Lymphatic endothelial cells derived from metastatic and non-metastatic lymph nodes of human colorectal cancer reveal phenotypic differences in culture.

Colorectal cancer is one of the most frequent causes of death in Western countries. Most patients develop metastasis traveling through the lymphatic system, and regional lymph node metastasis is considered a marker for dissemination, increased stage, and worse prognosis. Despite rapid advances in tumor biology, the processes that underpin lymphatic invasion and lymph node metastasis remain poorly understood. The aim of this study was to establish an easy protocol for isolation of pure tumor lymphatic endothelial cells derived from lymph nodes to study differences compared with normal endothelial cells of uninvolved tissue from the same patients. Cells were isolated with very high purity via magnetic cell sorting and express the specific lymphatic markers Prox-1 and Lyve-1. They show differences in expression of adhesion molecules, chemokines, and growth factor secretion, and capability to form capillaries when seeded on basal membrane, thereby, revealing important differences between the two cell type. These cultures may provide a promising platform for the comparative analysis of both cell types at the molecular and biological level and to optimize treatment strategies.

Newly produced T and B lymphocytes and T-cell receptor repertoire diversity are reduced in peripheral blood of fingolimod-treated multiple sclerosis patients.

BACKGROUND: Fingolimod inhibits lymphocyte egress from lymphoid tissues, thus altering the composition of the peripheral lymphocyte pool of multiple sclerosis patients. OBJECTIVE: The objective of this paper is to evaluate whether fingolimod determines a decrease of newly produced T- and B-lymphocytes in the blood and a reduction in the T-cell receptor repertoire diversity that may affect immune surveillance. METHODS: Blood samples were obtained from multiple sclerosis patients before fingolimod therapy initiation and then after six and 12 months. Newly produced T and B lymphocytes were measured by quantifying T-cell receptor excision circles and K-deleting recombination excision circles by real-time PCR, while recent thymic emigrants, naive CD8(+) lymphocytes, immature and naive B cells were determined by immune phenotyping. T-cell receptor repertoire was analyzed by complementarity determining region 3 spectratyping. RESULTS: Newly produced T and B lymphocytes were significantly reduced in peripheral blood of fingolimod-treated patients. The decrease was particularly evident in the T-cell compartment. T-cell repertoire restrictions, already present before therapy, significantly increased after 12 months of treatment. CONCLUSIONS: These results do not have direct clinical implications but they may be useful for further understanding the mode of action of this immunotherapy for multiple sclerosis patients.

Modulation of the central memory and Tr1-like regulatory T cells in multiple sclerosis patients responsive to interferon-beta therapy.

BACKGROUND: Interferon-beta is used to reduce disease activity in multiple sclerosis, but its action is incompletely understood, individual treatment response varies among patients, and biological markers predicting clinical benefits have yet to be identified. Since it is known that multiple sclerosis patients have a deficit of the regulatory T-cell subsets, we investigated whether interferon-beta therapy induced modifications of the two main categories of regulatory T cells (Tregs), natural and IL-10-secreting inducible Tr1 subset, in patients who are biologically responsive to the therapy. METHODS: T-cell phenotype was determined by flow cytometry, while real-time PCR was used to evaluate interferon-beta bioactivity through MxA determination, and to measure the RNA for IL-10 and CD46 molecule in peripheral blood mononuclear cells stimulated with anti-CD46 and anti-CD3 monoclonal antibodies, which are known to expand a Tr1-like population. RESULTS: Interferon-beta induced a redistribution of natural Treg subsets with a shift of naive Tregs towards the 'central memory-like' Treg population that expresses the CCR7 molecule required for the in vivo suppressive activity. Furthermore, in a subgroup of treated patients, the CD46/CD3 co-stimulation, probably through the Tr1-like subset modulation, increased the production of RNA for IL-10 and CD46. The same group showed a lower median EDSS score after two years of therapy. CONCLUSIONS: The selective increase of 'central memory-like' subset and the involvement of the Tr1-like population may be two of the mechanisms by which interferon-beta achieves its beneficial effects. The quantification of RNA for IL-10 and CD46 could be used to identify patients with a different response to interferon-beta therapy.

Adult-type hypolactasia genotyping in Northern Italy: prevalence of C/T-13910 polymorphism and questions after comparison with existing data.

A genotyping assay was setup to assess the prevalence, in the population of a Northern Italian city, of the C/T-13910 single nucleotide polymorphism, closely associated to lactose malabsorption in many world areas including Sardinia. The results were compared to published Italian data, in order to evaluate the worth of a future validation of the assay for use in routine practice. DNA was extracted from blood samples of 123 randomly chosen healthy blood donors coming from the same city area, and was analyzed by a real-time polymerase chain reaction (PCR) genotyping assay; the frequency of the hypolactasia-associated CC-genotype was compared to the weighted average of results extracted from studies reporting the frequency of hypolactasic phenotype or genotype in nearby or distant Italian regions. Sixty-five percent of donors carried the CC-genotype, a percentage similar to other northern Italian cities, but significantly higher than what previously determined in surrounding Italian regions at the phenotype level, i.e. by breath test. This discrepancy parallels recent reports of non concordance between results of genotyping and hypolactasic phenotype in some world areas, including a neighbouring Northern Italian city. A north-south gradient of CC-prevalence was also observed. These results reinforce the notion of wide inter-regional variations in the frequency of C/T-13910 polymorphism and of incostant concordance with hypolactasic phenotype, even in subjects from the same country. Given the unsatisfactory results recently obtained from validation of a related assay in a neighbouring city, the authors decided not to proceed further and keep the assay only as a diagnostic aid in special situations.

Alternatives to animal experimentation for hormonal compounds research.

Alternatives to animal testing and the identification of reliable methods that may decrease the need for animals are currently the subject of intense investigation worldwide. Alternative testing procedures are particularly important for synthetic and natural chemicals that exert their biological actions through binding nuclear receptors, called nuclear receptors-interacting compounds (NR-ICs), for which research is increasingly emphasizing the limits of several models in the accurate estimation of the physiological consequences of exposure to these compounds. In particular, estrogen receptor interacting compounds (ER-ICs) have a great impact on human health from the therapeutic, nutritional, and toxicological point of view due to the highly permissive nature of the estrogen receptors towards a large number of natural and synthetic compounds. Similar to in vitro systems, recently generated animal models (e.g., animal models generated for the study of estrogen receptor ligands) may fulfill the 3R principles: refine, reduce, and replace. If used correctly, NR-regulated models, such as reporter mice, xenopus, or zebrafish, and models obtained by somatic gene transfer in reporter systems, combined with imaging technologies, may contribute to strongly decreasing the overall number of animals required for NR-IC testing and research. With these models, flexible and highly standardized parameters and reporter marker quantification can be obtained. Here, we highlight the need for the substitution of currently used testing models with more appropriate ones that can reproduce the features and reactivity of specific mammalian target tissue/organs. We consider the promotion of this advancement a research priority bearing scientific, economic, social, and ethical relevance.

Kinetics of Th1/Th2 cytokines and lymphocyte subsets to predict chronic GVHD after allo-SCT: results of a prospective study.

The role of different cytokines and cells of immune system in the pathogenesis of chronic GVHD (cGVHD) is still controversial. Earlier studies, which were either retrospective or analysed one or a few factors, did not show unequivocal results. We prospectively evaluated cytokine levels and lymphocyte subsets in 30 patients who underwent Allo-SCT to investigate their possible correlation with cGVHD. Levels of IL-4, IL-6, IL-10, IFN-gamma, tumour necrosis factor-alpha (TNF-alpha) and its soluble receptors were assessed by ELISA in 30 patients at different times after SCT. Lymphocyte subsets were evaluated by flow cytometry in peripheral blood at the same times as cytokines. A multivariate analysis was performed using principal component analysis and multi-factor ANOVA (analysis of variance). Eighteen patients developed cGVHD at a median time of 6 months (range, 5-9) after SCT. In multivariate analysis, we observed a correlation between cGVHD and clusters of cytokines and lymphocyte subsets from the third to the sixth month after SCT. These clusters changed their composition over time, but they constantly included natural killer (NK) and CD152+ T cells as negative predictors of cGVHD. TNF-alpha prevailed among other cytokines before the onset of cGVHD. This prevalence could be related partly to the defect of immunoregulatory cells.

Interferon-beta therapy monitoring in multiple sclerosis patients.

Interferon-beta (IFN-beta) therapy has a central place in the management of multiple sclerosis (MS). The three recombinant IFN-beta preparations currently available have shown benefit on activity measures (relapses and active lesions apparent on magnetic resonance imaging), while therapy advantages on progression measures (disability and total lesion burden) are less consistent. Moreover, IFN-beta is effective only in a percentage of patients, since in many of them neutralizing anti-IFN-beta antibodies develop after 6-18 months of treatment, leading to loss of drug bioactivity. Comparative data across studies made with different IFN-beta preparations suggest that the optimal choice of IFN-beta subtype, preparation and dose regimen are important determinants of efficacy. Because IFN-beta actions depend on the activation of IFN-inducible genes, in addition to the direct quantification of anti-IFN-beta antibodies, several other methods for the measure of IFN-beta biologic activity have been recently developed. Among these, the determination of the IFN-beta-inducible gene product Myxovirus protein A (MxA) has proven to be the most reliable one. Another still open point is the role of the differential expression of IFN-beta receptor (IFNAR) components, since IFNAR2 subunit can be synthesized in three isoforms: functional, truncated non-functional and soluble. While this and other important issues require further studies, this article reviews and discusses the importance, potential and limits of the methods currently available to monitor IFN-beta therapy in MS patients.

Role of BDNF Val66Met functional polymorphism in Alzheimer's disease-related depression.

BACKGROUND: The gene encoding brain-derived neurotrophic factor (BDNF) has been suggested as a candidate for major depression, and for depression susceptibility in different neurological and psychiatric diseases. No study has investigated the role of BDNF genetic variation and depressive symptoms in Alzheimer's disease (AD). OBJECTIVE: The aim of this study was to assess the genetic contribution of BDNF Val66Met functional polymorphism to AD-related depression. METHODS: Two-hundred and sixty-four AD patients underwent clinical and neuropsychological examination as well as an evaluation of behavioral and psychiatric disturbances. They were subsequently divided into two subgroups according to the presence (AD-D) or the absence (AD-nD), based on DSM-IV criteria for depression in AD. In each subject, BDNF Val66Met functional polymorphism and apolipoprotein E (APOE) genotype were evaluated. RESULTS: In our sample, 35.2% of patients (n=93) reported AD-related depressive symptoms. Compared to patients bearing no polymorphisms (BDNF G/G), BDNF G/A carriers showed more than twofold-time risk (OR=2.38; 95%CI=1.38-4.13), and BDNF A/A carriers had a threefold-time risk (OR=3.04; 95%CI=1.15-8.00) for depression in AD. Accordingly, considering the allele frequencies, BDNF A allele was significantly over-represented in AD-D (32.8%) compared to AD-nD (19.0%) (OR=2.08; 95%CI=1.38-3.13). An association between the number of carried A allele and the severity of depressive symptoms was observed (P<0.002). No effect of APOE genotype on risk for depression was found. CONCLUSIONS: The present findings provide evidence of BDNF genetic variation role in the susceptibility to AD-related depression. This study puts emphasis on the usefulness of considering genetic background for better defining individualized risk profiles in AD.

Serum leptin levels are higher in females affected by frontotemporal lobar degeneration than Alzheimer's disease.

Frontotemporal lobar degeneration (FTLD) includes different heterogeneous conditions, mainly characterised by personality changes, along with cognitive deficits in language and executive functions. Movement disorders are variably represented. Behavioural disturbances constitute the core feature of FTLD, and eating disorders represent one of the most distinguishing symptoms between FTLD and Alzheimer's disease (AD). The biochemical correlates of such dysfunctions remain to be defined. The adipocyte derived hormone leptin is known to play a foundamental role in food intake and energy balance. To understand whether leptin could be involved in FTLD eating abnormalities, we measured serum leptin levels in 59 patients with FTLD compared with 25 with AD. Serum leptin levels in patients with FTLD were comparable with those in patients with AD. Nevertheless, females with FTLD showed significantly higher leptin levels compared with females with AD. No difference was found between FTDL and AD males or within the spectrum of patients with FTLD. Hyperphagic FTLD females showed higher circulating leptin levels in comparison with those without eating abnormalities; no differences were found between males with FTLD with respect to serum leptin and food intake disturbances. The present study showed a selective gender difference in leptin levels between females with FTLD and AD, which may suggest specific cognitive and behavioural networks need to be investigated.

Haplotypes in cathechol-O-methyltransferase gene confer increased risk for psychosis in Alzheimer disease.

BACKGROUND: The gene encoding catechol-O-methyltransferase (COMT) has been suggested as a candidate for Alzheimer-related psychosis (AD-P) susceptibility, and an association between AD-P and a functional valine to methionine polymorphism has been reported. OBJECTIVE: The aim of this study was to assess the genetic contribution of other COMT variants to the risk of AD-P. METHODS: Two hundred and forty-six AD patients underwent clinical and neuropsychological examination as well as an evaluation of behavioural and psychiatric disturbances. They were subsequently divided into two subgroups according to the presence (AD-P) or the absence (AD-nP) of psychotic symptoms. Four single-nucleotide polymorphisms (SNPs) within COMT gene were evaluated, i.e. rs737865, rs737864, intron 1 C2754delC, and the well-known valine/methionine variant (rs4680). Analyses were performed on the single locus and pairwise disequilibrium of loci, and multi-locus haplotype. RESULTS: The individual SNP analysis confirmed an association for the valine/methionine variant with AD-P. Haplotype analyses revealed that the alleles at four loci (rs737865, rs737864, intron 1 C2754delC, rs4680) interacted to create the risk of psychosis in AD, as A-C-C-G haplotype (OR=2.08, 95% CI=1.02-4.27, P=0.044) and G-C-delC-G haplotype (OR=2.54, 95% CI=1.32-4.90, P=0.006) in respect to the most common and not-at-risk A-C-C-A haplotype which was significantly overrepresented in AD-P. CONCLUSIONS: The present findings provide evidence of COMT genetic variations' role in the susceptibility to AD-related psychosis. The observation of a haplotype effect of different polymorphisms within the COMT gene puts emphasis on the usefulness of haplotype analysis in better defining individualized genetic risk profiles in AD.

Characterization of T-cell population in children with prolonged fetal exposure to dexamethasone for anti-Ro/SS-A antibodies associated congenital heart block.

The objectives of the study were to characterize the production, function and survival of T lymphocytes of children with prolonged fetal exposure to dexamethasone for anti-Ro/SS-A antibodies associated congenital complete heart block. The analysis of thymic function, studied by measuring the level of T-cell receptor excision circles, was performed by real time PCR, the composition of T-cell subpopulation was evaluated by flow cytometry and the T-cell diversity was assayed by heteroduplex analysis. T-cell competence was gauged at two functional levels by determining the proliferation and the number of T-cell divisions and by measuring gamma-interferon production after mitogenic stimulation. We observed that the thymic output, distribution of T-cell subsets, thymidine incorporation, number of T-cell divisions, and y-interferon production were comparable to those of age-matched control. On the contrary, heteroduplex analysis demonstrated the presence of both polyclonal and oligoclonal peripheral T-cell repertoires. In conclusion, the analysis of the T-cell compartment in children with prolonged intrauterine exposure to high dose dexamethasone did not disclose any relevant abnormality, except a restriction of T-cell receptor diversity in some patients.

Interaction of homocysteine and conventional predisposing factors on risk of ischaemic stroke in young people: consistency in phenotype-disease analysis and genotype-disease analysis.

BACKGROUND AND OBJECTIVES: Whether the association between mild hyperhomocysteinaemia and ischaemic stroke is the consequence of a predisposing genetic background or is due to the confounding influence of established predisposing factors remains to be determined. METHODS: Plasma total homocysteine (tHcy) concentration and the distribution of the C677T genotypes of the methylenetetrahydrofolate reductase gene (MTHFR) were compared in 174 consecutive patients with stroke aged <45 years and 155 age and sex-matched controls. The effect of conventional risk factors on the relationship between phenotype-disease and genotype-disease was analysed by two-way and three-way interaction analysis and by the classification and regression trees (CART) model. RESULTS: tHcy concentrations were markedly higher in patients with ischaemic stroke (median 11.9 micromol/l, range 2.0-94.0) than in controls (median 9.8 micromol/l, range 4.7-49.6). An increased risk was also associated with the TT677 genotype (odds ratio (OR) 1.98; 95% confidence interval (CI) 1.04 to 3.78) and with the T allele (1.40; 95% 1.03 to 1.92) of the MTHFR gene. A differential effect of Hcy levels on risk of stroke was observed according to the distribution of environmental-behavioural risk factors, with a stronger influence in the subcategory of people with hypertension and smokers (OR 24.8; 95% CI 3.15 to 196). A comparable environmental-dependent TT677 MTHFR genotype-stroke association was observed in the genotype-disease analysis. CONCLUSIONS: A consistency of phenotype-disease analysis and genotype-disease analysis is indicated by analysing specific subcategories of patients, defined by the distribution of established risk factors. The assumption that the Hcy-stroke relationship is unlikely due to a reverse-causality bias is indirectly supported by our data.

Genetic correlates of behavioral endophenotypes in Alzheimer disease: role of COMT, 5-HTTLPR and APOE polymorphisms.

Several studies have been conducted to understand the genetic correlates of Alzheimer disease (AD)-related behavioral and psychological symptoms in dementia (BPSD). However, given that BPSD rarely occur in isolation, it has been suggested that targeting BPSD individually is too narrow of an approach if one wants to accurately define all the associated risk factors. To date, we know of no work on genetic polymorphisms related to behavioral endophenotypes in AD. The present study sought to evaluate the relationship between such behavioral endophenotypes in AD and genetic variations in dopamine- or serotonin-related genes, such as catechol-O-methyltransferase (COMT) or 5-HTT gene-linked promoter region (5-HTTLPR), and apolipoprotein E (APOE). Among 232 AD patients who underwent clinical and neuropsychological examination, a behavioral and psychiatric evaluation, and genotyping at COMT, 5-HTTPLR, and APOE; 66.4% showed more than one behavioral symptom. By Principal Component Analysis of Neuropsychiatric Inventory (NPI) symptoms four endophenotypes were identified, these were termed "psychosis", "moods", "apathy", and "frontal". Modeling NPI symptom-endophenotype-genotype relationships, and taking into account possible confounds (i.e. demographic characteristics, comorbidities, concomitant pharmacological treatments, and disease severity) by latent variable models, COMT and 5-HTTLPR genetic variations correlated with "frontal" and "psychosis" endophenotypes. APOE genotype did not correlate with any endophenotype. These findings suggest that the possibility of identifying distinct phenotypes on a genetic basis among AD patients exists, and suggest that clustering of BPSD into endophenotypes might provide a new strategy for guiding future research on this issue.

Target-specific action of organochlorine compounds in reproductive and nonreproductive tissues of estrogen-reporter male mice.

Organochlorines are lipophylic molecules that accumulate in the fat where they remain for years. During weight loss, they are mobilized and their concentration increases in blood. The present work tests, in transgenic estrogen-reporter mice (ERE-tK-LUC), whether this increase is sufficient to modulate the estrogen receptors (ERs) in the whole body. Three weak estrogens were studied: p,p'DDT [1,1,1-trichloro2,2-bis(p-chlorophenyl) ethane], p,p'DDE [1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene], and betaBHC [beta-benzene-hexachloride]. Dose-dependent analysis of reporter expression (luciferase) were performed in tissues of acutely treated mice. A body map of ER activation was obtained. All these chemicals modulated the reporter, although with a different efficiency and depending upon the tissue analyzed. Induction was confirmed in the liver by determining the expression of the endogenous progesterone receptor (PR) gene, at the dose and time point at which the luciferase gene was maximally induced. After experimental accumulation in the fat tissue, followed by a 48-h period of fasting, we tested whether these compounds could be mobilized to reach sufficient levels to activate the ERs in selected reproductive and nonreproductive tissues (testicle, prostate, liver, and lung). This experimental setting produced results that were different than those obtained following acute treatments. In loaded mice, fasting induced betaBHC mobilization resulted in strong ER activation in the liver and the lung, which was blocked by ICI-182780. p,p'DDT mobilization had no effect in these tissues, but it acted efficiently in the prostate and testis. betaBHC inhibited the ERE-mediated reporter in the testicle and induced the reporter in the prostate. In this tissue, betaBHC action was not inhibited by the anti-estrogen ICI-182780. During fasting, betaBHC, p,p'DDT, and metabolite p,p'DDE increased in blood concentration, from 2.25 +/- 0.25, 0.51 +/- 0.09, and 0.38 +/- 0.06 microg/ml to 8.24 +/- 0.95, 4.52 +/- 0.68, and 5.06 +/- 0.57 microg/ml, respectively. The effect produced by these organochlorines in the liver correlates with the modulation of the ERalpha protein. We conclude that these organochlorines modulate differently the expression of estrogen-regulated genes in male mice. Their effect is tissue- and compound-specific and is dependent on the energetic balance.

Whole body action of xenoestrogens with different chemical structures in estrogen reporter male mice.

The present work tested the estrogenic activity of three weak environmental estrogens p,p'DDT [1,1,1-trichloro-2,2-bis(p-chlorophenyl) ethane], p,p'DDE [1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene] and betaBHC [beta-benzene-hexachloride] in the transgenic estrogen-reporter mouse model (ERE-tK-LUC). By a time dependent analysis of the transgenic reporter expression (luciferase), we showed that all these chemicals modulated the estrogen receptors (ERs) in the whole body, although with a different efficacy and depending upon the tissue analyzed. Peak activity was registered at 16 h of treatment with 5000 microg/kg of each compound. Organochlorines are lipophylic molecules that accumulate in fat. During weight loss they are mobilized and their concentration increases in blood. We tested whether after experimental accumulation in fat tissue, followed by a 48 h period of fasting, these compounds could be modulated to reach sufficient levels to activate the ERs in target tissues. This experimental setting produced results that were different from those obtained following acute treatments. In loaded mice, fasting induced betaBHC mobilization resulted in strong ER activation in the liver, lung, eye, cerebellum, hypothalamus and cortex. p,p'DDT mobilization had no effect in these tissues, but efficiently acted in the testis, where, on the contrary, betaBHC inhibited reporter expression. During fasting, betaBHC, p,p'DDT and the metabolite p,p'DDE increased in blood concentration, from 2.7 +/- 0.36, 0.65 +/- 0.01 and 0.48 +/- 0.06 microg/ml to 9.51 +/- 1.1, 4.98 +/- 0.77 and 6.0 +/- 0.71 microg/ml, respectively. We conclude that these organochlorines modulate differently the expression of estrogen regulated genes in a tissue- and compound-specific manner and that their action is dependent on the energy balance. Moreover, we show that this mouse model is suitable to detect the estrogenic activity of chemicals with variable structures such as alkyl phenols and polychlorobiphenyls.

Seroprevalence of HTLV-I and HTLV-II infection among immigrants in northern Italy.

To assess the prevalence of human T cell leukemia virus type I (HTLV-I) and 2 (HTLV-II) infection and the associated risk factors among immigrants living in Northern Italy, we surveyed 3017 open-population subjects from three geographical areas and 371 prisoners. In the open population, the overall prevalence was 0.3% for HTLV-I and 0.1% for HTLV-II, while among prisoners, HTLV-I and HTLV-II infection were detected in 1.4 and 0.8% of subjects, respectively. HTLV-I prevalence was higher in subjects with multiple sexual partners or sexually transmitted diseases. This association was significant in the open-population group and close to significance in prisoners. Multivariate analysis showed that human immunodeficiency virus (HIV) seropositivity remained significantly associated with HTLV-I infection in both targeted populations (OR: 11.2 in the open population; OR: 9.9 among prisoners), whereas sexual exposure was associated with HTLV-I seropositivity only for prisoners (OR: 14.3). No independent variable was related to HTLV-II infection.

Peripheral blood abnormalities in Alzheimer disease: evidence for early endothelial dysfunction.

Clinical and epidemiologic studies demonstrate that vascular risk factors may be involved in Alzheimer disease (AD). To evaluate whether vascular abnormalities are an early feature of AD, several parameters of coagulation and fibrinolysis were assessed. Thirty patients with mild AD and 30 age-matched control subjects entered the study. All subjects performed a standardized clinical and laboratory protocol. Persons with vascular risk factors and systemic diseases were excluded. AD patients present significant increased levels of thrombomodulin (p < 0.0001) and sE-selectin (p < 0.03). In contrast, no difference was found between the two diagnostic groups in the levels of beta-thromboglobulin, prothrombin fragment 1+2, fibrinogen, and von Willebrand factor. No other association but diagnosis was found with thrombomodulin and sE-selectin. These findings suggest that endothelial dysfunction is an early event in AD patients.

Comparison between different laboratory tests for the detection and prevention of heparin-induced thrombocytopenia.

BACKGROUND: Heparin-induced thrombocytopenia (HIT) is a possible complication of heparin therapy that can evolve with life-threatening thromboembolism, for which early diagnosis is essential. However, the specific laboratory approach to the diagnosis of HIT is still controversial. METHODS: Sera from 13 patients with HIT, from 15 patients with non-HIT thrombocytopenia, and from 10 normal subjects were used to compare nonfunctional and functional methods to detect anti-heparin:PF-4 antibodies and platelet activation. We used three enzyme-linked immunosorbent assays (ELISAs) and the particle gel immunoassay as nonfunctional tests, and platelet aggregometry, CD62p (p-selectin) phenotypical expression, and Annexin V binding as functional assays. RESULTS: CD62p expression was positive in 85% of the cases and Annexin V was positive in 40% of the HIT cases examined. Aggregometry gave variable results that depend strongly on the donor. CONCLUSION: Functional tests for platelet activation are more reliable for HIT diagnosis than the nonfunctional tests. We conclude that the phenotypical expression of p-selectin detected by flow cytometry on activated platelets appears to be a good functional marker for the diagnosis of HIT and its possible thromboembolic complications.

Platelet amyloid precursor protein forms in AD: a peripheral diagnostic tool and a pharmacological target.

Alzheimer Disease (AD) is characterized by the progressive deposition of beta-amyloid in the parenchyma and cerebral microvasculature. The beta-amyloid peptide derives from the metabolism of a larger precursor, Amyloid Precursor Protein (APP). This protein is present in central nervous system, but it is also expressed in peripheral tissues such as circulating cells. An alteration of the APP forms pattern in platelets has been recently reported in AD patients when compared to platelets both of control subjects or non AD patients (NADD). The accuracy of the assay to identify AD is high and decreased levels are found throughout the course of AD with a significant association with severity of symptoms. Moreover, a recent study has demonstrated that AD patients on donepezil (5 mg daily) for 4 weeks displayed two-fold increase in their APPr baseline levels up to normal range. Thus, platelet APP ratio (APPr) holds the potential to be a clinical marker, which might be of helpful and adjunctive value in the diagnosis of AD and in tracking the course of illness, also in the early stages when pharmacological treatment has the greatest potential of being effective.