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Hepatitis C virus (HCV) is still one of the major causes of chronic viral infection worldwide, and hepatic steatosis is a frequent pathological finding in patients with chronic HCV-related diseases. It is unclear whether the steatosis is associated with host factors or the virus itself, although a consistent relationship has been found between steatosis and a necro-inflammatory reaction with the increased secretion of immuno-regulators. A primary sources of inflammatory mediators are mast cells (MCs) bone marrow-derived cells that are detected in both normal and diseased livers. We determined MC density and correlated it with the fibrosis, inflammatory reaction and steatosis observed in the liver biopsies of patients affected by HCV with or without steatosis. All the histological features were assessed using a computer-aided image analysis system. There was a statistically significant difference in MC density between the HCV-infected patients with and without steatosis, with the lower mean value being detected in those without (P < 0.02). Furthermore, a nonstatistically significant difference in fibrosis and inflammation between the two patient groups was found. In conclusion, this is the first study showing a significant increase in MC density in the tissues of patients with chronic HCV infection and histologically documented steatosis.

Hepatocellular carcinoma (HCC) remains one of the major public health problems throughout the world. Although originally associated with tumorigenic processes, liver angiogenesis has also been observed in the context of different liver inflammatory, fibrotic, and ischemic conditions. Here we investigate the fractal dimension as a quantitator of non-Euclidean two-dimensional vascular geometry in a series of paired specimens of primary HCC and surrounding non-tumoral tissue, and discuss why this parameter might provide additional information regarding cancer behavior. The application of fractal geometry to the measurement of liver vascularity and the availability of a computer-aided quantitative method can eliminate errors in visual interpretation, and make it possible to obtain closer-to-reality numerals that are compulsory for any measurement process.

BACKGROUND Incomplete information in food consumption is a relevant topic in agricultural economics. This paper proposes a theoretical model describing consumer behavior, market equilibrium and public intervention in an industry where consumers must rely on the information of interested parties such as producers or associations. METHODS We provide simple game theory model showing the link between price competition and the strategic use of information. If information are unverifiable (as in the case of credence attributes) firms may have no incentive to advertise true claims and consumer decisions may be biased. Our model incorporates the opportunistic behavior of self-interested information providers. RESULTS The result is a model of competition in prices and information finding a potential for market failure and public intervention. In the paper we discuss the efficiency of three possible regulations: banning false claims, subsidizing advertising campaigns, and public statement if favor of true claims. CONCLUSION In that context, some recent patents related to both the regulatory compliance in communication and to the reduction of asymmetric information between producers and consumers have been considered. Finally, we found that the efficiency of these policy tools is affected by the reputation of trustworthiness of the firms.

The olive oil industry is a significant productive sector in the European Union and the related production process is characterised by a variety of different practices and techniques for the agricultural production of olives and for their processing into olive oil. Depending on these different procedures, olive oil production is associated with several adverse effects on the environment, both in the agricultural and in the olive oil production phase. As a consequence, tools such as LCA are becoming increasingly important for this type of industry. Following an overview of the characteristics of the olive oil supply chain and its main environmental problems, the authors of this chapter provide a description of the international state of the art of LCA implementation in this specific sector, as well as briefly describing other life cycle thinking methodologies and tools (such as simplified LCA, footprint labels and Environmental Product Declarations). Then, the methodological problems connected with the application of LCA in the olive oil production sector are analysed in depth, starting from a critical comparative analysis of the applicative LCA case studies in the olive oil production supply chain. Finally, guidelines for the application of LCA in the olive oil production sector are proposed.

Mast cells (MCs) are multifunctional effector cells of the immune system. MCs were originally thought to be involved in IgE-associated immediate hypersensitivity and allergic disorders, but it is now known that they contain or elaborate an array of mediators with a multitude of effects on many other cells. A number of studies have found that MCs are involved in various liver diseases. Although still controversial, they seem to be involved in the liver's fibrotic response to chronic inflammation and parasitic infection. Hepatic fibrosis is the most frequent liver response to toxic, infectious, or metabolic agents. During the establishment of this pathological condition, there is an increase in the components of the basement membrane that leads to continuous basement membrane-like structures being raised within Disse's space and a decrease in the number of sinusoid endothelial fenestrae. This leads to a complex process called "sinusoidal capillarization." At the cellular level, liver fibrogenesis is initiated by hepatocyte necrosis, which induces the recruitment of a large number of inflammatory cells, including MCs, which can be considered the primary effectors of the process changing sinusoidal endothelial cells into capillary-type endothelial cells. We review the roles played by MCs in hepatic chronic diseases and describe a biopsy section of hepatic tissue taken from a patient with chronic C virus-related hepatitis showing diffuse sinusoidal capillarization and a high density of MCs. This observation has led us to hypothesize a relationship between these highly specialized cells and sinusoidal capillarization.

Hereditary angioedema (HAE) is a potentially life-threatening autosomal dominant deficiency affecting 1 in 50,000 individuals from all ethnic groups worldwide. More than 99% of HAE cases are caused by a deficiency in functional plasma C1 esterase inhibitor (C1E-INH, a serine protease inhibitor) due to mutations in the SERPING1 gene. Low plasma C1E-INH activity dysregulates the contact, complement, and fibrinolytic systems, resulting in unpredictable, recurrent submucosal edema of cutaneous tissues, gastrointestinal and respiratory tracts. If not treated in a timely manner, laryngeal edema can result in death by asphyxiation. Current HAE treatments consist of management of acute attacks, repeated prophylactic therapy with C1E-INH or long term prophylaxis with attenuated androgens, each complicated by a high economic burden, limited compliance, drug side effects and contraindications. To circumvent this challenge, we hypothesized that a one-time administration of an adeno-associated virus (AAV) gene transfer vector expressing the genetic sequence of C1 esterase-inhibitor (serotype 10 expressing the human CIE-INH coding sequence, AAVrh.10hC1EI) would provide sustained C1E-INH activity levels in plasma, sufficient to prevent angioedema episodes. To study the efficacy of AAVrh.10hC1EI, using CRISPR/Cas9 technology we created a novel C1E-INH deficient mouse model analogous to human HAE disease, and characterized the resulting SERPING1 gene mutations by genome sequencing. The heterozygous mouse model shares characteristics associated with HAE in humans including decreased C1E-INH and C4 levels in plasma and increased vascular permeability. Administration of AAVrh.10hC1EI to the heterozygous mice resulted in sustained human C1E levels above the predicted therapeutic levels. In order to demonstrate that the increased vascular permeability observed in heterozygote C1E-INH+/- mice was a direct result of C1E-INH deficiency, Evans blue dye was injected intravenously and extravasation of dye from the vasculature evaluated. Compared to wild type mice under baseline conditions, the C1EINH+/- mice had increased extravasation of the dye into the hind paws quantitated by optical absorbance at 600 nm. Strikingly, AAVrh10.hC1EI-treated (1011 gc) mice displayed a marked decrease in dye extravasation, whereas non-treated C1E-INH +/- mice had markedly increased dye extravasation. These results demonstrate that a single treatment with AAVrh.10hC1EI has the potential to provide long term protection from angioedema attacks in the affected population, representing a paradigm shift in current therapeutic approaches.

The scope of this chapter is to calculate the net energy of the production chain for virgin olive oil. Therefore, the determination was carried out for the direct and indirect energy inputs and the energy present as feedstock in the outputs (products and by-products). To perform this analysis, all of the production processes for olives and for oil extraction were studied. For the agricultural phase, three systems of cultivation were taken into consideration: the centenary olive grove (COO), the “intensive” olive grove (HDO) and, the more recently introduced, “super-intensive” olive grove (HSDO). The last two models are distinguished by the high number of trees per hectare and by an intense mechanization of agricultural practices. Regarding the oil extraction phase, four different technologies were compared: the pressure system (PS), the two-phase system (2PS), the three-phase (3PS), and the system, called “de-pitted”, which provides for the separation of the pits before the oil is extracted (DPS). The analysis showed that the production of olives needs more than 90% of energy requirements, much of which is met by non-renewable sources of energy. The production of fertilizers, and also irrigation, are the production factors that require a considerable amount of energy. Among the three agricultural systems analyzed, the COO system of cultivation is the one that requires less energy as compared to the other systems. The scenario that enables the most energy return, however, is the SHDO system of cultivation, due to the greater amount of pruning residues that can be obtained.

Cytokines play critical roles in regulating the antibody response to vaccines. We sought to understand the role of endogenous cytokines in the determination of antibody production in the elderly, a group of subjects known to have a lower response rate to vaccination. We found that in a healthy elderly group, only 52% of whom responded to the influenza vaccine, endogenous levels of interleukin 6 (IL-6), IL-10 and gamma interferon (IFNgamma) did not differ statistically significantly between responders and non-responders (responders: n = 27, IL-6 = 293 +/- 101 pg/ml, IL-10 = 882 +/- 240 pg/ml; nonresponders: n = 26, IL-6 = 223 +/- 71 pg/ml, P = 0.57, IL-10 = 445 +/- 148 pg/ml, mean +/- SE, P = 0.14, respectively, and undetectable IFNgamma). Serum levels of these three cytokines were not changed significantly four weeks after vaccination (P0.05 for IL-6 and P0.01 for IL-10). In addition, there were also no age-dependent differences in serum IL-6 and IL-10 levels.

Excessive leverage is one of the most important problems facing Italian agricultural cooperatives as the competition in the EU intensifies. An empirical study of 500 agricultural cooperatives supports the hypothesis that cooperatives characterized as having “powerful managers” have a capital structure that is significantly different from the “nonpowerful manager” cooperatives. Powerful manager cooperatives were less leveraged and had a long term strategy that focused on minimizing financial risk by increasing their equity/asset ratio. The result of this strategy is an increased probability of long term international competitiveness.

The last few years have brought rapid growth in the number of Virtual Microscopes (VM) and the promotion of digital histology. In this digital era, it is natural to assist in the transportation of histology imaging into computer technology support. We have also assisted in a quick and precise race towards improved technology capable of acquiring detailed digital images which realistically report the original slide for easy consultation [1,2]. The advanced technology herein proposed aims at being a sophisticated imaging archive. Digitally scanning slides however, does not give additional information to the vision of tissue structures, even with high resolution or improved colour and image precision. We can now say that high enlargement microscopic observation can only give same details to structures visible to the eye directly through the microscope. This means that when VMs are used in a correct manner they are capable of giving significant progress to highlighting, not quantifying, a critical field in applying technology specific to computerised measuring. The potential to improve precision and objectivity of measurements can be achieved with additional technical equipment. The aim of this study is to present a machine invented with a calculation potential to facilitate the work of the observer in a medical practice, not only in terms of easy retrieval of images but also as an instrument for the automatic analysis of digital histology. The “Metrizer” aims at supplying precise and objective descriptions and measurements of the specimen under observation. These descriptions do not substitute the pathologist but they should assist him/her to assert with objectivity and safety the entity of the observed pathology [3-8]. Material and methods The “Metrizer” (Figure 1) is an automatic, compact machine composed of a lens for microscopic observation, digital cameras, a movement device and a computer complete with software to control machine movement and image analysis. With these components the machine facilitates consultation of histological preparations (virtual microscop e-s lide scanner function) and supplies objective numeric data regarding the state of the tissue harbouring diverse diseases without causing fatigue or human error. Using the Slide Scanner function it enables to automatically digitalise the entire histological slide in high definition and makes it easily accessible in the digital archive.

© A m Agricultural and Food Economics (AFE) is an on line peer-reviewed open-access journal supported by the Italian Association of Agricultural Economics (SIDEA) and published by SpringerOpen. This open access formula is an innovative and valuable opportunity for readers and authors who are interested in deepening their knowledge and keeping up to date on international agri-food policy and economic issues of the agricultural and food sector in a globalised world. In a fast-changing world, research in agri-food economics faces the important challenge of balancing the need of quickly publishing papers with that of following the rigorous scientific method. This e-journal tries to fill this gap by publishing peer-reviewed research articles that are open-access, and thus accessible to users immediately and free of charge for readers. AFE welcomes research articles from scholars and researchers from all over the world to publish problem-oriented, high quality and refereed papers. AFE seeks clearly written articles to promote insightful understanding of the current trends in the agri-food system. AFE publishes only original articles from a wide variety of economic perspectives that address current and relevant issues related to the agricultural and food system. Published papers focus on applied analysis, the discussion of innovative results, and relevant policy and managerial implications. Theoretical and methodological papers should be accessible to the journal’s variety of international readers. Qualitative papers and meta-analyses are welcome if they comply with the journal’s rigorous standards. Topics of specific interest of AFE include agricultural and food market analysis, agri-food firm management and marketing, organization of the agri-food chains, consumer behaviour, food quality and safety issues, economics of nutrition and food security, food and health economics, agri-food policy and trade, sustainable rural development, natural and marine resource economics, and land economics. Papers for publication in AFE are selected through a peer review process to ensure soundness, originality, relevance, and readability. AFE adopts a double-blind peer review process to ensure independent assessment of the papers. Each paper, with no exception, is reviewed by at least two referees of different nationalities. Manuscripts that are not within the scope of the journal may be returned to the authors without a formal review. The editorial board of AFE is composed, in addition to the editors in chief, by many internationally renowned economists that guarantee the high quality level of the papers published.

One hurdle in the translation of adeno-associated virus (AAV) vectors for the treatment of human disease is the challenge in demonstrating drug function in a potency assay, a requirement in the clinical translation to product. In vitro measurements of AAV potency suffer from poor efficiency of infectivity and are insensitive measures of potency. To address this issue, we have developed a robust in vivo assay for the measurement of vector potency. Using as the model vector the AAVrh.10 serotype coding for human frataxin (FXN, a mitochondrial protein essential for cellular function and that is deficient in Friedreich's ataxia), and based on the knowledge that intravenous administration of all AAV vectors primarily transduce the liver, we have developed a reproducible in vivo potency assay that can be used to set quality control standards for vector production. The assay is based on administration of the AAV vector (2.5 × 1010 genome copies) administered intravenously to 6 to 8 wk old Balb/c male mice with the liver harvested 2 wk later following PBS perfusion. Liver homogenates are processed to assess vector genome copies, transgene mRNA, and expressed protein. In order to establish acceptance criteria for assays of vector genome and mRNA levels, separate specifications were set for DNA sample load using the mouse housekeeping gene Tfrc, and RNA sample load using mouse 18S RNA, both based on data from quantitative PCR analysis of mouse livers (n=35) assayed in duplicate. Specifications were set as the median ± 2 standard deviations based on these assay results; for the potency assay results to be accepted, each of these specifications must be met by the test sample. For the AAVrh. 10 vector expressing FXN(AAVrh.10hFXN), liver vector genome levels (a measure of reproducibility of delivery) were 8.2 × 104 ± 2.2 × 104 genome copies/µg genomic DNA (n=10 mice, mean ± SD), liver human FXN mRNA levels (a measure of vector potency at the transcription level) were 2.2 × 103 ± 0.7 × 103 copies/µg total RNA (n=10 mice, mean ± SD) and liver human FXN protein levels (ELISA; a measure of vector potency at the protein level) were 54.8 ± 17.52 ng/mg protein (n=10 mice, mean ± SD). From this data, we established the following quality control specifications for AAVrh.10hFXN vectors: vector genome range 6 × 104 - 1 × 105 (copies/µg DNA), hFXN mRNA level range 1.6 × 103 - 2.9 × 103 (copies/µg RNA) and the hFXN protein levels 37 - 72 (ng/mg protein). This approach is adaptable to any AAV vector, providing an in vivo quality control for vector function.

The blood transfusion is a complex activity subject to a high risk of eventually fatal errors. The development and application of computer-based systems could help reducing the error rate, playing a fundamental role in the improvement of the quality of care. This poster presents an under development eLearning tool formalizing the guidelines of the transfusion process. This system, implemented in YAWL (Yet Another Workflow Language), will be used to train the personnel in order to improve the efficiency of care and to reduce errors.

Choosing the wrong theoretical model to describe an industry’s behavior may lead to biased estimates of the degree of market power. This paper presents a two-step, data-driven methodology to reduce the risk of mis-specification bias. In the first step, a sliced inverse regression identifies the significant factors that affect the industry’s pricing behavior. In the second step, a non-parametric regression of price on the SIR factors estimates the link functions. The output of the algorithm offers useful information to identify the model that best approximates the industry’s pricing behavior. In addition, the output of the algorithm is used to develop a post-estimation test for model specification.

Most agricultural policy analysis assumes that markets are perfectly competitive, despite increasing evidence to the contrary. We demonstrate that the interaction of market power and government intervention may lead to outcomes that are counter to key results of policy analysis for perfectly competitive markets. We show that market power may reduce or eliminate entirely the net welfare benefits from removing two traditional support mechanisms, price floors and deficiency payments, and may increase considerably the government's cost of implementing either of them. Accordingly, optimally designed price support measures may improve welfare in the presence of downstream oligopoly and/or oligopsony power. Copyright 2011, Oxford University Press.

This paper examines interactions between market power and agricultural policy in the U.S. wheat flour milling industry using a non-parametric approach. The analysis focuses on marketing loan and pre-1986 deficiency payment programs; farmers' payments from these programs are dependent on whether or not the market price exceeds a "policy" price. It assesses if the payments trigger a change in the underlying economic behavior of the milling industry, and any resulting change in the flour-wheat price margin. The analysis compares the outcomes of using constrained and unconstrained sliced inverse regressions in order to identify the significant factors affecting millers' pricing behavior. In both cases, the link functions are then estimated using a non-parametric regression of prices on these factors. Constraining the factors in the sliced inverse regression in order to generate coefficients that are easily interpreted using economic theory does not affect the results. Based on the SIR factors, millers were able to extract an additional $0.24/cwt. of flour by increasing their marketing margins in years farmers received program payments. Based on the CIR factors, the increase in the marketing margin was $0.23/cwt. In both cases the increase was approximately 10 percent of the estimated marketing margin in years farmers received program payments.

The aim of the present study is to analyze the most common method used for processing ripe table olives: the “California style”. Life Cycle Assessment (LCA) was applied to detect the “hot spots” of the system under examination. The LCA results also allowed us to compare the traditional “California style”, here called “method A”, with another “California style”, here called “method B”. We were interested in this latter method, because the European Union is considering introducing it into the product specification of the Protected Denomination of Origin (PDO) “La Bella della Daunia”. It was also possible to compare the environmental impacts of the two “California style” methods with those of the “Spanish style” method. From the comparison it is clear that “method B” has a greater environmental impact than “method A” because greater amounts of water and electricity are required, whereas “Spanish style” processing has a lower environmental impact than the ”California style” methods.El objetivo de este estudio es analizar el método más común utilizado para el procesamiento de la aceituna negra de mesa “estilo California” (Californian Style). La metodología LCA se aplicó para detectar los puntos calientes del sistema estudiado. Los resultados LCA también nos permitieron comparar el estilo californiano tradicional, aquí llamado “método A”, con otro estilo californiano, llamado “método B”. Nosotros estábamos interesados en el segundo método, porque la Unión Europea está considerando introducirlo en la Denominación de Origen Protegida (DOP) “La Bella della Daunia”. También fue posible comparar los impactos medioambientales de los dos mètodos californianos con los impactos del método español. Observando la comparación, está claro que el “método B” tiene un mejor impacto ambiental que el “método A” porque este último requiere más cantidad de agua y electricidad, por lo que el “Estilo Español” para procesar nos da un mejor procesamiento desde el punto de vista ambiental que los métodos del “Estilo Californiano”

The primary purpose of this paper is to provide updated estimates of domestic own-price, cross-price and income elasticities of demand and estimated price elasticities of supply for various California commodities. Flexible functional forms including the Box-Cox specification and the nonlinear almost ideal demand system are estimated and bootstrap standard errors obtained. Partial adjustment models are used to model the supply side. These models provide good approximations in which to obtain elasticity estimates.The six commodities selected represent some of the highest valued crops in California. The commodities are: almonds, walnuts, alfalfa, cotton, rice, and tomatoes (fresh and processed). All of the estimated own-price demand elasticities are inelastic and, in general, the income elasticities are all less than one. On the supply side, all the short-run price elasticities are inelastic. The long-run price elasticities are all greater than their short-run counterparts. The long-run price supply elasticities for cotton, almonds, and alfalfa are elastic, i.e., greater than one.Policy makers can use these estimates to measure the changes in welfare of consumers and producers with respect to changes in policies and economic variables.

It is well known that angiogenesis is a complex process that accompanies neoplastic growth, but pituitary tumours are less vascularized than normal pituitary glands. Several analytical methods aimed at quantifying the vascular system in two-dimensional histological sections have been proposed, with very discordant results. In this study we investigated the non-Euclidean geometrical complexity of the two-dimensional microvasculature of normal pituitary glands and pituitary adenomas by quantifying the surface fractal dimension that measures its space-filling property. We found a statistical significant difference between the mean vascular surface fractal dimension estimated in normal versus adenomatous tissues (P = 0.01), normal versus secreting adenomatous tissues (P = 0.0003), and normal versus non-secreting adenomatous tissues (P = 0.047), whereas the difference between the secreting and non-secreting adenomatous tissues was not statistically significant. This study provides the first demonstration that fractal dimension is an objective and valid quantitator of the two-dimensional geometrical complexity of the pituitary gland microvascular network in physiological and pathological states. Further studies are needed to compare the vascular surface fractal dimension estimates in different subtypes of pituitary tumours and correlate them with clinical parameters in order to evaluate whether the distribution pattern of vascular growth is related to a particular state of the pituitary gland.

Background Cancer remains one of the most complex diseases affecting humans and, despite the impressive advances that have been made in molecular and cell biology, how cancer cells progress through carcinogenesis and acquire their metastatic ability is still widely debated. Conclusion There is no doubt that human carcinogenesis is a dynamic process that depends on a large number of variables and is regulated at multiple spatial and temporal scales. Viewing cancer as a system that is dynamically complex in time and space will, however, probably reveal more about its underlying behavioural characteristics. It is encouraging that mathematicians, biologists and clinicians continue to contribute together towards a common quantitative understanding of cancer complexity. This way of thinking may further help to clarify concepts, interpret new and old experimental data, indicate alternative experiments and categorize the acquired knowledge on the basis of the similarities and/or shared behaviours of very different tumours.

AIM: To provide the accurate alternative metrical means of monitoring the effects of new antiviral drugs on the reversal of newly formed collagen. METHODS: Digitized histological biopsy sections taken from 209 patients with chronic C virus hepatitis with different grade of fibrosis or cirrhosis, were measured by means of a new, rapid, user-friendly, fully computer-aided method based on the international system meter rectified using fractal principles. RESULTS: The following were described: geometric perimeter, area and wrinkledness of fibrosis; the collation of the Knodell, Sheuer, Ishak and METAVIR scores with fractal-rectified metric measurements; the meaning of the physical composition of fibrosis in relation to the magnitude of collagen islets; the intra- and inter-biopsy sample variability of these parameters; the “staging” of biopsy sections indicating the pathway covered by fibrosis formation towards its maximum known value; the quantitative liver tissue architectural changes with the Hurst exponent. CONCLUSION: Our model provides the first metrical evaluations of the geometric properties of fibrosis and the quantitative architectural changes of the liver tissue. The representativeness of histological sections of the whole liver is also discussed in the light of the results obtained with the Hurst coefficient.

Despite the fact that all anatomical forms are characterised by non-polyhedral volumes, rough surfaces and irregular outlines, it has been suggested that sophisticated computer-aided analytical systems based on the Euclidean principles of regularity, smoothness and linearity can be used in human quantitative anatomy. However, the new fractal geometry is a more powerful means of quantifying the spatial complexity of real objects. The present study introduces the surface fractal dimension as a numerical index of the complex architecture of the corneal stroma, and investigates its behaviour during computer-simulated changes in keratocyte density and distribution, and in the heterogeneous composition of the extracellular matrix. We found that the surface fractal dimension depends on keratocyte density and distribution, as well as on the different concentrations of the constituents making up the extracellular matrix. Our results show that the surface fractal dimension could be widely used in ophthalmology not only because of its ability to quantify drug-correlated architectural changes, but also because it can stage corneal stroma alterations and predict disease evolution.

Our non-representative sample of 245 undergraduates had significantly lower scores on questions presented in the standard heterogeneous form (i.e., Direct Demand equation and Inverse Demand graph) than on questions presented in non-standard homogenous forms. This result, which holds for advanced students, highlights one reason why 95 percent of students in economics principles classes do not enter the major - economics can be gratuitously mathematical. We argue that the Inverse Demand standard hurts rather than helps economics when it is used in early courses, but that professors have no incentive to change their methods. We recommend that early classes use either no graphs or a homogenous combination of graph and equation. The "standard" should be introduced later, when benefits outweigh costs.

The earliest time at which serum antibody levels peak following administration of an influenza virus vaccine in elderly persons is not clearly defined. We compared the time intervals of 1 and 2 weeks after vaccination. A commercial trivalent vaccine containing the hemagglutinins of influenza viruses A/Texas/36/91 (H1N1), A/Johannesburg/33/94 (H3N2), and B/Harbin/7/94 was used. The hemagglutination inhibition (HAI) antibody titers at 1 week after vaccination were significantly lower than the HAI titers at 2 weeks postvaccination for all three vaccine components.

Primary biliary cirrhosis, is an autoimmune disease, semiquantitatively described by four stages obtained from evaluating the stage of hepatic structures. The follovng pathologic events, inflammation, destruction-regeneration of biliary tissue CK7+ and fibrosis determine this disease.

AIM: To describe a quantitative analysis method for liver biopsy sections with a machine that we have named “Dioguardi Histological Metriser” which automatically measures the residual hepatocyte mass (including hepatocytes vacuolization), inflammation, fibrosis and the loss of liver tissue tectonics. METHODS: We analysed digitized images of liver biopsy sections taken from 398 patients. The analysis with Dioguardi Histological Metriser was validated by comparison with semi-quantitative scoring system. RESULTS: The method provides: (1) the metrical extension in two-dimensions (the plane) of the residual hepatocellular set, including the area of vacuoles pertinent to abnormal lipid accumulation; (2) the geometric measure of the inflammation basin, which distinguishes intra-basin space and extra-basin dispersed parenchymal leukocytes; (3) the magnitude of collagen islets, (which were considered truncated fractals and classified into three degrees of magnitude); and (4) the tectonic index that quantifies alterations (disorders) in the organization of liver tissue. Dioguardi Histological Metriser machine allows to work at a speed of 0.1 mm2/s, scanning a whole section in 6-8 min. CONCLUSION: The results are the first standardized metrical evaluation of the geometric properties of the parenchyma, inflammation, fibrosis, and alterations in liver tissue tectonics of the biopsy sections. The present study confirms that biopsies are still valuable, not only for diagnosing chronic hepatitis, but also for quantifying changes in the organization and order of liver tissue structure.

AIM: To assess the sampling variability of computer-aided, fractal-corrected measures of fibrosis in liver biopsies. METHODS: Samples were derived from six to eight different parts of livers removed from 12 patients with clinically and histologically proven cirrhosis undergoing orthotopic liver transplantation. Sirius red-stained sections with a thickness of 2 μm were digitized using a computer-aided image analysis system that automatically measures the surface of fibrosis, as well as its outline perimeter, fractal surface and outline dimensions, wrinkledness, and Hurst coefficient. RESULTS: We found a high degree of inter-sample variability in the measurements of the surface [coefficient of variation (CV) = 43% ± 13%] and wrinkledness (CV = 28% ± 9%) of fibrosis, but the inter-sample variability of Hurst’s exponent was low (CV = 14% ± 2%). CONCLUSION: This study suggests that Hurst’s exponent might be used in clinical practice as the best histological estimate of fibrosis in the whole organ, and evidences the fact that biopsy sections, which are fundamental for the qualitative diagnosis of chronic hepatitis, play a key role in the quantitative estimate of architectural changes in liver tissue.

AIM: To introduce a computer-aided morphometric method for quantifying the necro-inflammatory phase in liver biopsy specimens using fractal geometry and Delaunay’s triangulation. METHODS: Two-micrometer thick biopsy sections taken from 78 chronic hepatitis C virus-infected patients were immunohistochemically treated to identify the inflammatory cells. An automatic computer-aided image analysis system was used to define the inflammatory cell network defined on the basis of Delaunay’s triangulation, and the inflammatory cells were geometrically classified as forming a cluster (an aggregation of a minimum of three cells) or as being irregularly distributed within the tissue. The phase of inflammatory activity was estimated using Hurst’s exponent. RESULTS: The proposed automatic method was rapid and objective. It could not only provide rigorous results expressed by scalar numbers, but also allow the state of the whole organ to be represented by Hurst’s exponent with an error of no more than 12%. CONCLUSION: The availability of rigorous metrical measures and the reasonable representativeness of the status of the organ as a whole raise the question as to whether the indication for hepatic biopsy should be revised by establishing clear rules concerning the contraindications suggested by its invasiveness and subjective interpretation.

QUANTITATIVE ANALYSIS OF TUMOR INFILTRATING LYMPHOCYTES IN CUTANEOUS MELANOMA Stefania Motta, PhD, University of Milan-Istituto Clinico Humanitas, 20089 Rozzano Milano, Italy, Marcello Monti, MD, University of Milan-Istituto Clinico Humanitas, 20089 Rozzano Milano, Italy, Barbara Franceschini, PhD, University of Milan-Istituto Clinico Humanitas, 20089 Rozzano Milano, Italy, Carlo Russo, PhD, University of Milan-Istituto Clinico Humanitas, 20089 Rozzano Milano, Italy

Streptococcal mitogen (SM), an extracellular product of group A streptococci, is non specifically mitogenic for both B and T lymphocytes. The mitogenic activity of SM is resistant to digestion with trypsin, to heating at 100 °C for 5 min, and to treatment with dithiothreitol. The proliferative response of lymphocytes from patients with a history of rheumatic fever is similar to that of lymphocytes from healthy donors when stimulated with optimal concentrations of SM, but is significantly reduced when low doses of SM are used. T lymphocytes stimulated with SM acquire Ia antigens and the ability to stimulate allogeneic and autologous lymphocytes in mixed lymphocyte reactions. An involvement of Ia antigens in these reactions is indicated by the specific block by monoclonal antibodies to human Ia antigens.

Evolution qualitative et quantitative des lipides totaux et des acides gras de six cultivars d'oranges lors de la maturation et de la surmaturation des fruits, en relation avec les proprietes organoleptiques des fruits et des jus. Les fruits trop murs ont une teneur en acide linoleique et linolenique plus elevee et presentent une alteration de l'odeur et de la saveur

A complement (C)-dependent antiglobulin assay was utilized to determine the reactivity of non-C-fixing monoclonal antibodies (MoAb) with human granulocyte-macrophage progenitor cells (CFU-GM). The variables of the assay were analyzed with non-C-fixing MoAb against Ia antigens, including CR11–462, which recognizes the same (or spatially close) determinant identified by the C-fixing anti-Ia MoAb Q5/13. The sensitivity of the antiglobulin assay was influenced by dilutions of anti-mouse Ig xenoantiserum and of rabbit C. Five non-C-fixing MoAb to Ia antigens, seven non-C-fixing MoAb to HLA-A,B antigens, and one non-C- fixing MoAb to beta 2-microglobulin induced marked inhibition of human CFU-GM in the antiglobulin assay. The activity of non-C-fixing MoAb in the antiglobulin assay was comparable to that of C-fixing anti-Ia and anti-HLA-A,B MoAb in the standard cytotoxicity assay. In addition, the cytotoxic effect of dilute C-fixing anti-Ia MoAb was enhanced when the antiglobulin technique was employed. The results of this study indicate that the antiglobulin assay is a rapid and simple technique for the characterization of antigens on human hematopoietic progenitors. Our data also indicate that Ia antigens are expressed on most CFU-GM and that the conflicting results in the literature (that is, those suggesting that Ia antigens are expressed on a smaller proportion of CFU-GM) may reflect differences in the cytolytic activity of the MoAb and rabbit C used.

T lymphocytes from patients with solid tumors and from aged donors are abnormal in their expression of Ia antigens following in vitro stimulation with phytohemagglutinin (PHA). Ia antigens were not detected on PHA-activated T lymphocytes from 15 of 27 patients with solid tumors. The abnormality in T lymphocytes from 25 donors older than 60 years was evidenced by a reduction in the percentage of T cells acquiring Ia antigens following stimulation with suboptimal amounts of PHA and by a delayed appearance of those antigens. In both groups of donors the defect in Ia antigen expression by PHA-activated T cells did not correlate with the reduced [3H]thymidine uptake. PHA-activated T cells from aged donors and from patients with solid tumors were poorly stimulatory in autologous and allogeneic mixed lymphocyte reactions. Furthermore, T lymphocytes from these two groups of donors displayed a reduced proliferative response to autologous non-T cells, but a normal proliferative response to allogeneic PHA-activated T cells and to non-T cells from control subjects.

T cells of two donors, JR (HLA-A23, 29; B7,7; G; DRw5) and HG (HLA-A2, 23; B40, w44; Cw4), were stimulated with cells from an HLA homozygous lymphoblastoid cell line JY (HLA-A2, 2; B7,7, C-, DRw4, 6) and cloned by limiting dilution after the third stimulation. Two cytotoxic T-cell (CTL) clones, JR-2-16 (from donor JR) and HG-31 (from donor HG), were used for detailed studies. The results of a panel study using lymphocytes from HLA-typed individuals and a study with two HLA recombinant families indicate that the antigens recognized by the CTL clones JR-2-16 and HG-31 were highly associated with HLA-A2 and HLA-B7, respectively. Blocking studies with a monoclonal antibody recognizing a framework determinant on HLA-A, -B and-C antigens and a monoclonal antibody reacting with HLA-A2 support the notion that JR-2-16 and HG-31 interact with the HLA-A2 and the HLA-B7 antigens per se. However, these clones did not recognize the HLA-A2 and HLA-B7 of all donors typed for these antigens, suggesting that the HLA-A2 and HLA-B7 antigens of these particular donors are variants of the serologically defined HLA antigens. These results indicate that in vitro-derived human CTL clones detect variations in the serologically defined allospecificities and can be used as reagents to elucidate the polymorphism of HLA antigens further.

Human null cells were isolated from peripheral blood lymphocytes (PBL) by differential centrifugation on a Ficoll-Hypaque gradient of the PBL following simultaneous rosetting with erythrocyte indicators specific for B and T lymphocytes. Specifically, the T lymphocytes were rosetted with 2-aminoethylisothiuronium bromide-treated sheep erythrocytes (ShE), whereas the B lymphocytes were either rosetted with ShE coated with xenoantibodies against human gamma globulin or first sensitized with monoclonal antibodies to human Ig-like antigens and then rosetted with ShE coated with xenoantibodies against mouse gamma globulin. Approximately 90% of the lymphocytes isolated were null cells that did not bear detectable B-cell markers-that is, surface immunoglobulin and/or Ia-like antigens-or T-cell markers-that is, ShE receptors. The large majority of the null cells expressed receptors for the IgG Fc fragment (53-93%), C3 component (65-92%) and monkey erythrocytes (60-91%) but lacked receptors for the IgM Fc fragment and murine erythrocytes. The null cells exhibited high natural killer cell activity and antibody-dependent cellular cytotoxicity and were two- to four-fold active than the total PBL and the T-enriched cell fractions. The null cells, however, did not respond to stimulation with phytohaemagglutinin and failed to function as either stimulators or responders in an unidirectional mixed lymphocyte reaction.

Analysis of the tissue distribution of human Ia-like antigens has shown that they have a wider distribution than originally reported. Furthermore, the expression of Ia-like antigens may change when cells undergo malignant transformation: for instance, melanoma cells acquire Ia-like antigens, while breast carcinoma cells lose them. Serological and immunochemical analysis of Ia-like antigens with monoclonal antibodies has shown a cellular and molecular heterogeneity of these molecules which had not been previously recognized with conventional allo- and xenoantisera. The functional significance of this heterogeneity is not known. Monoclonal antibodies to human Ia-like antigens cross-react with lymphocytes from other animal species indicating that portions of the molecules have been conserved during evolution. The biological implications of these findings are discussed.

The monoclonal antibody (MoAb) 6/31, an IgG2a, and the MoAb Cr-1, an IgGl, identify determinants expressed on the heavy chain of subpopulations of HLA-A and B antigens while the MoAb NAMB-1, an IgGl, reacts with human 2-microglobulin (2-m). The MoAb 6/31 and CR-1 reacted with lymphocytes from all of

Human T lymphocytes activated with PHA express Ia-like antigens and acquire the ability to stimulate autologous T lymphocytes in mixed lymphocyte reaction. This reaction is immunological in nature since it has specificity and memory. Ia-like antigens play a role in the stimulation of T lymphocytes by autologous PHA-T lymphocytes since monoclonal antibodies to Ia-like antigens can significantly, although not completely, inhibit the stimulation.

Four human T lymphocyte clones exhibiting proliferative responses to class I HLA antigens were isolated from an in vitro mixed lymphocyte culture (MLC). Three clones expressed the Leu-2+3- phenotype and demonstrated proliferation in response to HLA-B8, while the fourth clone expressed the Leu-2-3+ phenotype and proliferated in response to HLA-A2. These clones were also cytotoxic towards cells bearing the same target antigens. Blocking studies utilizing monoclonal antibodies demonstrated that proliferation was triggered by determinants on the class I molecule itself, and these determinants appear to be spatially close to those which determine serologic allospecificity. These findings support the concept that the class I molecules themselves are the weak MLC stimulating determinants previously mapped to the HLA-A and B regions of the major histocompatibility complex.

A non-complement fixing monoclonal antibody (Q2/70) to framework determinants of human Ia-like antigens was used to develop a method for isolating Ia-like antigen bearing, i.e., Ia-like (+) cells and cells lacking these antigens, i.e. Ia-like (−), from human peripheral blood lymphocytes (PBL). The method was based on sensitization of PBL with the antibody Q2/70, followed by rosetting with sheep (ShE) coated with purified rabbit anti-mouse Ig antibodies, differential centrifugation on a Ficoll-Hypaque gradient, and finally recovery of Ia-like (+) and Ia-like (−) cells from the bottom and the interface of the gradient respectively. Marker analysis of the two cell subpopulations showed that the majority of the bottom cell fraction were Ia-like (+) and carried B cell markers such as membrane bound immunoglobulins (MbIg) and C3 receptors. On the other hand, the majority of the interface cell fraction were Ia-like (−) and carried T cell markers such as receptors for 2-aminoethylisothiouronium treated sheep erythrocytes (AETShE) and goat erythrocytes (GoE). Serological and functional studies showed that the Ia-like (+) cells (1) were useful targets in complement mediated cytotoxicity assays for HLA-DR typing; (2) served as stimulator but not as responder in unidirectional mixed lymphocyte reactions (MLRs); (3) did not display lytic activity in natural killer (NK) cell cytotoxicity and in antibody dependent cellular cytotoxicity (ADCC); and (4) proliferated in response to pokeweed mitogen (PWM) stimulation in the presence of helper T cells. On the other hand, the Ia-like (−) cells (1) responded to but failed to stimulate allogeneic lymphocytes in the MLRs; (2) were highly active in NK and ADCC assays; and (3) provided helper activity in PWM stimulation of purified B cells.

The reactivity patterns of several monoclonal antibodies specific for monomorphic determinants of human Ia antigens were studied using flow cytometric techniques. We observed differential reactivity of these antibodies with human lymphoid cell lines, normal fresh human mononuclear cells, and lymphoblasts from PHA-activated cultures. The molecular heterogeneity of Ia antigens previously identified with immunochemical techniques was accompanied by heterogeneity of cell surface expression as identified by an immunofluorescent probe. The determinants identified by these anti-Ia monoclonal antibodies may provide useful markers in the isolation of cellular subpopulations responsive in the immune system.

Serological and immunochemical studies showed that monoclonal antibody Q2/70 (MoAb Q2/70), produced by the hybridoma technique, is specific for human Ia-like antigens. This antibody recognizes an antigenic determinant which is different from those defining the serologic polymorphism of Ia-like antigens, and is expressed on subsets of human Ia-like molecules and on lymphoid cells from other species. MoAb Q2/70 inhibits unidirectional MLRs ∗ between allogenic human lymphocytes, but not between murine and human lymphocytes. In ADCC ∗ assays, MoAb Q2/70 mediates lysis of cultured human B lymphoid cells RPMI 4098, effected by murine splenocytes. The antibody is suitable to isolate immunologically functional B lymphocytes from human peripheral blood.