Human immune system (HIS)-engrafted mice are new tools to investigate human immune responses. Here, we used HIS mice to study human immune responses against human HER-2-positive cancer cells and ...their ability to control tumour growth and metastasis.
BALB/c Rag2(-/-), Il2rg(-/-) mice were engrafted with CD34(+) or CD133(+) human cord blood hematopoietic stem cells (HSC) and vaccinated with human HER-2-positive cancer cells SK-OV-3 combined to human IL-12.
Both CD34(+) or CD133(+) human HSC gave long-term engraftment and differentiation, both in peripheral blood and in lymphoid organs, and production of human antibodies. Vaccinated mice produced specific anti-HER-2 human IgG. An s.c. SK-OV-3 challenge was significantly inhibited (but not abolished) in both vaccinated and non-vaccinated HIS mice. Tumours were heavily infiltrated with human and murine cells, mice showed NK cells and production of human interferon-γ, that could contribute to tumour growth inhibition. Vaccinated HIS mice showed significantly inhibited lung metastases when compared with non-vaccinated HIS mice and to non-HIS mice, along with higher levels of tumour-infiltrating human dendritic cells.
Anti-HER-2 responses were elicited through an adjuvanted allogeneic cancer cell vaccine in HIS mice. Human immune responses elicited in HIS mice effectively inhibited lung metastases.
The c-MET oncogene encodes the receptor for the Hepatocyte Growth Factor/Scatter Factor (HGF), a cytokine that stimulates the invasive growth of normal and neoplastic cells. The Met/HGF receptor is ...expressed by epithelial cells and its ligand by cells of mesenchymal origin. Receptor-ligand interaction occurs via a paracrine circuit. We studied the expression of the Met/HGF receptor and of its ligand in mesenchymal human tumours by examining 39 clinical samples of bone tumours. The Met/HGF receptor was not detectable in the majority of bone tumours, as expected from their mesenchymal origin. Notably, the receptor was overexpressed in 60% of the osteosarcomas examined. In 12 osteosarcoma cell lines the Met/HGF receptor was overexpressed, phosphorylated by HGF stimulation and fully functional. HGF was detected in two out of seven clinical specimens of osteosarcoma. The ligand and the receptor are co-expressed in two clonal osteosarcoma cell lines. In these lines the Met/HGF receptor was constitutively phosphorylated; phosphorylation was suppressed by suramin treatment, a known blocker of autocrine loops. These data suggest that activation of the Met/HGF receptor by a paracrine or an autocrine mechanism might play a role in the particularly aggressive behaviour of osteosarcomas.
While much experimental data shows that vaccination efficiently inhibits a subsequent challenge by a transplantable tumor, its ability to inhibit the progress of autochthonous preneoplastic lesions ...is virtually unknown. In this article, we show that a combined DNA and cell vaccine persistently inhibits such lesions in a murine HER-2/neu mammary carcinogenesis model. At 10 weeks of age, all of the ten mammary gland samples from HER-2/neu-transgenic mice displayed foci of hyperplasia that progressed to invasive tumors. Vaccination with plasmids coding for the transmembrane and extracellular domain of rat p185neu followed by a boost with rp185neu+ allogeneic cells secreting IFN-gamma kept 48% of mice tumor free. At 22 weeks, their mammary glands were indistinguishable from those of 10-week-old untreated mice. Furthermore, the transcription patterns of the two sets of glands coincided. Of the 12,000 genes analyzed, 17 were differentially expressed and related to the antibody response. The use of B cell knockout mice as well as the concordance of morphologic and gene expression data demonstrated that the Ab response is the main mechanism facilitating tumor growth arrest. This finding suggests that a new way can be found to secure the immunologic control of the progression of HER-2/neu preneoplastic lesions.
BALB/c mammary adenocarcinoma cells engineered to express TNF-related apoptosis-inducing ligand (TRAIL)/APO-2 ligand (APO-2L) on their membrane (TSA-TRAIL) grow with kinetics similar to that of ...parental cells (TSA-pc) in vitro and in nu/nu mice. In contrast, TSA-TRAIL cells grow faster than TSA-pc in normal BALB/c mice. In DBA/2 mice, which differ from BALB/c mice at minor histocompatibility Ags, they also grow faster and display a higher percentage of tumor takes than TSA-pc. In fully histoincompatible C57BL/6 (B6) mice, TSA-TRAIL cells form evident tumors that are slowly rejected by most mice, but outgrow in a few. In contrast, TSA-pc cells are rejected at once by B6 mice. Since TRAIL/APO-2L induces apoptosis by interacting with a variety of specific receptors, this rapid growth in both syngeneic and allogeneic mice may be the result of an immunosuppressive mechanism. The following evidence supports this hypothesis: 1) TSA-TRAIL cells overcome the strong immunity against TSA-pc cells elicited in BALB/c mice by preimmunization with TSA cells engineered to release IL-4; 2) their rejection by B6 mice does not prime a CTL-mediated memory; 3) thymidine uptake by T lymphocytes unstimulated or stimulated by allogeneic cells is inhibited when TSA-TRAIL cells are added as third party cells; 4) CTL kill TSA-pc but not TSA-TRAIL cells in 48-h assays; and 5) activated lymphocytes interacting with TSA-TRAIL cells in vivo and in vitro undergo apoptosis.
The MET oncogene encodes the receptor for HGF/Scatter Factor, known to control cell motility and invasion in epithelial cells. We report that the Met/HGF receptor, absent in differentiated adult ...skeletal muscles, is aberrantly expressed in clinical samples and in established cell lines of human rhadbomyosarcomas. In both the embryonal and alveolar histotypes the oncogene is overexpressed and, in some cases, amplified. The Met receptor is exposed at the cell surface and is functionally active in response to HGF/Scatter Factor. Accordingly, rhabdomyosarcoma cells exhibit an invasive phenotype in vitro in response to exogenous HGF/Scatter factor. As the factor is known to be produced by connective tissues, a paracrine stimulation of rhabdomyosarcoma invasiveness in vivo is hypothesized. Two alveolar rhabdomyosarcomas were found in co-express the ¿two-kringle' alternatively-spliced HGF/Scatter Factor variant, which has been previously shown to stimulate cell motility and matrix invasion in vitro. These cells displayed the invasive phenotype in the absence of exogenous HGF/Scatter Factor, suggesting an autocrine mechanism in vivo. These data indicate that aberrant expression of the MET proto-oncogene provides rhabdomyosarcoma cells with the same property as embryonal myoblasts to migrate into the surrounding connective tissues.
Cathodic protection (CP) has become a successful method for the rehabilitation of concrete structures affected by chloride‐induced corrosion of reinforcing steel. CP involves applying an electrical ...current from an external anode through the concrete to the reinforcement. The current causes steel polarisation, electrochemical reactions and ion transport. Normally the anode is placed over relatively large surface areas, including those where the steel is passive. Conventional views assume that protection current will not significantly flow outside the anode area. In many cases this results in a conservative design. This paper presents principles and first results of numerical calculations for design of an example CP system by finite element modelling. The final objective is to develop a tool for more economical CP system design. In particular, a CP system for the protection of local damage in bridges (e.g. at leaking joints) has been simulated. The corroding area with respect to the size of the anode is varied. Current and potential distributions and depolarisation values are predicted, both close to and more distant from the anode. It appears that current densities required to achieve sufficient polarisation are much higher than those usually found in the field. Neglecting time‐dependent repassivation processes is likely to be the main cause and further work is needed to include them. The present model can be used with reasonable confidence for preventive application to passive steel.
Recombinant human nerve growth factor (rhNGF) was delivered for up to 6 months by continuous intracerebroventricular (icv) infusion to CD (Sprague– Dawley derived) rats and cynomolgus monkeys. Rats ...(n=15/sex/group) received doses of 0 (vehicle), 6, 60, or 300 ng/day; monkeys (n=5/sex/group) received 0, 0.6, 6, or 60 μg/day of rhNGF. Animals tolerated icv infusion with no behavioral signs attributable to rhNGF. Body weight was transiently decreased in female rats at the highest dose. At the completion of dosing, histological examination in both species revealed an increase in the thickness of the leptomeninges along the ventral and lateral surfaces of the hindbrain and extending over the dorsal aspect of the spinal cord. The change was present to varying degrees at all doses of rhNGF and tended to be more severe at higher doses. At the light microscopic level, the leptomeninges contained layers of well-differentiated, spindle-shaped cells and a plexus of axonal fibers. Cells were immunoreactive for S-100 protein and were associated with an accumulation of Type IV collagen, suggesting Schwann cell origin. Electron microscopy revealed numerous fine caliber axons ensheathed by the presumptive Schwann cells, with myelination of individual axonal segments. These findings suggest that chronic icv delivery of rhNGF has stimulated axonal sprouting and secondary hyperplasia of Schwann or Schwann-like support cells within the pia-arachnoid.
The HER-2/neu proto-oncogene is overexpressed in 20-30% of human breast cancers and is associated with high recurrence risk. The oncogenic potential of HER-2/neu, together with its elevated ...expression in tumors, cell surface localization, and immunogenicity in some patients, make this oncoprotein an ideal target for immunotherapeutic approaches. To test the efficacy of immune-based strategies in eliciting an antitumor response, we used the N#202 transgenic mouse model engineered to overexpress the rat neu proto-oncogene under the control of the mouse mammary tumor virus promoter; females of this line develop spontaneous focal mammary tumors by 6 months of age. Transgenic mice immunized intramuscularly with a HER-2 cDNA ligated into the VR1012 (VICAL) expression vector under the control of the cytomegalovirus promoter developed significantly fewer spontaneous tumors as compared with mice injected with the empty vector (P < 0.0001) or not injected (P = 0.0006). However, this protection was observed only when immunization was started in 3-month-old but not in 6-month-old mice. These data suggest that the xenogeneic HER-2 DNA sequence can break immune tolerance to rat neu in transgenic N#202 mice and induce protective immunity that impairs the neu oncogene-driven progression of mammary carcinogenesis. The preventive effect achieved by our immunological approach appeared not to be based on anti-neu specific B and T cell immune attacks but was more possibly based on different mechanisms including aspecific and inflammatory immunological responses.
Expression of the T cell receptor (TCR) genes is not restricted to T lymphocytes. Human prostate and breast express a truncated TCR gamma transcript. In the mouse, TCR alpha (TCRA) and beta partial ...transcripts are expressed by mesenchymal cells and TCRA transcripts by epithelial cells of the kidney. We show now that TCRA constant region expression is common in normal and neoplastic human cells of mesenchymal and neuroectodermal origin. TCR transcripts are derived from an unrearranged TCRA locus. Moreover, rhabdomyosarcoma cells highly expressed a specific J49–C splicing product deriving from the assembly of J49 segment and constant region. TCRA ectopic transcripts/proteins negatively regulate rhabdomyosarcoma cell growth as suggested by TCRA gene expression downmodulation effects using a specific duplex small interfering RNA.
A number of drugs and drug candidates, including fenfluramine and ergot derivatives, are associated with valvulopathy in humans; however, these responses are poorly predicted from animal studies.
In ...vitro and
in vivo evidence suggests that these compounds exert their pathological effect through activation of serotonin 2B receptor (5HT2BR) signaling. However, the variable effect of fenfluramine and other 5HT2BR agonists in rodents has cast doubt on the relevance of animal findings to predicting human risk. Herein, a candidate compound, RO3013, induced subendocardial cell proliferation in the mitral and tricuspid valves in rats after only 3 days of daily dosing. Additionally, there was a treatment-related increase in immunostaining of the proliferation marker Ki67, and phosphorylated Smad3 in the heart indicative of TGFβ signaling co-localized with 5HT2BR expression. To substantiate the hypothesis that RO3013-induced valvular proliferation is secondary to 5HT2BR activation, the compound was evaluated
in vitro and found to bind to the human 5HT2BR with a
K
i of 3.8
μM; however, it was virtually devoid of agonist activity in a functional assay in human cells. By contrast, RO3013 bound to the rat 5HT2BR with a
K
i of 1.2
μM and activated the receptor with an EC50 of 0.5
μM. This agonist potency estimate is in good agreement with the free plasma concentrations of RO3013 at which valvular proliferation was observed. These results suggest that the rat may be susceptible to 5HT2BR-mediated valvular proliferation similar to humans; yet, the significant differences between binding and functional activities can be a possible explanation for the observed species-selective receptor responses.
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