A coreceptor-independent transgenic human TCR mediates anti-tumor and anti-self immunity in mice.

Recent advancements in T cell immunotherapy suggest that T cells engineered with high-affinity TCR can offer better tumor regression. However, whether a high-affinity TCR alone is sufficient to control tumor growth, or the T cell subset bearing the TCR is also important remains unclear. Using the human tyrosinase epitope-reactive, CD8-independent, high-affinity TCR isolated from MHC class I-restricted CD4(+) T cells obtained from tumor-infiltrating lymphocytes (TIL) of a metastatic melanoma patient, we developed a novel TCR transgenic mouse with a C57BL/6 background. This HLA-A2-restricted TCR was positively selected on both CD4(+) and CD8(+) single-positive cells. However, when the TCR transgenic mouse was developed with a HLA-A2 background, the transgenic TCR was primarily expressed by CD3(+)CD4(-)CD8(-) double-negative T cells. TIL 1383I TCR transgenic CD4(+), CD8(+), and CD4(-)CD8(-) T cells were functional and retained the ability to control tumor growth without the need for vaccination or cytokine support in vivo. Furthermore, the HLA-A2(+)/human tyrosinase TCR double-transgenic mice developed spontaneous hair depigmentation and had visual defects that progressed with age. Our data show that the expression of the high-affinity TIL 1383I TCR alone in CD3(+) T cells is sufficient to control the growth of murine and human melanoma, and the presence or absence of CD4 and CD8 coreceptors had little effect on its functional capacity.

Contributions of viral oncogenes of HPV-18 and hypoxia to oxidative stress and genetic damage in human keratinocytes.

Infection with high-risk human papillomaviruses like HPV-16 and HPV-18 is highly associated with the development of cervical and other cancers. Malignant transformation requires viral oncoproteins E5, E6 and E7, which promote cell proliferation and increase DNA damage. Oxidative stress and hypoxia are also key factors in cervical malignant transformation. Increased levels of reactive species of oxygen (ROS) and nitrogen (RNS) are found in the hypoxic tumor microenvironment, promoting genetic instability and invasiveness. In this work, we studied the combined effect of E5, E6 and E7 and hypoxia in increasing oxidative stress and promoting DNA damage and nuclear architecture alterations. HaCaT cells containing HPV-18 viral oncogenes (HaCaT E5/E6/E7-18) showed higher ROS levels in normoxia and higher levels of RNS in hypoxia compared to HaCaT parental cells, as well as higher genetic damage in hypoxia as measured by γH2AX and comet assays. In hypoxia, HaCaT E5/E6/E7-18 increased its nuclear dry mass and both cell types displayed marked heterogeneity in nuclear dry mass distribution and increased nuclear foci. Our results show contributions of both viral oncogenes and hypoxia to oxidative stress, DNA damage and altered nuclear architecture, exemplifying how an altered microenvironment combines with oncogenic transformation to promote tumor progression.

Individual differences in the effects of midazolam on anxiety-like behavior, learning, reward, and choice behavior in male mice.

Introduction: The aim of the present study was to investigate the behavioral effects of the benzodiazepine midazolam in male mice, in models of anxiolysis, learning, and abuse-related effects. Methods: In a first set of experiments, male Swiss mice were submitted to the training session of a discriminative avoidance (DA) task on the elevated plus maze to evaluate anxiety-like behavior and learning after vehicle or midazolam (1, 2 or 5 mg/kg, i.g.) administration. The same animals were submitted to a conditioned place preference (CPP) protocol with midazolam (1, 2 or 5 mg/kg, i.g.). In a second experiment, outbred (Swiss) and inbred (C57BL/6) male mice were submitted to a two-bottle choice (TBC) oral midazolam drinking procedure. Animals were exposed to one sucrose bottle and one midazolam (0.008, 0.016 or 0.032 mg/ml) plus sucrose bottle. Results: Midazolam (1 and 2 mg/kg) induced anxiolytic-like effects, and all doses of midazolam prevented animals from learning to avoid the aversive closed arm during the DA training session. Assessment of midazolam reward via the CPP procedure and choice via the TBC procedure showed notable variability. A 2-step cluster analysis for the CPP data showed that midazolam data were well-fitted to 2 separate clusters (preference vs. aversion), albeit with the majority of mice showing preference (75%). Correlational and regression analyses showed no relationship between midazolam reward and anxiolytic-like effects (time spent in the open arms in the DA test) or learning/memory. Two-step cluster analysis of the TBC data also demonstrated that, regardless of strain, mice overall fell into two clusters identified as midazolam-preferring or midazolam-avoiding groups. Both midazolam preference and avoidance were concentration-dependent in a subset of mice. Discussion: Our findings show that midazolam preference is a multifactorial behavior, and is not dependent solely on the emergence of therapeutic (anxiolytic-like) effects, learning impairments, or on genetic factors (inbred vs. outbred animals).

The association between resting and mild-to-moderate exercise pulmonary artery pressure.

The mean pulmonary artery pressure (P(pa)) achieved on mild-to-moderate exercise is age related and its haemodynamic correlates remain to be documented in patients free of pulmonary hypertension (PH). Our retrospective study involved patients free of PH investigated in our centre for possible pulmonary vascular disease between January 1, 2007 and October 31, 2009 who underwent right heart catheterisation at rest and during supine exercise up to 60 W. The 38 out of 99 patients aged <50 yrs were included and a P(pa) of 30 mmHg was considered the upper limit of normal on exercise. The 24 subjects who developed P(pa)>30 mmHg on exercise had higher resting P(pa) (19±3 versus 15±4 mmHg) and indexed pulmonary vascular resistance (PVRi; 3.4±1.5 versus 2.2±1.1 WU·m(2); p<0.05) than the remaining 14 subjects. Resting P(pa) >15 mmHg predicted exercise P(pa) >30 mmHg with 88% sensitivity and 57% specificity. The eight patients with resting P(pa) 22-24 mmHg all had exercise P(pa) >30 mmHg. In subjects aged <50 yrs investigated for possible pulmonary vascular disease and free of PH, patients with mild-to-moderate exercise P(pa) >30 mmHg had higher resting PVRi and higher resting P(pa), although there was no resting P(pa) threshold value that could predict normal response on mild-to-moderate exercise. The clinical relevance of such findings deserves further long-term follow-up studies.

Role of 5-HT2A receptors in the effects of ayahuasca on ethanol self-administration using a two-bottle choice paradigm in male mice.

RATIONALE: Ayahuasca has been proposed as a potential treatment of alcohol (ethanol) use disorder (AUD). The serotonin 5-HT2A receptor agonist N,N-dimethyltryptamine (DMT) is the main psychoactive component of ayahuasca, suggesting that its therapeutic effects may be mediated by 5-HT2A receptors. OBJECTIVES: The aim of the present study was to investigate the effects of ayahuasca on the expression of ethanol self-administration using a two-bottle choice procedure and the role of 5-HT2A receptors in those effects. METHODS: Male mice had intermittent access to ethanol (10% v/v) in a two-bottle choice procedure for 30 days. Animals were then submitted to 3 treatment phases, each followed by ethanol re-exposure tests. During the treatment phase, every 3 days, animals received i.p. injections of either vehicle or the 5-HT2A receptor antagonist M100907 (M100, 1 mg/kg) followed by an i.g. (gavage) administration of vehicle or ayahuasca (100 mg/kg) and were exposed to the self-administration apparatus with no ethanol availability. During re-exposure tests, animals were submitted to the same conditions as during acquisition, with no treatments prior to those sessions. RESULTS: Treatment with ayahuasca blocked the expression of ethanol self-administration, decreasing ethanol intake and preference during re-exposure tests. Pretreatment with M100 blocked the effects of ayahuasca on ethanol drinking without significantly attenuating ethanol self-administration. CONCLUSIONS: Treatment with ayahuasca during alcohol abstinence blocked the expression of alcohol self-administration in mice, and 5-HT2A receptor activation is critical for those effects to emerge. Our findings support a potential for ayahuasca and other 5-HT2A receptor agonists as adjunctive pharmacotherapies for the treatment of AUD.

Aripiprazole and topiramate, alone or in combination, block the expression of ethanol-induced conditioned place preference in mice.

BACKGROUND: Ethanol is the most largely consumed drug in the world. Because of its complex mechanisms of action, studies suggest that the combination of drugs with distinct pharmacological effects may be a promising alternative for treating ethanol use disorder. In the present study, we aimed to investigate the effects of topiramate, alone and in combination with aripiprazole, on ethanol-induced conditioned place preference (CPP). METHODS: Adult male mice were conditioned with ethanol (1.8 g/kg, i.p.) in the conditioned place preference (CPP) apparatus. Animals were then treated with vehicle, topiramate (2.5, 5 or 10 mg/kg, i.p.), aripiprazole (0.025, 0.05, 0.075 or 0.1 mg/kg, i.p.) or a combination of subthreshold doses of topiramate and aripiprazole (5 and 0.075 mg/kg, respectively) in the ethanol-paired compartment for 8 consecutive days. The expression of ethanol-induced CPP was then evaluated during a drug-free test performed 24 h after a re-exposure to ethanol in the ethanol-paired compartment. RESULTS: Treatment with 10 mg/kg topiramate or 0.1 mg/kg aripiprazole blocked the expression of ethanol-induced CPP. Combined treatment with 5 mg/kg topiramate and 0.075 mg/kg aripiprazole, doses that alone were not effective, also blocked the expression of CPP to ethanol. CONCLUSIONS: Topiramate and aripiprazole, alone or in combination, blocked the expression of ethanol-induced CPP. By showing that a combination of lower, subthreshold doses or topiramate and aripiprazole was effective in blocking the conditioned reinforcing properties of the ethanol-paired environment in mice, our current findings provide important insights into the therapeutic use of these drugs in ethanol use disorder.

Sex differences in the development of conditioned place preference induced by intragastric alcohol administration in mice.

BACKGROUND: The present study aimed to identify for the first time sex differences in the development of CPP induced by intragastric alcohol administration in mice. METHODS: Male and female adult Swiss mice were submitted to 16 days of conditioning with alcohol (0.5-3.0 g/kg, N = 8/dose/sex), with 2 post-conditioning tests (after 8 and 16 sessions) during the protocol. RESULTS: 8 days of conditioning (4 alcohol sessions, 4 saline sessions) with intragastric alcohol administration were sufficient to induce CPP in male mice at the doses of 1.0, 1.5 and 2.0 g/kg. However, only higher doses (2.0, 2.5 and 3.0 g/kg) induced CPP in female mice using an 8-day conditioning protocol, while a 16-day conditioning protocol was necessary for the development of intragastric alcohol-induced CPP at the doses of 1.0 and 1.5 g/kg. Regardless of the conditioning protocol, higher doses or alcohol that had rewarding effects in females (2.5 and 3.0 g/kg) did not induce CPP in males, with a significant difference between males and females at those doses. Analysis of the potency (EC50) and efficacy (Emax) of alcohol in inducing CPP when administered intragastrically in male and female mice showed significant sex differences with 8 conditioning sessions. CONCLUSIONS: Our data show a clear protocol (8 vs 16 days) and dose difference between male and female Swiss mice regarding the development of CPP induced by intragastric alcohol administration. Intragastric alcohol administration is closer to human drinking, and our protocol provides a more translational approach to studying the rewarding effects of alcohol in mice.

Survival in incident and prevalent cohorts of patients with pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a progressive, fatal disease. We studied 674 consecutive adult patients who were prospectively enrolled in the French PAH registry (121 incident and 553 prevalent cases). Two survival analyses were performed. First, the cohort of 674 patients was followed for 3 yrs after study entry and survival rates described. Then, we focused on the subset with incident idiopathic, familial and anorexigen-associated PAH (n = 56) combined with prevalent patients who were diagnosed <3 yrs prior to study entry (n = 134). In the cohort of 674 patients, 1-, 2-, and 3-yr survival rates were 87% (95% CI 84-90), 76% (95% CI 73-80), and 67% (95% CI 63-71), respectively. In prevalent idiopathic, familial and anorexigen-associated PAH, 1-, 2-, and 3-yr survival rates were higher than in incident patients (p = 0.037). In the combined cohort of patients with idiopathic, familial and anorexigen-associated PAH, multivariable analysis showed that survival could be estimated by means of a novel risk-prediction equation using patient sex, 6-min walk distance, and cardiac output at diagnosis. This study highlights survivor bias in prevalent cohorts of PAH patients. Survival of idiopathic, familial and anorexigen-associated PAH can be characterised by means of a novel risk-prediction equation using patients' characteristics at diagnosis.

Ayahuasca blocks the reinstatement of methylphenidate-induced conditioned place preference in mice: behavioral and brain Fos expression evaluations.

RATIONALE: Accumulating evidence suggests that ayahuasca, a hallucinogenic beverage used in traditional Amazonian communities for ritualistic and curative purposes, has been associated with reduced rates of substance use disorders. However, the brain mechanisms underlying the therapeutic effects of ayahuasca have not yet been fully elucidated. OBJECTIVES: The aim of the present study was to investigate the effects of treatment with ayahuasca on the rewarding properties of the psychostimulant methylphenidate. METHODS: The rewarding properties of ayahuasca (100 mg/kg, orally) and methylphenidate (10 mg/kg, i.p.) were investigated using the conditioned place preference (CPP) model. Furthermore, we evaluated the effects of repeated treatment with ayahuasca on the reinstatement of methylphenidate-induced CPP. Fos expression was evaluated in different limbic structures (cingulate cortex-area 1, prelimbic cortex, infralimbic cortex, orbitofrontal cortex-lateral orbital area, nucleus accumbens core and shell, ventral tegmental area, dorsal striatum, and basolateral amygdala) upon each experimental phase. RESULTS: Both ayahuasca and methylphenidate induced CPP in mice. However, ayahuasca had limited effects on Fos expression, while methylphenidate altered Fos expression in several brain regions associated with the behavioral effects of drugs of abuse. Treatment with ayahuasca after conditioning with methylphenidate blocked the reinstatement of methylphenidate-induced CPP. Those behavioral effects were accompanied by changes in Fos expression patterns, with ayahuasca generally blocking the changes in Fos expression induced by conditioning with methylphenidate and/or reexposure to methylphenidate. CONCLUSIONS: Our findings suggest that ayahuasca restored normal brain function in areas associated with the long-term expression of drug wanting/seeking in animals conditioned to methylphenidate.

Synthesis of a versatile constrained analogue of dipeptide DG (Asp-Gly).

The synthesis of an orthogonally protected constrained analogue of dipeptide DG (Asp-Gly) is reported exploiting alkylation of a chiral lactam. The versatility of this analogue was proven by removal of t-Boc protecting group, followed by coupling under homogeneous conditions with t-Boc-Arg(Z(2))-Gly, to give a conformationally restricted analogue of RGDG tetrapeptide.

[Investigation of pulmonary hypertension]. / Exploration d'une hypertension pulmonaire.

Pulmonary hypertension (PH) is defined by a mean pulmonary artery pressure (PAPm) superior than 25mmHg at rest or superior than 30mmHg with exercise. The classification of PH differentiates between "secondary" PH which results from a well-known disease, such as PH due to thromboembolic disease (obstructive PH), left cardiac failure (passive PH), or chronic respiratory diseases (hypoxic PH), and pulmonary arterial hypertension (PAH). PAH is a rare disease characterized by a progressive increase of pulmonary vascular resistance leading to right ventricular failure. PAH is classified as idiopathic, familial, or associated with various conditions (connective tissue diseases, congenital heart diseases with systemic-to-pulmonary shunts, portal hypertension, infection with the human immunodeficiency virus, or appetite-suppressant drugs). Transthoracic Doppler echocardiography is the investigation of choice for non invasive detection of PAH but right-heart catheterization is necessary to confirm the diagnosis of PAH and determine its mechanism. Pulmonary function tests and chest CT scan may detect an underlying chronic pulmonary disease (hypoxic PH). Lung perfusion scan and contrast-enhanced chest spiral CT scan can lead to the diagnosis of thromboembolic PH, which is to be confirmed by pulmonary angiography. Assessment of the severity of PH is based on clinical parameters (NYHA, right heart failure), functional tests (six-minute walk test), echocardiography and hemodynamics. Characterization of PH is essential in the management of PH because it determines the appropriate treatment: an etiological treatment in passive, obstructive or hypoxemic PH, or vasodilatator and antiproliferative therapies in PAH.

[Eosinophilic pleural effusion associated to Churg and Strauss syndrome]. / Epanchement pleural éosinophile associé à un syndrome de Churg et Strauss.

Eosinophilic pleural effusion (EPE) is defined as pleural eosinophilia greater than 10%. EPE can be seen in almost all conditions that can cause pleural effusion, but some aetiologies have to be investigated due to their frequency or potential severity. The most common aetiology of EPE is the presence of air or blood in the pleural cavity. Other frequent aetiologies include bacterial pneumonia, tuberculosis, parasitic disease and certain drugs. Although often considered to be a sign of a benign condition, pleural eosinophilia may be associated with malignancies. EPE may also indicate the presence of Churg and Strauss syndrome. We report the case of a 27-year-old man, in whom the exploration of EPE led to the diagnosis of Churg and Strauss syndrome with the association of asthma, blood and alveolar eosinophilia, myopericarditis and positive antineutrophil cytoplasmic antibodies (ANCA). This case report enables us to discuss the different causes of EPE and to illustrate how it may be a manifestation of Churg and Strauss syndrome.

Ayahuasca and Its DMT- and ß-carbolines - Containing Ingredients Block the Expression of Ethanol-Induced Conditioned Place Preference in Mice: Role of the Treatment Environment.

Ayahuasca is a hallucinogenic beverage produced from the decoction of Banisteriopsis caapi (Bc) and Psychotria viridis (Pv), ß-carboline- and N,N-dimethyltryptamine(DMT)-containing plants, respectively. Accumulating evidence suggests that ayahuasca may have therapeutic effects on ethanol abuse. It is not known, however, whether its effects are dependent on the presence of DMT or if non-DMT-containing components would have therapeutic effects. The aim of the present study was to investigate the rewarding properties of ayahuasca (30, 100, and 300 mg/kg, orally), Bc (132, 440, and 1320 mg/kg, orally) and Pv (3.75, 12.5 and 37.5 mg/kg, i.p.) extracts and their effects on ethanol (1.8 g/kg, i.p.) reward using the conditioned place preference (CPP) paradigm in male mice. Animals were conditioned with ayahuasca, Bc or Pv extracts during 8 sessions. An intermediate, but not a high, dose of ayahuasca induced CPP in mice. Bc and Pv did not induce CPP. Subsequently, the effects of those extracts were tested on the development of ethanol-induced CPP. Ayahuasca, Bc or Pv were administered before ethanol injections during conditioning sessions. While Bc and Pv exerted no effects on ethanol-induced CPP, pretreatment with ayahuasca blocked the development of CPP to ethanol. Finally, the effects of a post-ethanol-conditioning treatment with ayahuasca, Bc or Pv on the expression of ethanol-induced CPP were tested. Animals were conditioned with ethanol, and subsequently treated with either ayahuasca, Bc or Pv in the CPP environment previously associated with saline or ethanol for 6 days. Animals were then reexposed to ethanol and ethanol-induced CPP was quantified on the following day. Treatment with all compounds in the ethanol-paired environment blocked the expression of ethanol-induced CPP. Administration of an intermediate, but not a high, dose of ayahuasca and Bc, as well as Pv administration, in the saline-paired compartment blocked the expression of ethanol-induced CPP. The present study sheds light into the components underlying the therapeutic effects of ayahuasca on ethanol abuse, indicating that ayahuasca and its plant components can decrease ethanol reward at doses that do not exert abuse liability. Importantly, the treatment environment seems to influence the therapeutic effects of ayahuasca and Bc, providing important insights into clinical practice.

Resin-based dentin restorative materials under accelerated ageing: bio-functional behavior.

OBJECT: The aim of the present study was the evaluation of the effect of different polishing and finishing procedures on Filtek Z250 FZ ESPE restorative material. Particularly, the consequence of artificial aging (UV-irradiation) on this resin-based dental material was investigated determining also its outcome on cell behavior. METHODS: 96 specimens of restorative material were prepared using a light emitting diode curing unit and randomly divided into four finishing and polishing groups: (I) No treatment (FZ); (II) Identoflex rubbers (ID); (III) Enhance System (EN) and (IV) Sof-Lex Pop-on XT discs (SF). The surface morphology of native and artificially aged materials was assessed with Atomic Force Microscopy (AFM) and Scanning Electron Microscopy (SEM). FTIR and biological (biocompatibility and bacterial adhesion) analyses were also performed. RESULTS: Among all, the ID procedure represented an acceptable compromise for efficiency of polymerization and biocompatibility both before and after artificial ageing. SF and EN techniques showed better interactions with the biological environment. CONCLUSION: UV artificial ageing of the tested specimens has shown an acceleration of the surface degrading processes, favoring a possible decrease in the mechanical properties and the release of toxic free radicals. Finishing and polishing procedure seemed to affect the photodegrading pathways, even though no differences among the techniques were observed. As the cytotoxicity of materials undergoing accelerated aging is relevant, further improvement of dental restorative materials are required to limit the long-term biological damage.

In vitro evaluation of bio-functional performances of Ghimas titanium implants.

Titanium is the most widely used material for dental implants. The natural formation, in presence of oxygen, of different oxide films (passivation films) is correlated to titanium implant biocompatibility, resistance to corrosion and is responsible for implant bacteriostatic action. Surface roughness is another surface property of Ti-implants that, affecting implant-to-bone contact, improves integration. In the present study data concerning composition, surface roughness and biocompatibility of Ghimas implants and mini-implants undergoing sandblasting with Calcium Magnesium Carbonate (CaMg(CO3)2) are reported. AFM, SEM/EDX, XRD analyses and morpho-functional tests (MTT and ALP) were performed. Cell actin cytoskeletal modification (fluorescence phalloidin staining) was also observed with confocal laser microscopy (CLSM). Data related to surface geometry and chemical properties, associated with evidence of high purity of all the tested materials (XRD and EDX), highlighted the elevated biocompatibility of tested implants and mini-implants. CLSM investigation confirmed osteoblast features of an active cell behavior able to fit cell to chemico-mechanical stimuli present at the bone/implant interface and suggests an effective implant/alveolar bone integration in vivo.

Tailoring biomaterial compatibility: in vivo tissue response versus in vitro cell behavior.

Biocompatibility relies essentially on surface phenomena, represented by cell-cell, cell-material and material (polymer)-protein interactions. An in vivo and in vitro experimental investigation was carried out on the biomaterials of two different classes with a good potential for in situ utilisation. Non-resorbable (Polypyrrole, Polyaniline, Polyimide) and resorbable (PLLA-PDXO-PLLA) materials for tissue engineering were studied for their overall tissue tolerance and cellular interactions. These non-resorbable polymers conceived for biosensor applications and implantable drug-delivery systems are intrinsically conductive. The PLLA-PDXO-PLLA triblock copolymer showed interesting tensile properties for bone and cartilage tissue engineering due to the presence of 1,5-dioxepan-2-one. In vitro and in vivo parallel studies showed an interesting correspondence: a) the cells in contact with the resorbable material that appeared to be capable of migratory-regenerative aspects in vitro exhibited good compatibility in vivo; whereas b) the non-resorbable materials, which are designed to remain in situ in vivo, were seen to have the potential to represent an adverse factor (inflammation, fibrotic reactions) that correlated with some aspects of cell behaviour in vitro.

Fully inkjet-printed organic photodetectors with high quantum yield.

Bulk-heterojunction based organic photodetectors are fabricated by means of drop-on-demand inkjet printing with vertical topology, inverted structure, and small footprint (about 100 µm x 100 µm). Due to optimization of the deposition technique, an external quantum efficiency in excess of 80% at 525 nm and a -3dB bandwidth of a few tens of kHz is achieved.

Implementing the ESC/ERS pulmonary hypertension guidelines: real-life cases from a national referral centre.

Pulmonary hypertension (PH) comprises a heterogeneous group of disorders characterised by increased pulmonary vascular resistance that results in progressive right ventricular failure. In order to translate current evidence into routine clinical practice, the European Society of Cardiology (ESC) and the European Respiratory Society (ERS) have recently jointly proposed evidence-based guidelines for the optimal management of different PH patient groups. This article describes a series of clinical cases of PH due to various aetiologies that were referred to a large national PH expert referral centre. In each case, the assessment and therapeutic approach undertaken is described in the context of the new ECS/ERS guidelines. The routine diagnostic work-up of suspected idiopathic pulmonary arterial hypertension (PAH) and recommended treatments for patients with functional class II, III and IV disease is emphasised. Familial screening and management of heritable PAH is discussed. Appropriate investigation and therapeutic strategies for patients with chronic thromboembolic disease and PH that is associated with congenital heart disease, pulmonary veno-occlusive disease and systemic sclerosis are also highlighted.

A modified method of prothrombin time/International Normalised Ratio determination in capillary blood and monitoring oral anticoagulant therapy.

BACKGROUND: Oral anticoagulant therapy is monitored by a prothrombin time (PT) assay. The PT is standardised by the International Normalised Ratio (INR). The purpose of this study was to work out a modified method of PT/INR measurement in capillary blood for monitoring anticoagulation treatment. METHODS: Healthy donors, subjects with high or low haematocrit values, and oral anticoagulant-treated patients were included in the study. Plasma and capillary blood PT/INRs were determined by the standard Quick clotting assay, by the modified approach and with the CoaguChek S analyser. RESULTS: The performance characteristics of the developed method were accuracy, due to taking into account whole capillary blood haematocrit values, and precision, due to a decrease in the viscosity of the analysed samples. Implementation of the modified method showed that it is possible to use PT values of normal plasma for capillary blood INR calculation. The developed method allowed the determination of PT in capillary blood within the haematocrit value range from 0.15 up to 0.7. For capillary blood, the results of the modified method closely correlated with PT/INR values determined by the reference Quick method in venous plasma (r=0.99) and with the CoaguChek S analyser (r=0.97). CONCLUSIONS: The modified method of capillary blood PT/INR determination could be recommended for oral anticoagulant therapy monitoring.

Prognostic role of Fuhrman grade and vascular endothelial growth factor in pT1a clear cell carcinoma in partial nephrectomy specimens.

PURPOSE: Conventional renal cell carcinoma (RCC) is characterized by rich neovascularization and shows a fine vascular network around tumor cells. Nephron sparing surgery has been established as a method of choice or necessity for localized tumors. We investigated the importance of microvessel density (MVD), vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor (Flk-1) immunohistochemical expression in a large series of small conventional clear cell renal carcinomas treated with partial nephrectomy and assessed the prognostic value of their expression in terms of patients survival at long-term followup. MATERIALS AND METHODS: A total of 48 patients with a mean age +/- SD of 58.2 +/- 9.5 years who had conventional single RCC were considered. Median tumor diameter was 2.92 +/- 0.82 cm (range 1.3 to 5). Disease was grades 1 to 4 in 15, 29, 2 and 2 patients, respectively. Median followup was 92.9 months (range 17 to 186). RESULTS: Four patients (3.9%) had died of metastatic renal cancer at a median followup of 23.5 months, of whom 1 had a grade 2, 1 had a grade 3 and 2 had grade 4 RCC. Patients with MVD expression higher than the median (44.4 vessels per mm) did not show a significant difference in survival compared to patients with MVD expression lower than the median. Patients with VEGF expression higher than 25% in the histological specimen showed worse survival than patients with VEGF expression lower than 25%. Different Flk-1 expression did not determine a significant difference in survival. On univariate analysis of patient survival in relation to the different considered factors Fuhrman grading was the most important factor for survival. CONCLUSIONS: Our study shows that recurrence and death are possible even in patients with small renal tumors. MVD, VEGF and Flk-1 expression do not depend on tumor size in pT1a RCC. Therefore, to date Fuhrman grading appears to be the only factor predictive of survival even in small RCC. Thus, Fuhrman grading is predictive of mortality. While VEGF is not predictive of survival as a single parameter, based on its percent of expression (lower or higher than 25%) it can determine 2 groups that are different from the prognostic point of view.