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Cattle tick control remains a serious problem in cattle farms worldwide, due to the limited success achieved with chemicals. Although the use of vaccines for tick control may open possibilities for an integrated approach, the search for other protective antigens is still necessary to improve control. Boophilus microplus is a rich source of BmTIs, serine proteinase inhibitors that are present throughout the tick's lifecycle. The present paper reports a pen trial conducted to evaluate the performance of a synthetic BmTI N-terminal fragment as antigen against the tick in bovines. The trial was conducted with two groups of eight crossbred cattle under controlled infestation: one group was vaccinated with BmTI peptide using saponin as adjuvant and the other was kept as control. Challenge was performed with 15,000 larvae. The specific IgG response was measured by ELISA, with successful outcomes for vaccinated animals. Vaccination resulted in an 18.4% level of efficacy when compared with the control group.

The ideal immobilization methods that are suitable for binding immuno-active materials with high efficiency onto the sensing surface are the key target to pursue in the current biosensor design. In this paper, a new hybrid material formed by assembling gold nanoparticles (GNP) onto nano-sized hydroxyapatite (HA) has been employed for the interface design of piezoelectric immunosensor, on which the antibodies were bound. The detection performances of the resulting immunosensor were investigated by use of the antibody-antigen model system of alpha-Fetoprotein (AFP), an important indicator in the diagnosis of clinical cancers. The hybrid material was characterized by the UV-vis spectroscopy, the SEM and TEM measurements. The frequency and electrochemical impedance responses characteristics for the processes of immobilization and immunoreaction of anchored anti-AFP antibodies were studied in detail. The immunoresponse of the proposed immunosensor was compared with those antibodies immobilized by using HA or GNP alone. It was found that the developed sensing interface has some advantages such as the activation-free immobilization and the high antigen-binding activities of antibodies. The as-prepared immunosensor can allow for the determination of AFP in the concentration range of 15.3-600.0ngml(-1). Such an interface design with the nano-sized hybrid materials should be tailored as a new alternative used for biosensor design.

The evaluation of potential adverse effects of pharmaceuticals on the immune system is part of the standard drug development procedures and needs to be available prior to the start of phase III clinical trials. Although the histopathological assessment of lymphoid organs/tissues is considered fundamental for the identification and characterization of immunotoxic reactions, additional investigations are now recommended by the European guidelines for repeated-dose toxicity testing of medicinal products in order to achieve an accurate assessment of immune system functionality with regard to immunomodulation. In the present paper, we describe and discuss a study design which permits the investigation of the immune function in a 4-week study in rats following immunization by subcutaneous administration of the T-dependent antigen Keyhole Limpet Hemocynin (KLH). This includes assessment of hematology parameters, titration of KLH-specific antibodies in serum, lymphocyte immunophenotyping in blood, thymus, spleen and lymph nodes, macroscopic pathology and histopathological evaluation of the lymphatic organs/tissues and of the injection sites.

Several prevalent and life-threatening agents enter the organism via the mucosa. In this case, a mucosal cellular immune response is essential for protection and is therefore considered the main objective of vaccination. The frequency of antigen-specific CD4+ and CD8+ T cells can be determined directly in human whole blood by a combination of surface marker and intracellular cytokine staining. Immune cells primed in the mucosal compartment also migrate through the blood and can be identified by expression of the gut-specific homing receptor alpha4beta7. Simultaneously, these lymphocytes can be functionally characterized regarding their differentiation status by analysis of CD45RO and CD27 expression and effector functions by measuring intracellular perforin or granzyme B content. Thus, the technique described in the paper is a powerful tool for clinical monitoring of the total cellular immune response to complex antigens during infection or vaccination.

We report on a novel technique to develop an optical immunosensor based on fluorescence resonance energy transfer (FRET). IgG antibodies were labeled with acceptor fluorophores while one of three carrier molecules (protein A, protein G, or F(ab')2 fragment) was labeled with donor fluorophores. The carrier molecule was incubated with the antibody to allow specific binding to the Fc portion. The labeled antibody-protein complex was then exposed to specific and nonspecific antigens, and experiments were designed to determine the 'in solution' response. The paper reports the results of three different donor-acceptor FRET pairs, fluorescein isothiocyanate/tetramethylrhodamine isothiocyanate, Texas Red/Cy5, and Alexa Fluor 546/Alexa Fluor 594. The effects of the fluorophore to protein conjugation ratio (F/P ratio) and acceptor to donor fluorophore ratios between the antibody and protein (A/D ratio) were examined. In the presence of specific antigens, the antibodies underwent a conformational change, resulting in an energy transfer from the donor to the acceptor fluorophore as measured by a change in fluorescence. The non-specific antigens elicited little or no changes. The Alexa Fluor FRET pair demonstrated the largest change in fluorescence, resulting in a 35% change. The F/P and A/D ratio will affect the efficiency of energy transfer, but there exists a suitable range of A/D and F/P ratios for the FRET pairs. The feasibility of the FRET immunosensor technique was established; however, it will be necessary to immobilize the complexes onto optical substrates so that consistent trends can be obtained that would allow calibration plots.

This paper provides an evaluation of a plant-derived HBsAg-specific antibody in the immunopurification of the recombinant HBsAg for vaccine purposes. This plant-derived antibody was obtained from different batches of 100-200kg of tobacco leaves and coupled to Sepharose CL-4B with high efficiency. The plant-derived antibody immunoaffinity matrix purification behavior (elution capacity, antigen purity, purification cycles, and ligand leakage) was comparable to that of its mouse-derived monoclonal antibody homolog. This result supports the feasibility of using this plant-derived antibody for the immunopurification of the Hepatitis B surface antigen for human use, opening a new possibility to overcome the constrain of monoclonal antibody production in mice.

Haemolysin A of Escherichia coli and listeriolysin of Listeria monocytogenes represent important bacterial virulence factors. While such cytolysins are usually the reason for morbidity and even mortality, vaccine researchers have turned haemolysin A and listeriolysin into tools for vaccine delivery. Both cytolysins have found widespread application in vaccine research and are highly suitable for the elicitation of cell-mediated immunity. In this paper, we will review vaccine delivery mediated by the haemolysin A secretion system and listeriolysin and will highlight their use in vaccination approaches against protozoan parasites.

In previous investigations we pursued the hypothesis that lifelong, chronic antigenic load (CAL) is the major driving force of immunosenescence, which impacts on human lifespan by reducing the number of virgin antigen-non experienced (ANE) T cells, and filling the immunological space with expanded clones of memory and effector, antigen-experienced (AE) T cells. A model has been proposed to relate CAL with the conversion rate from ANE to AE CD8+ T cells. In addition, in order to account for individual variations of immunosenescence and lifespan, a noise term has been introduced to describe the individual fluctuations of CAL. This model was able to follow with a reasonable approximation the age-related decrease of ANE CD8+ T cells, as well as human survival curves. In this paper we extend this approach to historical survival curves, starting from 1750 until present days, and show that the quality of the fit of historical demographic data improves as we approach the recent, quantitative and qualitative decrease of CAL. Indeed, the almost linearity in the increase of lifespan and in the decrease of the noise fluctuation amplitude within this historical period suggests that the improvement of life conditions has steadily lowered the intensity of CAL and restricted the variability which results from the interaction between the individuals and their immunological environment. On the whole, this approach allows to appreciate when and how immunosenescence has started to impact on survivorship, and predicts its increasing, crucial role in explaining human mortality in hygienized, economically developed societies.

In this paper we present the results of a series of experiments on the activity of antibodies in a vapor phase sensor. For these experiments the sensor component was a ST-Quartz resonator with a center frequency of approximately 250 MHz. Anti-FITC antibodies were attached to the electrodes on the device surface via a protein-A crosslinker. Surface acoustic wave (SAW) resonator devices with various coatings were mounted in TO-8 packages, inserted into our sensor head module and subjected to various fluorescent analyte gases. Numerous controls were performed including the use of coated and uncoated devices along with devices coated with antibodies which were not specific for the target analyte. The SAW immunosensor response was monitored and a baseline frequency shift was observed when the analyte being presented was the antigen for the immobilized antibody. To provide an independent measure of antibody/antigen binding, the devices were removed from the sensor head, washed with a buffer solution to remove any unbound analyte, and then inspected using a confocal laser scanning microscope (CLSM). Since all the analytes being used in these experiments were fluorescent this afforded us the opportunity to visualize the attachment of the analyte to the antibody film. Given the high resolution of the CLSM, we were able to identify the location of the attachment of the fluorescent analytes relative to the 1.5 microm wide electrodes of the SAW device. We believe that these experiments demonstrate that we have achieved real time molecular recognition of these small molecules in the vapor phase.

The Escherichia coli protein Tag is traditionally regarded as an archetype of one of four classes of N -alkylpurine DNA glycosylases. However, its structure and phylogenetic relationship to other glycosylases remains a mystery. Fold-recognition and sequence profile analyses suggest that Tag shares the catalytic domain with helix-hairpin-helix (HhH) glycosylases such as MutY, AlkA and EndoIII, but its N- and C-termini together form a unique His 2 Cys 2 cluster. The findings presented in this paper provide insight into sequence–structure–function relationships in the Tag family and should aid in a more precise definition of the common core of the HhH superfamily of glycosylases involved in DNA repair.

This paper describes the selection of Gongronella butleri strains producing higher chitosan yield using UV mutagenesis. We have devised an enzyme-linked immunosorbent assay for the selection of high chitin deacetylase (CDA) yielding strains. Mutant strains M+1, M+2 and M+7 could produce twice the extractable chitosan yield and double the CDA activity, as compared to the wild type strain.

Dendritic cells (DCs) are recognized as the sole professional antigen-presenting cells capable of priming naive T cells of the helper and cytotoxic phenotypes. This property is presently exploited with success in vaccinal strategies against pathogens or tumor cells that otherwise escape immune recognition, but the repeated infusions of ex vivo expanded and sensitized DCs are usually required to achieve protection. In this paper, we demonstrate that liposomal antigens can efficiently relay and propagate the action of DCs, inducing a strong long-term response against their associated antigen. Their effect is mainly achieved by improving the ex vivo loading of DCs and by efficiently channeling the activation stimulus into the induction of effector function. This is demonstrated by the sustained immunoglobulin production as well as by the sustained lymphoproliferation and the increased cytokine secretion that can be achieved upon restimulation of DC-primed immune cells with limited amount of liposomal antigenic material. Being well-tolerated and easily prepared, liposomal antigens could therefore be expected to significantly contribute to the efficiency and to a more general utilization of the highly promising but rather cumbersome DC-based immunotherapies.

DNA microarray technology is a new and powerful tool that allows the simultaneous analysis of a large number of nucleic acid hybridization experiments in a rapid and efficient fashion. The development of the DNA microarray chip has been driven by modern techniques of microelectronic fabrication, miniaturization and integration to produce what is referred to as “laboratory-on-chip” devices. The application of DNA chip technology includes the comprehensive analysis of multiple gene mutations and expressed sequences with regard to newer drug designs, host–pathogen interactions and the design of new vaccines. An advantage of microarray technology is that it can assist researchers to better define and understand the expression profile of a given genotype associated with disease, adverse effects from exposure to certain stimuli, or the ability to understand or predict immune responses to specific antigens. This paper briefly reviews DNA microarray technology and its implications with special reference to vaccine design. The technical aspects comprising array manufacturing and design, array hybridization, formatting, scanning and data handling are also briefly discussed. © 2001 Elsevier Science Ltd. All rights reserved.

The spotted wolffish Anarhichas minor Olafsen is a promising new species in aquaculture in the cold waters of northern Norway. In this paper, some basic immunological studies of this marine species are reported. Of comparative interest are the cDNA sequences of the immunoglobulin transcript and the antibody responses to model antigens. Of more practical importance are the humoral immune responses and antibody specificities to potentially pathogenic bacteria. Full length cDNA clones encoding the immunoglobulin heavy and light chains in the spotted wolffish were sequenced demonstrating variable degrees of similarity to other teleost fish species. Also in the spotted wolffish the CH4 domain was deleted in the transmembrane form of the immunoglobulin heavy chain (IgH) as a receptor on B cells, with the transmembrane exon spliced directly to the CH3 domain. The antibody responses to various antigens like hapten-carrier molecules, protein antigens and bacterial pathogens were relatively high, but with some interesting exceptions. Anti-hapten responses to NIP and FITC were high while anti-DNS responses were low, but more surprisingly, there was hardly any B-cell response to the carrier molecule LPH. On the other hand, protein antigens like CGG and BSA were highly immunogenic in the spotted wolffish as were the bacterial antigens Vibrio anguillarum, V. salmonicida and Aeromonas salmonicida.

The use of spacers to distance an immobilized antibody from the surface of a support matrix introduces flexibility, which can reduce steric interferences between antibodies leading to a higher antigen capture efficiency. In this paper we investigated the use of a spacer molecule, poly-ethylene glycol (PEG), between the matrix surface and antibodies for the capture of Bacillus globigii, E. coli O157:H7, and ovalbumin. The antigen capture efficiency was determined using a surface ELISA method. Antibodies against the antigens were covalently immobilized either directly or via PEG to glass surfaces using a one-step EDC reaction. The amount of antibody immobilized was determined before blocking the nonspecific binding sites with bovine serum albumin. Antibodies immobilized via a PEG spacer showed a higher capture efficiency compared to direct immobilization, which was more pronounced with large antigens. Antibodies immobilized on glass supports were stable at 65 degrees C for at least 80 min, and the capture efficiency increased with heating at 65 degrees C for 20 min.

The recombinant Bm86-containing vaccine Gavac(TM) against the cattle tick Boophilus microplus has proved its efficacy in a number of experiments, especially when combined with acaricides in an integrated manner. However, tick isolates such as the Argentinean strain A, show low susceptibility to this vaccine. In this paper we report on the isolation of the Bm95 gene from the B. microplus strain A, which was cloned and expressed in the yeast Pichia pastoris producing a glycosylated and particulated recombinant protein. This new antigen was effective against different tick strains in a pen trial, including the B. microplus strain A, resistant to vaccination with Bm86. A Bm95-based vaccine was used to protect cattle against tick infestations under production conditions, lowering the number of ticks on vaccinated animals and, therefore, reducing the frequency of acaricide treatments. The Bm95 antigen from strain A was able to protect against infestations with Bm86-sensitive and Bm86-resistant tick strains, thus suggesting that Bm95 could be a more universal antigen to protect cattle against infestations by B. microplus strains from different geographical areas.

Veterinary vaccinology is a very interesting and rapidly developing field. In fact veterinary vaccines are not only used for the prevention of infectious diseases in the animal health sector, but also help to solve problems of public health, to reduce detrimental environmental impact of the use of some veterinary drugs and prevent the emergence of resistance of micro-organisms or parasites. After a short introduction, this paper will deal with the use of vaccines for animal health and welfare, including new developments in the veterinary field such as marker vaccines and vectored vaccines, the special case of equine influenza-inactivated vaccines and the use of veterinary vaccines in public health. The conclusions will analyse the reasons as to why develop veterinary vaccines and the obstacles to their development.

Fluorescent dyes that stain cell membranes or cytoplasm and then partition between daughter cells at division have been used in conjunction with flow cytometry to measure the proliferation of cells. In this paper, using peripheral blood mononuclear cells responding to tetanus toxoid, we describe an extension of this dye methodology to calculate the precursor frequency of antigen-specific T-cells. With mathematical deconvolution of the fluorescence histograms providing information about the proportion of cells in each of the daughter generations, information can be derived about the precursor frequency of cells in the original population that responded to the specific stimulus. Data from a model system with different proportions of fixed and viable cells indicate that the flow method returns accurate values for precursor frequency. Based on the characteristics of flow cytometric data acquisition, it is estimated that the flow method could detect proliferation of cells that represented, before addition of the stimulus, approximately 1/10(5) of the population. When comparing results to those from the limiting dilution technique, the flow cytometric method returns values that indicate higher precursor frequencies. Possible reasons for this discrepancy are discussed. The flow cytometric method offers the advantage of simplicity as well as the additional ability to phenotype the responding cells and determine their rate of proliferation. The flow method may find use as a simple, routine assay in the fields of allergy, transplant rejection, and autoimmunity and for quantitating responses to vaccination and cancer immunotherapy.

This paper reviews the application of flow cytometric techniques to the analysis and quantitation of mixed red cell populations. Such mixtures may arise in a variety of ways, for example: through transplacental hemorrhage during pregnancy or upon delivery; through iatrogenic procedures such as blood transfusion, bone marrow or peripheral blood stem cell transplantation; or through disease, when a loss of surface antigen expression may occur due to the expansion of abnormal progenitors lacking the appropriate gene or lacking the ability to translate the product of the gene. Flow cytometry can offer a simple and accurate means of identifying and quantitating mixed red cell populations through detection of antigens specific for either donor or recipient, and in many cases it is possible to distinguish homo- or heterozygous expression of a common antigen. Internal antigens may be accessed after fixation and permeabilisation of the red cells. These latter techniques are relatively new and expand the spectrum of antigenic differences available for the identification of individual species in mixed populations.

The necessity to make a Self(S)–NonSelf(NS) discrimination is the evolutionary selection pressure for specificity of the immune response. A new definition of paratopic specificity, which is heuristic and generalizable, can be derived from an understanding of this selection pressure. Specificity of the paratope is defined by a Specificity Constant,K,which is the probability that a functional change in recognition will be anti-Self. In an antigen-unselected population,Kis the proportion of cells that are anti-Self. This definition is unique in that it is derived from the function upon which evolution selects, namely the effector output. This paper describes how the concept of a Specificity Constant was derived, how it is estimated, what it can be used to explain, and how it impacts on repertoire and effectiveness of response.

Somatic cells, including immune cells such as T-cells have a limited capacity for proliferation and can only replicate for a finite number of generations (known as the Hayflick limit) before dying. In this paper we use mathematical models to investigate the consequences of introducing a Hayflick limit on the dynamics of T-cells stimulated with specific antigen. We show that while the Hayflick limit does not alter the dynamics of T-cell response to antigen over the short term, it may have a profound effect on the long-term immune response. In particular we show that over the long term the Hayflick limit may be important in determining whether an immune response can be maintained to a persistent antigen (or parasite). The eventual outcome is determined by the magnitude of the Hayflick limit, the extent to which antigen reduces the input of T-cells from the thymus, and the rate of antigen-induced proliferation of T-cells. Counter to what might be expected we show that the persistence of an immune response (immune memory) requires the density of persistent antigen to be less than a defined threshold value. If the amount of persistent antigen (or parasite) is greater than this threshold value then immune memory will be relatively short lived. The consequences of this threshold for persistent mycobacterial and HIV infections and for the generation of vaccines are discussed.

In murine splenocytes, a primary antigen-specific antibody response is stimulated by 2-O-alpha-D-glucopyranosyl-L-ascorbic acid (AA-2G), a stable form of ascorbate, as described in our previous paper. We examined here the effect of nerve growth factor (NGF) on the antigen-specific antibody production in vitro augmented by AA-2G. NGF (> or = 10 ng/ml), which alone had no effect, enhanced the anti-sheep-red-blood-cell (SRBC) antibody response stimulated by AA-2G. The effect of NGF plus AA-2G or AA-2G alone was abrogated by the presence of castanospermine, an alpha-glucosidase inhibitor, suggesting that the active form of AA-2G is ascorbate. The repeated additions; but not one addition, of ascorbate also resulted in a synergism with NGF on the antibody production. These results suggest that NGF might be a cytokine which functions as a regulatory factor for ascorbate-dependent immune responses.

Multidimensional liquid chromatography (MDLC) may be used in either (i) the profiling mode where it is the objective to fractionate all components in a mixture or (ii) the targeted component mode in which it is the objective to determine specific analytes. This paper focuses on targeted component analysis from complex mixtures, addressing the critical operations of analyte selection and transport from the first to the second dimension. Although the physical operation of switching a component into the second dimension with computer controlled valving is simple, it is shown that changes in analyte retention time and peak width with column age and fouling are a serious problem. The analyte moves out of the preselected time window for valve switching and quantitation is compromised in the second dimension. It is proposed that a solution to the “drifting peak” phenomenon in targeted component analysis is to use binary mobility elution in the first dimension. Binary mobility refers to those systems, such as affinity chromatography, in which analyte mobility is generally either 0 or 1 relative to mobile phase velocity. Coupling these binary changes in analyte mobility in the first dimension with valve switching eliminates the “drifting peak” phenomenon. In addition, it is shown that a wide time window may be used in affinity separations without compromising the separation or accumulating contaminants. Several cases are described in which immunosorbents were used with reversed phase columns to provide quantitative targeted component analyses from complex mixtures.

Interventional approaches that have been successful in delaying insulin-dependent diabetes mellitus (IDDM) using antigen-based immunotherapies include parenteral immunization. It has potential for clinical application provided that effective adjuvants suitable for human use can be found. We have previously shown that immunization with insulin and insulin B chain but not A chain in incomplete Freund's adjuvant (IFA) prevented diabetes by reducing IFN-gamma mRNA in the insulitis lesions. In this paper we show that the insulin B chain peptide (p9-23) contain the most protective epitope. Immunization with selected GAD peptides was ineffective. Immunization with B chain but not A chain using alum as adjuvant delayed diabetes onset (P = 0.012), whereas administration of alum alone was not protective. When Diphtheria-Tetanus toxoid-Acellular Pertussis (DTP) vaccine was used as the adjuvant vehicle, DTP itself induced significant protection (P0.003) which was associated with a Th2-like cytokine producing insulitis profile, IL-4 driven IgG1 antibody responses to insulin, GAD in the periphery and an augmentation of the autoimmune response to GAD. The anti-diabetic effect of DTP was enhanced when given with insulin B chain. These results encourage consideration of an approach using alum/DTP and insulin B chain immunization in clinical trials.

Stimulation of the mast cell line, RBL-2H3, with antigen via the tetrameric (alpha beta gamma 2) immunoglobulin E receptor (Fc epsilon R1) leads to the activation of cytosolic phospholipase A2 and the release of arachidonic acid. This pathway is dependent on the activation of the mitogen-activated protein (MAP) kinase. In this paper, we show that the MAP kinase/cytosolic phospholipase A2 pathway is linked to Fc epsilon R1 via the cytosolic tyrosine kinase, Syk, and that the GDP/GTP exchange factor, Vav, might be one candidate for accomplishing this link. Cross-linking of transmembrane chimeras containing the Fc epsilon R1 gamma motif, which is known to activate Syk, results in the tyrosine phosphorylation of Vav, activation of MAP kinase, and release of arachidonic acid. Cross-linking of chimeras containing the Fc epsilon R1 beta motif does not cause these events. Furthermore, stimulation of these events by antigen is enhanced by transient overexpression of a wild-type form of Syk and blocked by overexpression of a dominant negative form of Syk. By contrast, stimulation via the transfected, G protein-coupled, muscarinic m1 receptor is not influenced by either form of Syk and does not result in tyrosine phosphorylation of Vav. These data establish unequivocally that the two types of receptor are independently linked to the two types of receptor are independently linked to the MAP kinase/cytosolic phospholipase A2 pathway and demonstrate the existence of the Fc epsilon R1-Syk-MAP kinase pathway.

This paper discusses the assumed infinite numbers of antigens and antibodies, and the nature of their interactions, in relation to reproductive immunology. Classical models offered by Pauling, Burnet and Crick are discussed. The author's own model is described, based on selectionist principles, proposing that the numbers are defined, discrete and not infinite. The binding specificity of monoclonal antibodies of four to six amino acids limits the number on the total number of antibody-binding peptides. This limitation on size enables the total number of these peptides to define ‘one-hit' or ‘two-hit' protein sequences in databases of known proteins. Estimate of the combinations of variable heavy and light chains of different numerical magnitude is 2.5 × 10 6 . The author proposes the equivalence principle, where equivalent numbers of antigens and antibodies exist and theorizing a limited number of antigen–antibody pairs in the magnitude of 3.2 × 10 6 or slightly less. The nature of various protein–ligand interactions, indicated by their limited numbers of interactions, clarifies why a particular amino acid is encoded by a particular codon and the nature of encoding by tRNA. In relation to reproductive phenomena, specific proteins for contact with sperm ligands were designed using these parameters.

This paper provides selected personal insights on the development of vaccines against blood-sucking arthropods, with particular emphasis on vaccines against ticks. The emergence of novel or concealed antigens of haematophagous ectoparasites as candidate vaccine antigens is reviewed and the effect of feeding by the parasite on the expression of protective antigens is considered. The distribution of protective antigens through life cycle stages, the stage of the life cycle targeted by protective responses, and the nature of these responses, are commented on briefly. Concealed antigens of the gut, including the peritrophic membrane, and other internal organs, are evaluated for the role they play in induction of immunity artificially. Some of the work carried out to purify and characterise protective antigens of tick guts is described. A commentary is developed on vaccines that combine both "concealed" and "exposed" antigens. Some of the problems associated with the infestation and challenge of vaccinated hosts in the field are identified and the delivery of parasite antigens as vaccines that are both protective and "user-friendly" is emphasised as a major problem to be solved.

Enteric disease remains one of the greatest causes of mortality and morbidity in both human and veterinary species. There has been a remarkable lack of success in vaccination to control mucosal disease and it is therefore apparent that novel strategies are required to achieve effective mucosal immunity. Basic studies described in this paper have addressed problems associated with antigen handling and the induction of an immune response in the intestine, and the subsequent dissemination of effector cells and molecules to intestinal and extra-intestinal submucosal regions. Effective induction of IgA responses is dependent on T-cell help and requires cognate interactions between T cells and B cells within organized gut-associated lymphoid tissue (GALT). The delivery of an IgA antibody response to mucosal sites is also a T cell dependent but antigen driven process. The normal route by which antigen is taken up by GALT is via the epithelial surface but antigen presented in this way via villus epithelial cells generates predominantly a suppressor response. Strategies designed to overcome this effect include the use of powerful adjuvants (such as cholera toxin, muramyldipeptide and phorbol esters), the use of immunogenic carriers, or delivery via liposomes, microspheres or genetically engineered viral or bacterial vectors. Alternatively, the feasibility of accessing GALT via the serosal surface by administration of intraperitoneal antigen in oil emulsion has been explored and a vaccine formulation (Auspharm (patent pending)) has been developed which is suitable for IP delivery in commercial applications.

A nasal lavage model and a new filter paper disk model have been used to measure biologic and physiologic responses to antigen challenge in patients with allergic rhinitis. Pretreatment of subjects with cetirizine reduced the number of sneezes induced by antigen challenge but did not significantly reduce levels of histamine or prostaglandin D2 in a double-blind, placebo-controlled trial with the lavage model. Pretreatment with 60 or 300 mg of terfenadine did significantly reduce levels of histamine, kinin, albumin, and TAME-esterase activity in a double-blind, placebo-controlled study with this model. Again with the nasal lavage model, a double-blind, placebo-controlled comparison of pretreatment with 60 mg of terfenadine or 10 mg of loratadine showed that both agents significantly reduced sneezing. Both drugs also lowered levels of antigen-induced histamine and TAME-esterase, but only terfenadine did so significantly. In a double-blind, placebo-controlled study, the new disk method showed that terfenadine reduced sneezing but not nasal congestion in eight patients with allergic rhinitis. Terfenadine significantly reduced the weight of nasal secretions on both sides of the nose and significantly reduced histamine on the ipsilateral side.

The elevation of free intracellular Ca 2+ activity ([Ca 2+ ] i ) is widely recognised as a central event in many signal transduction processes in cellular physiology. Recent advances in optical techniques for measuring [Ca 2+ ] i as well as developments in quantitative low light level fluorescence microscopy have led to the application of these methods to the study of subcellular [Ca 2+ ] i in many biological systems. In the following paper we describe some techniques in our laboratory to provide quantitative high spatio-temporal resolution measurements of [Ca 2+ ] i in individual living cells during the signal transduction of cell surface receptor ligand interactions. In particular, we are studying the changes in [Ca 2+ ] i induced by the micro-aggregation of immunoglobulin E (IgE) receptor complexes on the surface of rat basophilic leukemia (RBL) cells (a tumor mast cell line) by multivalent antigen [1]. We seek to understand the mechanisms which are involved in the detection of these cell surface events which lead to changes in [Ca 2+ ] i as well as the interactions between the various subcellular components which impart the delicate control of [Ca 2+ ] i during cellular stimulation. The limitations and properties of the technology used for these studies will be discussed, and some illustrative examples of the type of [Ca 2+ ] i changes found in this biological system will be given.

which, if suitable for centrifugation, are not suitable for other biochemical manipulations. This necessitates switching to centrifuge tubes for the centrifugation steps and back again, involving unnecessary delay. Many commercial makes of tubes may be adapted for centrifugation if a suitable adapter can be found. This paper describes a quick and economical method for making custom-made adapters which are suitable for the numerous low-speed centrifugation tasks required for biochemical techniques. Materials and methods. The adapters were made for a colleague who required sucrose step gradients to be set up in numerous plastic centrifuge tubes. These tubes were of an unknown make but were required to fit into a Hettich universal benchtop centrifuge (Hettich Zentrifugen, D-7200 Tuttlingen, Germany) fitt.ed with four swing-out buckets. The material used was silicone rubber 3120RTV red (Dow Corning Corp., Midland, MI). This comes supplied in a l-lb tin together with a catalyst (S standard curve). The insides of the swing-out buckets were covered with a rolled layer of transparent acetate film to allow some clearance. To add air channels, V-shaped struts may be lightly tacked into place onto the transparent film. Uhu adhesive or Cow gum is suitable for these temporary joints. The silicone rubber was thoroughly mixed with the catalyst at a ratio of lO:l, using a wooden spatula, and poured into the buckets to a predetermined height, allowing for displacement of some of the silicone rubber by the cent,rifuge tubes. Because numerous centrifuge tubes were required, it was decided to fit three tubes into one bucket. These tubes were taped together and inserted into the silicone rubber. The buckets were placed close to each other. A glass plate with a weight was placed on top of the centrifuge tubes to align and keep

Although the phenomenon of immunodominance of epitopes within protein antigens is well established, immune responses in which antigen persists are characterized by a great diversity of Tand B-cell responses. This is observed especially in chronic infection and autoimmunity. In this brief paper, we would like to propose a mechanism for diversification of immune responses based on the unique ability of the B cell to present the diversity of peptides from an antigen or antigenic complex required for a diverse CD4 T-cell response based on our recent studies on the role of B cells in antigen presentation to CD4 T cells in vivo (Refs 1,2 and M.J. Mamula, S. Fatehnejad and J. Craft, submitted). The activation of CD4 T cells by an exogenous protein antigen requires preseotation by a cell that can internalize the antigen, process it, and present it on the surface bound to major histocompatibility complex (MHC) class II molecules. The same APC must provide suitable costimulatory signals, the best characterized of which is the CD28 ligand B7/BB1 ~'a. Much recent data supports a role for dendritic cells (DC) in the initiation of CD4 T-cell responses to protein antigens in vWO s-7. However, earlier studies suggested that B cells were important for the induction of recall proliferative responses to protein antigens, in that mice treated from ~-;'h --';'~" anti-la ~'-: . . . . :,._a.. o-co. . . . . . . . . . . .s~a~t~ a ~ t ~ u o u y tO suppress n __,, development had defective T-cell proliferative responses that could be restored with antigen-specific B cells 8-1°. It is important to note, however, that several studies have shown that such B-cell deficient mice have normal levels of MHC class II restricted CD4 helper T cells n. Thus, these studies suggest that B cells are required for some but not all aspects of CD4 T-cell priming. Dendritic cells have been shown to be the most potent stimulators of CD4 T-cell responses to non-self MHC molecules 5, and as they constitutively express B7/BB112 and high levels of MHC class II molecules s, they are also excellent candidates for initiating CD4 T-cell responses to extrinsic antigens. However, DC are quite inefficient at antigen uptake and lack a means for discriminating between extrinsic protein antigens and self proteins whose peptides compete with them for presentation. As it is estimated that 100-200 MHC class II peptide complexes are required to signal a CD4 T cell, and as a DC has around 10 s MHC class II molecules, a maximum of 1000 peptides could be presented by such a cell. Moreover, direct evidence shows that some peptides are very much overexpressed on DC ~3, suggesting even less complexity. Thus, it seems likely that extrinsic antigens will be presented relatively inefficiently by DC, consistent with the very high protein doses required for effective presentation of peptides by the MHC class II molecules of DC 6. Thus, only those peptides that bind extremely well to the MHC class II alleles of the DC are likely to achieve levels on the cell surface sufficient to prime a helper CD4 T cell. This probably accounts for the phenomenon of immunodominant peptide epitopes in primary CD4 T-cell responses. Once helper CD4 T cells are primed to immunodominant peptides on DC, they will in turn activate specific antigen-binding B cells that internalize antigen 10 000-fold more efficiently than non-specific cell¢ 4"1~. When these antigen-binding B cells are activated by the primed antigen-specific helper T cells, they become competent to prime naive CD4 T cells. Because of the large amounts of protein internalized by the antigen-specific B cell, a much greater range of peptides from the original protein, or from proteins associated with it, can be presented to these naive CD4 T cells. Thus, the antigen that binds to the B cell can diversify the CD4 T-cell response. Recent studies from Eynon and Parker I~ and from Fuchs and Matzinger ~7 demonstrate that when resting B cells present antigen to CD4 T cells, tolerance resu!ts, an observation thought to reflect the absence of co-stimuiatory activity on resting B cells. By contrast, B cells activated with a variety of microbial substances can stimulate naive CD4 T-cell proliferative responses in vitro ~s, although Fuchs and Matzinger ~ find that even LPS-acti

As will be apparent from a reading of the papers included in this issue, one reagent that has been used extensively in assessing the effects of the beta/A4 peptide on rodent neurons is the monoclonal antibody Alz 50. A variety of model systems have been employed, in which beta/A4 is delivered to neurons both in vitro, and in several different ways, in vivo. While Alz 50 can be a useful reagent in these studies, there are limitations to the utility of this antibody, and it is important to recognize that appropriate controls must be performed to establish the specificity of observed immunoreactivity.

Because crossed immunoelectrophoresis (X-IEP) is quantitative and offers more information than immunoelectrophoresis (IEP), we selected X-IEP to study various physiologic and pathologic effects on the antigen composition of mouse serum. We therefore needed and X-IEP map of normal mouse serum. This paper presents one that we have developed, along with brief descriptions of how the mouse serum antigens were identified. Cross-reactivity between several mouse and human serum antigens was especially helpful. Some data from using ratios of the areas of precipitation of detected antigens to that of an internal standard antigen, alpha-macroglobulin, are presented. They show that X-IEP readily detects quantitative abnormalities among any of the several serum antigens detected by this technique, and that 'normal' values for these antigens in one population of mice can be established in the same way of using ratios with just a few analyses.

We have isolated poly(A)+ RNA from four antigenic variants (117, 118, 121, 221) of one clone of Trypanosoma brucei. Translation of these poly(A)+ RNAs in a rabbit reticulocyte lysate gave rise to proteins that could be precipitated with antisera against homologous variant surface glycoprotein, the protein responsible for antigenic variation in trypanosomes. From the electrophoretic mobility of these in vitro products in sodium dodecyl sulphate (SDS) gels we infer that variant surface glycoproteins (VSGs) are made as pre-proteins, which require trimming to yield mature VSGs. The total translation products from the four poly(A)+ RNAs produced a complex set of bands on SDS gels, which only differed in the region where the variant pre-glycoproteins migrated. The only detectable variation in the messenger RNA populations of these variants is, therefore, in the messenger RNA for variant pre-glycoproteins. We have made duplex DNA copies of these poly(A)+ RNAs, linked the complementary DNA to plasmid pBR322 by GC tailing and cloned this recombinant DNA in Escherichia coli. Colony hybridization with complementary DNA made on poly(A)+ RNA showed that 7--10% of the colonies contained DNA that hybridized only with the homologous probe. Plasmid DNA was isolated from ten such colonies (two or three of each variant complementary DNA), bound to diazobenzyloxymethyl-cellulose (DBM) paper and used to select complementary messenger RNA from total poly(A)+ RNA by hybridization. In eight cases the RNA recovered from the filter gave variant pre-glycoprotein as the predominant product of in vitro translation. Poly(A)+ RNA from each of the variants only hydridized to the homologous complementary DNA in filter hybridizations. Each trypanosome variant, therefore, contains no detectable messenger RNAs for the three heterologous variant-specific glycoproteins tested. We conclude from this lack of cross-hybridization that antigenic diversity in trypanosomes, unlike antibody diversity in mammals, does not involve the linkage of a repertoire of genes for the variable N-terminal half to a single gene for the C-terminal half of the VSGs.

The probability distributions for the number of bound antigens and antibodies during immune response are obtained in this paper. Biological significance of this work and directions for further application are discussed along with some illustrative numerical results.

This is a continuation of our earlier studies on antigen handling and the ageing process which have shown the liver to be a major site of antigen retention in the body. This paper reports on the uptake and cellular distribution in relatively pure isolates of the principal cell types of the liver of [35S]sulfanilate-azo-bovine serum albumin (35S-BSA) injected into F-344 rats of ages 4, 12 and 28 months. In all three cell types [hepatocytes (H), Kupffer cells (K) and endothelial cells (E)] antigen retention peaks in the 12-month age class and then exhibits a significant decline in the 28-month age class; K and E cells retain more antigen per cell than do H cells. All three cell types metabolize antigen to nucleopeptides and this transformed antigen is immunologically active; K and E cells metabolize antigen to nucleopeptides more effectively than do H cells. A significant age-related difference is found when nucleopeptides from either H, K or E cells are used to stimulate an antigen-specific lymphoproliferative response in vitro: nucleopeptides from 4-month and 12-month rats stimulate the proliferation of lymphocytes to a significantly higher degree than do nucleopeptides from 28-month old rats. The experiments therefore identify in all three cell populations an age-related defect in antigen uptake and in metabolism of antigen to bioactive nucleopeptides, and they demonstrate that the nonparenchymal cells are more effective in antigen retention and in metabolism of antigen to nucleopeptides than are parenchymal cells. Further, the experiments support the thesis that the liver must be considered to be a principal part of the total immune system, its role much more fundamental than heretofore recognized.

The experiments described in this paper demonstrate that the ability of primed T cells to provide helper function is irradiation resistant in situ and in vitro, but not in vivo following transfer to another animal. Mice primed with antigen and then lethally irradiated developed humoral immune responses when injected with bone marrow cells and erythrocytes as antigens. Since reconstitution of immunocompetence was not dependent on injection of T cells, this would indicate the existence of irradiation-resistant T cell function in primed mice. Further experiments showed that priming to irradiation-resistant T cell function in situ could be accomplished with as few as 102 sheep red blood cells or 104 goat red blood cells injected intravenously. Priming appears to optimal when done 3–10 days before irradiation, and it appears to be ineffective 25 days or more before irradiation. Primed mice developed both direct and indirect plaque-forming cells. These data are compatible with the idea that irradiation interferes with the ability of primed T cells to migrate or home correctly, but not with their ability to provide helper function.

Summary Blood group antigens are widely distributed in human tissues. Numerous studies were carried out on adult kidneys, the aim of this work being to compare the distribution of ABH, I, i, Lewis and Pr antigens in adult kidneys with that of three month embryos. In actual fact, at this stage of development, the same structures as those observed in the adult can be seen. The study was carried out by means of the indirect immunofluorescence technique. In the adult, ABH antigens were evidenced on the cells of the vascular endothelium and in the distal, and collecting tubules of secretor subjects only. Lewis antigen was absent in the vascular structures but present in the cells of the distal and collecting tubules of all the subjects with the same Le gene. A high marker heterogeneity was noted inside a same tubule. I and i antigens were observed in the membrane and cytoplasm of some cells from the thick part of the loop of Henle and in some distal and collecting tubule cells. Pr antigen was present in the glomerulus and the distal and collecting tubules. In the three month fetus, ABH and Pr antigens were observed in the same structures. I antigen was absent, whereas i antigen was present in the cells of the distal and collecting tubules. However, the expression of the Lewis specificities was virtually inexistent. Therefore, at the age of three months, when the development of the organs is finished, the embryonic tissues possess the same markers as the adult with the exception of I and Lewis antigens. The results observed in the kidneys of the 14 week fetuses were similar to those observed in the adults (8, 10). Nevertheless, the staining of the apical membrane of the cells appeared stronger than in the adults. Lewis antigens were not shown on the endothelial cells of all the vessels. These antigens were only present in the membrane and cytoplasm of distal and collecting tubules [5]. Whilst the Lewis enzyme was clearly demonstrated in the glomerulus from Lewis positive kidneys, Lewis antigen was not found in the glomerulus [5, 9]. Although A, B, and H antigens were present on some kidney structures of the three month fetuses, Lewis antigens were only found in a small quantity. I and i antigens were only studied on the adult kidneys. In a former study, Feizi [2], using the method of inhibiting precipitation, did not evidence I and i antigens in the kidney. Moreover, in a recent paper, using indirect immunofluorescent techniques, Lenhart et al. [4] showed I and i antigens in different structures of adult kidney. As regards localization, our results come close to those observed by Lenhart et al. [4]. I and i antigen were also found mainly in the cytoplasm and on the surface of the epithelium of distal tubules ; no I and i antigens were found in the glomerulus. The fetuses of three months were completely lacking in I antigen. Whilst i antigen was expressed, its strength seems almost identical to that found in the adult. In contrast to Lenhart et al. [4], Pr antigens were found in the collecting and distal tubules. Nevertheless, some anti-Pr gave a very slight staining of the tubule, whereas the glomerulus were distinctly positive. Thus the main difference between the adult and fetus kidneys was the absence of I and Lewis antigens on the epithelium of the distal and collecting tubules.

This paper deals with the specificity of the anti 3′,5′-cyclic AMP antibodies which can be obtained with 2′- O -succinyl cyclic AMP-albumin as an immunogen. The binding of the hapten and its analogs was measured by equilibrium dialysis. Rat and rabbit antibodies were compared. In both cases the best ligands for the anti-hapten antibodies are 2′- O -acylated derivatives of cyclic AMP: the dissociation constants are below 10 −10 m . Cyclic AMP itself and its 6 N, 2′- O -diacylated derivatives are recognized less efficiently; their dissociation constants lie around 10 −8 m , similar to that of natural cyclic AMP binding proteins. Other nucleotides lacking either adenine or the 3′,5′-phosphate ring are not recognized. Three different populations of antibodies were detected by a more detailed analysis of the equilibrium curves.

There is an antigenic glycoprotein (Mr 48 kD) present on the surfaces of erythrocytes of embryonic and young chickens that cannot be detected on the circulating erythrocytes in adult birds. This antigen, generally defined by this differential expression, has been thought to be associated with the maturation of hematopoietic tissues. We now present evidence, based on the use of a monoclonal antibody, maEE1, and the characteristic pattern of this glycoprotein on two-dimensional (2D) gels, that this antigen, which we have named chickEE, is expressed in a number of other embryonic and adult tissues. Immunofluorescent labeling of cryosections and flow-cytometric analysis of cells labeled with maEE1 have revealed the presence of chickEE in the retina (present in all layers), in muscle tissues (present in the endomysium and within the vascular endothelium), in the liver (especially evident on the lateral surface of hepatocytes and within the sinusoids), on epithelia such as the gut and kidney tubule epithelium and within lymphoid organs (present on bursacytes, splenocytes, thymocytes and peripheral leukocytes, and again within the endothelium) of young and adult animals. The 2D gel patterns of chickEE derived from embryonic tissues (retina, hind limb, thymus and bursa) and the adult tissues (retina and spleen) are very similar to that of the embryonic erythrocyte. Thus, the extended reactivity of the monoclonal antibody to chickEE, maEE1, with additional tissues is, in at least the tissues examined, based on the presence of the chickEE glycoprotein and not on incidental cross-reactivity. The evidence presented in this paper for the widely-shared expression of chickEE antigen makes it necessary to reconsider the function of this component of the cell surface.

Antibodies directed against centromeric chromatin characteristically occur in the sera of patients with the CREST variant of scleroderma. We have studied the in situ enzymatic sensitivity and solubility of the centromeric antigen and have isolated an antigenic moiety that reacts with anticentromere antibodies. The centromeric antigen in the human epithelial cell line, HEp-2, was sensitive to DNAase I and micrococcal nuclease but not affected by RNAase A, trypsin or amylase. It was insoluble in 0.15-4 M NaCl but was extracted from the HEp-2 cells by 4 M urea/2 M NaCl. Antigenic activity in a 4 M urea/2 M NaCl extract of rabbit thymus was demonstrated by immunoabsorption. Indirect immunofluorescence of the extract separated by polyacrylamide gel electrophoresis revealed a fluorescent band with a mol. wt of 33,000. Calf thymus and trout testis histone preparations were fractionated by gel electrophoresis and transferred by blotting techniques to diazobenzyloxymethyl cellulose paper for autoradiography. Anticentromere antibodies bound to and were absorbed by trout testis histone 1. We propose that the centromeric antigen may be a variant of histone 1 that is associated with condensed chromatin.

The interaction between the antigens of Fasciola hepatica and the host immune response are reviewed. This paper evaluates not only more recent work, but the older literature as well. Antigens from each stage in the life cycle are considered with the idea of identifying those antigens with a potential for use in an effective vaccine. Antigens which cross-react with other parasite species are detailed as well as those that cross-react between different stages in the life cycle of F. hepatica. The objective of the review is to demonstrate for other investigators that vaccination against F. hepatica is a distinct possibility. We hope to encourage more investigators to initiate work on this aspect of an economically-important cosmopolitan parasite.

Tubulointerstitial nephritis (TIN), as studied experimentally, can be induced by many of the same humoral immune mechanisms now recognized to cause immune forms of glomerular injury [reviewed in 1–5] (Table 1). In addition, there is compelling evidence of direct cellular immune involvement in some forms of TIN. In general, however, less is known about the immunopathogenesis of human TIN than its glomerular counterpart. For example, the specific mechanisms responsible for many of the instances of TIN associated with drug hypersensitivity remain unclear. Much of what is known comes from the study of experimental models of TIN. In addition, these model systems are being used to forward the understanding of control of nephritogenic immune reactions [6–10]. This paper will focus on a discussion of the different immune mechanisms that have been shown to contribute to TIN in experimental models and their extension to human TIN as appropriate.

The epidermo-dermal junction ultrastructurally appears to be high organized, presenting 4 layers: (1) the basal cell plasma membrane, a trilaminar layer with special attachment structures (hemidesmosomes); (2) lamina lucida (rara) an electron-lucent zone with 'anchoring' filaments (vide infra); (3) basal lamina (densa) electron-dense and relatively thick (30-50 nm) and (4) the subbasal fibrous area, composed of 3 different fibrils: (a) 'anchoring fibrils' meshing with the lamina densa upwards and forming and interlocking (a) meshwork with the adjacent anchoring fibrils downwards; (b) dermal microfibril bundles starting from the basal lamina and passing deep in the dermis. The dermal part of these, are in connection with the elastic fibers and ultrastructurally they are identical and; (c) collagen fibers, randomly oriented without connection with the basal lamina. The biochemical composition comprises collagenous and noncollagenous substances: collagens can be divided into 3 groups designated as type IV collagen, type V(AB collagen) and 7S collagen. The non-collagenous materials as yet identified are: bullous pemphigoid antigen (reactive with the bullous pemphigoid antibodies), laminin, heparan-sulfate containing proteoglycan and amyloid P-component (a normal plasma constituent). Functional aspects of basement membrane components: (1) support of a caffolding function assumed by the collagens; (2) attachment of cells of different embryonic origin (fibronectin, PB antigen and laminin); (3)regulation of the permeability (heparan-sulfate proteoglycan and other glycosaminoglycans); and (4) a role in development and morphogenesis (laminin) and type IV collagen). This paper must be read in extenso by those interested in the microstructure and functions of the skin. (I. Capusan - Cluj)

The practical realization of the concept of specific immunotherapy for systemic lupus erythematosus (SLE) has been hampered, thus far, by an inability to link DNA fragments to carrier protein. In this paper, a novel technique is described, in which glutaraldehyde is the linking agent. A 2-stage method was used to link oligonucleotides to a soluble protein carrier, such as keyhole limpet hemocyanin (KLH) or human gamma globulin (HGG), whereas a 1-stage technique was sufficient to link oligonucleotides to sheep red cells. Both the ultraviolet absorbance spectrum and diphenylamine assay demonstrated that oligonucleotides were coupled to soluble protein. The conjugate of oligonucleotide to protein carrier appears to be recognized by anti-DNA antibody since oligonucleotide linked to either KLH or HGG inhibited the binding of anti-DNA antibody in vitro, and oligonucleotide-coupled sheep cells are agglutinating by seropositve sera from lupus patients. In addition, oligonucleotide-KLH raised hemagglutinating antibody to denatured DNA in C57BL/6, DBA/2 or NZB mice, as well as IgG antibody as detected by SPRIA in C57BL/6 and DBA/2 mice. The significance of this new method for the development of an antigen specific therapy of SLE is discussed.

This paper describes a radioimmunoassay for the B beta 15-42 peptide derived from human fibrinogen or fibrin. Iodinated B beta 15-42 is bound by specific antiserum and binding can be completely inhibited by excess of the non-iodinated B beta 15-42, B beta 1-42 or B beta 1-118. These peptides cannot be distinguished in this assay. Furthermore, fibrinogen can also completely inhibit binding of iodinated B beta 15-42 peptide. Due to the cross-reaction with fibrinogen, clinical blood samples require a processing step prior to their use in this radioimmunoassay. Ethanol precipitation allows for fibrinogen removal and a near quantitative recovery of both added B beta 15-42 peptide as well as of the endogenous blood peptide(s) containing the B beta 15-42 sequence. The mean level of B beta 15-42 immunoreactive material in normal individuals was found to be 0.41 pmol/ml while that in one group of patients was 20-40 times this value.

This paper summarises the evidence for the involvement of the immune response in the development of neoplasia, discusses the immunological abnormalities found in patients with head and neck carcinoma, and outlines recent attempts at treatment of patients with head and neck carcinoma by modulation of the immune response (immunotherapy).

The use of Sepharose beads in the defined antigen substrate sphere (DASS) immuno-fluorescent system has been restricted by the need for sophisticated equipment for fluorescence quantitation since reliable subjective reading is not possible. This paper describes a method for visual assessment of results in this system. Antigen is coupled to sodium metaperiodate activated cross-linked Sepharose 4B beads and blocked with rhodamine-BSA prior to reduction. This procedure eliminates the use of the hazardous cyanogen bromide activation process and is potentially suitable for use in both direct systems for evaluation of specificity and potency of fluorescent conjugates and in indirect systems for serological screening of sera for specific antibody.

Secondary delayed type hypersensitivity (DTH) to sheep red blood cells (SRBC) in mice is a long-lived memory phenomenon which is characterized by the accelerated reappearance of the state of DTH after a booster injection of the antigen. In this paper the nature of the DTH-related T memory cells accounting for secondary DTH was investigated. Parabiosis of primed and nonprimed mice for a period of 4 weeks resulted in an equally large secondary DTH responsiveness in both partners. This ability was maintained in both members for at least 6 months after termination of the parabiosis. These results indicate that (a) DTH-related T memory cells are potentially circulating cells, and (b) the persistence of these memory cells is not dependent on the presence of the antigen which induced their generation. Subcutaneous (sc) injection of intravenously (iv) primed mice with a small dose of antithymocyte serum before boosting did prevent the development of secondary DTH responsiveness in sc boosted mice, but not in iv boosted mice. Treatment of primed mice with vinblastine or azathioprine did not decrease the capacity of adoptive transfer of secondary DTH by means of spleen cells, but passive transfer of secondary DTH was completely abolished by this treatment. These results suggest that (a) SRBC-induced DTH-related T memory cells are nonproliferating, partially sessile, partially recirculating cells, and (b) these memory cells proliferate before they become DTH-related effector cells.