Generation of Endotoxin-Specific Monoclonal Antibodies by Phage and Yeast Display for Capturing Endotoxin.

Endotoxin, a synonym for lipopolysaccharide (LPS), is anchored in the outer membranes of Gram-negative bacteria. Even minute amounts of LPS entering the circulatory system can have a lethal immunoactivating effect. Since LPS is omnipresent in the environment, it poses a great risk of contaminating any surface or solution, including research products and pharmaceuticals. Therefore, monitoring LPS contamination and taking preventive or decontamination measures to ensure human safety is of the utmost importance. Nevertheless, molecules used for endotoxin detection or inhibition often suffer from interferences, low specificity, and low affinity. For this reason, the selection of new binders that are biocompatible, easy to produce, and that can be used for biopharmaceutical applications, such as endotoxin removal, is of high interest. Powerful techniques for selecting LPS-binding molecules in vitro are display technologies. In this study, we established and compared the selection and production of LPS-specific, monoclonal, human single-chain variable fragments (scFvs) through two display methods: yeast and phage display. After selection, scFvs were fused to a human constant fragment crystallizable (Fc). To evaluate the applicability of the constructs, they were conjugated to polystyrene microbeads. Here, we focused on comparing the functionalized beads and their LPS removal capacity to a polyclonal anti-lipid A bead. Summarized, five different scFvs were selected through phage and yeast display, with binding properties comparable to a commercial polyclonal antibody. Two of the conjugated scFv-Fcs outperformed the polyclonal antibody in terms of the removal of LPS in aqueous solution, resulting in 265 times less residual LPS in solution, demonstrating the potential of display methods to generate LPS-specific binding molecules.

Efficacy and morbidity of biodegradable versus titanium osteosyntheses in orthognathic surgery: A systematic review with meta-analysis and trial sequential analysis.

Titanium osteosynthesis is currently the gold standard in orthognathic surgery. Use of biodegradable osteosyntheses avoids removal of plates/screws in a second operation. This systematic review aimed to assess the efficacy and morbidity of biodegradable vs. titanium osteosyntheses in orthognathic surgery (PROSPERO CRD42018086477). Patients with syndromic disorder(s) and/or cleft lip/palate were excluded. Randomised, prospective and retrospective controlled studies were searched for in nine databases (February 2021). The time periods perioperative, short-term, intermediate, long-term, and overall follow-up were studied. Meta-analyses were performed using random-effects models. A total of 9073 records was assessed, of which 33 were included, comprising 2551 patients. Seven RCTs had 'some concerns' while another seven RCTs had 'high' risk of bias (Cochrane-RoB2). No differences in malunion (qualitative analyses), mobility of bone segments [RR 1.37 (0.47; 3.99)], and malocclusion [RR 0.93 (0.39; 2.26)] were found. The operative time was longer in the biodegradable group [SMD 0.50 (0.09; 0.91)]. Symptomatic plate/screw removal was comparable among both groups [RR 1.29 (0.68; 2.44)]. Skeletal stability was similar in most types of surgery. Biodegradable osteosyntheses is a valid alternative to titanium osteosyntheses for orthognathic surgery, but with longer operation times. Since the quality of evidence varied from very low to moderate, high-quality research is necessary to elucidate the potential of biodegradable osteosyntheses.

Reversible Immobilization of Proteins in Sensors and Solid-State Nanopores.

The controlled functionalization of surfaces with proteins is crucial for many analytical methods in life science research and biomedical applications. Here, a coating for silica-based surfaces is established which enables stable and selective immobilization of proteins with controlled orientation and tunable surface density. The coating is reusable, retains functionality upon long-term storage in air, and is applicable to surfaces of complex geometry. The protein anchoring method is validated on planar surfaces, and then a method is developed to measure the anchoring process in real time using silicon nitride solid-state nanopores. For surface attachment, polyhistidine tags that are site specifically introduced into recombinant proteins are exploited, and the yeast nucleoporin Nsp1 is used as model protein. Contrary to the commonly used covalent thiol chemistry, the anchoring of proteins via polyhistidine tag is reversible, permitting to take proteins off and replace them by other ones. Such switching in real time in experiments on individual nanopores is monitored using ion conductivity. Finally, it is demonstrated that silica and gold surfaces can be orthogonally functionalized to accommodate polyhistidine-tagged proteins on silica but prevent protein binding to gold, which extends the applicability of this surface functionalization method to even more complex sensor devices.

Single nucleotide polymorphisms in AREG and EREG are prognostic biomarkers in locally advanced gastric cancer patients after surgery with curative intent.

OBJECTIVE: Amphiregulin (AREG) and epiregulin (EREG) are important ligands to the epithelial growth factor receptor, which is involved in the regulation of progression and stemness in gastric cancer (GC). This study investigated whether frequent single nucleotide polymorphisms (SNPs) in genes of AREG and EREG are associated with recurrence-free survival and overall survival in patients with locally advanced GC. METHODS: SNPs with a minor allele frequency of at least 10% were analyzed using direct DNA sequencing in two independent study populations. RESULTS: The minor allele of AREG rs1615111 was associated with a significantly higher 3-year recurrence rate and lower 3-year survival rate [hazard ratio (HR)=2.21 and 2.35, respectively] compared with patients homozygous for the dominant allele G. The value for overall survival could be validated with a HR of 2.54 (P=0.018) in an independent cohort. Patients homozygous for the minor allele A of EREG rs12641042 had a significantly higher 3-year survival rate than patients with allele C (HR 0.48; P=0.034), but significance was lost in multivariable analysis (P=0.066). The value of rs12641042 could not be validated (P=0.98). Exploratory multivariable subgroup analysis showed the strongest prognostic value for rs1615111 in tumors with a diffuse histology (Pfor interaction=0.004). CONCLUSION: AREG rs1615111, located in the AREG genomic region, can significantly define different prognostic cohorts in locally advanced GC. This value is most evident in GC patients with diffuse histology, which might be relevant as none of the trials testing epithelial growth factor receptor inhibitors has been enriched for diffuse histology or a molecularly defined population.

Intra-Articular Injection of Adipose-Derived Stromal Vascular Fraction in Osteoarthritic Temporomandibular Joints: Study Design of a Randomized Controlled Clinical Trial.

INTRODUCTION: Temporomandibular joint (TMJ) osteoarthritis is a degenerative disease of the TMJ. It is characterized by progressive degradation of the extracellular matrix components of articular cartilage, with secondary inflammatory components leading to pain in the temporomandibular region and reduced mouth opening. Current treatments do not halt disease progression, hence the need for new therapies to reduce inflammation and, consequently, improve symptoms. The aim of our randomized controlled clinical trial protocol is to investigate the efficacy of adjuvant intra-articular injections of autologous tissue-like stromal vascular fraction (tSVF), compared to arthrocentesis alone, in reducing pain and improving mouth opening in TMJ osteoarthritis patients. MATERIALS AND METHODS: The primary endpoint analysis will consist of the visual analogue scale (VAS) for pain. The secondary endpoint analyses will include maximal interincisal mouth opening measurements; assessment of oral health and mandibular function based on the oral health impact profile (OHIP) questionnaire and mandibular functional impairment questionnaire (MFIQ); complications during the follow up; synovial cytokine analysis at baseline and after 26 weeks; and nucleated cells and tSVF (immuno)histochemistry analyses of the intervention group. DISCUSSION: Our randomized clinical trial protocol will be applied to evaluate the efficacy of a new promising tSVF injection therapy for TMJ osteoarthritis. The safety of intra-articular injections of tSVF has been proven for knee osteoarthritis. However, since a tSVF injection is considered a heterologous application of cell therapy, the regulatory requirements are strict, which makes medical ethical approval challenging.

Cohesiveness tunes assembly and morphology of FG nucleoporin domain meshworks - Implications for nuclear pore permeability.

Nuclear pore complexes control the exchange of macromolecules between the cytoplasm and the nucleus. A selective permeability barrier that arises from a supramolecular assembly of intrinsically unfolded nucleoporin domains rich in phenylalanine-glycine dipeptides (FG domains) fills the nuclear pore. There is increasing evidence that selective transport requires cohesive FG domain interactions. To understand the functional roles of cohesive interactions, we studied monolayers of end-grafted FG domains as a bottom-up nanoscale model system of the permeability barrier. Based on detailed physicochemical analysis of the model films and comparison of the data with polymer theory, we propose that cohesiveness is tuned to promote rapid assembly of the permeability barrier and to generate a stable and compact pore-filling meshwork with a small mesh size. Our results highlight the functional importance of weak interactions, typically a few kBT per chain, and contribute important information to understand the mechanism of size-selective transport.

Distinct defects in collagen microarchitecture underlie vessel-wall failure in advanced abdominal aneurysms and aneurysms in Marfan syndrome.

An aneurysm of the aorta is a common pathology characterized by segmental weakening of the artery. Although it is generally accepted that the vessel-wall weakening is caused by an impaired collagen metabolism, a clear association has been demonstrated only for rare syndromes such as the vascular type Ehlers-Danlos syndrome. Here we show that vessel-wall failure in growing aneurysms of patients who have aortic abdominal aneurysm (AAA) or Marfan syndrome is not related to a collagen defect at the molecular level. On the contrary our findings indicate similar (Marfan) or even higher collagen concentrations (AAA) and increased collagen cross-linking in the aneurysms. Using 3D confocal imaging we show that the two conditions are associated with profound defects in collagen microarchitecture. Reconstructions of normal vessel wall show that adventitial collagen fibers are organized in a loose braiding of collagen ribbons. These ribbons encage the vessel, allowing the vessel to dilate easily but preventing overstretching. AAA and aneurysms in Marfan syndrome show dramatically altered collagen architectures with loss of the collagen knitting. Evaluations of the functional characteristics by atomic force microscopy showed that the wall has lost its ability to stretch easily and revealed a second defect: although vascular collagen in normal aortic wall behaves as a coherent network, in AAA and Marfan tissues it does not. As result, mechanical forces loaded on individual fibers are not distributed over the tissue. These studies demonstrate that the mechanical properties of tissue are strongly influenced by collagen microarchitecture and that perturbations in the collagen networks may lead to mechanical failure.

Viscoelasticity of thin biomolecular films: a case study on nucleoporin phenylalanine-glycine repeats grafted to a histidine-tag capturing QCM-D sensor.

Immobilization of proteins onto surfaces is useful for the controlled generation of biomolecular assemblies that can be readily characterized with in situ label-free surface-sensitive techniques. Here we analyze the performance of a quartz crystal microbalance with dissipation monitoring (QCM-D) sensor surface that enables the selective and oriented immobilization of histidine-tagged molecules for morphological and interaction studies. More specifically, we characterize monolayers of natively unfolded nucleoporin domains that are rich in phenylalanine-glycine repeats (FGRDs). An FGRD meshwork is thought to be responsible for the selectivity of macromolecular transport across the nuclear pore complex between the cytosol and the nucleus of living cells. We demonstrate that nucleoporin FGRD films can be formed on His-tag Capturing Sensors with properties comparable to a previously reported immobilization platform based on supported lipid bilayers (SLB). Approaches to extract the film thickness and viscoelastic properties in a time-resolved manner from the QCM-D response are described, with particular emphasis on the practical implementation of viscoelastic modeling and a detailed analysis of the quality and reliability of the fit. By comparing the results with theoretical predictions for the viscoelastic properties of polymer solutions and gels, and experimental data from an atomic force microscopy indentation assay, we demonstrate that detailed analysis can provide novel insight into the morphology and dynamics of FG repeat domain films. The immobilization approach is simple and versatile, and can be easily extended to other His-tagged biomolecules. The data analysis procedure should be useful for the characterization of other ultrathin biomolecular and polymer films.

Ultrathin nucleoporin phenylalanine-glycine repeat films and their interaction with nuclear transport receptors.

Nuclear pore complexes (NPCs) are highly selective gates that mediate the exchange of all proteins and nucleic acids between the cytoplasm and the nucleus. Their selectivity relies on a supramolecular assembly of natively unfolded nucleoporin domains containing phenylalanine-glycine (FG)-rich repeats (FG repeat domains), in a way that is at present poorly understood. We have developed ultrathin FG domain films that reproduce the mode of attachment and the density of FG repeats in NPCs, and that exhibit a thickness that corresponds to the nanoscopic dimensions of the native permeability barrier. By using a combination of biophysical characterization techniques, we quantified the binding of nuclear transport receptors (NTRs) to such FG domain films and analysed how this binding affects the swelling behaviour and mechanical properties of the films. The results extend our understanding of the interaction of FG domain assemblies with NTRs and contribute important information to refine the model of transport across the permeability barrier.

Titanium or Biodegradable Osteosynthesis in Maxillofacial Surgery? In Vitro and In Vivo Performances.

Osteosynthesis systems are used to fixate bone segments in maxillofacial surgery. Titanium osteosynthesis systems are currently the gold standard. However, the disadvantages result in symptomatic removal in up to 40% of cases. Biodegradable osteosynthesis systems, composed of degradable polymers, could reduce the need for removal of osteosynthesis systems while avoiding the aforementioned disadvantages of titanium osteosyntheses. However, disadvantages of biodegradable systems include decreased mechanical properties and possible foreign body reactions. In this review, the literature that focused on the in vitro and in vivo performances of biodegradable and titanium osteosyntheses is discussed. The focus was on factors underlying the favorable clinical outcome of osteosyntheses, including the degradation characteristics of biodegradable osteosyntheses and the host response they elicit. Furthermore, recommendations for clinical usage and future research are given. Based on the available (clinical) evidence, biodegradable copolymeric osteosyntheses are a viable alternative to titanium osteosyntheses when applied to treat maxillofacial trauma, with similar efficacy and significantly lower symptomatic osteosynthesis removal. For orthognathic surgery, biodegradable copolymeric osteosyntheses are a valid alternative to titanium osteosyntheses, but a longer operation time is needed. An osteosynthesis system composed of an amorphous copolymer, preferably using ultrasound welding with well-contoured shapes and sufficient mechanical properties, has the greatest potential as a biocompatible biodegradable copolymeric osteosynthesis system. Future research should focus on surface modifications (e.g., nanogel coatings) and novel biodegradable materials (e.g., magnesium alloys and silk) to address the disadvantages of current osteosynthesis systems.

Reliability and accuracy of the torque applied to osteosynthesis screws by maxillofacial surgeons and residents.

Applying the right torque to osteosynthesis screws is important for undisturbed bone healing. This study aimed to compare test-retest and intra-individual reliabilities of the torque applied to 1.5 mm and 2.0 mm osteosynthesis screws by residents and oral and maxillofacial surgeons (OMF-surgeons), to define the reference torque intervals, and to compare reference torque interval compliances. Five experienced OMF-surgeons and 20 residents, 5 of each 4 residency years, were included. Each participant inserted six 1.5 × 4 mm and six 2.0 × 6 mm screws into a preclinical model at two test moments 2 weeks apart (T1 and T2). Participants were blinded for the applied torque. Descriptive statistics, reference intervals, and intra-class correlation coefficients (ICC) were calculated. The OMF-surgeons complied more to the reference intervals (1.5 mm screws: 95% and 2.0 mm screws: 100%) than the residents (82% and 90%, respectively; P = 0.009 and P = 0.007) with the ICCs ranging between 0.85-0.95 and 0.45-0.97, respectively. The residents' accuracy and reliability were inadequate regarding the 1.5 mm screws but both measures improved at T2 for both screw types compared to T1, indicating a learning effect. Training residents and/or verifying the applied torque by experienced OMF-surgeons remains necessary to achieve high accuracy and reliability, particularly for 1.5 mm screws.

Biocompatibility and degradation comparisons of four biodegradable copolymeric osteosynthesis systems used in maxillofacial surgery: A goat model with four years follow-up.

Applying biodegradable osteosyntheses avoids the disadvantages of titanium osteosyntheses. However, foreign-body reactions remain a major concern and evidence of complete resorption is lacking. This study compared the physico-chemical properties, histological response and radiographs of four copolymeric biodegradable osteosynthesis systems in a goat model with 48-months follow-up. The systems were implanted subperiosteally in both tibia and radius of 12 Dutch White goats. The BioSorb FX [poly(70LLA-co-30DLLA)], Inion CPS [poly([70-78.5]LLA-co-[16-24]DLLA-co-4TMC)], SonicWeld Rx [poly(DLLA)], LactoSorb [poly(82LLA-co-18GA)] systems and a negative control were randomly implanted in each extremity. Samples were assessed at 6-, 12-, 18-, 24-, 36-, and 48-month follow-up. Surface topography was performed using scanning electron microscopy (SEM). Differential scanning calorimetry and gel permeation chromatography were performed on initial and explanted samples. Histological sections were systematically assessed by two blinded researchers using (polarized) light microscopy, SEM and energy-dispersive X-ray analysis. The SonicWeld Rx system was amorphous while the others were semi-crystalline. Foreign-body reactions were not observed during the complete follow-up. The SonicWeld Rx and LactoSorb systems reached bone percentages of negative controls after 18 months while the BioSorb Fx and Inion CPS systems reached these levels after 36 months. The SonicWeld Rx system showed the most predictable degradation profile. All the biodegradable systems were safe to use and well-tolerated (i.e., complete implant replacement by bone, no clinical or histological foreign body reactions, no [sterile] abscess formation, no re-interventions needed), but nanoscale residual polymeric fragments were observed at every system's assessment.

Evaluation of potential synergistic action of a combined treatment with alpha-methyl-prednisolone and taurine on the mdx mouse model of Duchenne muscular dystrophy.

AIMS: Glucocorticoids are the sole drugs clinically used in Duchenne muscular dystrophy, in spite of the relevant side effects. Combination of glucocorticoids with synergistic drugs may be one strategy to lower doses and control side effects, meanwhile providing wider control of the complex pathology. This study is a preclinical evaluation of the effect of a combined treatment of α-methyl-prednisolone (PDN) with taurine, a safe aminoacid with positive effects on some pathology-related events. METHODS: PDN (1 mg/kg/day i.p.) and taurine (1 g/kg/day orally) were administered either alone or in combination, for 4-8 weeks to male dystrophic mdx mice chronically exercised on a treadmill. Effects were assessed in vivo and ex vivo with a variety of methodological approaches. RESULTS: In vivo, each treatment significantly increased fore limb strength, a marked synergistic effect being observed with the combination PDN + taurine. Ex vivo, PDN + taurine completely restored the mechanical threshold, an electrophysiological index of calcium homeostasis, of extensor digitorum longus myofibres and the benefit was greater than for PDN alone. In parallel, the overactivity of voltage-independent cation channels in dystrophic myofibres was reduced. No effects were observed on plasma levels of creatine kinase, while lactate dehydrogenase was decreased by taurine and, to a minor extent, by PDN + taurine. A similar histology profile was observed in PDN and PDN + taurine-treated muscles. PDN + taurine significantly increased taurine level in fast-twitch muscle and brain, by high-pressure liquid chromatography analysis. CONCLUSIONS: The combination PDN + taurine has additive actions on in vivo and ex vivo functional end points, with less evident advantages on histopathology and biochemical markers of the disease.

Comparison of the mechanical properties of biodegradable and titanium osteosynthesis systems used in oral and maxillofacial surgery.

To guide the selection of osteosynthesis systems, this study compared the mechanical properties of biodegradable and titanium osteosynthesis systems. SonicPins Rx and xG were subjected to pull-out tests. Additionally, 15 biodegradable (Inion CPS 2.0 and 2.5 mm; LactoSorb 2.0 mm; Macropore 2.0 mm; Polymax 2.0 mm; BioSorb FX 2.0 mm; ResorbX 2.1 mm; Osteotrans-MX 2.0 mm with plate thicknesses 1.0 and 1.4 mm; SonicWeld Rxplate/Rxpins, xGplate/Rxpins and xGplate/xGpins 2.1 mm without and with tapping the burr hole) and six titanium (CrossDrive (2006), CrossDrive (2018), MaxDrive; all 1.5 and 2.0 mm) straight, four-hole osteosynthesis systems were evaluated. All systems were subjected to tensile, bending and torsion tests. Pull-out loads of the SonicPins were comparable (P = 0.423). Titanium systems' tensile loads were higher than biodegradable systems (P < 0.001). CrossDrive (2018) and MaxDrive systems' tensile and torsional stiffness were lower, accompanied with higher ductility, than corresponding CrossDrive (2006) systems (P < 0.001). Bending stiffness of 1.5 mm titanium systems was comparable to, and of the 2.0 mm systems higher than, all biodegradable systems (P < 0.001). Regarding biodegradable systems, Inion CPS 2.5 mm had highest tensile load and torsional stiffness, SonicWeld 2.1 mm highest tensile stiffness, and BioSorbFX 2.0 mm highest bending stiffness (P < 0.001). On the basis of the results of this study, the CrossDrive (2018) and MaxDrive 1.5 mm titanium systems are recommended for midface fractures (e.g., zygomatic or maxillary fractures) and osteotomies (e.g., Le Fort I osteotomy), and the CrossDrive (2018) and MaxDrive 2.0 mm titanium systems for mandibular fractures and osteotomies when a titanium osteosynthesis system is used. When there is an indication for a biodegradable osteosynthesis system, the SonicWeld 2.1 mm or BioSorbFX 2.0 mm are recommended for midface fractures and osteotomies, and the Inion CPS 2.5 mm biodegradable system for mandibular osteotomies and non-load bearing mandibular fractures, especially when high torsional forces are expected (e.g., mandibular symphysis fractures).

Polymorphisms within Immune Regulatory Pathways Predict Cetuximab Efficacy and Survival in Metastatic Colorectal Cancer Patients.

Cetuximab, an IgG1 EGFR-directed antibody, promotes antibody-dependent cell-mediated cytotoxicity. We hypothesized that single-nucleotide polymorphisms (SNPs) in immune regulatory pathways may predict outcomes in patients with metastatic colorectal cancer treated with cetuximab-based regimens. A total of 924 patients were included: 105 received cetuximab in IMCL-0144 and cetuximab/irinotecan in GONO-ASL608LIOM01 (training cohort), 225 FOLFIRI/cetuximab in FIRE-3 (validation cohort 1), 74 oxaliplatin/cetuximab regimens in JACCRO CC-05/06 (validation cohort 2), and 520 FOLFIRI/bevacizumab in FIRE-3 and TRIBE (control cohorts). Twelve SNPs in five genes (IDO1; PD-L1; PD-1; CTLA-4; CD24) were evaluated by PCR-based direct sequencing. We analyzed associations between genotype and clinical outcomes. In the training cohort; patients with the CD24 rs52812045 A/A genotype had a significantly shorter median PFS and OS than those with the G/G genotype (PFS 1.3 vs. 3.6 months; OS 2.3 vs. 7.8 months) in univariate (PFS HR 3.62; p = 0.001; OS HR 3.27; p = 0.0004) and multivariate (PFS HR 3.18; p = 0.009; OS HR 4.93; p = 0.001) analyses. Similarly; any A allele carriers in the JACCRO validation cohort had a significantly shorter PFS than G/G carriers (9.2 vs. 11.8 months; univariate HR 1.90; p = 0.011; multivariate HR 2.12; p = 0.018). These associations were not demonstrated in the control cohorts. CD24 genetic variants may help select patients with metastatic colorectal cancer most likely to benefit from cetuximab-based therapy.

Angiogenesis in asthma.

Asthma is a chronic inflammatory disease of the airways characterized by infiltration and activation of inflammatory cells and by structural changes, including subepithelial fibrosis, smooth muscle cells hypertrophy/hyperplasia, epithelial cell metaplasia and angiogenesis. These structural changes are thought to correlate with asthma severity and to account for the development of progressive lung function deterioration. The mechanism underlying airway angiogenesis in asthma and its precise clinical relevance have not yet been completely elucidated. This review provides recent data showing the contribution of allergic inflammation in increased airway vascularity and potential therapeutical approaches in asthma treatment by acting on bronchial microvascular changes.

A novel cell type-specific role of p38alpha in the control of autophagy and cell death in colorectal cancer cells.

Cancer develops when molecular pathways that control the fine balance between proliferation, differentiation, autophagy and cell death undergo genetic deregulation. The prospects for further substantial advances in the management of colorectal cancer reside in a systematic genetic and functional dissection of these pathways in tumor cells. In an effort to evaluate the impact of p38 signaling on colorectal cancer cell fate, we treated HT29, Caco2, Hct116, LS174T and SW480 cell lines with the inhibitor SB202190 specific for p38alpha/beta kinases. We report that p38alpha is required for colorectal cancer cell homeostasis as the inhibition of its kinase function by pharmacological blockade or genetic inactivation causes cell cycle arrest, autophagy and cell death in a cell type-specific manner. Deficiency of p38alpha activity induces a tissue-restricted upregulation of the GABARAP gene, an essential component of autophagic vacuoles and autophagosomes, whereas simultaneous inhibition of autophagy significantly increases cell death by triggering apoptosis. These data identify p38alpha as a central mediator of colorectal cancer cell homeostasis and establish a rationale for the evaluation of the pharmacological manipulation of the p38alpha pathway in the treatment of colorectal cancer.

Clodronate inhibits angiogenesis in vitro and in vivo.

The effects of amino-bisphosphonate clodronate on endothelial cell functions involved in angiogenesis, namely proliferation and morphogenesis on matrigel were tested in vitro, whereas its effects on angiogenesis were studied in vivo. This was performed by using the chick embryo chorioallantoic membrane (CAM) assay. In vitro, clodronate inhibited the endothelial cell proliferation in a dose-dependent fashion, peaking at 30 microM. At the same concentration, clodronate inhibited the fibroblast growth factor-2 (FGF-2)-induced capillary-like tube formation in the morphogenesis assay on matrigel. In vivo, when tested with the CAM assay, clodronate again displayed the capability to inhibit FGF-2-induced angiogenesis. Overall, these results suggest that antiangiogenesis by clodronate can be used to treat a wide spectrum of angiogenesis-dependent diseases, including certain chronic inflammatory diseases and cancer.

Angiogenesis in vasculitides.

Vasculitides, including Wegener's granulomatosis, Takayasu's arteritis, giant cell arteritis, Kawasaki disease, Behçet disease, thromboangiitis obliterans and erythema elevatum diutinum, are inflammatory diseases of blood vessel wall characterized by myointimal proliferation, fibrosis and thrombus formation leading to stenosis or occlusion of the vascular lumen, and finally to tissue ischemia. In these diseases the hypoxic environment subsequent to stenosis or occlusion of the vascular lumen is a potent signal for the generation of new blood vessels. Angiogenesis may be a compensatory response to ischemia and to the increased metabolic activity and may be also a further inflammatory stimulus because endothelial cells of newly-formed vessels express adhesion molecules and produce colony-stimulating factors and chemokines for leukocytes.

The history of the angiogenic switch concept.

Spontaneously arising tumor cells are not usually angiogenic at first. The phenotypic switch to angiogenesis is usually accomplished by a substet that induces new capillaries that then converge toward the tumor. The switch clearly involves more than simple upregulation of angiogenic activity and is thought to be the result of a net balance of positive and negative regulators. Tumor growth is although to require disruption of this balance and hence this switch must turned on for cancer progression. Progenitor endothelial cells, the crosstalk between angiogenic factors and their receptors and the interaction between vasculogenesis and lymphangiogenesis are all factors that may contribute to the switch. Its promotion is also the outcome of genetic instability resulting in the emergence of tumor cell lines. This review describes the history of the angiogenic switch illustrated in the literature and with particular reference to the three transgenic mouse models, namely RIP1-TAG2, keratin-14 (K14) (human papilloma virus) HPV16 and papilloma virus, used for stage-specific assessment of the effects of antiangiogenic and antitumorigenic agents.