In vivo therapeutic synergism of anti-epidermal growth factor receptor and anti-HER2 monoclonal antibodies against pancreatic carcinomas.

PURPOSE: Pancreatic carcinoma is highly resistant to therapy. Epidermal growth factor receptor (EGFR) and HER2 have been reported to be both dysregulated in this cancer. To evaluate the in vivo effect of binding both EGFR and HER2 with two therapeutic humanized monoclonal antibodies (mAb), we treated human pancreatic carcinoma xenografts, expressing high EGFR and low HER2 levels. EXPERIMENTAL DESIGN: Nude mice, bearing xenografts of BxPC-3 or MiaPaCa-2 human pancreatic carcinoma cell lines, were injected twice weekly for 4 weeks with different doses of anti-EGFR (matuzumab) and anti-HER2 (trastuzumab) mAbs either alone or in combination. The effect of the two mAbs, on HER receptor phosphorylation, was also studied in vitro by Western blot analysis. RESULTS: The combined mAb treatment significantly inhibited tumor progression of the BxPC-3 xenografts compared with single mAb injection (P = 0.006) or no treatment (P = 0.0004) and specifically induced some complete remissions. The two mAbs had more antitumor effect than 4-fold greater doses of each mAb. The significant synergistic effect of the two mAbs was confirmed on the MiaPaCa-2 xenograft and on another type of carcinoma, SK-OV-3 ovarian carcinoma xenografts. In vitro, the cooperative effect of the two mAbs was associated with a decrease in EGFR and HER2 receptor phosphorylation. CONCLUSIONS: Anti-HER2 mAb has a synergistic therapeutic effect when combined with an anti-EGFR mAb on pancreatic carcinomas with low HER2 expression. These observations may open the way to the use of these two mAbs in a large panel of carcinomas expressing different levels of the two HER receptors.

Placental growth factor contributes to micro-vascular abnormalization and blood-retinal barrier breakdown in diabetic retinopathy.

OBJECTIVE: There are controversies regarding the pro-angiogenic activity of placental growth factor (PGF) in diabetic retinopathy (DR). For a better understanding of its role on the retina, we have evaluated the effect of a sustained PGF over-expression in rat ocular media, using ciliary muscle electrotransfer (ET) of a plasmid encoding rat PGF-1 (pVAX2-rPGF-1). MATERIALS AND METHODS: pVAX2-rPGF-1 ET in the ciliary muscle (200 V/cm) was achieved in non diabetic and diabetic rat eyes. Control eyes received saline or naked plasmid ET. Clinical follow up was carried out over three months using slit lamp examination and fluorescein angiography. After the control of rPGF-1 expression, PGF-induced effects on retinal vasculature and on the blood-external barrier were evaluated respectively by lectin and occludin staining on flat-mounts. Ocular structures were visualized through histological analysis. RESULTS: After fifteen days of rPGF-1 over-expression in normal eyes, tortuous and dilated capillaries were observed. At one month, microaneurysms and moderate vascular sprouts were detected in mid retinal periphery in vivo and on retinal flat-mounts. At later stages, retinal pigmented epithelial cells demonstrated morphological abnormalities and junction ruptures. In diabetic retinas, PGF expression rose between 2 and 5 months, and, one month after ET, rPGF-1 over-expression induced glial activation and proliferation. CONCLUSION: This is the first demonstration that sustained intraocular PGF production induces vascular and retinal changes similar to those observed in the early stages of diabetic retinopathy. PGF and its receptor Flt-1 may therefore be looked upon as a potential regulatory target at this stage of the disease.

Developmental effects of basic fibroblast growth factor and platelet-derived growth factor on glial cells in a three-dimensional cell culture system.

In order to study peptide growth factor action in a three-dimensional cellular environment, aggregating cell cultures prepared from 15-day fetal rat telencephalon were grown in a chemically defined medium and treated during an early developmental stage with either bovine fibroblast growth factor (bFGF) or platelet-derived growth factor (PDGF homodimers AA and BB). A single dose (5-50 ng/ml) of either growth factor given to the cultures on day 3 greatly enhanced the developmental increase of the two glia-specific enzyme activities, 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) and glutamine synthetase (GS), whereas it had relatively little effect on total protein and DNA content. Distinct patterns of dose-dependency were found for CNP and GS stimulation. At low concentrations of bFGF (0.5-5 ng/ml) and at all PDGF concentrations applied, the oligodendroglial marker enzyme CNP was the most affected. A relatively small but significant mitogenic effect was observed after treatment with PDGF, particularly at higher concentrations or after repetitive stimulation. The two PDGF homodimers AA and BB were similar in their biological effects and potency. The present results show that under histotypic conditions both growth factors, bFGF and PDGF, promote the maturation rather than the proliferation of immature oligodendrocytes and astrocytes.

Coactivated platelet-derived growth factor receptor {alpha} and epidermal growth factor receptor are potential therapeutic targets in intimal sarcoma.

Intimal sarcoma (IS) is a rare, malignant, and aggressive tumor that shows a relentless course with a concomitant low survival rate and for which no effective treatment is available. In this study, 21 cases of large arterial blood vessel IS were analyzed by immunohistochemistry and fluorescence in situ hybridization and selectively by karyotyping, array comparative genomic hybridization, sequencing, phospho-kinase antibody arrays, and Western immunoblotting in search for novel diagnostic markers and potential molecular therapeutic targets. Ex vivo immunoassays were applied to test the sensitivity of IS primary tumor cells to the receptor tyrosine kinase (RTK) inhibitors imatinib and dasatinib. We showed that amplification of platelet-derived growth factor receptor α (PDGFRA) is a common finding in IS, which should be considered as a molecular hallmark of this entity. This amplification is consistently associated with PDGFRA activation. Furthermore, the tumors reveal persistent activation of the epidermal growth factor receptor (EGFR), concurrent to PDGFRA activation. Activated PDGFRA and EGFR frequently coexist with amplification and overexpression of the MDM2 oncogene. Ex vivo immunoassays on primary IS cells from one case showed the potency of dasatinib to inhibit PDGFRA and downstream signaling pathways. Our findings provide a rationale for investigating therapies that target PDGFRA, EGFR, or MDM2 in IS. Given the clonal heterogeneity of this tumor type and the potential cross-talk between the PDGFRA and EGFR signaling pathways, targeting multiple RTKs and aberrant downstream effectors might be required to improve the therapeutic outcome for patients with this disease.

Glucose-dependent insulinotropic polypeptide (GIP) and its receptor (GIPR): cellular localization, lesion-affected expression, and impaired regenerative axonal growth.

Glucose-dependent insulinotropic polypeptide (GIP) was initially described to be rapidly regulated by endocrine cells in response to nutrient ingestion, with stimulatory effects on insulin synthesis and release. Previously, we demonstrated a significant up-regulation of GIP mRNA in the rat subiculum after fornix injury. To gain more insight into the lesion-induced expression of GIP and its receptor (GIPR), expression profiles of the mRNAs were studied after rat sciatic nerve crush injury in 1) affected lumbar dorsal root ganglia (DRG), 2) spinal cord segments, and 3) proximal and distal nerve fragments by means of quantitative RT-PCR. Our results clearly identified lesion-induced as well as tissue type-specific mRNA regulation of GIP and its receptor. Furthermore, comprehensive immunohistochemical stainings not only confirmed and exceeded the previous observation of neuronal GIP expression but also revealed corresponding GIPR expression, implying putative modulatory functions of GIP/GIPR signaling in adult neurons. In complement, we also observed expression of GIP and its receptor in myelinating Schwann cells and oligodendrocytes. Polarized localization of GIPR in the abaxonal Schwann cell membranes, plasma membrane-associated GIPR expression of satellite cells, and ependymal GIPR expression strongly suggests complex cell type-specific functions of GIP and GIPR in the adult nervous system that are presumably mediated by autocrine and paracrine interactions, respectively. Notably, in vivo analyses with GIPR-deficient mice suggest a critical role of GIP/GIPR signal transduction in promoting spontaneous recovery after nerve crush, insofar as traumatic injury of GIPR-deficient mouse sciatic nerve revealed impaired axonal regeneration compared with wild-type mice.

Regression of cardiac growth in kidney transplant recipients using anti-m-TOR drugs plus RAS blockers: a controlled longitudinal study.

BACKGROUND Left ventricular hypertrophy (LVH) is common in kidney transplant (KT) recipients. LVH is associated with a worse outcome, though m-TOR therapy may help to revert this complication. We therefore conducted a longitudinal study to assess morphological and functional echocardiographic changes after conversion from CNI to m-TOR inhibitor drugs in nondiabetic KT patients who had previously received RAS blockers during the follow-up. METHODS We undertook a 1-year nonrandomized controlled study in 30 non-diabetic KT patients who were converted from calcineurin inhibitor (CNI) to m-TOR therapy. A control group received immunosuppressive therapy based on CNIs. Two echocardiograms were done during the follow-up. RESULTS Nineteen patients were switched to SRL and 11 to EVL. The m-TOR group showed a significant reduction in LVMi after 1 year (from 62 ± 22 to 55 ± 20 g/m2.7; P=0.003, paired t-test). A higher proportion of patients showing LVMi reduction was observed in the m-TOR group (53.3 versus 29.3%, P=0.048) at the study end. In addition, only 56% of the m-TOR patients had LVH at the study end compared to 77% of the control group (P=0.047). A significant change from baseline in deceleration time in early diastole was observed in the m-TOR group compared with the control group (P=0.019). CONCLUSIONS Switching from CNI to m-TOR therapy in non-diabetic KT patients may regress LVH, independently of blood pressure changes and follow-up time. This suggests a direct non-hemodynamic effect of m-TOR drugs on cardiac mass.

PGE2-induced colon cancer growth is mediated by mTORC1.

The inflammatory prostaglandin E2 (PGE2) cytokine plays a key role in the development of colon cancer. Several studies have shown that PGE2 directly induces the growth of colon cancer cells and furthermore promotes tumor angiogenesis by increasing the production of the vascular endothelial growth factor (VEGF). The signaling intermediaries implicated in these processes have however not been fully characterized. In this report, we show that the mechanistic target of rapamycin complex 1 (mTORC1) plays an important role in PGE2-induced colon cancer cell responses. Indeed, stimulation of LS174T cells with PGE2 increased mTORC1 activity as observed by the augmentation of S6 ribosomal protein phosphorylation, a downstream effector of mTORC1. The PGE2 EP4 receptor was responsible for transducing the signal to mTORC1. Moreover, PGE2 increased colon cancer cell proliferation as well as the growth of colon cancer cell colonies grown in matrigel and blocking mTORC1 by rapamycin or ATP-competitive inhibitors of mTOR abrogated these effects. Similarly, the inhibition of mTORC1 by downregulation of its component raptor using RNA interference blocked PGE2-induced LS174T cell growth. Finally, stimulation of LS174T cells with PGE2 increased VEGF production which was also prevented by mTORC1 inhibition. Taken together, these results show that mTORC1 is an important signaling intermediary in PGE2 mediated colon cancer cell growth and VEGF production. They further support a role for mTORC1 in inflammation induced tumor growth.

Transforming growth factor beta 1 production by CD4+ CD25+ regulatory T cells in peripheral blood mononuclear cells from healthy subjects stimulated with Leishmania guyanensis.

Transforming growth factor beta (TGF-beta) has been shown to be a central immunomodulator used by leishmaniae to escape effective mechanisms of protection in human and murine infections with these parasites. However, all the information is derived from studies of established infection, while little is known about TGF-beta production in response to Leishmania stimulation in healthy subjects. In this study, TGF-beta1 production was demonstrated in peripheral blood mononuclear cells from healthy subjects never exposed to leishmaniae in response to live Leishmania guyanensis, and the TGF-beta1-producing cells were described as a distinct subpopulation of CD4(+) CD25(+) regulatory T cells. The suppressive properties of CD4(+) CD25(+) T cells were demonstrated in vitro by their inhibition of production of interleukin 2 (IL-2) and IL-10 by CD4(+) CD25(-) T cells in the presence of either anti-CD3 or L. guyanensis. Although neutralization of TGF-beta1 did not reverse the suppressive activity of CD4(+) CD25(+) T cells activated by anti-CD3, it reversed the suppressive activity of CD4(+) CD25(+) T cells activated by L. guyanensis. Altogether our data demonstrated that TGF-beta1 is involved in the suppressive activity of L. guyanensis-stimulated CD4(+) CD25(+) T cells from healthy controls.

APRIL, a new ligand of the tumor necrosis factor family, stimulates tumor cell growth.

Members of the tumor necrosis factor (TNF) family induce pleiotropic biological responses, including cell growth, differentiation, and even death. Here we describe a novel member of the TNF family designated APRIL (for a proliferation-inducing ligand). Although transcripts of APRIL are of low abundance in normal tissues, high levels of mRNA are detected in transformed cell lines, and in human cancers of colon, thyroid, and lymphoid tissues in vivo. The addition of recombinant APRIL to various tumor cells stimulates their proliferation. Moreover, APRIL-transfected NIH-3T3 cells show an increased rate of tumor growth in nude mice compared with the parental cell line. These findings suggest that APRIL may be implicated in the regulation of tumor cell growth.

Modelling changes in stable isotope compositions of minerals during net transfer reactions in a contact aureole: Wollastonite growth at the northern Hunter Mountain Batholith (Death Valley National Park, USA)

One of the world's largest wollastonite deposits was formed at the contact of the northern Hunter Mountain Batholith (California, USA) in Paleozoic sediments. Wollastonite occurs as zones of variable thickness surrounding layers or nodules of quartzite in limestones. A minimum formation temperature of 650 degrees C is estimated from isolated periclase-bearing lenses in that area. Contact metamorphism of siliceous carbonates has produced mineral assemblages that are consistent with heterogeneous, and partly limited infiltration of water-rich fluids, compatible with O-18/O-16 and C-13/C-12 isotopic patterns recorded in carbonates. Oxygen isotope compositions of wollastonites in the study area may also not require infiltration of large quantities of externally-derived fluids that were out of equilibrium with the rocks. 8180 values of wollastonite are high (14.8 parts per thousand to 25.0 parts per thousand; median: 19.7 parts per thousand) and close to those of the host limestone (19.7 parts per thousand to 28 parts per thousand; median: 24.9 parts per thousand) and quartz (18.0 parts per thousand. to 29.1 parts per thousand; median: 22.6 parts per thousand). Isotopic disequilibrium exists at quartz/wollastonite and wollastonite/calcite boundaries. Therefore, classical batch/Rayleigh fractionation models based on reactant and product equilibrium are not applicable to the wollastonite rims. An approach that relies on local instantaneous mass balance for the reactants, based on the wollastonite-forming reaction is suggested as an alternative way to model wollastonite reaction rims. This model reproduces many of the measured delta O-18 values of wollastonite reaction rims of the current study to within +/- 1 parts per thousand, even though the wollastonite compositions vary by almost 10 parts per thousand. (C) 2011 Elsevier B.V. All rights reserved.

Circulating insulin-like growth factor-I in pregnancy and maternal risk of breast cancer.

BACKGROUND: Elevated serum concentrations of insulin-like growth factor (IGF)-1 have been associated with increased risk of breast cancer. Previously, we reported a similar association in samples obtained during pregnancy. The current study was conducted to further characterize the association of IGF-1 during pregnancy with maternal breast cancer risk. METHODS: A case-control study was nested within the Finnish Maternity Cohort. The study was limited to primiparous women less than 40 years of age, who donated blood samples during early (median, 12 weeks) pregnancy and delivered a single child at term. Seven hundred and nineteen women with invasive breast cancer were eligible. Two controls (n = 1,434) were matched to each case on age and date at blood donation. Serum IGF-1 concentration was measured using an Immulite 2000 analyzer. Conditional logistic regression was used to estimate odds ratios (OR) and 95% confidence intervals (CI).RESULTS: No significant associations were observed between serum IGF-1 concentrations and breast cancer risk in both the overall analysis (OR 1.08 (95% CI 0.80-1.47) and in analyses stratified by histological subtype, lag-time to cancer diagnosis, age at pregnancy or age at diagnosis.CONCLUSIONS: There was no association between IGF-1 and maternal breast cancer risk during early pregnancy in this large nested case-control study.Impact:Serum IGF-1 concentrations during early pregnancy may not be related to maternal risk of breast cancer.

Light-regulated plant growth and development.

Plants are sessile and photo-autotrophic; their entire life cycle is thus strongly influenced by the ever-changing light environment. In order to sense and respond to those fluctuating conditions higher plants possess several families of photoreceptors that can monitor light from UV-B to the near infrared (far-red). The molecular nature of UV-B sensors remains unknown, red (R) and far-red (FR) light is sensed by the phytochromes (phyA-phyE in Arabidopsis) while three classes of UV-A/blue photoreceptors have been identified: cryptochromes, phototropins, and members of the Zeitlupe family (cry1, cry2, phot1, phot2, ZTL, FKF1, and LKP2 in Arabidopsis). Functional specialization within photoreceptor families gave rise to members optimized for a wide range of light intensities. Genetic and photobiological studies performed in Arabidopsis have shown that these light sensors mediate numerous adaptive responses (e.g., phototropism and shade avoidance) and developmental transitions (e.g., germination and flowering). Some physiological responses are specifically triggered by a single photoreceptor but in many cases multiple light sensors ensure a coordinated response. Recent studies also provide examples of crosstalk between the responses of Arabidopsis to different external factors, in particular among light, temperature, and pathogens. Although the different photoreceptors are unrelated in structure, in many cases they trigger similar signaling mechanisms including light-regulated protein-protein interactions or light-regulated stability of several transcription factors. The breath and complexity of this topic forced us to concentrate on specific aspects of photomorphogenesis and we point the readers to recent reviews for some aspects of light-mediated signaling (e.g., transition to flowering).

Relatedness among arbuscular mycorrhizal fungi drives plant growth and intraspecific fungal coexistence.

Arbuscular mycorrhizal fungi (AMF) form symbioses with most plant species. They are ecologically important determinants of plant growth and diversity. Considerable genetic variation occurs in AMF populations. Thus, plants are exposed to AMF of varying relatedness to each other. Very little is known about either the effects of coexisting AMF on plant growth or which factors influence intraspecific AMF coexistence within roots. No studies have addressed whether the genetics of coexisting AMF, and more specifically their relatedness, influences plant growth and AMF coexistence. Relatedness is expected to influence coexistence between individuals, and it has been suggested that decreasing ability of symbionts to coexist can have negative effects on the growth of the host. We tested the effect of a gradient of AMF genetic relatedness on the growth of two plant species. Increasing relatedness between AMFs lead to markedly greater plant growth (27% biomass increase with closely related compared to distantly related AMF). In one plant species, closely related AMF coexisted in fairly equal proportions but decreasing relatedness lead to a very strong disequilibrium between AMF in roots, indicating much stronger competition. Given the strength of the effects with such a shallow relatedness gradient and the fact that in the field plants are exposed to a steeper gradient, we consider that AMF relatedness can have a strong role in plant growth and the ability of AMF to coexist. We conclude that AMF relatedness is a driver of plant growth and that relatedness is also a strong driver of intraspecific coexistence of these ecologically important symbionts.

Microstructural, chemical and textural records during growth of snowball garnet

The growth history of two populations of snowball garnet from the Lukmanier Pass area (central Swiss Alps) was examined through a detailed analysis of three-dimensional geometry, chemical zoning and crystallographic orientation. The first population, collected in the hinge of a chevron-type fold, shows an apparent rotation of 360 degrees. The first 270 degrees are characterized by spiral-shaped inclusion trails, gradual and concentric Mn zoning and a single crystallographic orientation, whereas in the last 90 degrees, crenulated inclusion trails and secondary Mn maxima centred on distinct crystallographic garnet domains are observed. Microstructural, geochemical and textural data indicate a radical change in growth regime between the two growth sequences. In the first 270 degrees, growth occurred under rotational non-coaxial flow, whereas in the last 90 degrees, garnet grew under a non-rotational shortening regime. The second population, collected in the limb of the same chevron-type fold structure, is characterized by a spiral geometry that does not exceed 270 degrees of apparent rotation. These garnet microstructures do not record any evidence for a modification of the stress field during garnet growth. Concentric Mn zoning as well as a single crystallographic orientation are observed for the entire spiral. Electron backscatter diffraction data indicate that nearly all central domains in the snowball garnet are characterized by one [001] axis oriented (sub-)parallel to the symmetry axis and by another [001] axis oriented (sub-)parallel to the orientation of the internal foliation. These features suggest that the crystallographic orientation across the garnet spiral is not random and that a relation exists among the symmetry axis, the internal foliation and the crystallographic orientation.