Laboratory models of the thermal evolution of the mantle during rollback subduction.

The subduction of oceanic lithosphere plays a key role in plate tectonics, the thermal evolution of the mantle and recycling processes between Earth's interior and surface. Information on mantle flow, thermal conditions and chemical transport in subduction zones come from the geochemistry of arc volcanoes, seismic images and geodynamic models. The majority of this work considers subduction as a two-dimensional process, assuming limited variability in the direction parallel to the trench. In contrast, observationally based models increasingly appeal to three-dimensional flow associated with trench migration and the sinking of oceanic plates with a translational component of motion (rollback). Here we report results from laboratory experiments that reveal fundamental differences in three-dimensional mantle circulation and temperature structure in response to subduction with and without a rollback component. Without rollback motion, flow in the mantle wedge is sluggish, there is no mass flux around the plate and plate edges heat up faster than plate centres. In contrast, during rollback subduction flow is driven around and beneath the sinking plate, velocities increase within the mantle wedge and are focused towards the centre of the plate, and the surface of the plate heats more along the centreline.

Induction of type 1 immune pathology in the brain following immunization without central nervous system autoantigen in transgenic mice with astrocyte-targeted expression of IL-12.

IL-12, a cytokine produced by microglia, may regulate cellular immunity at a localized level in the CNS. To investigate this further, we examined the consequences of peripheral immune stimulation without specific autoantigen in wild-type or transgenic (termed GF-IL12) mice with astrocyte production of the bioactive IL-12 p75 heterodimer. Active immunization with CFA and pertussis toxin, a procedure known to stimulate a robust type 1-biased immune response, produced CNS immune pathology from which GF-IL12 but not wild-type mice developed signs of clinical disease consisting of loss of activity, piloerection, mild tremor, and motor change. All immunized mice had some degree of mononuclear cell infiltration into the brain; however, the severity of this was markedly increased in GF-IL12 mice where leukocytes accumulated in perivascular and parenchymal locations. Accumulating cells consisted of CD4(+) and CD8(+) T cells and macrophage/microglia. Moreover, expression of cytokines (IFN-gamma and TNF), chemokines (IFN-inducible protein-10 and RANTES), the immune accessory molecules, MHC class II, B7.2, ICAM-1 and VCAM-1, and NO synthase-2 was induced in the CNS of the GF-IL12 mice. Therefore, peripheral immunization of GF-IL12 but not wild-type mice can provoke active type 1 immunity in the brain-a process that does not require CNS-specific immunizing autoantigen. These findings indicate that the cytokine milieu of a tissue can dramatically influence the development of intrinsic immune responses and associated pathology.

Interferon-independent, human immunodeficiency virus type 1 gp120-mediated induction of CXCL10/IP-10 gene expression by astrocytes in vivo and in vitro.

The CXC chemokine gamma interferon (IFN-gamma)-inducible protein CXCL10/IP-10 is markedly elevated in cerebrospinal fluid and brain of individuals infected with human immunodeficiency virus type 1 (HIV-1) and is implicated in the pathogenesis of HIV-associated dementia (HAD). To explore the possible role of CXCL10/IP-10 in HAD, we examined the expression of this and other chemokines in the central nervous system (CNS) of transgenic mice with astrocyte-targeted expression of HIV gp120 under the control of the glial fibrillary acidic protein (GFAP) promoter, a murine model for HIV-1 encephalopathy. Compared with wild-type controls, CNS expression of the CC chemokine gene CCL2/MCP-1 and the CXC chemokine genes CXCL10/IP-10 and CXCL9/Mig was induced in the GFAP-HIV gp120 mice. CXCL10/IP-10 RNA expression was increased most and overlapped the expression of the transgene-encoded HIV gp120 gene. Astrocytes and to a lesser extent microglia were identified as the major cellular sites for CXCL10/IP-10 gene expression. There was no detectable expression of any class of IFN or their responsive genes. In astrocyte cultures, soluble recombinant HIV gp120 protein was capable of directly inducing CXCL10/IP-10 gene expression a process that was independent of STAT1. These findings highlight a novel IFN- and STAT1-independent mechanism for the regulation of CXCL10/IP-10 expression and directly link expression of HIV gp120 to the induction of CXCL10/IP-10 that is found in HIV infection of the CNS. Finally, one function of IP-10 expression may be the recruitment of leukocytes to the CNS, since the brain of GFAP-HIV gp120 mice had increased numbers of CD3(+) T cells that were found in close proximity to sites of CXCL10/IP-10 RNA expression.

Diapiric flow at subduction zones: a recipe for rapid transport.

Recent geochemical studies of uranium-thorium series disequilibrium in rocks from subduction zones require magmas to be transported through the mantle from just above the subducting slab to the surface in as little as approximately 30,000 years. We present a series of laboratory experiments that investigate the characteristic time scales and flow patterns of the diapiric upwelling model of subduction zone magmatism. Results indicate that the interaction between buoyantly upwelling diapirs and subduction-induced flow in the mantle creates a network of low-density, low-viscosity conduits through which buoyant flow is rapid, yielding transport times commensurate with those indicated by uranium-thorium studies.

Regulation of matrix metalloproteinases and their inhibitor genes in lipopolysaccharide-induced endotoxemia in mice.

An imbalance between matrix metalloproteinases (MMPs) and inhibitors of MMPs (TIMPs) may contribute to tissue destruction that is found in various inflammatory disorders. To determine in an in vivo experimental setting whether the inflammatory reaction in the course of lipopolysaccharide (LPS)-induced endotoxemia causes an altered balance in the MMP/TIMP system, we analyzed the expression of a number of MMP and TIMP genes as well as MMP enzymatic activity in the liver, kidney, spleen, and brain at various time points after systemic injection of different doses of LPS in mice. Injection of sublethal doses of LPS led to an organ- and time-specific pattern of up-regulation of several MMP genes and the TIMP-1 gene in the liver, spleen, and kidney, whereas in the brain only TIMP-1 was induced. Injection of a lethal dose of LPS caused similar but more prolonged expression of these MMP genes as well as the induction of additional MMP genes in all organs. In LPS-treated mice in situ hybridization revealed collagenase 3 gene induction in cells resembling macrophages whereas TIMP-1 RNA was detected predominantly in parenchymal cells. Finally, gelatin zymography revealed increased gelatinolytic activity in all organs after LPS treatment. These observations highlight a dramatic shift in favor of increased expression of the MMP genes over the TIMP genes during LPS-induced endotoxemia, and suggest that MMPs may contribute to the development of organ damage in endotoxemia.

Photographic assessment of the appearance of chronic pressure and leg ulcers.

The purpose of this paper was to examine the validity and reliability of using photographs of wounds to accurately assess wound status. The results of assessing wound appearance using wound photographs was compared to results obtained from a bedside assessment using the Pressure Sore Status Tool (PSST). The photographic wound assessment tool (PWAT) used in this comparison represents a modified version of the PSST and includes the six domains that can be determined from wound photographs. The PWAT was used on photographs of both chronic pressure ulcers (n = 56) and leg ulcers due to vascular insufficiency (n = 81). The photographic tool has excellent intrarater (ICC = 0.96) and interrater (ICC = 0.73) reliability and good concurrent validity (r = 0.70) compared with a full bedside assessment PSST. The PWAT has also shown to be sensitive to change in wound appearance of healing ulcers, but not nonhealing ulcers. These results would suggest that in the event that a full bedside assessment is not possible, wound photographs may be used to accurately assess wound appearance of both chronic pressure ulcers located on the trunk and vascular ulcers of the lower extremity. Establishing a valid and reliable assessment of wound healing using photographic images is of great relevance to the advancing fields of computer image analysis and telemedicine.

Astrocyte-targeted expression of IL-12 induces active cellular immune responses in the central nervous system and modulates experimental allergic encephalomyelitis.

The role of IL-12 in the evolution of immunoinflammatory responses at a localized tissue level was investigated. Transgenic mice were developed with expression of either both the IL-12 subunits (p35 and p40) or only the IL-12 p40 subunit genes targeted to astrocytes in the mouse CNS. Glial fibrillary acidic protein (GF)-IL-12 mice, bigenic for the p35 and p40 genes, developed neurologic disease which correlated with the levels and sites of transgene-encoded IL-12 expression. In these mice, the brain contained numerous perivascular and parenchymal inflammatory lesions consisting of predominantly CD4+ and CD8+ T cells as well as NK cells. The majority of the infiltrating T cells had an activated phenotype (CD44high, CD45Rblow, CD62Llow, CD69high, VLA-4 high, and CD25+). Functional activation of the cellular immune response was also evident with marked cerebral expression of the IFN-gamma, TNF, and IL-1alphabeta genes. Concomitant with leukocyte infiltration, the CNS expression of immune accessory molecules was induced or up-regulated, including ICAM-1, VCAM-1, and MHC class II and B7-2. Glial fibrillary acidic protein-p40 mice with expression of IL-12 p40 alone remained asymptomatic, with no inflammation evident at any age studied. The effect of local CNS production of IL-12 in the development of experimental autoimmune encephalomyelitis was studied. After immunization with myelin oligodendrocyte glycoprotein-peptides, GF-IL-12 mice had an earlier onset and higher incidence but not more severe disease. We conclude that localized expression of IL-12 by astrocytes can 1) promote the spontaneous development of activated type 1 T cell and NK cellular immunity and cytokine responses in the CNS, and 2) promote more effective Ag-specific T cell dynamics but not activity in experimental autoimmune encephalomyelitis.

Serotonin discontinuation syndrome: does it really exist?

Treatment guidelines for depression have typically focused on diagnosis, how to initiate antidepressants, and duration of therapy, while very little is discussed about discontinuing treatment. With the advent of the serotonin-specific reuptake inhibitors (SSRIs), there is now growing evidence to support a "discontinuation syndrome" associated with withdrawal of therapy. This article describes two cases and presents a review of the literature.

A role for apoE in regulating the levels of alpha-1-antichymotrypsin in the aging mouse brain and in Alzheimer's disease.

This study was designed to explore the possible functional relationships between apolipoprotein E (apoE) and the protease inhibitor alpha-1-antichymotrypsin in the aging mouse brain and in Alzheimer's disease. For this purpose, levels of EB22/5 (the mouse homologue to human alpha-1-antichymotrypsin) mRNA expression was studied in apoE-deficient mice. These mice showed an age-dependent increase of EB22/5 mRNA expression in the brain. Furthermore, overexpression of allele 3 of human APOE gene in transgenic mice (in an apoE-deficient background) resulted in normalization of levels of EB22/5 mRNA expression compatible with levels found in control mice. In contrast, overexpression of human APOE4 allele or down-regulation of the apoE receptor low density lipoprotein receptor-related protein by deletion of the receptor-associated protein was associated with elevated levels of EB22/5 similar to apoE-deficient mice. Consistent with the findings in murine models, human alpha-1-antichymotrypsin protein was increased in brain homogenates from patients with Alzheimer's disease, and levels of this serpin were the highest in patients with the APOE4 allele. In summary, the present study showed evidence supporting a role for apoE in regulating alpha-1-antichymotrypsin expression. This is relevant to Alzheimer's disease because these two molecules appear to be closely associated with the pathogenesis of this disorder.

Chemokines and the inflammatory response to viral infection in the central nervous system with a focus on lymphocytic choriomeningitis virus.

Leukocyte migration to the central nervous system (CNS) is a common process with often devastating consequences that follows infection of this tissue compartment with a variety of viruses. The mechanisms underlying this process are poorly defined but, it is hypothesized that chemokines may be important regulatory signals for the cerebral recruitment and extravasation of leukocytes. Here we discuss this hypothesis in the context of different viral infections of the CNS with emphasis on lymphocytic choriomeningitis virus (LCMV). In general, the pattern of chemokine gene expression in these CNS viral infections is dynamic and complex with often overlapping expression of a number of different subclasses of chemokine genes. In the case of CNS infection with LCMV, cerebral chemokine gene expression was observed in euthymic and to a lesser extent athymic mice and preceded increases in cytokine gene expression and in euthymic mice, CNS leukocyte recruitment. These observations together with the finding that CRG-2/IP-10, a prominently expressed chemokine gene in many different CNS viral infections, was expressed by cells intrinsic to the CNS e.g. astrocytes, suggest that activation of chemokine gene expression may be a direct, early and localized host response to viral infection. These findings are consistent with the proposed involvement of chemokines as key signaling molecules for the migration of leukocytes to the CNS following virus infection.

Analysis of Gene Expression by Multiprobe RNase Protection Assay.

RNase protection assay (RPA) is becoming an increasingly popular method for the detection and quantitation of RNA levels in cells and tissues (1-3). Hybridization is conducted in solution using an excess of a labeled antisense single-stranded RNA as probe. Thus, hybridization of the probe with target RNA results in the formation of stable, double-stranded RNA-RNA hybrids. After hybridization, the excess probe is removed by digestion with single-strand specific RNase, leaving behind only those probe molecules that were "protected" from digestion by virtue of having formed a duplex with their complementary mRNA target. These protected hybrids are denatured and separated from remaining labeled probe using standard sequencing polyacrylamide gel electrophoresis. The separated protected probe can then be visualized using routine autoradiography. In comparison with other RNA detection methods, such as Northern blot analysis or RT-PCR, the RPA has a number of advantages. These include: 1. High sensitivity and specificity. 2. Small sample requirement. 3. Tolerant of RNA degradation. 4. Easy quantitation. 5. Rapid and simultaneous analysis of multiple target transcripts. 6. High throughput analysis. 7. Construction and use of "designer" probe sets.

Late-onset chronic inflammatory encephalopathy in immune-competent and severe combined immune-deficient (SCID) mice with astrocyte-targeted expression of tumor necrosis factor.

To examine the role of tumor necrosis factor (TNF)-alpha in the pathogenesis of degenerative disorders of the central nervous system (CNS), transgenic mice were developed in which expression of murine TNF-alpha was targeted to astrocytes using a glial fibrillary acidic protein (GFAP)-TNF-alpha fusion gene. In two independent GFAP-TNFalpha transgenic lines (termed GT-8 or GT-2) adult (>4 months of age) animals developed a progressive ataxia (GT-8) or total paralysis affecting the lower body (GT-2). Symptomatic mice had prominent meningoencephalitis (GT-8) or encephalomyelitis (GT-2) in which large numbers of B cells and CD4+ and CD8+ T cells accumulated at predominantly perivascular sites. The majority of these lymphocytes displayed a memory cell phenotype (CD44high, CD62Llow, CD25-) and expressed an early activation marker (CD69). Parenchymal lesions contained mostly CD45+ high, MHC class II+, and Mac-1+ cells of the macrophage microglial lineage with lower numbers of neutrophils and few CD4+ and CD8+ T cells. Cerebral expression of the cellular adhesion molecules ICAM-1, VCAM-1, and MAdCAM as well as a number of alpha- and beta-chemokines was induced or upregulated and preceded the development of inflammation, suggesting an important signaling role for these molecules in the CNS leukocyte migration. Degenerative changes in the CNS of the GFAP-TNFalpha mice paralleled the development of the inflammatory lesions and included primary and secondary demyelination and neurodegeneration. Disease exacerbation with more extensive inflammatory lesions that contained activated cells of the macrophage/microglial lineage occurred in GFAP-TNFalpha mice with severe combined immune deficiency. Thus, persistent astrocyte expression of murine TNF-alpha in the CNS induces a late-onset chronic inflammatory encephalopathy in which macrophage/microglial cells but not lymphocytes play a central role in mediating injury.

DNA immunization with minigenes: low frequency of memory cytotoxic T lymphocytes and inefficient antiviral protection are rectified by ubiquitination.

Our previous studies have shown that isolated cytotoxic T lymphocyte (CTL), B-cell, and T-helper epitopes, for which we coined the term minigenes, can be effective vaccines; when expressed from recombinant vaccinia viruses, these short immunogenic sequences confer protection against a variety of viruses and bacteria. In addition, we have previously demonstrated the utility of DNA immunization using plasmids encoding full-length viral proteins. Here we combine the two approaches and evaluate the effectiveness of minigenes in DNA immunization. We find that DNA immunization with isolated minigenes primes virus-specific memory CTL responses which, 4 days following virus challenge, appear similar in magnitude to those induced by vaccines known to be protective. Surprisingly, this vigorous CTL response fails to confer protection against a normally lethal virus challenge, although the CTL appear fully functional because, along with their high lytic activity, they are similar in affinity and cytokine secretion to CTL induced by virus infection. However this DNA immunization with isolated minigenes results in a low CTL precursor frequency; only 1 in approximately 40,000 T cells is epitope specific. In contrast, a plasmid encoding the same minigene sequences covalently attached to the cellular protein ubiquitin induces protective immunity and a sixfold-higher frequency of CTL precursors. Thus, we show that the most commonly employed criterion to evaluate CTL responses-the presence of lytic activity following secondary stimulation-does not invariably correlate with protection; instead, the better correlate of protection is the CTL precursor frequency. Recent observations indicate that certain effector functions are active in memory CTL and do not require prolonged stimulation. We suggest that these early effector functions of CTL, immediately following infection, are critical in controlling virus dissemination and in determining the outcome of the infection. Finally, we show that improved performance of the ubiquitinated minigenes most probably requires polyubiquitination of the fusion protein, suggesting that the enhancement results from more effective delivery of the minigene to the proteasome.

Differential expression of matrix metalloproteinase and tissue inhibitor of matrix metalloproteinase genes in the mouse central nervous system in normal and inflammatory states.

Matrix metalloproteinases (MMPs) are implicated in the pathogenesis of inflammatory disorders of the central nervous system (CNS) whereas the contribution of the major endogenous counter-regulators of MMPs, the tissue inhibitors of the matrix metalloproteinases (TIMPs), is unclear. We investigated the temporal and spatial expression patterns in the CNS of nine MMP genes and three TIMP genes in normal mice, in mice with EAE, and in transgenic mice with astrocyte (glial fibrillary acidic protein)-targeted expression of the cytokines interleukin-3 (macrophage/microglial demyelinating disease), interleukin-6 (neurodegenerative disease), or tumor necrosis factor-alpha (lymphocytic encephalomyelitis). In normal mice, the MMPs MT1-MMP, stromelysin 3, and gelatinase B were expressed at low levels, whereas high expression of TIMP-2 and TIMP-3 was observed predominantly in neurons and in the choroid plexus, respectively. In EAE and the transgenic mice, significant induction or up-regulation of various MMP genes was observed, the pattern of which was somewhat specific for each of the models, and there was significant induction of TIMP-1. In situ localization experiments revealed a dichotomy between MMP expression that was restricted to leukocytes and possibly microglia within inflammatory lesions and TIMP-1 expression that was observed in activated astrocytes circumscribing the lesions. These findings demonstrate specific spatial and temporal regulation in the expression of individual MMP and TIMP genes in the CNS in normal and inflammatory states. The distinct localization of TIMP-1 and MMP expression during CNS inflammation suggests a dynamic state in which the interplay between these gene products may determine both the size and resolution of the destructive inflammatory focus.

Lipopolysaccharide-induced IL-12 expression in the central nervous system and cultured astrocytes and microglia.

We examined whether the cytokine IL-12 could be induced locally in the brain or in glial cell cultures following LPS treatment. In the brain, expression of IL-12 p35 mRNA was constitutive and did not alter following i.p. injection of LPS. In contrast, IL-12 p40 mRNA was only detectable in the brain of mice given two staggered injections of LPS. Dual labeling in situ analysis revealed IL-12 p40 RNA-positive cells scattered throughout the brain parenchyma, with a small number of these cells being identified as astrocytes, while the majority of IL-12 p40 RNA-expressing cells appeared to be microglia. In cultured microglia or astrocytes, LPS and to a much lesser degree IL-1beta, but not IFN-gamma or TNF-alpha, induced the expression of IL-12 p40 mRNA. Numerous glial fibrillary acidic protein-immunopositive cells colabeled for IL-12 p40 RNA; indicating that LPS-stimulated astrocytes expressed IL-12 in vitro. Immunoblot analysis of lysates from LPS-treated astrocytes revealed the presence of multiple species of 40, 43, 75, and 120 kDa containing the IL-12 p40 protein. Finally, secretion of the IL-12 p75 heterodimer was detectable by ELISA from astrocytes treated with LPS plus IFN-gamma, but not with LPS alone. The findings indicate that IL-12 gene expression can be activated in the brain, with the resident glial cells being a prodigious source of this cytokine. The localized production of IL-12 may have a significant impact on the development of cell-mediated immune responses within the central nervous system.

Definition of two angiogenic pathways by distinct alpha v integrins.

Angiogenesis depends on cytokines and vascular cell adhesion events. Two cytokine-dependent pathways of angiogenesis were shown to exist and were defined by their dependency on distinct vascular cell integrins. In vivo angiogenesis in corneal or chorioallantoic membrane models induced by basic fibroblast growth factor or by tumor necrosis factor-alpha depended on alpha v beta 3, whereas angiogenesis initiated by vascular endothelial growth factor, transforming growth factor-alpha, or phorbol ester depended on alpha v beta 5. Antibody to each integrin selectively blocked one of these pathways, and a cyclic peptide antagonist of both integrins blocked angiogenesis stimulated by each cytokine tested. These pathways are further distinguished by their sensitivity to calphostin C, an inhibitor of protein kinase C that blocked angiogenesis potentiated by alpha v beta 5 but not by alpha v beta 3.

Inhibition of bacterial growth in vitro following stimulation with high voltage, monophasic, pulsed current.

Low-intensity direct current has been reported to be effective in promoting healing of infected wounds, and these results have been assumed to apply to stimulation of wound tissue with monophasic high voltage pulsed current (HVPC). The purpose of this study was to determine whether HVPC has an inhibitory effect on growth in vitro of three bacterial species--Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa--commonly isolated from open wounds. Following exposure to HVPC, the measured zone of inhibition of bacterial growth was not significantly different between bacterial species. Inhibition at the anode (positive pole) occurred secondary to build-up of toxic end products, and inhibition at the cathode (negative pole) resulted from exposure to HVPC. Duration of exposure and voltage showed a highly significant linear relationship. Exposure to more than 250 V of HVPC for at least two hours resulted in some degree of inhibition of growth in all three bacterial species.

Financial aspects of clinical education to facilities.

The financial aspects of clinical education for full-time junior and senior physical therapy students as related to facilities offering clinical affiliatins were determined. A questionnaire concerning supervisory expenses, student support, direct and indirect costs returned by 35 facilities of all types provided the basic information. Comparisons of costs and revenues were made for junior and senior students separately and for facilities according to type, size,and personnel involved in student supervision.