A nitinol "U-Clip" versus sutured arteriovenous anastomosis: local tissue response and intimal hyperplasia development in a sheep model.

OBJECTIVES: This study sought to compare the local tissue response and subsequent volume of intimal hyperplasia (IH) that develops throughout the maturation of an arteriovenous fistula created using continuous/interrupted polypropylene with that of a novel, metal-alloy, penetrating anastomotic clip device. MATERIALS AND METHODS: Forty-six fistulae were created in 23 sheep under a paired design using the nitinol U-Clip (n = 23) in one hind limb and continuous (n = 20) or interrupted (n = 3) polypropylene suture for the other. Animals were killed at 4 (n = 3), 14 (n = 3), 28 (n = 10), 42 (n = 3), and 180 (n = 4) days. Histological sections were evaluated for quantitative histology and immunohistochemistry. RESULTS: Compared with continuous polypropylene, U-Clip specimens demonstrated less intima-media area per unit length (IMA/L), proliferating cells, and tissue necrosis at all time points (MANOVA, F = 9.8-24.1, all p ≤ .005; observed power >82%). Specifically, values of IMA/L were reduced by 5% (p = .97), 37% (p = .02), 33% (p < .01), 9% (p = .42), and 14% (p = .22) at the time points of 4, 14, 28, 42, and 180 days respectively. Proliferating cells were reduced by 75% (p < .01), 72% (p = .03), 76% (p = .03), 27% (p = .31), and 60% (p = .01) and tissue necrosis by 67% (p < .01), 58% (p = .02), 40% (p = .33), 21% (p = .43), 77% (p = .11). In a 28-day comparison between U-Clip and interrupted polypropylene the U-Clip group demonstrated a 4% (p = .65) reduction in IMA/L, 74% (p < .01) in proliferating cells and 49% (p < .05) in tissue necrosis. CONCLUSIONS: These results provide evidence of reduced local tissue necrosis, proliferating cells, and IH, favouring arteriovenous fistulae created using the U-Clip anastomotic device over conventional polypropylene suture techniques most evident over the first 4 weeks.

An arteriovenous fistula model of intimal hyperplasia for evaluation of a nitinol U-Clip anastomosis.

OBJECTIVES: The aim of this study was to create an ovine arteriovenous fistula (AVF) model which would closely replicate a human forearm fistula and use this to quantify the degree of intimal hyperplasia in those created with the U-Clip compared to a conventional sutured anastomosis. MATERIALS AND METHODS: Twenty AVFs were created in 10 Border Leicester-Merino sheep between the superficial femoral artery and vein of each hind limb. On one side the U-Clip and on the other a continuous polypropylene suture was used to perform the anastomosis. The animals were sacrificed at 2 (n = 3), 4 (n = 4), 6 (n = 3) weeks and histological slices were taken of each AVF in cross section to determine the intimal media area per unit length (IMA/L). RESULTS: Intimal hyperplasia (IH) was observed at all time points with one AVF found occluded with thrombus at the time of harvest. The IMA/L was significantly lower in the U-Clip groups by 24% at 2 weeks, 32% at 4 weeks and 23% at 6 weeks (Two-way ANOVA, p = 0.019, observed power = 0.825, time or side p ≥ 0.766, type p = 0.001; Paired t-test, p < 0.001 between matched anastomotic types). Time taken to perform the anastomosis was similar between the two anastomotic techniques (Polypropylene 14(8-18) vs. U-Clip 15.3(11-23) min; p = 0.47). CONCLUSION: This ovine AVF model results in IH similar to that seen in a human AVF. The IH that occurs with the U-Clip is less than that of continuous polypropylene suture.

Pain management in spinal metastases: the role of percutaneous vertebral augmentation.

Recent technological advances combined with innovative interventional radiology techniques can now offer an alternative less invasive treatment option for many patients with malignant vertebral body infiltration. Percutaneous vertebral augmentation procedures offer less invasive but effective pain relief to many patients with symptomatic spinal metastatic disease. The procedures are image guided and involve the injection of polymethylmethacrylate bone cement into the effected vertebral body. This technique can also be combined with radiofrequency ablation, which may accelerate vertebral stability. In this review, we examine the recent literature surrounding this topic and provide an overview of these emerging techniques.

A metalloprotease inhibitor blocks shedding of the 80-kD TNF receptor and TNF processing in T lymphocytes.

TNF is synthesized as a 26-kD membrane-anchored precursor and is proteolytically processed at the cell surface to yield the mature secreted 17-kD polypeptide. The 80-kD tumor necrosis factor (TNF) receptor (TNFR80) is also proteolytically cleaved at the cell surface (shed), releasing a soluble ligand-binding receptor fragment. Since processing of TNF and TNFR80 occurs concurrently in activated T cells, we asked whether a common protease may be involved. Here, we present evidence that a recently described inhibitor of TNF processing N-(D,L-[2-(hydroxyaminocarbonyl)methyl]-4-methylpentanoyl)L- 3-(2'naphthyl)- alanyl-L-alanine, 2-aminoethyl amide (TAPI) also blocks shedding of TNFR80, suggesting that these processes may be coordinately regulated during T cell activation. In addition, studies of murine fibroblasts transfected with human TNFR80, or a cytoplasmic deletion form of TNFR80, reveal that inhibition of TNFR80 shedding by TAPI is independent of receptor phosphorylation and does not require the receptor cytoplasmic domain.

Pelvic congestion syndrome: the role of interventional radiology in the treatment of chronic pelvic pain.

Chronic pelvic pain is a common problem for female patients and is defined as pain that has been present for 6 months or more. Chronic pelvic pain with associated ovarian vein varicosities is termed pelvic congestion syndrome (PCS) and is an important but under-diagnosed condition. The aetiology of pelvic varicosities is reflux of blood in the ovarian veins due to the absence of functioning valves, resulting in retrograde blood flow and eventual venous dilatation. The cardinal presenting symptom of PCS is pelvic pain, usually described as a dull ache, without evidence of inflammatory disease. Clinical signs may include vulval varicosities extending on to the medial thigh and long saphenous territory as well as tenderness on deep palpation at the ovarian point; however, such signs are not always present. Non-invasive imaging (ultrasound, CT and magnetic resonance venography) plays a central role in establishing the diagnosis, excluding alternative causes of pelvic pain and providing a road map for novel minimally invasive treatment options that are now available. Day-case percutaneous-directed venous embolisation is now accepted as a valuable treatment option for PCS with promising results from early clinical trials and is fast becoming the first-line treatment option for this condition. This paper aims to raise awareness of PCS among clinicians and reviews the pathogenesis, imaging assessment and minimally invasive treatment options that are now available.

Transcatheter coil embolisation: a novel definitive treatment option for intralobar pulmonary sequestration.

Pulmonary sequestrations have been conventionally treated surgically with removal of the tissue mass and ligation of its feeding vessels. There is established evidence to support the use of transcatheter arterial coil embolisation as an effective definitive treatment option for extralobar sequestration especially in the paediatric literature describing good long-term clinical outcomes. We present a case of an adult with intralobar sequestration in whom the diagnosis was established with multi-detector computed tomography (MDCT) and in whom transcatheter arterial coil embolisation was successfully performed as a definitive treatment option to support the growing body of evidence of transcatheter arterial coil embolisation as a safe and effective treatment option for both form of pulmonary sequestrations.

A lymphotoxin-beta-specific receptor.

Tumor necrosis factor (TNF) and lymphotoxin-alpha (LT-alpha) are members of a family of secreted and cell surface cytokines that participate in the regulation of immune and inflammatory responses. The cell surface form of LT-alpha is assembled during biosynthesis as a heteromeric complex with lymphotoxin-beta (LT-beta), a type II transmembrane protein that is another member of the TNF ligand family. Secreted LT-alpha is a homotrimer that binds to distinct TNF receptors of 60 and 80 kilodaltons; however, these receptors do not recognize the major cell surface LT-alpha-LT-beta complex. A receptor specific for human LT-beta was identified, which suggests that cell surface LT may have functions that are distinct from those of secreted LT-alpha.

A disease-associated cellular immune response in type 1 diabetics to an immunodominant epitope of insulin.

The 9-23 amino acid region of the insulin B chain (B9-23) is a dominant epitope recognized by pathogenic T lymphocytes in nonobese diabetic mice, the animal model for type 1 diabetes. We describe herein similar (B9-23)-specific T-cell responses in peripheral lymphocytes obtained from patients with recent-onset type 1 diabetes and from prediabetic subjects at high risk for disease. Short-term T-cell lines generated from patient peripheral lymphocytes showed significant proliferative responses to (B9-23), whereas lymphocytes isolated from HLA and/or age-matched nondiabetic normal controls were unresponsive. Antibody-mediated blockade demonstrated that the response was HLA class II restricted. Use of the highly sensitive cytokine-detection ELISPOT assay revealed that these (B9-23)-specific cells were present in freshly isolated lymphocytes from only the type 1 diabetics and prediabetics and produced the proinflammatory cytokine IFN-gamma. This study is, to our knowledge, the first demonstration of a cellular response to the (B9-23) insulin epitope in human type 1 diabetes and suggests that the mouse and human diseases have strikingly similar autoantigenic targets, a feature that should facilitate development of antigen-based therapeutics.

P53 expression is associated with malignant potential in xenograft tissues of a fibrosarcoma mouse model.

BACKGROUND: The expression of wild-type and mutant p53 was studied in two fibrosarcoma cell lines in a mouse xenograft model. MATERIALS AND METHODS: Human cell lines HT1080 and Hs913(D)T were implanted in athymic mice via intramuscular (i.m.) or subcutaneous (s.c.) routes. After eight weeks, liver, lung and primary inoculation sites were harvested. Sections were stained using two methods: a) haematoxylin and eosin to detect tumour at implantation site, liver and lung; b) immunohistochemistry using monoclonal antibodies to detect expression of wild-type (wt) and mutant p53. RESULTS: Both cell lines had similar implantation rates via either route but Hs913(D)T had a higher metastatic rate than HT1080. The Hs913(D)T cells exhibited greater expression of mutant and wild-type p53 than the HT1080 cells. CONCLUSION: The expression of wild-type and mutant p53 is associated with a cell line of greater malignant potential. The inoculation route does not affect primary tumour uptake or metastatic rate.

Expression of HER1/EGFR protein in human soft tissue sarcomas.

AIM: To measure epidermal growth factor receptor (HER1/EGFR) expression in a range of soft tissue sarcoma (STS) patient samples. METHOD: HER1/EGFR expression was examined by immunohistochemistry in archival tissues of 46 STS patients. RESULTS: HER1/EGFR was positively expressed in 36/46 of STS samples distributed among different histological types. The levels of HER1/EGFR in STS tumour tissues in positive samples were higher compared to those in nearby normal tissues. CONCLUSION: HER1/EGFR is significantly expressed in soft tissue sarcomas, which is a finding reflected in other series. The significance of this finding for targeted therapy is as yet unknown.

Active hexose transport across cultured human Caco-2 cells: characterisation and influence of culture conditions.

Human Caco-2 cells (passage 80 to 100) were seeded onto collagen-coated Millipore filter assemblies and these were maintained in culture either (a) floated on the surface of the medium or (b) submerged within the body of the medium. Structural and functional assessments were made over a 30-day period. After seeding, all cells assumed a flattened, squamous configuration and rapidly became confluent. Cells submerged within the medium formed polarised monolayers with well developed junctional complexes, abundant apical microvilli and increasing levels of alkaline phosphatase activity. Cells grown floated on the surface of the medium formed complex multilayers in which polarisation was confined to the surface layer. Junctional complexes and apical microvilli were similar to those seen in submerged monolayers but alkaline phosphatase activities were higher. Transepithelial electrical resistance increased rapidly from day 1, as the layers became confluent. Electrical resistance was higher and short-circuit current and potential differences were lower across monolayers than across multilayers. After 10 days in culture, the addition of D-glucose to the apical bathing solution, of all cell layers, caused a rapid rise in short-circuit current and potential difference. These changes were sodium-dependent and phlorizin-sensitive. Galactose and 3-O-methylglucose induced similar changes and the affinity constants for these hexoses ranked in the order reported for rat jejunum (Km glucose 2.44 +/- 0.52 mM; Km galactose 8.05 +/- 1.33 mM; Km 3-O-methylglucose 22.0 +/- 5.2 mM). Culture conditions had a marked effect on hexose maximum transport rates (glucose Vmax: submerged 2.94 +/- 0.20 microA/cm2; floated 9.94 +/- 0.82 microA/cm2, P less than 0.05) but affinity constants were unchanged. Apical to basolateral mannitol fluxes, used as an index of paracellular permeability, decreased from day 1 to day 5 and then remained steady. Fluxes across monolayers and multilayers were not significantly different. We conclude that sodium-dependent hexose transport occurs in cultured Caco-2 cell layers grown on permeable supports. Culture conditions, however, have a marked effect on both cell layer structure and function, and should be an important factor when considering Caco-2 cells as an in vitro model of enterocyte function.

Location of osteoclast precursors in fetal rat calvaria cultured on collagen gels.

Although osteoclasts are derived from hematopoietic cells, the exact identity of their precursors and the mechanism for their recruitment onto bone surfaces remain unclear. We wished to study their differentiation in the fetal rat calvaria and to locate its source of osteoclast precursor cells. Osteoclasts were detected by neutral red staining or cytochemical reaction for acid phosphatase of intact bone (cell number and area measured by computerized image analysis) or in cryostat sections of bone (enzyme activity measured by quantitative cytochemistry). Histology of semithin sections of fixed bones was also examined. The 19 day calvariae contained few mature osteoclasts. After 48 h culture on gels of type 1 collagen (1.5 mg/ml) supplemented with 5 mM calcium beta-glycerophosphate, 10 mM proline, and 2 micrograms/ml ascorbic acid, numerous large osteoclasts were seen on their endocranial surfaces. In contrast, cell morphology and enzyme activity deteriorated in bones cultured in liquid medium. The cells that formed in vitro rapidly responded to calcitonin by contraction. Stripping of endocranial membranes from the calvariae prevented osteoclast formation in culture, but these cells were seen when "stripped" bones had been cocultured with their membranes for 48 h or with intact 16 day calvariae (well before the onset of osteogenesis). Few osteoclasts were found when an 0.22 micron filter was inserted between the stripped calvaria and the endocranial membranes. We conclude that the endocranial membranes, which contain the meningeal blood vessels, are a major source of osteoclast precursors and that these cells are present in calvarial tissue even before the onset of osteogenesis.

Nephroblastoma overexpressed gene (NOV) codes for a growth factor that induces protein tyrosine phosphorylation.

NOV (nephroblastoma overexpressed gene) is a member of the CCN (connective tissue growth factor [CTGF], Cyr61/Cef10, NOV) family of proteins. These proteins are cysteine-rich and are noted for having growth-regulatory functions. We have isolated the rat NOV gene, and the DNA sequence shares 90% identity with the mouse and 80% identity with the human sequences. The rat NOV gene was expressed in all rat tissues examined, including brain, lung, heart, kidney, liver, spleen, thymus and skeletal muscle. Higher levels of rat NOV mRNA were seen in the brain, lung and skeletal muscle compared to the other tissues. Examination of NOV expression in various human cell lines revealed that NOV was expressed in U87, 293, T98G, SK-N-MC and Hs683 but not in HepG2, HL60, THP1 and Jurkat. The human NOV gene was transfected into 293 cells and the expressed protein purified. When 3T3 fibroblasts were treated with this recombinant NOV protein, a dose-dependent increase in proliferation was observed. Analysis of tyrosine-phosphorylated proteins revealed that when 3T3 cells were treated with NOV, a 221 kDa protein was phosphorylated. These data suggest that NOV can act as a growth factor for some cells and binds to a specific receptor that leads to the phosphorylation of a 221 kDa protein.

Production of lymphotoxin (LT alpha) and a soluble dimeric form of its receptor using the baculovirus expression system.

Human LT alpha and a fusion protein (p60:Fc) comprised of the extracellular domain of the 60 kDa TNF receptor (TNFR60) fused to the Fc portion of human IgG1 were produced in insect cells infected with recombinant baculoviruses. The p60:Fc fusion produced in insect cells accumulates in culture supernatants to levels > 2 mg/l. Purified p60:Fc binds human TNF and LT alpha with high affinity (200-600 pM) and neutralizes TNF cytolytic activity at equimolar stoichiometric concentration. The data show that p60:Fc is an effective ligand-precipitating reagent which recognizes recombinant LT alpha produced in mammalian or insect cells and naturally occurring LT alpha produced in T cells. The levels of human LT alpha produced in baculovirus-infected insect cells is estimated to be approximately 20 mg/l. Insect cell-derived human LT alpha is biologically active in an L929 cytotoxicity assay and is efficiently neutralized by p60:Fc. These data demonstrate that the baculovirus system is useful for overexpressing biologically active LT alpha and p60:Fc and therefore, may be applicable to other oligomeric cytokines and soluble dimeric cytokine receptors.

Amelioration of relapsing experimental autoimmune encephalomyelitis with altered myelin basic protein peptides involves different cellular mechanisms.

T-cells specific for a region of human myelin basic protein, amino acids 87-99 (hMBP87-99), have been implicated in the development of multiple sclerosis (MS) a demyelinating disease of the central nervous system. Administration of soluble altered peptide ligand (APL), made by substituting native residues with alanine at either positions 91(91K > A or A91) or 97 (97R > A or A97) in the hMBP87-99 peptide, blocked the development of chronic relapsing experimental autoimmune encephalomyelitis (R-EAE), in the SJL mouse. The non-encephalitogenic APL A91, appears to induce cytokine shifts from Th1 to Th2 in the target T-cells, whereas the encephalitogenic superagonist APL A97 causes deletion of the MBP87-99 responsive cells. Thus, single amino acid changes at different positions in the same peptide epitope can lead to APL capable of controlling auto-immune disease by different mechanisms.

Cloning of a cDNA encoding a novel interleukin-1 receptor related protein (IL 1R-rp2).

We have identified and isolated both the rat and human cDNAs for a novel putative receptor related to the interleukin-1 type 1 receptor. We have named this protein interleukin 1 receptor related protein two (IL 1R-rp2). The rat cDNA for IL1R-rp2 was first identified using oligonucleotides of degenerate sequence in a polymerase chain reaction (PCR) paradigm with rat brain mRNA as the template. The protein encoded by both of these cDNAs are 561 amino acids long and exhibit 42% and 26% overall identity with the interleukin-1 type 1 and type 2 receptors, respectively. RNase protection assays from rat tissues revealed a predominant expression for IL 1R-rp2 in the lung and epididymis with lower levels detected in the testis and cerebral cortex. By in situ hybridization we were able to determine that the expression in rat brain appeared to be non-neuronal and associated with the cerebral vasculature. When expressed transiently in COS-7 cells the receptor was incapable of high affinity binding to either [125I]-recombinant human IL 1 alpha or [125I]-recombinant human IL 1 beta. Together, these data demonstrate the existence of a novel protein that is related to the interleukin-1 receptor but does not bind IL-1 by itself.

Differential signaling and hierarchical response thresholds induced by an immunodominant peptide of myelin basic protein and an altered peptide ligand in human T cells.

Changes in peptide antigen concentration or structure can have a profound effect on T cell responsiveness by inducing selected T cell effector functions. In this study, we have compared the biological responses of an MBP83-99-specific human Th0 T cell clone (TCC) stimulated with increasing concentrations of native peptide or an altered peptide ligand (APL). Our results show that the hierarchy of response thresholds for proliferation and cytokine secretion is similar for native peptide and APL. However, because a much higher concentration of the APL is required to evoke the same degree of response, the cytokine profile is shifted towards a Th2-like response relative to the same concentration of native peptide. In addition, we observed qualitative differences in TCR signal transduction triggered by native peptide and a weak agonist APL even at concentrations that elicit similar biological responses. Thus, the relationship between TCR signaling and biological responses may be more complex than previously recognized.

Atenolol and sustained release nifedipine alone and in combination in hypertension. A randomised, double-blind, crossover study.

In this randomised, double-blind, crossover trial, the efficacy in hypertension of atenolol and nifedipine as single agents or in combination was compared. 81 patients with mild to moderate essential hypertension (sitting diastolic blood pressure 100-120 mm Hg, aged 20-70 years) from 6 outpatient clinics entered the study. By use of a Latin-square design, patients received, in randomised fashion, sustained release nifedipine 20mg twice daily, atenolol 50mg in the morning and then placebo in the evening, or sustained release nifedipine 20mg plus atenolol 50mg in the morning and then placebo in the evening. Each schedule was followed for 4 weeks. All treatments lowered systolic and diastolic blood pressure in the supine and standing positions compared with pretreatment values. The combination regimen significantly reduced supine and standing systolic (p less than 0.01 and p less than 0.001, respectively) and diastolic (p less than 0.001) blood pressure compared with nifedipine alone, and it also significantly reduced supine and standing systolic (p less than 0.01 and p less than 0.03, respectively) and diastolic (p less than 0.01) blood pressure compared with atenolol alone. Heart rate was significantly decreased by atenolol and the combination compared with nifedipine alone. 15 patients withdrew because of side effects: 9 during nifedipine treatment, 2 during atenolol treatment and 4 during combination treatment. Side effects were typical of those associated with nifedipine or atenolol. Flushes and hot sweats, which were frequent with nifedipine, were significantly less (p less than 0.001) with atenolol or the combination.(ABSTRACT TRUNCATED AT 250 WORDS)

Prolactin activates protein kinase C and stimulates growth-related gene expression in rat liver.

We have examined the effect of prolactin (PRL) on growth-related gene expression, protein kinase C (PKC) activity and diacylglycerol (DAG) mass in rat liver. Hepatic levels of messenger (m)RNA for c-myc, ornithine decarboxylase (ODC) and beta-actin increased in a dose-dependent manner within 1 h after PRL administration. Prolactin also caused a transient elevation of liver DAG levels and particulate-associated PKC activity. The PRL-provoked increases in DAG mass and particulate PKC activity were coincident and maximal at 20 min and began declining toward control levels by 30 min. These results suggest a temporal relationship between PRL-stimulated DAG accumulation and PKC activation. Furthermore, the subsequent rapid induction of growth-related gene expression provides new information on the role of PRL as a hepatic mitogen.