Alexandru C. Stan

Dietary l-arginine decreases myointimal cell proliferation and vascular monocyte accumulation in cholesterol-fed rabbits

L-arginine, the precursor of endogenous nitric oxide (NO), has been shown to enhance endothelial function and to reduce the progression of atherosclerosis in cholesterol-fed rabbits. In the present study, we investigated whether myointimal cell proliferation is enhanced in hypercholesterolaemic rabbit aorta and whether chronic treatment of the rabbits with L-arginine or with the NO synthase inhibitor L-NAME influences this proliferative response and vascular monocyte accumulation. Rabbits were fed 1% cholesterol or normal rabbit chow for 12 weeks. Subgroups of cholesterol-fed rabbits were treated with oral L-arginine (2.25%) or L-NAME (3 mg/dl) in drinking water. Myointimal cell proliferation was quantified in aortic segments by immunohistochemical detection of bromodeoxyuridine (BrdU) incorporation into nuclear DNA; vascular monocyte accumulation was assessed by immunohistochemistry using a monoclonal anti-macrophage/monocyte antibody (RAM-11). Plasma levels of L-arginine and the endogenous NO synthase inhibitor, ADMA, were quantified by high-performance liquid chromatography (HPLC). Cholesterol feeding increased the aortic intima/media (I/M) ratio, which was not measurable in the control group, to 1.9 +/- 0.3. This was paralleled by enhanced cell proliferation (cholesterol, 2.4 +/- 0.2%; P < 0.05; control, 0.02 +/- 0.001% BrdU-positive cells per 72 h) and vascular monocyte accumulation. Double immunostaining for BrdU and alpha-actin showed that about two thirds of the proliferating cells were smooth muscle cells. ADMA levels increased from 0.8 +/- 0.1 micromol/l to 2.2 +/- 0.2 micromol/l in cholesterol-fed rabbits, but were unchanged by L-arginine or L-NAME treatment. Myointimal proliferation and intima/media ratios were correlated with ADMA plasma levels. Dietary L-arginine reduced monocyte accumulation by 85 +/- 2% (P < 0.05 vs cholesterol), myointimal cell proliferation (1.8 +/- 0.3% per 72 h; P < 0.05) and intimal thickening (I/M ratio: 0.7 +/- 0.2), whereas the inhibitor of NO synthase, L-NAME, further increased cell proliferation to 3.1 +/- 0.4% per 72 h (P < 0.05). No significant difference was observed in vascular monocyte infiltration between the cholesterol and L-NAME groups. We conclude that cell proliferation and vascular monocyte accumulation are enhanced in hypercholesterolaemic rabbit aorta. These atherogenic effects can be attenuated by dietary L-arginine. Decreased NO formation might underlie the enhanced monocyte accumulation and cell proliferation in hypercholesterolaemic rabbit aorta. The observed inhibition of cell proliferation adds to our understanding of the antiatherosclerotic effects of L-arginine in vivo.

CpG motifs of DNA vaccines induce the expression of chemokines and MHC class II molecules on myocytes

Determining how an immune response is initiated after in vivo transfection of myocytes with plasmids encoding foreign antigens is essential to understand the mechanisms of intramuscular (i. m.) genetic immunization. Since myocytes are facultative antigen‐presenting cells lacking MHC class II and co‐stimulatory molecules, it was assumed that their unique role upon DNA vaccination is to synthesize and secrete the protein encoded by the plasmid. Here we describe that i. m. injection of unmethylated CpG motifs induced the expression of chemokines (monocyte chemotactic protein‐1) and MHC class II molecules on myocytes. Our results indicate that immunostimulatory DNA sequences (CpG motifs) of DNA vaccines augment synthesis of chemokine by myocytes with subsequent recruitment of inflammatory cells secreting IFN‐γ, a potent cytokine that up‐regulates the expression of MHC class II molecules on myocytes. A myoblast cell line triple transfected with plasmids encoding MHC class II molecules and an immunodominant CD4 T cell epitope of influenza virus presented the endogenously synthesized peptide and activated specific T cells. These findings suggest that one mechanism for the immunogenicity of DNA vaccines consists in the presentation of peptides to CD4 T cells by in vivo plasmid‐transfected myocytes.

Characterization of mutated protein encoded by partially duplicated fibrillin-1 gene in tight skin (TSK) mice

Fibrillin-1 (Fbn-1) is a ubiquitous protein present in the extracellular matrix of various organs and it is a major component of microfibrils embedded in the core of elastic fibers. In humans, mutations or deletions of the Fbn-1 gene are associated with several genetic diseases. In addition, several microsatellite alleles near Fbn-1 gene were found associated with diffuse scleroderma. In TSK/+ mice, which develop a scleroderma-like syndrome, the Fbn-1 gene exhibits an inframe duplication of exons 17-40. In this study, we report that the synthesis and secretion of wild-type Fbn-1 in TSK/+ is higher than that of the mutated Fbn-1 protein excluding the possibility that TSK genetic defect is due to a loss of the wild allele. We also demonstrate for the first time that TGF-beta, which plays a crucial role in skin fibrosis, binds to both wild-type and mutated Fbn-1. The amount of bound TGF-beta was higher in mutated than wild-type Fbn-1 and appears related to the number of TGF-beta binding motifs.

Antigen-specific downregulation of T cells by doxorubicin delivered through a recombinant MHC II--peptide chimera.

As the number of drugs with potential therapeutic use for T-cell-mediated diseases increases, there is a need to find methods of delivering such drugs to T cells. The major histocompatibility complex (MHC)–peptide complexes are the only antigen-specific ligands for the T-cell receptor (TCR) expressed on T cells, and they may be an appropriate drug delivery system. We engineered a soluble bivalent MHC class II–peptide chimera on the immunoglobulin scaffold (I-Edαβ/Fcγ2a/HA110-120, DEF) that binds stably and specifically to CD4 T cells recognizing the HA110-120 peptide. Doxorubicin, a powerful antimitogenic anthracycline, was enzymatically assembled on the galactose residues of a DEF chimera. The DEF-gal-Dox construct preserved both the binding capacity to hemagglutinin (HA)-specific T cells, and the drug toxicity. Brief exposure of HA-specific T cells to DEF-gal-Dox construct in vitro was followed by drug internalization in the lysosomes, translocation to the nucleus, and apoptosis. Administration of DEF-gal-Dox to mice expressing the TCR-HA transgene reduced the frequency of TCR-HA T cells in the spleen and thymus by 27% and 42%, and inhibited HA proliferative capacity by 40% and 60%, respectively. It has not been demonstrated previously that pharmacologically active drugs able to modulate T-cell functions can be delivered to T cells in an antigen-specific manner by soluble, bivalent MHC II–peptide chimeras.

Auditory midbrain implant: histomorphologic effects of long-term implantation and electric stimulation of a new deep brain stimulation array.

Hypothesis: Chronic implantation and electric stimulation with a human prototype auditory midbrain implant (AMI) array within the inferior colliculus achieves minimal neuronal damage and does not cause any severe complications. Background: An AMI array has been developed for patients with neural deafness and, based on animal studies, has shown to possess potential as an auditory prosthesis in humans. To investigate the safety of the AMI for clinical use, we characterized the histomorphologic effects of chronic implantation and stimulation within its target structure, the inferior colliculus. Methods: Eight cats were chronically implanted for 3 months, and histologic sections were analyzed to assess long-term tissue effects. Four of the 8 cats were additionally stimulated for 60 days (4 h/d) starting 4 weeks after implantation to assess if clinically relevant stimuli further affected the tissue response. Results: In general, both neurons and neuropil surrounding the implant track were apparently unaffected, whereas a fibrillary sheath (~50 µm thick) developed around the array. There was a significant decrease in neuron density 50 to 100 µm away from the track with a significantly elevated number of glial cells out to approximately 250 to 350 µm. Chronic stimulation seemed to improve the tissue response and neuronal survival around the implant, although further studies are needed to confirm this finding. Conclusion: The histomorphologic effects and extent of neuronal damage observed for our AMI array are similar to those of other neural implants currently and safely used in humans. The minimal tissue damage surrounding the implanted array is encouraging with regard to the safety of the array for human use.

Evidence of recurrent atypical meningioma with rhabdoid transformation and expression of pyrogenic cytokines in a child presenting with a marked acute-phase response: case report and review of the literature

Children presenting with acute systemic illnesses that lack specific clinical or serological defining features may be diagnosed as having a chronic infection, an atypical systemic vasculitis or a connective tissue disease, but often turn out to have occult neoplasias. Cytokines have been implicated in causing many of the systemic effects in such cases. In this study, we describe the case of a 9-year-old boy presenting at an interval of 18 months with a marked acute-phase response due to a recurrent atypical meningioma with rhabdoid transformation of the tentorium cerebelli. Resection of the recurrent tumor was curative. We evaluated in detail the local and systemic production of cytokines released by the primary and the recurrent tumor. Blood and CSF samples were taken pre-, intra-, and postoperatively, and the production of IL-6, IL-1beta, and TNF-alpha was measured by enzyme-linked immunosorbent assays (ELISA). The level of IL-6 in CSF was about 150-fold increased before tumor resection, normalizing postoperatively. On the contrary, the levels of IL-1beta and TNF-alpha in CSF and of IL-6, IL-1beta, and TNF-alpha in serum were pre-, intra-, and postoperatively within normal limits. Cytokine production was also evaluated immunohistochemically, and confirmed strong IL-6 and TNF-alpha expression in the primary and the recurrent tumor, while expression of IL-1beta was lacking. The scattered MHC class II- and leukocyte common antigen (LCA)-expressing inflammatory cells, which were infiltrating exclusively the tumoral stroma, had no detectable cytokine immunoreactivity. We conclude that chronic IL-6 and TNF-alpha production by the tumor cells in this patient was responsible for the severe systemic illness with which he presented.

BILATERAL OSTEOMAS ARISING FROM THE INTERNAL AUDITORY CANAL : CASE REPORT

OBJECTIVE Osteomas arising from the internal auditory canal and developing in the cerebellopontine angle have rarely been reported. We present the first case of bilateral osteomas in this region and describe our management strategy. CLINICAL PRESENTATION A 30-year-old woman presented with strong vertigo, tinnitus, and hypacusis on the left side. Brain magnetic resonance imaging and computed tomographic scans displayed bilateral cerebellopontine angle osteomas arising from the internal auditory canal. The larger tumor on the left side was found to be compressing the vestibulocochlear nerve. INTERVENTION Surgery was performed on the symptomatic side via the retrosigmoid approach, and the tumor was removed completely. The vertigo resolved completely after surgery, and the patient had no further tinnitus attacks. An audiogram showed slightly improved hearing with a mean of 20 dB in the main speech area. CONCLUSION Osteomas should be considered in patients with bilateral cerebellopontine angle tumors. Surgical removal might provide resolution of symptoms.

Bilateral malignant melanoma metastases to the internal auditory canal/cerebellopontine angle: surgical management and preservation of function

Although intracranial metastases of malignant melanomas are common, localization at the cerebellopontine angle (CPA) or in the internal auditory canal (IAC) is rare, and bilateral presentation especially so. We present the case of a 46-year-old Caucasian woman with bilateral IAC/CPA lesions and a prior history of malignant melanoma on the right leg. During preoperative investigations, the presence of the bilateral IAC/CPA lesions along with several radiologically identified lesions along the neural axis led to the suspicion that she had neurofibromatosis Type 2 despite her history of malignant melanoma and the lack of characteristic skin lesions and family history. Histopathological analysis of the resected lesion confirmed the intraoperative diagnosis of bilateral CPA malignant melanoma metastases. Surgical removal of the tumors via the retrosigmoid approach with preservation of normal bilateral facial nerve function and unilateral serviceable hearing, combined with control of the systemic disease, provided this patient with a near-normal quality of life for at least 42 months after the initial diagnosis of melanoma.

PepT2 transporter protein expression in human neoplastic glial cells and mediation of fluorescently tagged dipeptide derivative β-Ala-Lys-Nε-7-amino-4-methyl-coumarin-3-acetic acid accumulation

OBJECT The present study was aimed at analyzing the accumulation of the fluorescently tagged dipeptide derivative, beta-Ala-Lys-N(epsilon)-7-amino-4-methyl coumarin-3-acetic acid (AMCA), in primary cultures of human neoplastic glial cells. This molecule is a highly specific reporter used to investigate the dipeptide transport system hPepT2. METHODS In this study the authors used immunocytochemical methods to determine the cell-specific accumulation of a small and fluorescently tagged reporter molecule named beta-Ala-Lys-N(epsilon)-AMCA to detect dipeptide transport capacity of neoplastic glial cells. Furthermore, specific mRNA levels were quantified using Northern blot analysis and the tissue distribution of hPepT2 mRNA transcripts was demonstrated with in-situ hybridization histochemical analysis. RESULTS Recent fluorescent immunocytochemical analyses have revealed that beta-Ala-Lys-N(epsilon)-AMCA specifically accumulates within anaplastic cells of astrocytic lineage but not in anaplastic oligodendrocytes or neurons. Northern blot analysis demonstrated that human hPepT2 mRNA is specifically detected in primary cell cultures of human glioblastoma but not in oligodendroglioma. Moreover, in situ hybridization analyses revealed an astrocytic localization of hPepT2 transcripts in human glioblastoma and astrocytoma cells. The hPepT2 transcription levels were clearly dependent on the grade of glial cell differentiation: within low-grade gliomas (WHO Grade II), more hPepT2 mRNA was detected compared with tumors of a higher grade of dedifferentiation (WHO Grade IV). Analysis of expression levels of hPepT2 mRNA in human neoplastic glial cells xenografted into the brains of athymic rats (han rnu(+/+)) showed a markedly increased expression of hPepT2 after 2 weeks of growth in vivo compared with the primary counterparts grown in vitro. CONCLUSIONS The authors concluded that expression of the hPepT2 transporter protein is a characteristic of glial cells of astrocytic lineage, and is dependent on the grade of astroglial cell differentiation and the extracellular matrix (here brain neuropil). The authors found that beta-Ala-Lys-N(epsilon)-AMCA is as an excellent reporter molecule for assessing neoplastic glial cell function and physiological characteristics.

Expression of granulocyte colony-stimulating factor in recurrent glial tumors is inversely correlated with tumor progression

We have previously reported that in vivo-expression of granulocyte colony-stimulating factor (G-CSF) is a characteristic feature of reactive and neoplastic astrocytes. The aim of the present study was to analyze the expression of G-CSF protein in primary and recurrent astroglial, oligodendroglial and mixed glial-differentiated tumors. G-CSF expression was present in all GFAP-positive tumors excepting glioblastomas. G-CSF expression was significantly reduced in recurrent tumors more dedifferentiated than their primary counterparts. G-CSF expression was absent in all GFAP-negative tumors such as oligodendrogliomas. Our results demonstrate that G-CSF is a highly sensitive differentiation marker of neoplastic astrocytes, which is lost during tumor progression.