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Supplementary Figure S3. Association of the cell cycle/immune signature with B, lactate dehydrogenase levels.

Purpose: The association of tumor gene expression profiles with progression-free survival (PFS) outcomes in patients with BRAFV600-mutated melanoma treated with vemurafenib or cobimetinib combined with vemurafenib was evaluated.Experimental Design: Gene expression of archival tumor samples from patients in four trials (BRIM-2, BRIM-3, BRIM-7, and coBRIM) was evaluated. Genes significantly associated with PFS (P < 0.05) were identified by univariate Cox proportional hazards modeling, then subjected to unsupervised hierarchical clustering, principal component analysis, and recursive partitioning to develop optimized gene signatures.Results: Forty-six genes were identified as significantly associated with PFS in both BRIM-2 (n = 63) and the vemurafenib arm of BRIM-3 (n = 160). Two distinct signatures were identified: cell cycle and immune. Among vemurafenib-treated patients, the cell-cycle signature was associated with shortened PFS compared with the immune signature in the BRIM-2/BRIM-3 training set [hazard ratio (HR) 1.8; 95% confidence interval (CI), 1.3–2.6, P = 0.0001] and in the coBRIM validation set (n = 101; HR, 1.6; 95% CI, 1.0–2.5; P = 0.08). The adverse impact of the cell-cycle signature on PFS was not observed in patients treated with cobimetinib combined with vemurafenib (n = 99; HR, 1.1; 95% CI, 0.7–1.8; P = 0.66).Conclusions: In vemurafenib-treated patients, the cell-cycle gene signature was associated with shorter PFS. However, in cobimetinib combined with vemurafenib-treated patients, both cell cycle and immune signature subgroups had comparable PFS. Cobimetinib combined with vemurafenib may abrogate the adverse impact of the cell-cycle signature. Clin Cancer Res; 23(17); 5238–45. ©2017 AACR.

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Purpose: The association of tumor gene expression profiles with progression-free survival (PFS) outcomes in patients with BRAFV600-mutated melanoma treated with vemurafenib or cobimetinib combined with vemurafenib was evaluated. Experimental Design: Gene expression of archival tumor samples from patients in four trials (BRIM-2, BRIM-3, BRIM-7, and coBRIM) was evaluated. Genes significantly associated with PFS (P < 0.05) were identified by univariate Cox proportional hazards modeling, then subjected to unsupervised hierarchical clustering, principal component analysis, and recursive partitioning to develop optimized gene signatures. Results: Forty-six genes were identified as significantly associated with PFS in both BRIM-2 (n = 63) and the vemurafenib arm of BRIM-3 (n = 160). Two distinct signatures were identified: cell cycle and immune. Among vemurafenib-treated patients, the cell-cycle signature was associated with shortened PFS compared with the immune signature in the BRIM-2/BRIM-3 training set [hazard ratio (HR) 1.8; 95% confidence interval (CI), 1.3–2.6, P = 0.0001] and in the coBRIM validation set (n = 101; HR, 1.6; 95% CI, 1.0–2.5; P = 0.08). The adverse impact of the cell-cycle signature on PFS was not observed in patients treated with cobimetinib combined with vemurafenib (n = 99; HR, 1.1; 95% CI, 0.7–1.8; P = 0.66). Conclusions: In vemurafenib-treated patients, the cell-cycle gene signature was associated with shorter PFS. However, in cobimetinib combined with vemurafenib-treated patients, both cell cycle and immune signature subgroups had comparable PFS. Cobimetinib combined with vemurafenib may abrogate the adverse impact of the cell-cycle signature. Clin Cancer Res; 23(17); 5238–45. ©2017 AACR.

Interleukin (IL)-25 is a member of the IL-17 family of cytokines. However, unlike the other members of this family, IL-25 promotes T helper (Th) 2 responses. We now show that IL-25 also regulates the development of autoimmune inflammation mediated by IL-17–producing T cells. We have generated IL-25–deficient (il25−/−) mice and found that they are highly susceptible to experimental autoimmune encephalomyelitis (EAE). The accelerated disease in the il25−/− mice is associated with an increase of IL-23 in the periphery and a subsequent increase in the number of inflammatory IL-17–, IFNγ-, and TNF-producing T cells that invade the central nervous system. Neutralization of IL-17 but not IFNγ in il25−/− mice prevented EAE, suggesting that IL-17 is a major disease-promoting factor. IL-25 treatment at several time points during a relapse-remitting model or chronic model of EAE completely suppressed disease. IL-25 treatment induced elevated production of IL-13, which is required for suppression of Th17 responses by direct inhibition of IL-23, IL-1β, and IL-6 expression in activated dendritic cells. Thus, IL-25 and IL-17, being members of the same cytokine family, play opposing roles in the pathogenesis of organ-specific autoimmunity.

Summary Innate immunity is the first line of defense against infection, protecting the host during the development of adaptive immunity and critically affecting the nature of the adaptive response. We show that, in contrast to tumor necrosis factor α (TNF-α), the related protein TWEAK attenuates the transition from innate to adaptive mechanisms. TWEAK −/− mice had overabundant natural killer (NK) cells and displayed hypersensitivity to bacterial endotoxin, with their innate immune cells producing excess interferon (IFN)-γ and interleukin (IL)-12. TWEAK inhibited stimulation of the transcriptional activator STAT-1 and induced p65 nuclear factor (NF)-κB association with histone deacetylase 1, repressing cytokine production. TWEAK −/− mice developed oversized spleens with expanded memory and T helper 1 (T H 1) subtype cells upon aging and mounted stronger innate and adaptive T H 1-based responses against tumor challenge. Thus, TWEAK suppresses production of IFN-γ and IL-12, curtailing the innate response and its transition to adaptive T H 1 immunity.

CD4(+)CD25(+) regulatory T (T reg) cells play a pivotal role in control of the immune response. Transforming growth factor-beta (TGF-beta) has been shown to be required for T reg cell activity; however, precisely how it is involved in the mechanism of suppression is poorly understood. Using the T cell transfer model of colitis, we show here that CD4(+)CD45RB(high) T cells that express a dominant negative TGF-beta receptor type II (dnTbetaRII) and therefore cannot respond to TGF-beta, escape control by T reg cells in vivo. CD4(+)CD25(+) T reg cells from the thymus of dnTbetaRII mice retain the ability to inhibit colitis, suggesting that T cell responsiveness to TGF-beta is not required for the development or peripheral function of thymic-derived T reg cells. In contrast, T reg cell activity among the peripheral dnTbetaRII CD4(+)CD25(+) population is masked by the presence of colitogenic effector cells that cannot be suppressed. Finally, we show that CD4(+)CD25(+) T reg cells develop normally in the absence of TGF-beta1 and retain the ability to suppress colitis in vivo. Importantly, the function of TGF-beta1(-/-) T reg cells was abrogated by anti-TGF-beta monoclonal antibody, indicating that functional TGF-beta can be provided by a non-T reg cell source.

Engagement of the T-cell receptor (TCR) initiates a signaling cascade that ultimately results in activation of the transcription factor NF-kappaB, which regulates many T-cell functions including proliferation, differentiation and cytokine production. Herein we demonstrate that Rip2, a caspase recruitment domain (CARD)-containing serine/threonine kinase, plays an important role in this cascade and is required for optimal TCR signaling and NF-kappaB activation. Following TCR engagement, Rip2 associated with Bcl10, a CARD-containing signaling component of the TCR-induced NF-kappaB pathway, and induced its phosphorylation. Rip2-deficient mice were defective in TCR-induced NF-kappaB activation, interleukin-2 production, and proliferation in vitro and exhibited defective T-cell-dependent responses in vivo. The defect in Rip2-/- T-cells correlated with a lack of TCR-induced Bcl10 phosphorylation. Furthermore, deficiency in Bcl10-dependent NF-kappaB activation could be rescued in Rip2-/- embryonic fibroblasts by exogenous wild-type Rip2 but not a kinase-dead mutant. Together these data define an important role for Rip2 in TCR-induced NF-kappaB activation and T-cell function and highlight the significance of post-translational modification of Bcl10 by Rip2 in T-cell signaling.

We have biologically characterized two new members of the IL-17 cytokine family: IL-17F and IL-25. In contrast to conventional in vitro screening approaches, we have characterized the activity of these new molecules by direct in vivo analysis and have compared their function to that of other IL-17 family members. Intranasal administration of adenovirus expressing IL-17, IL-17C, or IL-17F resulted in bronchoalveolar lavage neutrophilia and inflammatory gene expression in the lung. In contrast, intranasal administration of IL-25-expressing adenovirus or IL-25 protein resulted in the production of IL-4, IL-5, IL-13, and eotaxin mRNA in the lung and marked eosinophilia in the bronchoalveolar lavage and lung tissue. Mice given intranasal IL-25 also developed epithelial cell hyperplasia, increased mucus secretion, and airway hyperreactivity. IL-25 gene expression was detected following Aspergillus and Nippostrongylus infection in the lung and gut, respectively. IL-25-induced eosinophilia required IL-5 and IL-13, but not IL-4 or T cells. Following IL-25 administration, the IL-5+ staining cells were CD45R/B220+, Thy-1+/−, but were NK1.1-, Ly-6G(GR-1)-, CD4-, CD3-, and c-kit-negative. γ-common knockout mice did not develop eosinophilia in response to IL-25, nor were IL-5+ cells detected. These findings suggest the existence of a previously unrecognized cell population that may initiate Th2-like responses by responding to IL-25 in vivo. Further, these data demonstrate the heterogeneity of function within the IL-17 cytokine family and suggest that IL-25 may be an important mediator of allergic disease via production of IL-4, IL-5, IL-13, and eotaxin.

Ocular inflammation leads to vision loss through the destruction and scarring of delicate tissues along the visual axis. To identify inflammatory mediators involved in this process, we used real time RT-PCR to quantify the expression of mRNA transcripts of 34 cytokines, 26 chemokines, and 14 chemokine receptors at certain time points during T cell-mediated ocular inflammation. We induced disease by adoptive transfer of Ag-specific Th1 or Th2 cells into recipients expressing the target Ag in their eyes. We also compared the mediator expression patterns seen in adoptive transfer-induced inflammation with that seen in mouse eyes developing experimental autoimmune uveoretinitis. In addition, we used laser capture microdissection to examine chemokine mRNA production by both retinal pigment epithelium cells and infiltrating leukocytes in inflamed eyes. Major findings included the following: 1) Three patterns of expression of the inflammation-related molecules were seen in recipients of adoptively transferred Th cells: preferential expression in Th1 recipients, or in Th2 recipients, or similar expression in both recipient groups. 2) In experimental autoimmune uveoretinitis, the inflammatory mediator expression pattern largely paralleled that seen in Th1-induced disease. 3) Both retinal pigment epithelium and infiltrating leukocytes expressed chemokine transcripts in distinct, but overlapping patterns in inflamed eyes. 4) Interestingly, trancripts of multiple cytokines, chemokines, and chemokine receptors were constitutively expressed in high levels in mouse eyes. Seven of these molecules have not been previously associated with the eye. These data underscore the multiplicity of mediators that participate in the pathogenesis of eye inflammation and point to upstream cytokines as potential therapeutic targets.

The oceanic core complex comprising Atlantis Massif was formed within the past 1.5–2 Myr at the intersection of the Mid-Atlantic Ridge, 30° N, and the Atlantis Transform Fault. The corrugated, striated central dome prominently displays morphologic and geophysical characteristics representative of an ultramafic core complex exposed via long-lived detachment faulting. Sparse volcanic features on the massif's central dome indicate that minor volcanics have penetrated the inferred footwall, which geophysical data indicates is composed predominantly of variably serpentinized peridotite. In contrast, the hanging wall to the east of the central dome is comprised of volcanic rock. The southern part of the massif has experienced the greatest uplift, shoaling to less than 700 m below sea level, and the coarsely striated surface there extends eastward to the top of the median valley wall. Steep landslide embayments along the south face of the massif expose cross sections through the core complex. Almost all of the submersible and dredge samples from this area are deformed, altered peridotite and lesser gabbro. Intense serpentinization within the south wall has likely contributed to the uplift of the southern ridge and promoted the development of the Lost City Hydrothermal Field near the summit. Differences in the distribution with depth of brittle deformation observed in microstructural analyses of outcrop samples suggest that low-temperature strain, such as would be associated with a major detachment fault, is concentrated within several tens of meters of the domal surface. However, submersible and camera imagery show that deformation is widespread along the southern face of the massif, indicating that a series of faults, rather than a single detachment, accommodated the uplift and evolution of this oceanic core complex.

The normal response to inhaled Ag is the absence of Ag-specific IgE and cytokine production to later Ag challenges. Although the mechanism of this aerosol-induced IgE tolerance is not completely understood, it may prevent sensitization to inhaled Ags, which could otherwise lead to allergy and asthma. We examined the consequences of ongoing Th1 and Th2 responses in the lungs of mice during OVA inhalation to mimic conditions that may subvert tolerance and lead to sensitization. We found that concurrent, secondary Th2 lung responses to keyhole limpet hemocyanin or primary responses to Nippostrongylus larvae or Asperigillus fumagatus extract prevented establishment of IgE tolerance to aerosolized OVA. Intranasal rIL-4 given before OVA aerosolization also prevented establishment of tolerance, whereas concurrent Th1 responses to influenza virus or Mycobacterium bovis bacillus Calmette-Guérin had no effect. However, once established, aerosol tolerance to OVA could not be completely broken by OVA rechallenge concurrent with a secondary Th2 response to keyhole limpet hemocyanin or A. fumagatus extract, or by intranasal rIL-4. These data suggest that the immune status of the lung of an individual may profoundly influence the initial response to inhaled Ag, and that aerosol-induced IgE tolerance may not be appropriately established in individuals undergoing concurrent, Th2-mediated responses to Ags or pathogens.

Cytokine networking in the lung in response to inhaledAspergillus fumigatuswas assessed using a murine model of primary pulmonary aspergillosis in immunocompetent Crl:CF-1 mice. Inhalation of virulentA. fumigatus(6 × 106CFU) resulted in the induction of interleukin 18 (IL-18), tumor necrosis factor alpha (TNF-α), IL-12, and gamma interferon (IFN-γ) protein in bronchoalveolar lavage fluid and/or lung tissue. Induction of immunoreactive IL-18 preceded induction of TNF-α protein, which preceded induction of immunoreactive IL-12 and IFN-γ. Real-time reverse transcriptase (RT) PCR analysis of infected lung tissue demonstrated that induction of IL-18 protein also preceded induction of pulmonary TNF-α, IL-12, and IFN-γ mRNAs. Mice were subsequently treated with cytokine-specific neutralizing monoclonal antibodies (MAbs) to the IL-18 receptor (anti-IL-18R MAb), TNF-α (anti-TNF-α MAb), IL-12 (anti-IL-12 MAb), and/or IFN-γ (anti-IFN-γ MAb), and effects on intrapulmonary cytokine activity and growth ofA. fumigatuswere assessed in infected lung homogenates. Simultaneous neutralization of IL-12 and IL-18 resulted in decreased levels of immunoreactive TNF-α, while neutralization of IL-18, TNF-α, or IL-12 alone or of IL-18 and IL-12 together resulted in decreased levels of immunoreactive IFN-γ. Simultaneous neutralization of IL-12 and IL-18 or neutralization of TNF-α alone or in combination with IL-12, IL-18, or IFN-γ also resulted in a significant increase inA. fumigatusCFU in lung tissue. Taken together, these results demonstrate that endogenous IL-18, IL-12, and TNF-α, through their modulatory effects on both intrapulmonary cytokine activity and growth ofA. fumigatus, play key roles in host defense against primary pulmonary aspergillosis.

Intestinal lamina propria (LP) CD4+ T cells are memory-like effector cells that proliferate at relatively low levels and require high levels of TCR signaling and costimulation for full activation in vitro. To study LP CD4+ T cell functional potential we used DO11.10 TCR transgenic (Tg) mice specific for the class II MHC-restricted OVA323–339 peptide and nontransgenic BALB/c mice. Activation of LP Tg+ T cells with Ag using mucosal explants induced high levels of IL-2, IL-4, and IFN-γ. Culturing isolated LP cells with IL-12 enhanced IFN-γ production and down-regulated IL-4 and IL-2, whereas addition of IL-4 maintained IL-4 production without inhibiting IFN-γ production. Systemic administration of relatively high dose (HD; 100 nM) OVA323–339 peptide induced similar levels of bromodeoxyuridine (BrdU) incorporation by LP and splenic Tg+ T cells in vivo, whereas low dose (LD; 4.5 nM) peptide injections induced 4-fold greater levels of BrdU incorporation for LP compared with splenic Tg+ T cells. Coadministration of CTLA-4Ig reduced BrdU incorporation for splenic cells by 70% with HD and LD stimulation, but had little effect on LP responses to HD stimulation. Results of in vivo studies were confirmed in nontransgenic BALB/c mice using HD (200 μg) and LD (10 μg) anti-CD3 mAb+/− CTLA-4Ig. These results suggest that LP T cells are differentiated effector cells that respond at high levels when activated with relatively low levels of Ag- and B7-mediated costimulation in vivo. The reduced activation threshold of LP T cells may facilitate responses to low levels of Ag derived from mucosal pathogens.

We describe a novel mouse CXC chemokine that is selectively expressed in lung epithelial cells and up-regulated in various lung inflammation models. Although this chemokine clusters with other ELR-CXC chemokines, none of them can confidently be assigned to be its human homologue based on sequence identity. In addition, the highly restricted mRNA tissue distribution of this chemokine differentiates it from all previously described chemokines: Lungkine could not be detected in any of the 70 cDNA libraries analyzed corresponding to specific murine cell populations and tissues. High levels of Lungkine mRNA were specifically detected in the lung and at lower levels in fetal lung tissue by Northern blot and in situ hybridization, suggesting a potential role for this chemokine during lung development. Moreover, Lungkine protein is secreted into the airway spaces and induces the in vitro and in vivo migration of neutrophils, suggesting that it is involved in lung-specific neutrophil trafficking. Using fluorescent in situ hybridization, we show that Lungkine maps to mouse chromosome 5.

Approximately 600 km2 of Deep-Tow side-looking sonar data were collected at the eastern intersection of the Mid-Atlantic Ridge with the Kane Transform. The merged digital image mosaic provides a synoptic view of the oceanic crust exposed across major escarpments at this ridge-transform intersection (RTI). We characterized this large acoustic data set using textural attributes extracted from the backscatter image by means of a gray level co occurrence matrix method and Fourier fractal analysis. False-color texture attribute maps created from these methods aid in the interpretation and discrimination of surficial deposits and bedrock units on the seafloor. Principal components analysis performed on the textural attributes reduced the dimensionality of the textural feature vector and optimized imago texture discrimination. The textural attributes were calibrated to ground truth geologic data and extrapolated to regions with no ground data. The resultant texture and classification maps are consistent with the results of submersible studies but reveal more details of the seafloor geology than the conventional visual interpretation of the backscatter image and ground geologic observations. The inferred distribution of gabbros, basalts, surficial sediments, and rubble deposits shown in the classification map provides new constraints on seafloor geology at the eastern Kane RTI. Over 60% of the southern transform valley wall is covered by pelagic sediments and talus or rubble deposits, with intermittent exposures of coarse-grained gabbroic rocks and basaltic volcanic rocks. The western rift valley wall is classified as mainly massive outcrops of gabbroic rocks. The contact between the gabbroic outcrops on the western rift valley wall and the pillow basalt terrane on the rift valley floor can be traced for several kilometers along the base of the median valley wall. This new perspective supports the interpretation of the “inside-corner massif” as an “oceanic core complex” which is being degraded by mass wasting along tectonically active escarpments of the bounding median valley and transform valley walls.

We designed a series of case studies for Introductory Geology Laboratory courses using computer visualization techniques integrated with traditional laboratory materials. These consist of a comprehensive case study which requires three two-hour long laboratory periods to complete, and several shorter case studies requiring one or two, two-hour laboratory periods. Currently we have prototypes of the Yellowstone National Park, Hawaii volcanoes and the Mid-Atlantic Ridge case studies. The Yellowstone prototype can be used to learn about a wide variety of rocks and minerals, about geothermal activity and hydrology, about volcanic hazards and the hot-spot theory of plate tectonics. The Hawaiian exercise goes into more depth about volcanoes, volcanic rocks and their relationship to plate movements. The Mid-Atlantic Ridge project focuses on formation of new ocean crust and mineral-rich hydrothermal deposits at spreading centers. With new improvements in visualization technology that are making their way to personal computers, we are now closer to the ideal of a “virtual” field trip. We are currently making scenes of field areas in Hawaii and Yellowstone which allow the student to pan around the area and zoom in on interesting objects. Specific rocks in the scene will be able to be “picked up” and studied in three dimensions. This technology improves the ability of the computer to present a realistic simulation of the field area and allows the student to have more control over the presentation. This advanced interactive technology is intuitive to control, relatively cheap and easy to add to existing computer programs and documents.

The inter-graben zone between the Solea and Mitsero grabens in the northern part of the Troodos ophiolite (Cyprus) displays igneous, tectonic, and physiographic features that are characteristic of seamount volcanism at modern oceanic spreading centers. In this zone the 200-m-high Alestos hill volcanic edifice consisting of pillow lavas with preserved lava tubes and cone sheet intrusions is interpreted to have developed as a seamount fed by a late-stage, off-axis shallow-level gabbroic pluton that was intrusive into young oceanic crust. Concentric cone sheet intrusions and associated faults and shear zones in and around the Alestos hill seamount collectively form a seafloor caldera complex, which probably formed during the extrusion of lavas and subsequent cauldron subsidence over the gabbroic pluton. The ENE-trending elongated massive sulfide ore deposit formation in the Alestos–Memi area represents a focused upflow zone for hydrothermal fluids driven by the same off-axis gabbroic intrusion responsible for formation of the Alestos hill seamount. The ENE-trending and up to 150-m-wide Alestos–Memi fault zone dissects the elongated ore body in a dextral sense and might have provided a high permeability conduit in the upper crust for upwelling hydrothermal flow at the time of magmatism. The geometry of this fault zone and its kinematic relations with a N–S-striking Alestos Forest normal fault in the west are suggestive of its transfer fault origin, reminiscent of mineralized transfer faults found at the Mid-Atlantic Ridge. The active TAG hydrothermal mound with massive sulfide deposits that is located on a small volcanic dome 3 km off-axis and along a transfer fault in the Trans-Atlantic Geotraverse (TAG) area at 26°N provides a modern analogue for the Alestos hill seamount and associated faulting and mineralization in the ancient oceanic crust of the Troodos ophiolite. These findings indicate that off-axis magmatism played a major role in crustal accretion, hydrothermal alteration, and ore mineralization during the late-stage evolution of the Troodos ophiolite.

The first paleomagnetic results from oriented dike samples collected on the Mid-Atlantic Ridge shed new light on the complex interplay between magmatic accretion and mechanical extension at a slow spreading ridge segment. An upper crustal section about 1.5 km thick is exposed along a west-dipping normal fault zone that defines the eastern median valley wall of the southern segment of the Mid-Atlantic Ridge south of the Kane fracture zone (MARK area). Two distinct groups of dikes are differentiated on the basis of orientation and paleomagnetic characteristics. One group, on the basis of the paleomagnetic data, appears to be in its original intrusion orientation. This group includes both ridge-parallel, vertical dikes as well as dikes in other orientations, calling into question assumptions about uniform dike orientations at oceanic spreading centers. The second group consists of dikes that have paleomagnetic directions that are distinct from the predicted dipole direction, and we interpret them to have been tectonically rotated. These also occur in many orientations. The spatial relations between rotated and nonrotated dikes indicate that intrusion, faulting, and block rotation were contemporaneous beneath the median valley floor. Nonrotated dikes exposed on the eastern median valley wall indicate that there has been no net rotation of this upper crustal assemblage since magmatic construction ceased. Hence slip and associated uplift probably occurred in the fault zones' present orientation. These results provide the basis for a general model of mechanical extension and dike intrusion for this segment of the Mid-Atlantic Ridge. Initially, a portion of crust forms beneath the median valley by synkinematic dike intrusion into laterally discontinuous fault blocks. Slip and associated uplift along a cataclastic normal fault zone later exposes this crustal section on the valley margin. As spreading continues, this valley-bounding cataclastic normal fault zone is abandoned in favor of a new fault system thus passively moving the exposed crustal section away from the median valley.

The intestinal lamina propria (LP) is a major effector site of the mucosal immune system where antigen-specific and antigen-nonspecific factors shape the functional responses of CD4+T helper cells. To study the functional differentiation of LP T helper cells we utilized DO11.10 T cell receptor (TCR) transgenic (Tg) mice that expressed a clonotypic TCR specific for a class II major histocompatibility complex-restricted peptide of chicken ovalbumin. The majority of cells expressing Tg TCR (Tg+) in peripheral lymphoid tissue expressed naive surface phenotypes whereas nearly all Tg+T cells in the intestinal LP expressed an activated/memory-like phenotype. Flow cytometric analysis of Tg+T cell populations revealed that a small proportion of cells in peripheral lymphoid tissue but nearly all cells in the LP expressed dual (Tg plus non-Tg) TCRs. In Tg × recombinase-activating-gene-1-deficient (Tg ×RAG-1−/−) mice, splenic and LP T cells expressed naive surface phenotypes and produced cytokines equivalent to naive splenic cells from Tg ×RAG-1+/+mice. In contrast, Tg LP cells from Tg ×RAG-1+/+mice produced 35-fold greater levels of interferon-γ and 5-fold greater levels of interleukin 4 compared with naive splenic cells. These findings suggested that activation of Tg+T cells through endogenous non-Tg TCR had promoted the localization and differentiation of memory-like effector T helper cells in the intestine.

Human papillomavirus (HPV) E2 and E1 proteins are required for the replication of viral genomes in vivo. We have examined the effects of increasing the level of E2 on viral and cellular replication using recombinant adenoviruses. Infection of cells which maintain HPV 31 DNA episomally with E2 recombinant adenoviruses resulted in a 5-fold increase in genome copy number as well as an S phase arrest allowing for the continued replication of cellular DNA. Similar effects on cell cycle progression were seen following infection of normal human foreskin keratinocytes, the natural host cell. The DNA content of these cells increased beyond 4N indicating that multiple rounds of replication had occurred without an intervening mitotic event. In addition, increased cyclin A and E associated kinase activity was observed, while no change was detected in cyclin B associated kinase activity or in the activation state of cdc2 kinase. Interestingly, the levels of the p53 tumor suppresser protein were dramatically reduced through a post-transcriptional mechanism following infection. These data suggest a role for E2 in regulating viral and cellular replication by abrogation of a mitotic checkpoint, which is, at least in part, controlled by p53.

Tilted dikes occur within the pillow lava, sheeted dike, and gabbroic sections of 1 million-year-old, fast spread oceanic crust exposed on the walls of the Hess Deep Rift. During recent Alvin dives, oriented samples from both tilted and vertical dikes were collected from several of the outcrops. These are the first oriented paleomagnetic samples ever collected directly from outcrops on the ocean floor and were obtained with the help of a specially constructed geological compass. Remanent magnetic inclinations measured on the dike samples are significantly different from the near-horizontal inclinations expected. The present attitudes of the dikes and their remanent magnetizations indicate that the dikes have been technically rotated since their intrusion. Submersible observations from other dive programs that examined parts of nearby escarpments suggest that the structural complexity observed in the Alvin study areas may be only a local phenomenon, or at least, not a pervasive characteristic of oceanic crust. Although tectonic rotations such as those observed may have taken place as a result of the opening of the Hess Deep Rift, crosscutting vertical dikes and other geologic considerations suggest that substantial tectonic rotations occurred at or very near the East Pacific Rise axis, probably as a result of intermittent axial collapse. The observed structures suggest that tectonic processes beneath fast spreading ridges and the internal structure of crust produced at them may be significantly more complex than previously assumed.

The Solea graben, on the northern flank of the Troodos ophiolite, Cyprus, is a 15–20-km-wide asymmetric structure primarily defined by dike attitudes in the sheeted dike complex. The wider western portion of the graben consists mainly of gently dipping dikes while the eastern portion is narrow and consists of blocks of dikes with widely varying attitudes. Using a method which restores the sampled dikes to vertical and the primary remanent magnetizations to a previously known stable remanent magnetization direction for the Troodos complex, we have calculated rotation axes and amounts of rotation for the dikes. The rotation axes for dikes in the western portion of the Solea graben are subhorizontal and subparallel to the original strikes of the dikes. In the eastern portion the rotation axes have varying attitudes, including some which are very steep. Our results are consistent with field observations that suggest that the western Solea graben formed by listric normal faulting near the axis of a spreading center, with rotation of the dikes above a detachment surface. Across the graben axis on the eastern portion, more intense intrusive activity and minor normal faulting resulted in less rotation about subhorizontal axes and some rotations about vertical axes.

Brittle fault zones commonly are characterized by a multitude of lineated shear planes having a wide distribution of orientations. In our model, we assume the faulted rock comprises an aggregate of rigid blocks whose surfaces are the shear planes. We define two independent scales of motion to distinguish the local rigid rotation of the blocks with their shear planes (the “micromotion”) from the average deformation of the material on a macroscopic scale (the “macromotion”). The macromotion is defined by the macrovelocity gradient tensor, which is separated into symmetric and antisymmetric parts, the deformation rate and the macrospin tensors respectively; the micromotion is defined by a microspin tensor. On any shear plane, slickenlines form parallel to the direction of the maximum rate of shear, which we assume is determined by the direction of the maximum component of the macrovelocity gradient tensor tangent to the shear plane and by a component of the microspin in the shear plane. For a restricted kinematic model, patterns of slickenline orientations are defined on a uniform distribution of shear plane orientations by the values of D and W . D is a ratio of differences in the principal values of the deformation rate tensor, and W is a ratio of the net spin to the maximum rate of macroshear. We distinguish between instantaneous slickenline patterns and finite-deformation patterns. The two are different if the shear planes rotate relative to the principal deformation rate axes, in which case finite-deformation slickenlines form curved or crossing lineations. If W = 0, the instantaneous slickenline patterns have orthorhombic symmetry (0 D D = 1 or 0). If W ≠ 0, the instantaneous slickenline patterns have monoclinic symmetry. For pure shear in an isotropic body, instantaneous slickenline patterns have orthorhombic symmetry ( W = 0, D = 0.5) whereas for pure shear accommodated by shear on a planar anisotropy ( W = −1 , D = 0.5), the pattern is monoclinic. For simple shear with the rigid rotation of local shear planes defined by the shear-induced spin ( W = 0, D = 0.5), the instantaneous slickenline pattern is orthorhombic but the finite-deformation pattern is monoclinic. For simple shear with no shear plane rotation ( W = −1, D = 0.5), the instantaneous pattern is monoclinic. Special cases of our hypothesis are equivalent to the hypothesis that slickenlines are parallel to the direction of maximum resolved shear stress (e.g. Angelier, 1979, 1984). The slickenline patterns predicted for this case are always of orthorhombic or higher symmetry, and are equivalent to our instantaneous patterns for which W = 0 and to our finite-deformation patterns for which, in addition, there is no microspin.

Objective Ex vivo and in vivo micro–computed tomography (micro-CT) combined with a novel image analysis algorithm were used to quantify cortical bone loss and periosteal new bone formation for therapeutic evaluation in a murine model of collagen-induced arthritis. Methods An automated algorithm was created to locate 5 metatarsophalangeal and 3 metacarpophalangeal joints in 3-dimensional micro-CT images of mouse paws for evaluation of joint cortical bone volume (JCBV) within close proximity of the joints as well as cortical bone mineral density and periosteal new bone formation within the paws. For validation, automated estimates of JCBV were compared with radiographic visual scores (RVS) in 4 treatment groups (n = 9 per group): rat anti-mouse CD11a monoclonal antibody, methotrexate (MTX), anti-CD11a plus MTX, and saline only. In a separate study, serial images of hind limbs were evaluated in 2 treatment groups: murine tumor necrosis factor receptor II–Fc fusion protein (mTNFRII; n = 10) and control antibody (n = 7). Results Automated estimates of the JCBV were significantly correlated with the RVS (hind paws R = −0.94, front paws R = −0.81, combined R = −0.87). The anti-CD11a group had significantly higher JCBV compared with controls. In the serial study, the automated estimate of JCBV detected significant treatment effects in the mTNFRII-Fc group compared with controls. Cortical bone mineral density was significantly higher in all treatment groups compared with controls. Conclusion Micro-CT combined with a novel image analysis technique (estimation of JCBV) provides a fully automated means to quantify bone destruction in a mouse model of rheumatoid arthritis.

[1] Side-scan sonar data collected along the steep, faulted north wall of the Hess Deep Rift provide images of a cross section of the upper oceanic crust. These data are integrated with ground truth from the submersible Alvin and the Argo II remotely operated vehicle (ROV) to evaluate faulting and mass wasting associated with the opening of the rift and to help trace geologic contacts between widely spaced dives and ROV surveys. Initial shipboard and subsequently processed data show excellent backscatter returns with well-imaged textures and distinctive patterns corresponding to specific rock units and surficial deposits. Four backscatter textures are widely developed and found to correspond to outcrops of basaltic lavas, outcrops of sheeted dikes and gabbroic rocks, surficial talus and rubble deposits, and pelagic sedimentary material. Using test areas identified by Alvin dives, the side-scan sonar data were processed to automatically classify the backscatter returns in terms of major rock units. The resultant processed image is used for further manual and computer-aided classification of the scarp geology. These techniques are evaluated for mapping the geology in rugged terrains similar to the steep walls of Hess Deep Rift.

The poor success in controlling small bowel (SB) allograft rejection is partially attributed to the unique immune environment in the donor intestine. We hypothesized that Ag-induced activation of donor-derived T cells contributes to the initiation of SB allograft rejection. To address the role of donor T cell activation in SB transplantation, SB grafts from DO11.10 TCR transgenic mice (BALB/c, H-2Ld+) were transplanted into BALB/c (isografts), or single class I MHC-mismatched (Ld-deficient) BALB/c H-2dm2 (dm2, H-2Ld−) mutant mice (allografts). Graft survival was followed after injection of control or antigenic OVA323–339 peptide. Eighty percent of SB allografts developed severe rejection in mice treated with antigenic peptide, whereas 30 days regardless of OVA323–339 administration. Activation of donor T cells increased intragraft expression of proinflammatory cytokine (IFN-γ) and CXC chemokine IFN-γ-inducible protein-10 mRNA and enhanced activation and accumulation of host NK and T cells in SB allografts. Treatment of mice with neutralizing anti-IFN-γ-inducible protein-10 mAb increased SB allograft survival in Ag-treated mice (67%; p < 0.05) and reduced accumulation of host T cells and NK cells in the lamina propria but not mesenteric lymph nodes. These results suggest that activation of donor T cells after SB allotransplantation induces production of a Th1-like profile of cytokines and CXC chemokines that enhance infiltration of host T cells and NK cells in SB allografts. Blocking this pathway may be of therapeutic value in controlling SB allograft rejection.

The uppermost 2 km of the oceanic crust created at the fast spreading (135 mm yr−1, full rate) equatorial East Pacific Rise (EPR) is exposed for tens of kilometers along escarpments bounding the Hess Deep Rift. Mosaics of large-scale digital images from the remotely operated vehicle (ROV) Argo II and direct observations from the submersible Alvin document a degree of geological complexity and variability that is not evident from most studies of ophiolites or prevailing models of seafloor spreading. Dramatic variations in the thickness and internal structure are documented in both the basaltic volcanic and sheeted dike rock units. These rock units are characterized by extensive faulting, fine-scale fracturing, and rotations of coherent crustal blocks meters to tens of meters across. The uppermost basaltic lavas are essentially undeformed and have overall gently inclined flow surfaces. Through most of the basaltic lava unit, however, lava flow contacts dip (20°–70°W) toward the EPR and generally increase in dip downward in the section. Dikes cutting the lavas and in the underlying sheeted dike unit generally dip (90°–40°E) away from the EPR. Deeper level gabbroic rocks show little evidence of the intense fracturing typical of the overlying units. We interpret this upper crustal structure as the result of subaxial subsidence within 1–2 km of the EPR that accommodated the thickening of the basaltic lava unit to ∼500 m. Variations in the thickness of lava and dike units and spatially related structures along the rift escarpments suggest temporal fluctuations in magma supply. These results indicate that substantial brittle deformation accompanied waxing and waning volcanism during the accretion of the crustal section exposed at the Hess Deep Rift. If this type of structure is typical of uppermost oceanic crust generated at the EPR, these processes may be common along fast spreading mid-ocean ridges.

Soluble foreign antigen usually leads to a transient clonal expansion of antigen-specific T cells followed by the deletion and/or functional inactivation of the cells. As interleukin (IL)-10 is a key immunoregulatory cytokine, we questioned whether neutralization of IL-10 during priming with soluble antigen could prime for a subsequent T helper cell type 1 (Th1) effector recall response. By using an adoptive transfer model to track the fate of antigen-specific T cell receptor (TCR)-transgenic CD4+ T cells, we show that administration of soluble ovalbumin (OVA) protein, but not OVA323–339 peptide antigen, together with an anti–IL-10 receptor (R) mAb led to the enhancement of a Th1 response upon rechallenge. Lipopolysaccharide (LPS) present in the protein was necessary for priming for Th1 recall responses in the presence of anti–IL-10R mAb, as removal of LPS abrogated this effect. Moreover, addition of LPS to the peptide did not itself allow priming for recall Th1 effector responses unless endogenous levels of IL-10 were neutralized with an anti–IL-10R mAb. A significant increase in OVA-specific IgG1 and IgG2a isotypes was observed when the protein antigen was administered with anti–IL-10R mAb; however, this was not the case with peptide antigen administered together with anti–IL-10R and LPS. Our data, showing that LPS receptor signaling and neutralization of endogenous immunosuppressive cytokines is essential for Th1 priming, has important implications for the design of relevant vaccines for effective in vivo immunotherapy.

Background & Aims: Previous data suggest that oral antigen induces interferon (IFN)-γ production in intestinal T cells. However, oral tolerance is associated with decreased production of IFN-γ by T cells after antigen sensitization. The aim of this study was to examine the role of IFN-γ in oral tolerance. Methods: Oral tolerance was examined in BALB/c mice after the adoptive transfer of T cells from chicken ovalbumin (OVA 323-339 )-specific, DO11.10 × RAG-1 −/− T-cell receptor transgenic mice. Results: OVA feeding induced systemic tolerance of delayed-type hypersensitivity (DTH) and antibody responses. OVA feeding up-regulated IFN-γ production by transgenic T cells in Peyer's patch and mesenteric lymph node but not splenic tissues. Treatment of OVA-fed mice with neutralizing monoclonal antibody to IFN-γ prevented tolerance of DTH responses. Analysis of transgenic T-cell numbers in DTH sites by immunohistochemical staining suggested that induction of IFN-γ by oral antigen decreased accumulation of transgenic T cells in cutaneous sites of antigen injection. IFN-γ–deficient or wild-type DO11.10 and BALB/c mice were used to show that IFN-γ production by donor transgenic T cells was critical for oral tolerance. Conclusions: These data suggest that the induction of IFN-γ by oral antigen contributes to systemic tolerance by decreasing migration of T cells to peripheral sites of inflammation. GASTROENTEROLOGY 2000;119:129-138

This paper introduces a transgenic (Tg) mouse in which the majority of the CD4-bearing T cells have T-cell receptors that react with ovalbumin (OVA) as a model for ethanol research. Although these Tg animals were bred onto the BALB/c genetic background, a strain generally considered to be nonpreferring in ethanol consumption, we determined that BALB/c mice would consume an ethanol-containing liquid diet, without significant mortality, and assessed alteration of specific immune responses. BALB/c, C57BL/6 (B6), or (BALB/c x C57BL/6)F-1 hybrid (CB6F1) mice were fed LED containing 35, 30, 25, or 20% ethanol-derived calories. Significant mortality (> 40%) was seen only in BALB/c and pronounced weight loss was seen in BALB/c, B6, CB6F1 mice when they were fed the diet containing the greatest ethanol concentration (LED35). Diets containing lesser amounts of ethanol did not cause mortality. Liquid diets containing > or = 30% ethanol-derived calories significantly impaired the chicken gamma-globulin-specific delayed hypersensitivity responses in BALB/c, B6, and CB6F1mice without significantly affecting the humoral immune response to sheep red blood cells. We show that immunization of the Tg mice is not required for the development of a vigorous "delayed hypersensitivity" response to OVA or the I-Ad-restricted peptide OVA323-339 in mice fed standard solid lab chow or liquid control diet. In marked contrast, OVA Tg mice fed ethanol show a profound inhibition of this immune response, indicating that ethanol-induced inhibition of cell-mediated immunity occurs independently of antigen priming.