Reversing immunosuppression in the tumor microenvironment of fibrolamellar carcinoma via PD-1 and IL-10 blockade.

Fibrolamellar carcinoma (FLC) is a rare liver tumor driven by the DNAJ-PKAc fusion protein that affects healthy young patients. Little is known about the immune response to FLC, limiting rational design of immunotherapy. Multiplex immunohistochemistry and gene expression profiling were performed to characterize the FLC tumor immune microenvironment and adjacent non-tumor liver (NTL). Flow cytometry and T cell receptor (TCR) sequencing were performed to determine the phenotype of tumor-infiltrating immune cells and the extent of T cell clonal expansion. Fresh human FLC tumor slice cultures (TSCs) were treated with antibodies blocking programmed cell death protein-1 (PD-1) and interleukin-10 (IL-10), with results measured by cleaved caspase-3 immunohistochemistry. Immune cells were concentrated in fibrous stromal bands, rather than in the carcinoma cell compartment. In FLC, T cells demonstrated decreased activation and regulatory T cells in FLC had more frequent expression of PD-1 and CTLA-4 than in NTL. Furthermore, T cells had relatively low levels of clonal expansion despite high TCR conservation across individuals. Combination PD-1 and IL-10 blockade signficantly increased cell death in human FLC TSCs. Immunosuppresion in the FLC tumor microenvironment is characterized by T cell exclusion and exhaustion, which may be reversible with combination immunotherapy.

The role of soils in the regulation of ocean acidification.

Soils play an important role in mediating chemical weathering reactions and carbon transfer from the land to the ocean. Proposals to increase the contribution of alkalinity to the oceans through 'enhanced weathering' as a means to help prevent climate change are gaining increasing attention. This would augment the existing connection between the biogeochemical function of soils and alkalinity levels in the ocean. The feasibility of enhanced weathering depends on the combined influence of what minerals are added to soils, the formation of secondary minerals in soils and the drainage regime, and the partial pressure of respired CO2 around the dissolving mineral. Increasing the alkalinity levels in the ocean through enhanced weathering could help to ameliorate the effects of ocean acidification in two ways. First, enhanced weathering would slightly elevate the pH of drainage waters, and the receiving coastal waters. The elevated pH would result in an increase in carbonate mineral saturation states, and a partial reversal in the effects of elevated CO2. Second, the increase in alkalinity would help to replenish the ocean's buffering capacity by maintaining the 'Revelle Factor', making the oceans more resilient to further CO2 emissions. However, there is limited research on the downstream and oceanic impacts of enhanced weathering on which to base deployment decisions. This article is part of the theme issue 'The role of soils in delivering Nature's Contributions to People'.

Improving therapeutic efficacy of IL-12 intratumoral gene electrotransfer through novel plasmid design and modified parameters.

The use of immunomodulatory cytokines has been shown effective in regressing a wide range of tumors. However, systemic delivery of recombinant cytokines results in serious, potentially life-threatening, adverse effects. By contrast, nucleic acid transfer via electroporation (EP) is a safe and effective method of delivering plasmid-encoded cytokines to tumors. Intratumoral delivery of IL-12 plasmid DNA by electroporation (IT-pIL12-EP) produced objective response rates in Phase 2 clinical trials in metastatic melanoma. However, only 17.9% of patients receiving IT-pIL12-EP show a complete therapeutic response. Here, we sought to improve the antitumor efficacy of our clinical IT-pIL12-EP plasmid electroporation platform. We evaluated multiple plasmid designs for IL-12 expression. IL-12 expression from a plasmid incorporating a picornavirus-derived co-translational P2A site was the most effective in expressing IL-12p70. In addition, modifying the electroporation parameters improved transfection efficiency and expression of plasmid-derived IL-12p70, as well as its downstream effector IFN-γ in vivo. Finally, using a murine melanoma model that is representative of the intended target patient population, we show that combining modified electroporation conditions with the pIL12-P2A plasmid expression enhances the systemic antitumor response. These improvements to the IT-pIL12-EP platform may improve patient clinical response rates and survival when translated to clinical trials.

Mathematical methods for diffusion MRI processing.

In this article, we review recent mathematical models and computational methods for the processing of diffusion Magnetic Resonance Images, including state-of-the-art reconstruction of diffusion models, cerebral white matter connectivity analysis, and segmentation techniques. We focus on Diffusion Tensor Images (DTI) and Q-Ball Images (QBI).

Isolation of multipotent progenitor cells from human fetal liver capable of differentiating into liver and mesenchymal lineages.

Little is known about the differentiation capabilities of nonhematopoietic cells of the human fetal liver. We report the isolation and characterization of a human fetal liver multipotent progenitor cell (hFLMPC) population capable of differentiating into liver and mesenchymal cell lineages. Human fetal livers (74-108 days of gestation) were dissociated and maintained in culture. We treated the colonies with geneticin and mechanically isolated hFLMPCs, which were kept in an undifferentiated state by culturing on feeder layers. We derived daughter colonies by serial dilution, verifying monoclonality using the Humara assay. hFLMPCs, which have been maintained in culture for up to 100 population doublings, have a high self-renewal capability with a doubling time of 46 h. The immunophenotype is: CD34+, CD90+, c-kit+, EPCAM+, c-met+, SSEA-4+, CK18+, CK19+, albumin-, alpha-fetoprotein-, CD44h+, and vimentin+. Passage 1 (P1) and P10 cells have identical morphology, immunophenotype, telomere length, and differentiation capacity. Placed in appropriate media, hFLMPCs differentiate into hepatocytes and bile duct cells, as well as into fat, bone, cartilage, and endothelial cells. Our results suggest that hFLMPCs are mesenchymal-epithelial transitional cells, probably derived from mesendoderm. hFLMPCs survive and differentiate into functional hepatocytes in vivo when transplanted into animal models of liver disease. hFLMPCs are a valuable tool for the study of human liver development, liver injury, and hepatic repopulation.

Protection of human and murine hepatocytes against Fas-induced death by transferrin and iron.

Recent studies in a murine model show that transferrin (Tf) interferes with Fas-mediated hepatocyte death and liver failure by decreasing pro-apoptotic and increasing anti-apoptotic signals. We show here in vitro in murine and human hepatocyte cell lines and in vivo in mice that Fas-induced apoptosis is modulated by exogenous Tf and iron. The results obtained with iron-free Tf (ApoTf), iron-saturated Tf (FeTf), and the iron chelator salicylaldehyde isonicotinoyl hydrazone (SIH) in its iron-free and iron-saturated (FeSIH) forms indicate that apoptosis-modulating effects of Tf are not mediated by iron alone. Both the Tf molecule and iron affect multiple aspects of cell death, and the route of iron delivery to the cell may be critical for the final outcome of cellular Fas signaling. Survival of hepatocytes 'stressed' by Fas signals can be manipulated by Tf and iron and may be a target for prophylactic and therapeutic interventions.

Expression of suppressors of cytokine signaling during liver regeneration.

The cytokines TNF and IL-6 play a critical role early in liver regeneration following partial hepatectomy (PH). Since IL-6 activates signal transducers and activators of transcription (STATs), we examined whether the suppressors of cytokine signaling (SOCS) may be involved in terminating IL-6 signaling. We show here that SOCS-3 mRNA is induced 40-fold 2 hours after surgery. SOCS-2 and CIS mRNA are only weakly induced, and SOCS-1 is not detectable. SOCS-3 induction after PH is transient and correlates with a decrease in STAT-3 DNA binding and a loss of tyrosine 705 phosphorylation. This response is markedly reduced in IL-6 knockout (KO) mice. TNF injection induces SOCS-3 mRNA in wild-type mice (albeit weakly compared with the increase observed after PH) but not in TNF receptor 1 or IL-6 KO mice. In contrast, IL-6 injection induces SOCS-3 in these animals, demonstrating a requirement for IL-6 in SOCS-3 induction. IL-6 injection into wild-type mice also induces SOCS-1, -2, and CIS mRNA, in addition to SOCS-3. Together, these results suggest that SOCS-3 may be a key component in downregulating STAT-3 signaling after PH and that SOCS-3 mRNA levels in the regenerating liver are regulated by IL-6.

Tuberin phosphorylation regulates its interaction with hamartin. Two proteins involved in tuberous sclerosis.

Hamartin and tuberin are products of the tumor suppressor genes, TSC1 and TSC2, respectively. When mutated, a characteristic spectrum of tumor-like growths develop resulting in the syndrome of tuberous sclerosis complex. The phenotypes associated with TSC1 and TSC2 mutations are largely indistinguishable suggesting a common biochemical pathway. Indeed, hamartin and tuberin have been shown to interact stably in vitro and in vivo. Factors that regulate their interaction are likely critical to the understanding of disease pathogenesis. In this study, we showed that tuberin is phosphorylated at serine and tyrosine residues in response to serum and other factors, and it undergoes serial phosphorylation that can be detected by differences in electrophoretic mobilities. A disease-related TSC2 mutation (Y1571H) nearly abolished tuberin phosphorylation when stimulated with pervanadate. Expression of this mutant tuberin caused a marked reduction in TSC1-TSC2 interaction compared with wild-type protein and significantly curtailed the growth inhibitory effects of tuberin when overexpressed in COS1 cells, consistent with a loss of function mutation. Examination of a second pathologic mutation, P1675L, revealed a similar relationship between limited phosphorylation and reduced interaction with hamartin. Our data show for the first time that 1) tuberin is phosphorylated at tyrosine and serine residues, 2) TSC1-TSC2 interaction is regulated by tuberin phosphorylation, and 3) defective phosphorylation of tuberin is associated with loss of its tumor suppressor activity. These findings suggest that phosphorylation may be a key regulatory mechanism controlling TSC1-TSC2 function.

Disruption of redox homeostasis in tumor necrosis factor-induced apoptosis in a murine hepatocyte cell line.

Tumor necrosis factor (TNF) is a mediator of the acute phase response in the liver and can initiate proliferation and cause cell death in hepatocytes. We investigated the mechanisms by which TNF causes apoptosis in hepatocytes focusing on the role of oxidative stress, antioxidant defenses, and mitochondrial damage. The studies were conducted in cultured AML12 cells, a line of differentiated murine hepatocytes. As is the case for hepatocytes in vivo, AML12 cells were not sensitive to cell death by TNF alone, but died by apoptosis when exposed to TNF and a small dose of actinomycin D (Act D). Morphological signs of apoptosis were not detected until 6 hours after the treatment and by 18 hours approximately 50% of the cells had died. Exposure of the cells to TNF+Act D did not block NFkappaB nuclear translocation, DNA binding, or its overall transactivation capacity. Induction of apoptosis was characterized by oxidative stress indicated by the loss of NAD(P)H and glutathione followed by mitochondrial damage that included loss of mitochondrial membrane potential, inner membrane structural damage, and mitochondrial condensation. These changes coincided with cytochrome C release and the activation of caspases-8, -9, and -3. TNF-induced apoptosis was dependent on glutathione levels. In cells with decreased levels of glutathione, TNF by itself in the absence of transcriptional blocking acted as an apoptotic agent. Conversely, the antioxidant alpha-lipoic acid, that protected against the loss of glutathione in cells exposed to TNF+Act D completely prevented mitochondrial damage, caspase activation, cytochrome C release, and apoptosis. The results demonstrate that apoptosis induced by TNF+Act D in AML12 cells involves oxidative injury and mitochondrial damage. As injury was regulated to a larger extent by the glutathione content of the cells, we suggest that the combination of TNF+Act D causes apoptosis because Act D blocks the transcription of genes required for antioxidant defenses.

MKK7 is a stress-activated mitogen-activated protein kinase kinase functionally related to hemipterous.

Exposure of mammalian cells to stressful stimuli results in activation of the c-Jun NH2-terminal kinase (JNK)/stress-activated protein kinases (SAPKs), a family of protein kinases related to mitogen-activated protein (MAP) kinase. JNK/SAPKs are activated by specific MAP kinase kinases (MKKs), one of which, MKK4/SEK1, has been characterized extensively. In Drosophila, the JNK/SAPK Basket (Bsk) and the MKK Hemipterous (Hep), are important for embryonic development. Loss of function of either gene inhibits dorsal closure, a morphogenetic movement in which the edges of the embryonic ectoderm move together over the amnioserosa. There is evidence that the Rho GTPases Rac and Cdc42 are also required for dorsal closure, suggesting that Rac or Cdc42 may regulate Hep and Bsk. We have identified MKK7, a murine homolog of Hep. MKK7 functionally rescues hep mutant flies. In fibroblasts, MKK7 is activated by stress and by the GTPase Rac1. MKK7 directly phosphorylates and activates JNK/SAPK. Thus, MKK7 is a homolog of hep and functions in a conserved signaling pathway involving JNK/SAPK and the GTPase Rac1.

Rapid membrane effects of steroids in neuroblastoma cells: effects of estrogen on mitogen activated protein kinase signalling cascade and c-fos immediate early gene transcription.

Rapid effects of steroid hormones have been observed in neuronal cells for many years. We show here, that in the human neuroblastoma cell line SK-N-SH, the membrane impermeable conjugated 17beta-estradiol (E2BSA) activates mitogen activated protein kinase kinase (MAPKK or MEK) and induces the phosphorylation and activation of both ERK-1 and ERK-2 (mitogen activated protein kinase or MAPK). Additionally, E2BSA induces the transcription of a reporter gene construct driven by the promoter of the mouse c-fos proto-oncogene. The effects of this membrane impermeable estrogen on c-fos transcription are not inhibited by the estrogen receptor antagonists Tamoxifen or ICI 182,780, further excluding the involvement of the intracellular estrogen receptor. This is also illustrated by the observation that E2BSA does not activate estrogen response element (ERE) mediated transcription. This is the first report of rapid membrane effects of 17beta-estradiol on growth factor related signalling pathways in neuronal cells, and indicates a potential mechanism by which 17beta-estradiol might affect the expression of genes whose promoters do not contain EREs but are responsive to factors acting through other response elements such as AP-1 and SRE sites.

The mitogen-activated protein kinase pathway can mediate growth inhibition and proliferation in smooth muscle cells. Dependence on the availability of downstream targets.

Activation of the classical mitogen-activated protein kinase (MAPK) pathway leads to proliferation of many cell types. Accordingly, an inhibitor of MAPK kinase, PD 098059, inhibits PDGF-induced proliferation of human arterial smooth muscle cells (SMCs) that do not secrete growth-inhibitory PGs such as PGE2. In striking contrast, in SMCs that express the inducible form of cyclooxygenase (COX-2), activation of MAPK serves as a negative regulator of proliferation. In these cells, PDGF-induced MAPK activation leads to cytosolic phospholipase A2 activation, PGE2 release, and subsequent activation of the cAMP-dependent protein kinase (PKA), which acts as a strong inhibitor of SMC proliferation. Inhibition of either MAPK kinase signaling or of COX-2 in these cells releases them from the influence of the growth-inhibitory PGs and results in the subsequent cell cycle traverse and proliferation. Thus, the MAPK pathway mediates either proliferation or growth inhibition in human arterial SMCs depending on the availability of specific downstream enzyme targets.

Toxicological consequences of Aroclor 1254 ingestion by female rhesus (Macaca mulatta) monkeys and their nursing infants. Part 3: post-reproduction and pathological findings.

A group of 80 menstruating rhesus (Macaca mulatta) monkeys were randomly allocated to four similar rooms (20 monkeys/room) and then to one of five dose groups (four females/dose group/room). Each day the monkeys self-ingested capsules containing doses of 0, 5, 20, 40 or 80 microg Aroclor 1254/kg body weight. After 25 months of continuous dosing, approximately 90% of the treated females had attained a qualitative pharmacokinetic steady state with respect to the concentration of polychlorinated biphenyl (PCB) in their nuchal fat pad. Concurrently, sebaceous glands were being examined for changes analogous to chloracne. Subsequently, the females were paired with untreated males. The infants' blood PCB levels at birth were not correlated with its dam's dose or blood PCB level. However, there was an association between an infants preweaning blood PCB levels and its dam's dose and PCB milk levels. After weaning, the infants were not dosed with PCB. The half-life for the PCB in the infants' blood was determined and found to be slightly more than 15 wk. After 6 yr on test, three monkeys from the 0, 5, 20 and 40 microg dose groups were randomly allocated to a depletion study to ascertain the half-lives of specific PCB congeners (Mes et al., Chemosphere 1995, 30, 789-800). Concurrently, necropsies began of the remaining females, and of seven infants from the treated dams and four infants from the control dams, which had attained an age of 2 yr. Approximately 3 yr later, the depletion monkeys were necropsied. The only statistically significant treatment-related pathological changes found during the study were in the adult females, in which an involution of the sebaceous glands and a dose related increase in liver weight due to hyperplasia were evident.

A novel cytoplasmic dual specificity protein tyrosine phosphatase implicated in muscle and neuronal differentiation.

Dual specificity protein tyrosine phosphatases (dsPTPs) are a subfamily of protein tyrosine phosphatases implicated in the regulation of mitogen-activated protein kinase (MAPK). In addition to hydrolyzing phosphotyrosine, dsPTPs can hydrolyze phosphoserine/threonine-containing substrates and have been shown to dephosphorylate activated MAPK. We have identified a novel dsPTP, rVH6, from rat hippocampus. rVH6 contains the conserved dsPTP active site sequence, VXVHCX2GX2RSX5AY(L/I)M, and exhibits phosphatase activity against activated MAPK. In PC12 cells, rVH6 mRNA is induced during nerve growth factor-mediated differentiation but not during insulin or epidermal growth factor mitogenic stimulation. In MM14 muscle cells, rVH6 mRNA is highly expressed in proliferating cells and declines rapidly during differentiation. rVH6 expression correlates with the inability of fibroblast growth factor to stimulate MAPK activity in proliferating but not in differentiating MM14 cells. rVH6 protein localizes to the cytoplasm and is the first dsPTP to be localized outside the nucleus. This novel subcellular localization may expose rVH6 to potential substrates that differ from nuclear dsPTPs substrates.

The correlation of ankle oscillometric blood pressures and segmental pulse volumes to Doppler systolic pressures in arterial occlusive disease.

PURPOSE: This study was designed to evaluate the accuracy and failure rates of automatically collected oscillometric ankle systolic pressures (Psys) and pulse volumes (Pvol) using a new algorithm as compared with Psys obtained by standard manual Doppler-and-cuff technique. METHODS: One hundred ten consecutive patients at a vascular laboratory had brachial and ankle Psys measured with the two methods. Pvol at or near the mean arterial pressure was also obtained automatically by the oscillometric device. RESULTS: Both methods showed a 6.6% failure rate when measuring Psys at the ankle. Oscillometric Psys measurement was possible when Doppler Psys failed as a result of nonoccluding arteries. No difference was found between the two methods in occluding limbs with ankle-brachial indexes of 1.30 or more. Sequential brachial Psys values had a mean difference (Doppler-oscillometric) or 2 +/- 10.9 mm Hg and a correlation coefficient (r) of 0.92. Measurements at the ankle had a mean difference of -8.4 +/- 16.8 mm Hg and r = 0.90. These differences were not statistically significant. Mean arterial pressure Pvol recorded at the ankle also correlated with ankle Doppler Psys (r = 0.71) and showed a 1.9% failure rate. CONCLUSION: Both automatic oscillometric plethysmographic Psys and Pvol at the ankle are shown to correlate well with Doppler-and-cuff Psys in patients with vascular disease. Oscillometric measurements can replace Doppler measurements in most clinical situations.

Acoustic properties of some biocompatible polymers at body temperature.

In response to the many invasive applications of ultrasound which are developing, the acoustic properties of several aliphatic and aromatic polyurethanes and Polyether block amide (PEBA) copolymers are presented. These polymers were reported by their manufacturers as being biocompatible and are possibly suitable for short-term implantation in humans. Speed and attenuation of sound are measured at 37 degrees C as a function of frequency by use of a Fourier-transform method. These properties are reported in tabular and graphic form.

Differential activation of mitogen-activated protein kinase in response to basic fibroblast growth factor in skeletal muscle cells.

In the MM14 mouse myoblast cell line, fibroblast growth factor (FGF) stimulates proliferation and represses differentiation. However, the intracellular signaling pathways used by FGF to affect these cellular processes are unknown. The predominant FGF receptor present on MM14 cells, FGFR1, is a receptor tyrosine kinase capable of activating the mitogen-activated protein kinase (MAPK) cascade in fibroblast and neuronal cell lines. To determine whether the FGF signal is mediated via the MAPK cascade in myoblasts, MM14 cells were stimulated with basic FGF (bFGF) and activities of the various kinases were measured. After withdrawal from serum and bFGF for 3 hr, bFGF stimulated MAPK kinase (MAPKK) activity, but MAPK and S6 peptide kinase activities were not detected. In contrast, when serum and bFGF were withdrawn for 10 hr, the activities of MAPKK, MAPK, and S6 peptide kinase were all stimulated by bFGF treatment. The inability of bFGF to stimulate MAPK after 3 hr of withdrawal may be due, in part, to the presence of a MAPK phosphatase activity that was detected in MM14 cell extracts. This dephosphorylating activity diminishes during commitment to terminal differentiation and is inhibited by sodium orthovanadate. Thus, the ability of bFGF to stimulate MAPK in MM14 cells is correlated with the loss of a MAPK phosphatase activity. These results show that although bFGF activates MAPKK in proliferating myoblasts, the mitogenic signal does not progress to the downstream kinases, providing a physiological example of an uncoupling of the MAPK cascade.