred on fibronectin and laminin. Collagens (especially types I and III) promoted the dispersion of peripheral NBT-II cells with various speeds of locomotion, as revealed by time-lapse videomicroscopy. With the exception of cells at the periphery on collagens, cells inside the halo did not exchange neighbors, migrated transiently as an epithelial sheet during halo formation, and finally remained stationary. These effects were reproduced with NBT-II tumor fragments obtained from nude mice. Tumor cells were linked together with desmosomes (as revealed by immunoreactivity against desmoglein). Migration on collagens correlated with the mechanical disruption of intercellular contacts and consequently with the progressive disappearance of desmoglein immunoreactivity. Immunofluorescence studies also revealed a reduced expression of the epithelium-specific cell adhesion molecule liver cell adhesion molecule after contact with collagens. These results suggest that direct interactions with collagens may favor single cell infiltration by bladder carcinoma.

Mechanisms for pro matrix metalloproteinase activation.

The activation of pro matrix metalloproteinases (MMPs) by sequential proteolysis of the propeptide blocking the active site cleft is regarded as one of the key levels of regulation of these proteinases. Potential physiological mechanisms including cell-associated plasmin generation by urokinase-like plasminogen activator, or the action of cell surface MT1-MMPs appear to be involved in the initiation of cascades of pro MMP activation. Gelatinase A, collagenase 3 and gelatinase B may be activated by MT-MMP based mechanisms, as evidenced by both biochemical and cell based studies. Hence the regulation of MT-MMPs themselves becomes critical to the determination of MMP activity. This includes activation, assembly at the cell surfaces as TIMP-2 complexes and subsequent inactivation by proteolysis or TIMP inhibition.

GABA uptake by purified rat schwann cells in culture.

Purified rat Schwann cells maintained in culture for up to 6 months retained their ability to take up the neurotransmitter gamma-aminobutyric acid (GABA) by a high-affinity mechanism. Although cultured fibroblasts also accumulated GABA, they did so by a low affinity mechanism. These results indicate that Schwann cells continue to express a high affinity GABA transport system in the absence of signals from neurons.

Mitogens for glial cells: a comparison of the response of cultured astrocytes, oligodendrocytes and Schwann cells.

We have identified two growth factors for cultured rat astrocytes: fibroblast growth factor, a peptide derived from either whole bovine brain, or pituitaries, and a growth factor in extracts of bovine pituitary which was previously identified as a Schwann cell mitogen. Oligodendrocytes in primary cultures derived from neonatal rat central nervous system divide only rarely if at all. These growth factors did not stimulate primary oligodendrocytes to divide. Occasionally cells found in suspension in long-term cultures of the central nervous system were enriched for cells which were identified as oligodendrocytes by the presence of galactocerebroside on their surface and myelin basic protein in their cytoplasm. When provided with a monolayer of irradiated 3T3 cells, these oligodendrocytes were able to spread out and extend elaborate branched processes typical of oligodendrocytes in the primary cultures. Unlike their counterparts in the primary cultures, these suspension-derived oligodendrocytes are capable of cell division as demonstrated by the uptake of [3H]thymidine and autoradiography.

Dissemination of wear particles to the liver, spleen, and abdominal lymph nodes of patients with hip or knee replacement.

BACKGROUND: The importance of particles generated by wear and corrosion of joint replacement prostheses has been understood primarily in the context of the local effects of particle-induced periprosthetic osteolysis and aseptic loosening. We studied dissemination of wear particles in patients with total hip and knee replacement to determine the prevalence of and the histopathological response to prosthetic wear debris in the liver, spleen, and abdominal para-aortic lymph nodes. METHODS: Postmortem specimens from twenty-nine patients and biopsy specimens from two living patients with a failed replacement were analyzed. Specimens of tissue obtained from the cadavera of fifteen patients who had not had a joint replacement served as controls. The concentration of particles and the associated tissue response were characterized with the use of light microscopy of stained histological sections. Metallic particles were identified by electron microprobe analysis. Polyethylene particles were studied with the use of oil-red-O stain and polarized light microscopy. The composition of polyethylene particles was confirmed in selected cases by Fourier transform infrared spectroscopy and hot-stage thermal analysis. Twenty-one of the patients studied post mortem had had a primary total joint replacement. Eleven of them had had a hip prosthesis for a mean of sixty-nine months (range, forty-three to 171 months), and ten had had a knee replacement for a mean of eighty-four months (range, thirty-one to 179 months). The other eight patients studied post mortem had had a hip replacement in which one or more components had loosened and had been revised. The mean time between the initial arthroplasty and the time of death was 174 months (range, forty-seven to 292 months), and the mean time between the last revision procedure and the time of death was seventy-one months (range, one to 130 months). RESULTS: Metallic wear particles in the liver or spleen were more prevalent in patients who had had a failed hip arthroplasty (seven of eight) than in patients who had had a primary hip (two of eleven) or knee replacement (two of ten). The principal source of wear particles in the majority of these patients involved secondary nonbearing surfaces rather than wear between the two primary bearing surfaces as intended. In one living patient, dissemination of titanium alloy particles from a hip prosthesis with mechanical failure was associated with a visceral granulomatous reaction and hepatosplenomegaly, which required operative and medical treatment. Metallic wear particles were detected in the paraaortic lymph nodes in 68 percent (nineteen) of the twenty-eight patients with an implant from whom lymph nodes were available for study. In 38 percent (eleven) of all twenty-nine patients with an implant who were studied post mortem, metallic particles had been further disseminated to the liver or spleen, where they were usually found within small aggregates of macrophages occurring as infiltrates without apparent pathological importance. Polyethylene particles elicited a similar response. They were identified in the paraaortic lymph nodes of 68 percent (nineteen) of the twenty-eight patients and the liver or spleen of 14 percent (four) of the twenty-nine patients. The majority of the disseminated wear particles were less than one micrometer in size. Currently available methods lack the sensitivity and specificity necessary to detect very low concentrations of submicrometer polyethylene particles and probably underestimated the prevalence of polyethylene wear debris in the liver and spleen. CONCLUSIONS: In this study, systemic distribution of metallic and polyethylene wear particles was a common finding, both in patients with a previously failed implant and in those with a primary total joint prosthesis. The prevalence of particles in the liver or spleen was greater after reconstructions with mechanical failure. (ABSTRACT TRUNCATED)

The role of plasminogen in cell-mediated collagen degradation.

The ability of VX2 tumour cells and chondrocytes to degrade radiolabelled collagen films was shown to be dependent on the presence of the serum proteinase plasminogen. Degradation of collagen films in the presence of plasminogen was inhibited by addition of exogenous TIMP indicating that such lysis was mediated by collagenase. VX2 cells required ten times less plasminogen than chondrocytes to effect comparable degradation; this result was probably related to the observation that VX2 cells did not synthesize the specific tissue inhibitor of metalloproteinases, TIMP.

Expression of transfected transforming growth factor alpha induces a motile fibroblast-like phenotype with extracellular matrix-degrading potential in a rat bladder carcinoma cell line.

Acquisition of cell motility is often correlated with the malignant progression of a transformed cell. To investigate some of the mechanisms involved in the development of a migratory state, we transfected the NBTII rat carcinoma cell line, which forms stationary epithelial clusters in culture, with the gene encoding human transforming growth factor alpha (TGF alpha). Expression of TGF alpha in NBTII cells resulted in cells of motile and vimentin-positive phenotype with internalized desmosomal components, analogous to the treatment of cells with exogenous TGF alpha. The clones expressed a 5.2-kb TGF alpha message and synthesized an 18-kDa form of TGF alpha. Supernatants of TGF alpha-producing clones induced the internalization of desmosomal components, the production of vimentin, and increased motility in untransfected epithelial NBTII cells, indicating that the factor produced by the clones was in a biologically active form. TGF alpha-producing clones secreted significant levels of a 95-kDa gelatinolytic metal-loproteinase, virtually absent in untransfected cell supernatants. In contrast, levels of inhibitors of metalloproteinases and of a plasminogen activator were similar in untransfected and TGF alpha-transfected NBTII cells. These results suggest that expression of TGF alpha in an epithelial tumor cell results in the development of a motile, fibroblast-like phenotype with matrix-degrading potential, which could result in a more aggressive tumor in vivo.

Physiological mechanisms for metalloproteinase activation.

The activation of procollagenase and prostromelysin by mechanisms that might be functional in vivo has been investigated. Studies with cell monolayers plated onto collagen films have indicated key roles for plasmin and TIMP in these processes. Prostromelysin activation could be rapidly effected by fibroblast monolayers in the presence of plasminogen, with identical kinetics to plasminogen-streptokinase generated plasmin. Procollagenase activation by plasmin was shown to be poor, although an M(r) shift of 11,000 occurred. Activation was enhanced ten-fold by the presence of active stromelysin even at a very low molar ratio. A tumour cell line secreting procollagenase but not stromelysin was found to be dependent upon the addition of both stromelysin and plasminogen to effect degradation of collagen films. Biochemical studies of metalloproteinase activation were carried out using other purified proteinases synthesized by connective tissue cells including endopeptidase 24.11, endopeptidase-2, cathepsin B and cathepsin L. None was a particularly effective activator relative to plasmin, but cathepsin B was shown to activate stromelysin. By use of both cell model systems and biochemical studies of purified enzymes we have found that the role of plasmin as the major metalloproteinase activator in normal connective tissue cells remains unchallenged.

Collagen degradation and platelet-derived growth factor stimulate the migration of vascular smooth muscle cells.

Cell migration is a key event in many biological processes and depends on signals from both extracellular matrix and soluble motogenic factors. During atherosclerotic plaque development, vascular smooth muscle cells migrate from the tunica media to the intima through a basement membrane and interstitial collagenous matrix and proliferate to form a neointima. Matrix metalloproteinases have previously been implicated in neointimal formation and in this study smooth muscle cell adhesion and migration on degraded collagen have been evaluated. Vascular smooth muscle cells adhered to native intact collagen type I and to its first degradation by-product, 3/4 fragment (generated by collagenase-3 cleavage), unwound at 35 degrees C to mimic physiological conditions. PDGF-BB pre-treatment induced a fourfold stimulation of smooth muscle cell motility on the collagen 3/4 fragment whereas no increase in smooth muscle cell motility on collagen type I was observed. Cell migration on collagen type I was mediated by alpha2 integrin, whereas PDGF-BB-stimulated migration on the 3/4 collagen fragment was dependent on alphavbeta3 integrin. alphavbeta3 integrin was organised in clusters concentrated at the leading and trailing edges of the cells and was only expressed when cells were exposed to the 3/4 collagen fragment. Tyrphostin A9, an inhibitor of PDGF receptor-beta tyrosine kinase activity, resulted in complete abolition of migration of PDGF-BB treated cells on collagen type I and 3/4 fragment. These results strongly support the hypothesis that the cellular migratory response to soluble motogens can be regulated by proteolytic modification of the extracellular matrix.

Fibroblast growth factors and insulin growth factors combine to promote survival of rat Schwann cell precursors without induction of DNA synthesis.

In embryonic rat nerves, we recently identified an early cell in the Schwann cell lineage, the Schwann cell precursor. We found that when these cells were removed from contact with axons they underwent rapid apoptotic death, and that in a proportion of the cells this death could be prevented by basic fibroblast growth factor (bFGF, FGF-2). We now report that 100% of Schwann cell precursors isolated from peripheral nerves of 14-day-old-rat embryos can be rescued by a combination of insulin-like growth factor (IGF) 1 or 2 in combination with either acidic FGF (aFGF, FGF-1), bFGF or Kaposi's sarcoma FGF (K-FGF; FGF-4). The precursors display an absolute requirement for both an IGF and an FGF to achieve maximal survival. Elevation of intracellular levels of cAMP by forskolin does not result in a significant shift in the IGF/FGF dose-response curves. In contrast, the percentage of precursors rescued by FGF in the presence of insulin is dramatically increased by elevation of cAMP. These growth factor combinations did not stimulate DNA synthesis significantly in Schwann cell precursors. These findings show that cooperation between growth factors is required to suppress cell death in Schwann cell precursors, and suggest that survival and DNA synthesis are regulated by distinct growth factor combinations in these cells. The observations are consistent with the idea that survival regulation by FGFs and IGFs plays an important role in the development of glial cells in early embryonic nerves.

Inhibition of type I collagen film degradation by tumour cells using a specific antibody to collagenase and the specific tissue inhibitor of metalloproteinases (TIMP).

Rabbit VX2 tumour cells in culture produced a collagenolytic activity which was shown to be immunologically identical to collagenase from rabbit articular chondrocytes and bone. VX2 cells degraded type I collagen films spontaneously and did not produce detectable levels of the tissue inhibitor of metalloproteinases (TIMP). Chondrocytes, however, required both stimulation of collagenase synthesis and activation to effect lysis and were observed to make appreciable amounts of TIMP. The degradation of type I collagen films by VX2 tumour cells was significantly inhibited by both a specific antibody to rabbit collagenase and by purified TIMP, thus demonstrating the unequivocal role of collagenase in this model system.

Adhesion mechanisms in embryogenesis and in cancer invasion and metastasis.

Cell-substratum and cell-cell adhesion mechanisms contribute to the development of animal form. The adhesive status of embryonic cells has been analysed during epithelial-mesenchymal cell interconversion and in cell migrations. Clear-cut examples of the modulation of cell adhesion molecules (CAMs) have been described at critical periods of morphogenesis. In chick embryos the three primary CAMs (N-CAM. L-CAM and N-cadherin) present early in embryogenesis are expressed later in a defined pattern during morphogenesis and histogenesis. The axial mesoderm derived from gastrulating cells expresses increasing amounts of N-cadherin and N-CAM. During metamerization these two adhesion molecules become abundant at somitic cell surfaces. Both CAMs are functional in an in vitro aggregation assay; however, the calcium-dependent adhesion molecule N-cadherin is more sensitive to perturbation by specific antibodies. Neural crest cells which separate from the neural epithelium lose their primary CAMs in a defined time-sequence. Adhesion to fibronectins via specific surface receptors becomes a predominant interaction during the migratory process, while some primary and secondary CAMs are expressed de novo during the ontogeny of the peripheral nervous system. In vitro, different fibronectin functional domains have been identified in the attachment, spreading and migration of neural crest cells. The fibronectin receptors which transduce the adhesive signals play a key role in the control of cell movement. All these results have prompted us to examine whether similar mechanisms operate in carcinoma cell invasion and metastasis. In vitro, rat bladder transitional carcinoma cells convert reversibly into invasive mesenchymal cells. A rapid modulation of adhesive properties is found during the epithelial-mesenchymal carcinoma cell interconversion. The different model systems analysed demonstrate that a limited repertoire of adhesion molecules, expressed in a well-defined spatiotemporal pattern, is involved in tissue formation and in key processes of tumour spread.

Characterization of a plasma membrane protein present in non-myelin-forming PNS and CNS glia, a subpopulation of PNS neurons, perineurial cells and smooth muscle in adult rats.

A plasma membrane protein common to non-myelin-forming peripheral glia, including non-myelin-forming Schwann cells, satellite cells and enteric glia, is recognized and defined by monoclonal antibody A5E3. It is not detectable immunohistochemically on myelin-forming Schwann cells. The antigen is also present in large amounts on smooth muscle cells and perineurial cells, on some PNS neurons, and at lower levels on astrocytes of adult rat. In neonatal but not adult animals, the antigen is present on skeletal muscle fibres and myoblasts. In immunoblots and immune precipitation experiments on smooth muscle and Schwann cell extracts the antigen is a polypeptide with an apparent molecular weight of 130 kd. In being present in some non-neural tissues, albeit very highly restricted in cell type, this antigen resembles several other cell surface glycoproteins found in large amounts in the nervous system.

Secretion of metalloproteinases by stimulated capillary endothelial cells. II. Expression of collagenase and stromelysin activities is regulated by endogenous inhibitors.

Rabbit brain capillary endothelial cells treated with 12-O-tetradecanoylphorbol-13-acetate produce the metalloproteinases, procollagenase and prostromelysin, as up to 20% of their total secreted protein. However, little or no catalytic activity of these enzymes can be found after treatment with either trypsin or an organomercurial agent, which are able to activate the proenzymes in the medium from stimulated rabbit fibroblasts. We now have shown that enzyme activities of procollagenase and prostromelysin are revealed after conditioned medium is analyzed by gel filtration chromatography or by electrophoresis on sodium dodecyl sulfate-substrate gels. In both systems, the metalloproteinases were separated from metalloproteinase inhibitors. The major inhibitor of Mr = 30,000 from capillary endothelial cells was immunologically identical with the rabbit tissue inhibitor of metalloproteinases. Two additional inhibitors of metalloproteinases at Mr = 22,000 and 19,000 were also observed. Inhibitors were present in the conditioned medium from rabbit fibroblasts in much lower quantities and were also qualitatively different. When gel filtration chromatography was used to remove the tissue inhibitor of metalloproteinases from medium conditioned by stimulated capillary endothelial cells, both activatable procollagenase and prostromelysin were readily demonstrable. These data suggest that endogenous inhibitors regulate the expression of metalloproteinases secreted by endothelial cells.

Extracellular matrix and matrix metalloproteinases in sciatic nerve.

Although matrix metalloproteinases (MMPs) are increasingly being implicated in several pathologies of the nervous system, it is not yet clear what role they play in normal neurobiological processes. We review the expression of extracellular matrix (ECM) components as well as MMPs and tissue inhibitors of metalloproteinases (TIMPs) in the peripheral nervous system. We explore the expression of certain MMPs and the four TIMPs at the mRNA level in the postnatal mouse sciatic nerve. In addition, we have used substrate gel and in situ zymography to determine levels of MMP-2 and -9 and TIMP activity in rat sciatic nerve after crush and during regeneration. A rapid and transient increase in MMP-9 localised at and immediately distal to the site of injury was observed, whereas an increase in MMP-2 activity was delayed, prolonged, and extended proximal and distal to the injury site. This activity coincides with periods of axonal elongation, suggesting that it could act to facilitate axonal extension along the nerve matrix. We also detected multiple species of gelatinolytic inhibitory activity, including TIMP-1 and -3 in control and injured nerve. These activities probably act to prevent uncontrolled gelatinolytic activity, maintaining nerve integrity at the level essential for axonal regrowth.