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.

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.

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.

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.

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.

Synthesis and subcellular transport of sulfogalactosyl glycerolipids in the myelinating mouse brain.

In the 17-day-old myelinating mouse brain the site of sulfogalactosyl glycerolipid synthesis and the kinetics of its subcellular distribution were studied by a 2 h pulse-labeling with [35S]sulfate followed by a 4 h chase of [35S]sulfogalactosyl glycerolipid. At several time intervals after the intraperitoneal [35S]sulfate injection, subcellular fractions of brain were obtained by differential and discontinuous sucrose gradient centrifugation. The crude microsomal membrane fraction (17 500 X g supernatant) was further subfractionated into light myelin, plasma membranes, Golgi vesicles, endoplasmic reticulum membranes and heavy vesicles associated with acid hydrolase activities. The results of the [35S]sulfogalactosyl glycerolipid labeling kinetics indicate that these lipids are synthesized in the Golgi-endoplasmic reticulum complex and transferred in vesicles associated with lysosomes to the myelin membranes. During this transfer part of the sulfogalactosyl glycerolipids appears to be degraded, similarly as described for brain sulfatides. This double function of lysosomes may be part of a general regulation mechanism of brain myelin glycolipid content.

Optimal conditions for the assay of fibroblast neuraminidase with different natural substrates.

A method for the assay of neuraminidase in human cultured fibroblasts has been worked out. The substrates, all naturally occurring, were: sialyloligosaccharides (alpha(2 lead to 3)sialyllactose, alpha(2 leads to 6)sialyllactose, disialyllactose), sialylglycoplipids (disialogangliosides GD1a and GD1b), sialylglycoproteins and sialylglycopeptides (ovine submaxillary glycoprotein and its pronase-glycopeptides). The method was based on the determination of the enzymically liberated N-acetylneuraminic acid (NeuAc) by a chromatographic-colorimetric microprocedure. The enzyme acted on sialyloligosaccharides and, in the presence of Triton X-100, on gangliosides, while it did not appreciably affect sialylglycoproteins and sialylglycopeptides. The optimum pH was 4.0 for all tested substrates; the Km values were higher for sialyloligosaccharides (about 10(-3) M), lower for gangliosides (about 10(-4) M); the apparent maximum velocity was higher with alpha(2 leads to 3)sialyllactose (400 mU/mg protein); the reaction rate was linear with time for up to 2 h, and with up to 0.6 mg of enzymic protein. The assay method proved to be sufficiently sensitive (3-4 nmol liberated NeuAc), simple, and reproducible (mean activity on pooled fibroblasts with alpha(2 leads to 3)sialyllactose: 400 mU +/- 6 S.E.).

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.

Interactions of pig brain cytosolic sialidase with gangliosides. The formation of catalytically inactive enzyme-ganglioside complexes requires homogeneous ganglioside micelles and is a reversible phenomenon.

Cytosolic sialidase A, obtained from pig brain and purified, interacts with ganglioside GT1b giving two catalytically inactive enzyme-ganglioside complexes. Treatment of these complexes with Triton X-100 under given conditions (1% detergent; 1 h at 37 degrees C; 0.1 M acetic acid-sodium acetate buffer, pH 4.8) leads to the liberation of part of the enzyme (about 47%) in a free and fully active form. Reversible inactivation of cytosolic sialidase requires the presence of homogeneous micelles of GT1b or of mixed micelles (for instance Triton X-100 and GT1b) with a high GT1b content. Triton X-100/ganglioside mixed micelles with a molar ratio above 50, as well as small unilamellar vesicles of egg yolk lecithin and GT1b (7-15 mol%), did not inactivate the enzyme at all; on the contrary these forms of ganglioside dispersion behaved as excellent substrates for the enzyme. It is to be concluded that under in vitro conditions the ability of ganglioside to interact with cytosolic sialidase, giving rise to catalytically inactive complexes or to Michaelis-Menten enzyme-substrate complexes, depends on the supramolecular organization of the ganglioside molecules. Arrangements of tightly packed molecules with strong side-side interactions facilitate the formation of complexes with the enzyme; arrangement with separated and loosely interacting molecules facilitates binding at the catalytically active site of the enzyme.

A double-blind comparison of clebopride and placebo in dyspepsia secondary to delayed gastric emptying.

Seventy-six patients suffering from dyspeptic symptoms secondary to roentgenologically demonstrated delayed gastric emptying were treated with clebopride (0.5 mg TID) or with placebo during a three-month double-blind trial. Clebopride was more effective (P less than or equal to 0.001) than placebo in reducing or relieving symptoms and roentgenological findings associated with delayed gastric emptying. No interactions of clebopride with concomitant drugs or coexisting disorders were observed, and the incidence of side effects was low. We conclude that clebopride will be beneficial in the management of patients with delayed gastric emptying.

Angiokeratoma corporis diffusum and arteriovenous fistulas with dominant transmission in the absence of metabolic disorders.

BACKGROUND: A three-generation family with members affected by angiokeratoma corporis diffusum (ACD) and arteriovenous fistulas of the legs is described. Our purpose was to investigate possible lysosomal storage defects previously described in association with ACD. OBJECTIVE: Results of physical examination of both affected and unaffected family members were otherwise normal as was the life span. The inheritance pattern of both ACD and arteriovenous fistula traits was autosomal dominant, with variable expressivity and incomplete penetrance. Microscopic examination of ACD lesions showed dilated capillaries without vacuolation of cells. Ultrastructural studies failed to reveal lysosomal abnormalities. Normal levels of alpha-galactosidase, beta-galactosidase, alpha-fucosidase, and alpha-sialidase were detected in peripheral blood leukocytes and skin fibroblasts. CONCLUSIONS: The association of autosomal dominant ACD and arteriovenous fistulas might represent a novel syndrome. However, pathogenesis of these lesions remains unknown.

Enzymes of lysosomal origin in human plasma and serum: assay conditions and parameters influencing the assay.

The condition for maximal activity (pH, buffer, saturating substrate concentration, range of linear relationships between enzyme activity versus incubation time, and versus enzyme concentration) in the fluorimetric assay of several glycohydrolases of lysosomal origin in human plasma and serum have been established. The following enzymes were studied: alpha-galactosidase, beta-galactosidase, beta-N-acetylglucosaminidase, beta-glucosidase, beta-glucuronidase, alpha-mannosidase, alpha-fucosidase. All examined enzymes turned out to be more or less unstable upon storage at 37 degrees C, 4 degrees c, and -20 degrees C in both serum and plasma. The only exceptions were beta-glucuronidase, which was stable in plasma and serum, and alpha-fucosidase which was stable only in plasma. Generally the degree of instability was greater in serum than in plasma. The levels of some enzymes (alpha-galactosidase, beta-galactosidase, beta-N-acetyl glucosaminidase, beta=glucuronidase) were markedly higher in serum than in plasma; conversely the levels of the same enzymes in "platelet free" serum equalled those in plasma. This stresses the necessity to use freshly prepared plasma for lysosomal glycohydrolase assay. Under the procedural conditions recommended for the assay the methods for the determination of lysosomal glycohydrolases in plasma appeared to be simple, sensitive and reproducible.

Influence of age and sex on five human plasma lysosomal enzymes assayed by automated procedures.

Automated fluorimetric procedures for the assay of five lysosomal glycohydrolases-beta-N-acetylglucosaminidase; beta-galactosidase; beta-glucuronidase; alpha-mannosidase; alpha-fucosidase-in human plasma were set up. A Carlo Erba autoanalyser CLA 1500, provided with a sampler refrigerating unit and connected with a recording Turner Mod 111 fluorimeter was employed. The automated procedures, under the established optimal conditions, proved to be highly accurate and reproducible. Using the automated assay procedures the effect of sex and age on the plasma levels of the same enzymes was studied. 1273 randomly selected health subjects were studied. No sex differences were observed for all the enzymes studied with the exception of beta-glucuronidase which displayed higher values (about 30%) in males from 25 to 60 years. The developmental profiles of all enzymes in females and males were similar and characterised by: (a) absolute maximum level in the umbilical cord blood; (b) absolute minimum level at 10-14 years; (c) decrease to a second minimum occurring around 35 years (not displayed by beta-galactosidase and by beta-glucuronidase in males); (e) slow further increase up to the elderly level which was then maintained till the oldest age examined, 74 years.

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.

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.

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.