Detection of ABCB5 tumour antigen-specific CD8+ T cells in melanoma patients and implications for immunotherapy.

ATP binding cassette subfamily B member 5 (ABCB5) has been identified as a tumour-initiating cell marker and is expressed in various malignancies, including melanoma. Moreover, treatment with anti-ABCB5 monoclonal antibodies has been shown to inhibit tumour growth in xenotransplantation models. Therefore, ABCB5 represents a potential target for cancer immunotherapy. However, cellular immune responses against ABCB5 in humans have not been described so far. Here, we investigated whether ABCB5-reactive T cells are present in human melanoma patients and tested the applicability of ABCB5-derived peptides for experimental induction of human T cell responses. Peripheral blood mononuclear cells (PBMNC) isolated from blood samples of melanoma patients (n = 40) were stimulated with ABCB5 peptides, followed by intracellular cytokine staining (ICS) for interferon (IFN)-γ and tumour necrosis factor (TNF)-α. To evaluate immunogenicity of ABCB5 peptides in naive healthy donors, CD8 T cells were co-cultured with ABCB5 antigen-loaded autologous dendritic cells (DC). ABCB5 reactivity in expanded T cells was assessed similarly by ICS. ABCB5-reactive CD8+ T cells were detected ex vivo in 19 of 29 patients, melanoma antigen recognised by T cells (MART-1)-reactive CD8+ T cells in six of 21 patients. In this small, heterogeneous cohort, reactivity against ABCB5 was significantly higher than against MART-1. It occurred significantly more often and independently of clinical characteristics. Reactivity against ABCB5 could be induced in 14 of 16 healthy donors in vitro by repeated stimulation with peptide-loaded autologous DC. As ABCB5-reactive CD8 T cells can be found in the peripheral blood of melanoma patients and an ABCB5-specific response can be induced in vitro in naive donors, ABCB5 could be a new target for immunotherapies in melanoma.

Blood coagulation dissected.

Hemostasis is the physiological control of bleeding and is initiated by subendothelial exposure. Platelets form the primary vascular seal in three stages (localization, stimulation and aggregation), which are triggered by specific interactions between platelet surface receptors and constituents of the subendothelial matrix. As a secondary hemostatic plug, fibrin clot formation is initiated and feedback-amplified to advance the seal and stabilize platelet aggregates comprising the primary plug. Once blood leakage has been halted, the fibrinolytic pathway is initiated to dissolve the clot and restore normal blood flow. Constitutive and induced anticoagulant and antifibrinolytic pathways create a physiological balance between too much and too little clot production. Hemostatic imbalance is a major burden to global healthcare, resulting in thrombosis or hemorrhage.

Positional OSA part 2: retrospective cohort analysis with a new classification system (APOC).

BACKGROUND: In Part 1 of this two-part article, the Amsterdam Positional Obstructive Sleep Apnoea Classification (APOC) was recently introduced, a classification system aimed at facilitating the identification of suitable candidates for positional therapy (PT): patients who will benefit from a clinically significant improvement of their obstructive sleep apnoea (OSA) with PT. APOC was developed with new generation PT devices in mind rather than conventional PT (tennis ball technique). New generation PT can be defined as a well-tolerated device which prevents a patient from adopting the worst sleeping position (WSP) without negatively influencing sleep efficiency, as objectified by a full night polysomnography (PSG). PT is rapidly gaining momentum in the scope of OSA treatment. The objective of this manuscript is to measure the prevalence of position-dependent obstructive sleep apnoea (POSA) according to the APOC, in a consecutive series of patients referred for PSG as well as an investigation of associations between POSA and certain patient characteristics. METHODS: We performed a retrospective, single-centre cohort study including a consecutive series of patients who underwent a PSG during the period of April 2010 until October 2010. RESULTS: Within this OSA-cohort (n = 253), a prevalence of POSA of 69 % when applying APOC is measured, compared to 64 % when applying Cartwright's classification. An inverse relation between POSA and BMI was observed, likewise between POSA and apnoea hypopnoea index (AHI). CONCLUSION: We are of opinion that APOC is a suitable tool to identify patients who will or will not benefit from PT, thus resulting in more cost-efficient treatment.

The Co-Expression of Kallikrein 5 and Kallikrein 7 Associates with Poor Survival in Non-HPV Oral Squamous-Cell Carcinoma.

OBJECTIVE: Oral squamous-cell carcinoma (OSCC) still has a poor prognosis. Lymph node metastasis (LNM) is a major determinant of treatment decisions and prognosis. Serine protease inhibitor Kazal-type 5 (SPINK5) is the inhibitor of kallikrein 5 (KLK5) and KLK7. SPINK5, KLK5 and KLK7 are three of the genes of a recently validated LNM-predicting gene expression profile in OSCC. This study evaluates their clinicopathological role and value as biomarkers in OSCC. METHODS: Eighty-three patients with primary OSCC, treated surgically between 1996 and 2000, were included. Gene expression data were acquired from a previously reported study. Human papillomavirus (HPV) status was determined by an algorithm for HPV-16. Protein expression for KLK5, KLK7 and SPINK5 was semi-quantitatively determined in all 83 tumours by immunohistochemistry. All expression data were correlated with clinicopathological parameters. RESULTS: Concurrent loss of KLK5 and KLK7 correlates with worse disease-specific and overall survival (DSS and OS). Multivariate analysis proved that co-expression is an independent prognostic factor for DSS (p = 0.029) and OS (p = 0.001). CONCLUSION: This report demonstrates that concurrent loss of KLK5 and KLK7 associates with a poor clinical outcome in OSCC and could therefore serve as prognostic marker in this disease.

Positional OSA part 1: Towards a clinical classification system for position-dependent obstructive sleep apnoea.

BACKGROUND: In 1984, Cartwright suggested that physicians should differentiate between patients with either positional obstructive sleep apnoea (POSA) or non-positional OSA. Treatment of POSA has advanced dramatically recently with the introduction of a new generation of positional therapy (PT), a small device attached to either the neck or chest which corrects the patient from adopting the supine position through a vibrating stimulus. Encouraging data have been published suggesting that this simple therapy successfully prevents patients with POSA from adopting the supine position without negatively influencing sleep efficiency, as well as allowing for good adherence. Unfortunately, evaluating the efficacy of PT and comparing results are hindered by the fact that there are no universally used POSA criteria. In 1984, Cartwright introduced the arbitrary cut-off point of a difference of 50% or more in apnoea index between supine and non-supine positions. INTRODUCTION: The aim of this project was to introduce a new classification system, which ideally should identify suitable candidates for PT: patients that will benefit from a clinically significant improvement of their OSA with PT. The shared use of this classification can facilitate collection of data across multiple centres and comparison of results across studies. We report on the development and process that resulted in the Amsterdam Positional OSA Classification (APOC). METHOD: A panel of three field experts were instructed to independently assign the diagnosis POSA to 100 randomly selected patients they considered likely to benefit from a clinically significant improvement of their OSA with PT. In a group setting, the completed lists were compared. Discrepancies were discussed until consensus was met. This resulted in the consensus standard used to calibrate the new classification. Using the nominal group technique, the APOC was developed. RESULTS: The APOC criteria evolve around the percentage of total sleep time spent in either the worst sleeping position (WSP) or the best sleeping position (BSP) and the apnoea-hypopnoea index (AHI) in BSP. On applying APOC, one discriminates between the true positional patient, the non-positional patient and the multifactorial patient, whose OSA severity is influenced in part by sleep position. APOC has an increased sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) compared to previously applied POSA criteria in identifying patients that will benefit from positional therapy.

X-ray spectroscopic study of solvent effects on the ferrous and ferric hexacyanide anions.

We present an Fe Kα resonant inelastic X-ray scattering (RIXS) and X-ray emission (XES) study of ferrous and ferric hexacyanide dissolved in water and ethylene glycol. We observe that transitions below the absorption edge show that the solvent has a distinct effect on the valence electronic structure. In addition, both the RIXS and XES spectra show a stabilization of the 2p levels when dissolved in water. Using molecular dynamics simulations, we propose that this effect arises from the hydrogen-bonding interactions between the complex and nearby solvent molecules. This withdraws electron density from the ligands, stabilizing the complex but also causing a slight increase in π-backbonding.

Markers of circulating tumour cells in the peripheral blood of patients with melanoma correlate with disease recurrence and progression.

BACKGROUND: Multimarker quantitative real-time polymerase chain reaction (qRT-PCR) represents an effective method for detecting circulating tumour cells in the peripheral blood of patients with melanoma. OBJECTIVES: To investigate whether the phenotype of circulating melanoma cells represents a useful indicator of disease stage, recurrence and treatment efficacy. METHODS: Peripheral blood was collected from 230 patients with melanoma and 152 healthy controls over a period of 3years and 9months. Clinical data and blood samples were collected from patients with primary melanoma (early stages, 0-II, n=154) and metastatic melanoma (late stages, III-IV, n=76). Each specimen was examined by qRT-PCR analysis for the expression of five markers: MLANA, ABCB5, TGFß2, PAX3d and MCAM. RESULTS: In total, 212 of the patients with melanoma (92%) expressed markers in their peripheral blood. Two markers, MLANA and ABCB5, had the greatest prognostic value, and were identified as statistically significant among patients who experienced disease recurrence within our study period, being expressed in 45% (MLANA) and 49% (ABCB5) of patients with recurrence (P=0·001 and P=0·031, respectively). For patients administered nonsurgical treatments, MCAM expression correlated with poor treatment outcome. CONCLUSIONS: Circulating tumour cells were detectable at all stages of disease and long after surgical treatment, even when patients were considered disease free. Specifically, expression of ABCB5 and MLANA had significant prognostic value in inferring disease recurrence, while MCAM expression was associated with poor patient outcome after treatment, confirming multimarker qRT-PCR as a potential technique for monitoring disease status.

Solvent-induced luminescence quenching: static and time-resolved X-ray absorption spectroscopy of a copper(I) phenanthroline complex.

We present a static and picosecond X-ray absorption study at the Cu K-edge of bis(2,9-dimethyl-1,10-phenanthroline)copper(I) ([Cu(dmp)2](+); dmp = 2,9-dimethyl-1,10-phenanthroline) dissolved in acetonitrile and dichloromethane. The steady-state photoluminescence spectra in dichloromethane and acetonitrile are also presented and show a shift to longer wavelengths for the latter, which points to a stronger stabilization of the excited complex. The fine structure features of the static and transient X-ray spectra allow an unambiguous assignment of the electronic and geometric structure of the molecule in both its ground and excited (3)MLCT states. Importantly, the transient spectra are remarkably similar for both solvents, and the spectral changes can be rationalized using the optimized ground- and excited-state structures of the complex. The proposed assignment of the lifetime shortening of the excited state in donor solvents (acetonitrile) to a metal-centered exciplex is not corroborated here. Molecular dynamics simulations confirm the lack of complexation; however, in both solvents the molecules come close to the metal but undergo rapid exchange with the bulk. The shortening of the lifetime of the title complex and nine additional related complexes can be rationalized by the decrease in the (3)MLCT energy. Deviations from this trend may be explained by means of the effects of the dihedral angle between the ligand planes, the solvent, and the (3)MLCT-(1)MLCT energy gap.

[Peri-implantitis in the general oral and maxillofacial surgery practice. A pilot study]. / Peri-implantitis in de algemene kaakchirurgische praktijk. Een pilotonderzoek.

A peri-implant infection is a disorder with an annually increasing prevalence and incidence as a result of the increasing number of oral implants utilized. Based on existing literature, a patient-control study was carried out, as a pilot study, among patients who had been treated with oral implants at a department of oral and maxillofacial surgery in a general hospital. Significant relations were found between on the one hand the prevalence of peri-implantitis and on the other hand bone augmentation, a suprastructure of a fixed partial denture or a complete removable overdenture on a ball attachment mesostructure when compared to a crown, poor oral hygiene, poor periodontal condition, and the absence of keratinized mucosa surrounding the implant.

[Spontaneous dental abscesses in familial hypophosphatemic rickets]. / Multipele spontane odontogene abcessen bij familiaire hypofosfatemische rachitis.

Symptoms of familial hypophosphatemic rickets are growth retardation, the formation of O- or X-legs, pain of the joints, spontaneous dental abscesses, and delayed tooth eruption. The dental symptoms are predominantly attributable to the demineralization of dentin. In absence of adequate preventive measurements,familial hypophosphatemic rickets may lead to spontaneous pulpal necrosis. The prophylactic application of occlusal sealants might be effective in preventing abscess formation.

Consecutive dosing of UVB irradiation induces loss of ABCB5 expression and activation of EMT and fibrosis proteins in limbal epithelial cells similar to pterygium epithelium.

Pterygium pathogenesis is often attributed to a population of altered limbal stem cells, which initiate corneal invasion and drive the hyperproliferation and fibrosis associated with the disease. These cells are thought to undergo epithelial to mesenchymal transition (EMT) and to contribute to subepithelial stromal fibrosis. In this study, the presence of the novel limbal stem cell marker ABCB5 in clusters of basal epithelial pterygium cells co-expressing with P63α and P40 is reported. ABCB5-positive pterygium cells also express EMT-associated fibrosis markers including vimentin and α-SMA while their ß-catenin expression is reduced. By using a novel in vitro model of two-dose UV-induced EMT activation on limbal epithelial cells, we could observe the dysregulation of EMT-related proteins including an increase of vimentin and α-SMA as well as downregulation of ß-catenin in epithelial cells correlating to downregulation of ABCB5. The sequential irradiation of limbal fibroblasts also induced an increase in vimentin and α-SMA. Taken together, these data demonstrate for the first time the expression of ABCB5 in pterygium stem cell activity and EMT-related events while the involvement of limbal stem cells in pterygium pathogenesis is exhibited via sequential irradiation of limbal epithelial cells. The later in vitro approach can be used to further study the involvement of limbal epithelium UV-induced EMT in pterygium pathogenesis and help identify novel treatments against pterygium growth and recurrence.

Knowledge diffusion in the network of international business travel.

We use aggregated and anonymized information based on international expenditures through corporate payment cards to map the network of global business travel. We combine this network with information on the industrial composition and export baskets of national economies. The business travel network helps to predict which economic activities will grow in a country, which new activities will develop and which old activities will be abandoned. In statistical terms, business travel has the most substantial impact among a range of bilateral relationships between countries, such as trade, foreign direct investments and migration. Moreover, our analysis suggests that this impact is causal: business travel from countries specializing in a specific industry causes growth in that economic activity in the destination country. Our interpretation of this is that business travel helps to diffuse knowledge, and we use our estimates to assess which countries contribute or benefit the most from the diffusion of knowledge through global business travel.

The value of complementary co-workers.

As individuals specialize in specific knowledge areas, a society's know-how becomes distributed across different workers. To use this distributed know-how, workers must be coordinated into teams that, collectively, can cover a wide range of expertise. This paper studies the interdependencies among co-workers that result from this process in a population-wide dataset covering educational specializations of millions of workers and their co-workers in Sweden over a 10-year period. The analysis shows that the value of what a person knows depends on whom that person works with. Whereas having co-workers with qualifications similar to one's own is costly, having co-workers with complementary qualifications is beneficial. This co-worker complementarity increases over a worker's career and offers a unifying framework to explain seemingly disparate observations, answering questions such as "Why do returns to education differ so widely?" "Why do workers earn higher wages in large establishments?" "Why are wages so high in large cities?"

[Stem cell-based strategies in vascular surgery]. / Stammzell-basierter biologischer Gefäßersatz.

Critical chronic ischemia in patients with underlying arterial occlusive disease requires vascular reconstructive surgery. The limited supply of suitable small-diameter autologous vascular grafts in many patients and obvious disadvantages of synthetic bypass material demand the development of clinically usable tissue-engineered blood vessel substitutes. Despite substantial progress in the field over the last two decades, their implementation into the clinical routine has been challenging. The limited replicative life span of human adult vascular cells and their slow rate of collagenous matrix production in vitro have posed important problems in the development of mechanically robust and biologically functional engineered grafts. With recent advances in stem cell research, new cell sources for vascular tissue engineering have become available. In particular, the discovery of human induced pluripotent stem (iPS) cells derived from adult differentiated cells, as well as of human multipotent adult mesenchymal stem cells without gene modification requirements and related safety concerns, may advance the development of novel autologous cell-based tissue engineering approaches. Here we discuss recent developments in the field of vascular progenitor cells and opportunities and challenges for the clinical translation of stem cell-engineered vascular tissue substitutes.

UV light-blocking contact lenses protect against short-term UVB-induced limbal stem cell niche damage and inflammation.

UVB irradiation has been linked to pathogenesis of pterygium, a conjunctival tumor growing onto transparent cornea, the windscreen of the eye. Due to corneal anatomy, ambient UVB irradiation is amplified at the stem cell-containing nasal limbus. The aim of this study was to analyse the effect of a UV-blocking contact lens (UVBCL, senofilcon A, Class 1 UV blocker) on limbal epithelial cells and fibroblasts under UVB irradiation compared to a non-UVB-blocking contact lens. UVBCL prevented UVB-induced DNA damage (as assessed by cyclobutane pyrimidine dimer immunostaining) as well as a decrease in proliferation and scratch wound closure rate of both limbal epithelial and fibroblast cells. Similarly, UVBCL protected limbal epithelial cells from UVB-induced loss of their phenotype in terms of colony forming efficiency and stem cell marker expression (ABCB5, P63α, integrin ß1) compared to controls. Moreover, with UVBCL pro-inflammatory cytokines such as TNFα and MCP1 remained unchanged. These data demonstrate the significance of UV-protection in preserving the limbal niche in response to at least short-term UVB. Our data support the use of UVBCL in protecting limbal niche cells, especially after limbal stem cell transplantation and in patients after pterygium surgery, to help prevent recurrences.

[Coagulation abnormalities and the dentist]. / Stollingsstoornissen en de tandarts.

Defects in the coagulation system increase the risk of excessive bleeding during and after invasive dental procedures. This increased risk of bleeding is irrespective whether the coagulation disorder is inherited or acquired, for example by using anticoagulant medication. However, the cause of the coagulation disorder dictates preventive measures to be taken. Therefore, a careful medical history is crucial to identify and characterize patients with coagulation abnormalities. Recently, the insight in the mechanism of coagulation has increased considerably. This has resulted in specific procedures to adapt the level of coagulation required for specific invasive dental procedures. The dentist must discuss this temporary alteration of the coagulation level with the medical specialist of the patient.

P-glycoprotein--a novel therapeutic target for immunomodulation in clinical transplantation and autoimmunity?

P-glycoprotein, the human MDR1 gene product and cancer multidrug resistance-associated ATP-binding cassette transporter, is physiologically expressed on peripheral blood mononuclear cells, but its role in cellular immunity is only beginning to be elucidated. A role of P-glycoprotein in the secretion of several T cell- and antigen presenting cell-derived cytokines has been described, and additional functions of the molecule have been identified in lymphocyte survival and antigen presenting cell differentiation. Taken together, these findings provide compelling evidence that P-glycoprotein serves several distinct functions in the initiation of primary immune responses, and a critical role of the molecule in functional immune responses is now established. Here, we will review the current understanding of P-glycoprotein function in T cell activation and antigen presenting cell function, which are relevant to the fields of clinical transplantation and autoimmunity, and summarize the evidence for in vitro and in vivo immunomodulatory actions of several known P-glycoprotein-inhibiting agents currently in clinical use for other indications. We suggest that it is the P-glycoprotein-inhibitory function of many of these agents that underly their immunoregulatory capacities. Thus, the established immunoregulatory function of P-glycoprotein and the availability of P-glycoprotein-inhibitory drugs raise the possibility that P-glycoprotein may represent a promising novel therapeutic target for immune modulation in acute and chronic allograft rejection, and cell-mediated autoimmune disorders.

Interferon alpha2b differentially affects proliferation of two human renal cell carcinoma cell lines differing in the P-glycoprotein-associated multidrug-resistant phenotype.

PURPOSE: Interferon alpha (IFNalpha) has been used in the immunotherapy of renal cell carcinoma (RCC), but the various mechanisms of its antiproliferative effects are poorly understood. Recent evidence suggests that IFNalpha is involved in the up-regulation of multidrug resistance (MDR) gene expression, and that the MDR gene product, P-glycoprotein (Pgp). facilitates the transport of several cytokines, some of which have been implicated in mediating tumor antiproliferative effects. We hypothesized that IFNalpha-induced antiproliferative activity may require Pgp-mediated transport, and that susceptibility to IFNalpha may thus correlate with Pgp expression. METHODS: Pgp expression by the human RCC cell lines KTCTL-2 and KTCTL-26 was characterized by immunofluorescence staining, using the Pgp-specific primary antibodies C219 and JSB1. KTCTL-2 and KTCTL-26 cell lines were subsequently treated with IFNalpha2b, and growth kinetics of treated and control cell cultures were determined daily by cell counting. RESULTS: KTCTL-2 expresses Pgp at low levels, whereas KTCTL-26 is a highly expressing cell line. IFNalpha2b treatment abrogated cell proliferation in KTCTL-26, whereas proliferation of KTCTL-2 was only partially inhibited. CONCLUSIONS: We have identified two RCC cell lines that differ in the MDR phenotype and exhibit different responses to the antiproliferative activity of IFNalpha2b. These preliminary findings raise the possibility that susceptibility to the antiproliferative effects of IFNalpha2b may correlate with Pgp expression, and further studies are warranted.

Effect of ramp slope on determination of aerobic parameters from the ramp exercise test.

The effect of ramp slope on determination of aerobic parameters from the ramp exercise test. Med. Sci. Sports Exercise, Vol. 14, No. 5, pp. 339-343, 1982. We have previously demonstrated that the four parameters of aerobic function (maximal oxygen uptake (muVO2), VO2 at the anaerobic threshold (theta an), the time constant for VO2 kinetics (tau VO2), and work efficiency (eta)) may all be determined reliably from a single test in which the work rate increases continuously at a constant rate, i.e., ramp. That study, however, utilized a single ramp slope of 50 W X min-1, which may not be appropriate for subjects with very low or very high work tolerances. We therefore studied the effect of different ramp slopes on the determination of these parameters. Ramp slopes of 20, 30, 50, and 100 W X min-1 were generated on a cycle ergometer, and each was assigned randomly to 14 healthy subjects. Ventilatory and gas exchange variables were measured breath-by-breath utilizing on-line digital computation. Ramp slopes of 20, 30, and 50 W X min-1 yielded the same values for each aerobic parameter. The 100 W X min-1 ramp yielded muVO2 and eta an values that were the same as those found for the other ramp slopes, but tau VO2 and eta could not be discerned validly from this ramp slope. We conclude that valid assessment of the four parameters of aerobic function is possible with ramp slopes between 20 and 50 W X min-1; no further information on the parameters is to be gained by prolonging the tests with ramps slower than 20 W X min-1.