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By substituting the heavy chain constant region of IgM and IgD with that of IgG, IgA or IgE, immunoglobulin class switching endows antibodies with novel effector functions that enhance the ability of the immune system to effectively clear invading pathogens. Plasmacytoid dendritic cells critically link innate immunity with adaptive immunity by producing massive amounts of type 1 IFN in response to viruses. We have recently found that type 1 IFN triggers class switching by inducing myeloid dendritic cells to upregulate the expression of BAFF and APRIL, two powerful B cell-activating molecules. In this paper, we propose that IFN-producing plasmacytoid dendritic cells modulate class switching by activating B cells through both T cell-dependent and T cell-independent pathways. A better understanding of these pathways may facilitate the development of novel antiviral vaccine strategies and aid in identifying new therapies for antibody-mediated autoimmune disorders, such as lupus. [ABSTRACT FROM AUTHOR]

SummaryOver 20 years ago, Coutinho and Möller reported that high concentrations of LPS were paralytic for the development of antibody secreting cells (ASC). This data was used to explain bell-shaped dose–response curves observed for antihapten antibody formation in response to haptenated LPS. In turn, this bell curve was used to formulate the one-signal model of B cell activation, which argued that antigen signalling was generally unimportant to B cell responses. The present paper re-examines LPS dose–response curves and finds results that do not support the view that high doses of LPS inhibit B cell differentiation to ASC. If high-dose paralysis is not an attribute of LPS stimulation, then the bell-shaped dose curve for hapten-specific ASC originally observed by Coutinho and Möller required an alternative explanation. Through the use of haptenated Ficoll, it was possible to show that the generation of LPS-induced antitrinitrophenol ASC could be inhibited by antigen presented on an inert substrate. Thus, the transmission of surface Ig-mediated (antigen) signals at higher concentrations can explain the antihapten bell-shaped dose curves, in contradiction to the conclusions of the one-signal model. [ABSTRACT FROM AUTHOR]

Antibodies to hormones have been used extensively to study endocrine regulation of reproduction and in recent times they have been commercially exploited as therapeutic agents to manipulate reproduction. Despite the release of two commercial products, little is known about the mechanism of action of immunoneutralization. The presence of hormone-binding antibodies in the circulation and the detection of hormone antibody complexes in blood has led investigators to suggest a vascular site of action for immunoneutralization. However, a vascular site of action is difficult to reconcile with some of the known effects of immunoneutralization to the boar taint steroid (5 α-androst-16-en-3-one) to androstenedione and to gonadotrophin-releasing hormone. In this paper we review some of our recent research into the mechanism of action of androstenedione immunoneutralization. We suggest that hormonal immunoneutralization of androstenedione may take place in the extracellular compartment or within the plasma membrane where antibodies to androstenedione may bind to a specific steroid-binding protein in the plasma membrane of granulosa cells. [ABSTRACT FROM AUTHOR]

We have previously reported that erythropoiesis commences in the liver and spleen after malarial infection, and that newly generated erythrocytes in the liver are essential for infection of malarial parasites as well as continuation of infection. At this time, erythropoietin (EPO) is elevated in the serum. In the present study, we administered EPO or anti-EPO antibody into C57BL/6 (B6) mice to modulate the serum level of EPO. When mice were infected with a non-lethal strain (17NXL) of Plasmodium yoelii (blood-stage infection of 104 parasitized erythrocytes per mouse), parasitemia continued for 1 month, showing a peak at day 17. Daily injection of EPO (200 IU/day per mouse) from day five to day 14 prolonged parasitemia, whereas injection of anti-EPO antibody (1.5 mg/day per mouse) every second day from day five to day 28 decreased it. Erythropoiesis was confirmed in the liver, spleen and bone marrow by the appearance of nucleated erythrocytes (TER119+). When anti-EPO antibody was injected by the same protocol into mice infected with a lethal strain (17XL) of P. yoelii, all mice showed decreased parasitemia and recovered from the infection. These results suggest that the use of anti-EPO antibody after malarial infection may be of therapeutic value in severe cases of malaria. [ABSTRACT FROM AUTHOR]

The present paper describes the physiological properties of recombinant possum TNF-α and an adjuvant effect on antibody responses to the model protein antigen, keyhole limpet haemocyanin (KLH). For these studies recombinant possum TNF-α was produced in the yeast Pichia pastoris. The recombinant cytokine was secreted into the culture medium and purified by gel filtration. Possum TNF-α produced in this expression system was N-glycosylated and bioactive in two different assays. In a murine fibroblast L929 cytotoxicity assay, the possum TNF-α had lower specific activity compared to human TNF-α, while in a possum-specific assay, possum TNF-α enhanced the proliferation of PHA-stimulated possum thymocytes and was more active than human TNF-α. The physiological effect of the recombinant possum TNF-α was investigated in groups of possums administered doses of 6, 30 or 150 μg of cytokine. For each dose, TNF-α caused profound effects on the numbers of circulating leucocytes characterized by a three-to-four-fold increase in neutrophil numbers at 6–24 h after injection and an initial sharp decrease in lymphocyte numbers. The efficacy of TNF-α as an immunological adjuvant was determined in possums administered KLH (125 μg) in an aqueous or Al(OH)3-based formulation with or without added recombinant TNF-α (150 μg). Serum antibody responses to KLH were monitored by ELISA. The TNF-α stimulated two-fold and four-fold increases in antibody levels in aqueous and Al(OH)3-based vaccine formulations, respectively. The strongest antibody responses were observed in the group of possums that received KLH formulated in Al(OH)3 with addition of TNF-α. [ABSTRACT FROM AUTHOR]

This paper reports the production and characterization of three monoclonal antibodies (mAb) recognizing ovine vascular cell adhesion molecule-1 (VCAM-1). The mAb were raised against sheep umbilical vein endothelial cells (ShUVEC) and flow cytometric analysis demonstrated that one mAb, QE4G9, was crossreactive with human VCAM-I expressed on Chinese hamster ovary cell transfectants. Protein modulation studies on ShUVEC and immunoperoxidase staining of inflamed renal tissue further indicated the reactivity of the other two ovine mAb. QE1E3 and QE2G4, with ovine VCAM-1. The flow cytometric profile of the three mAb on stimulated ShUVEC was identical to that observed with the cross-reactive anti-human VCAM-1 mAb, HAE2-1. Peak expression occurred between 6-12 h after stimulation, followed by a slight decrease to a plateau extending beyond 48 h. In functional assays, all mAb inhibited adhesion of PMA-activated sheep PBMC to stimulated ShUVEC. In addition, QE4G9 inhibited proliferation of sheep PBMC in the mixed lymphocyteendothelial cell reaction (MLER) by 56%. The results demonstrate that the three anti-ovine VCAM-1 mAb recognize functional epitopes on sheep vascular endothelial cells. These mAb will be valuable tools in the investigation of VCAM-1 expression in various pathophysiological conditions using sheep models, and in the study of VCAM-1-mediated leucocyte-endothelial cell interactions, both in vitro and in vivo. [ABSTRACT FROM AUTHOR]

CD2 is expressed on murine B cells, probably as a result of a chromosomal translocation event during speciation. There are no activating antibodies to mouse CD2, but when activated non-specifically, only T cells up-regulate their expression of CD2. We investigated the expression and function of CD2 on B cells using mice transgenic for human CD2 under the control of a modified version of the autologous promoter/enhancer that included the immunoglobulin enhancer. This construction directs expression of human CD2 to the B cell compartment as well as to the T cell compartment. In this paper, we confirm that activating pairs of anti-CD2 monoclonal antibodies are mitogenic for mouse T cells transgenic for human CD2. In contrast, mouse B cells that express similar amounts of human CD2 are not stimulated to proliferate by equivalent doses of these antibodies. We were also unable to show any functional consequence for these B cells as a result of CD2 ligation. [ABSTRACT FROM AUTHOR]

The predominance of IgA antibodies in mucosal sites reflects a combination of high rate IgA isotype switching among precursor cells in induction sites, their selective localization in mucosal effector tissues and vigorous proliferation of these cells after extravasation. Each of these steps leading to IgA expression at the mucosa is under cytokine control. This paper will address the role of cytokines in induction and expression of IgA responses and strategies for manipulating cytokine expression. Therapeutic interventions based on this information may not only lead to improved vaccination responses and correction of immunodeficiencies but also, by invoking the phenomenon of oral tolerance, may assist in the management of autoimmune, allergic and alloreactive disease. [ABSTRACT FROM AUTHOR]

We have previously identified the major antigen of elastin-associated microfibrils as a 31kD glycoprotein which we named microfibril-associated glycoprotein or MAGP. Affinity-purified antibodies to MAGP were shown to localise specifically to elastin-associated microfibrils in sections of bovine foetal nuchal ligament (20). In the present paper we compare the localisation of anti-MAGP antibodies and antitropoelastin antibodies in a range of bovine elastic and non-elastic tissues. The results show that anti-MAGP antibodies invariably localised to immuno-reactive elastic fibres, wherever they occurred. Extensive additional localisation was observed in a number of tissues. This extra distribution of anti-MAGP antibodies was found to correspond to those structures exhibiting the oxytalan histochemical staining reaction in tissues such as skin, periodontal ligament and ocular zonule. Since these oxytalan fibres have been shown lo consist of 12 nm microfibrils which are morphologically similar to those of elastic fibres (and unpublished data from this laboratory confirm this conclusion), the results suggest that MAGP is a component of 12 nm microfibrils in both elastic and non-elastic tissues. Anti-tropoelastin antibodies did not localise to these oxytalan fibres, suggesting that tropoelastin is not a component of 12 nm microfibrils. MAGP was also detected in extracellular matrix regions of tissues such as skeletal muscle. Achilles tendon and spleen, suggesting that 12 nm microfibrils, containing one or more macromolecular constituents in common, make up an important structural system within the extracellular matrix in a wide range of elastic and non-elastic tissues. [ABSTRACT FROM AUTHOR]

Many parasitic infections stimulate antibody responses in their mammalian hosts. The ability of these antibodies to protect against disease varies markedly. Research has revealed that functional properties of antibodies determine their role in protection against parasites. Investigations of antibodies against Plasmodium spp. have demonstrated a variety of functional activities, ranging from invasion inhibition and parasite growth inhibition to antibody‐dependent cellular phagocytosis and antibody‐dependent cellular cytotoxicity. These activities have been demonstrated with a large variety of parasite molecules at multiple life cycle stages, highlighting the importance of functional antibody responses in malaria. Other parasitic infections have not yet been investigated in similar detail, but these mechanisms are likely to operate in nonmalarial parasitic infections as well. In this report, we review data on the role of functional antibody responses in protection from parasitic infections, highlighting discoveries in malaria, a parasite for which our knowledge base is the most advanced. [ABSTRACT FROM AUTHOR]

Follicular dendritic cells (FDC) play a crucial role in the regulation of humoral immunity. They are believed to be responsible for long-term persistence of antibody, due to their role in antibody response induction and their ability to retain antigen in immunogenic form for long periods. In this article, a regulatory control model is proposed which links persistence of humoral immunity with cellular processes associated with FDCs. The argument comprises three elements. The first is a literature review of population-level studies of post-vaccination antibody persistence. It is found that reciprocal-time (∝1/t) decay of antibody levels is widely reported, over a range of ages, observation times and vaccine types. The second element is a mathematical control model for cell population decay for which reciprocal-time decay is a stable attractor. Additionally, control effectors are easily identified, leading to models of homeostatic control of the reciprocal-time decay rate. The final element is a literature review of FDC functionality. This reveals a striking concordance between cell properties required by the model and those widely observed of FDCs, some of which are unique to this cell type. The proposed model is able to unify a wide range of disparate observations of FDC function under one regulatory principle, and to characterize precisely forms of FDC regulation and dysregulation. Many infectious and immunological diseases are increasingly being linked to FDC regulation, therefore a precise understanding of the underlying mechanisms would be of significant benefit for the development of new therapies. [ABSTRACT FROM AUTHOR]

The schistosome blood flukes are some of the largest global causes of parasitic morbidity. Further study of the specific antibody response during schistosomiasis may yield the vaccines and diagnostics needed to combat this disease. Therefore, for the purposes of antigen discovery, sera and antibody-secreting cell (ASC) probes from semi-permissive rats and sera from susceptible mice were used to screen a schistosome protein microarray. Following Schistosoma japonicum infection, rats had reduced pathology, increased antibody responses and broader antigen recognition profiles compared with mice. With successive infections, rat global serological reactivity and the number of recognized antigens increased. The local antibody response in rat skin and lung, measured with ASC probes, increased after parasite migration and contributed antigen-specific antibodies to the multivalent serological response. In addition, the temporal variation of anti-parasite serum antibodies after infection and reinfection followed patterns that appear related to the antigen driving the response. Among the 29 antigens differentially recognized by the infected hosts were numerous known vaccine candidates, drug targets and several S. japonicum homologs of human schistosomiasis resistance markers--the tegument allergen-like proteins. From this set, we prioritized eight proteins that may prove to be novel schistosome vaccine and diagnostic antigens. [ABSTRACT FROM AUTHOR]

Antibody-dependent phagocytosis (ADP) is a potentially important immune mechanism to clear HIV. How HIV-specific ADP responses mature during HIV infection or in response to vaccinations administered, including the partially successful RV144 HIV vaccine, is not known. We established a modified ADP assay to measure internalisation of HIV antibody (Ab)-opsonised targets using a specific hybridisation internalisation probe. Labelled beads were coated with both biotinylated HIV gp140 envelope protein and a fluorescent internalisation probe, opsonised with Abs and incubated with a monocytic cell line. The fluorescence derived from the fluorescent internalisation probe on surface-bound beads, but not from internalised beads, was quenched by the addition of a complementary quencher probe. HIV Env-specific ADP was measured in 31 subjects during primary infection and early chronic HIV infection. Although ADP responses were present early during HIV infection, a significant increase in ADP responses in all 31 subjects studied was detected (P<0.001). However, when we tested 30 HIV-negative human subjects immunised with the Canarypox/gp120 vaccine regimen (subjects from the RV144 trial) we did not detect HIV-specific ADP activity. In conclusion, a modified assay was developed to measure HIV-specific ADP. Enhanced ADP responses early in the course of HIV infection were observed but no ADP activity was detected following the vaccinations administered in the RV144 trial. Improved vaccine regimens may be needed to capitalise on ADP-mediated immunity against HIV. [ABSTRACT FROM AUTHOR]

Amyloid light chain (AL) amyloidosis is a lethal disorder characterized by the pathologic deposition of clonal plasma cell-derived, fibrillogenic immunoglobulin light chains in vital organs. Current chemotherapeutic regimens are problematic in patients with compromised organ function and are not effective for all patients. Here, a platform of computer-based prediction and preclinical mouse modeling was used to begin development of a complementary, immunotherapeutic approach for AL amyloidosis. Three peptide/MHC I-binding algorithms identified immunogenic peptides from three AL plasma cell-associated proteins: (1) amyloidogenic λ6 light chains, (2) CYP1B1, a universal tumor antigen hyper-expressed in AL plasma cells and (3) B lymphocyte-induced maturation protein 1 (Blimp-1), a transcription factor required for plasma cell differentiation. The algorithms correctly predicted HLA-A*0201-binding native and heteroclitic peptides. In HLA-A2 transgenic mice, these peptides, given individually or in combination, induced potent CTL which kill peptide-loaded human lymphoma cells and/or lymphoma cells producing target protein. Blimp-1 peptide-immunized mice exhibited a reduced percentage of splenic, lymph node and bone marrow plasma cells and a decrease in the absolute number of splenic plasma cells demonstrating (1) presentation of target peptide by endogenous plasma cells and (2) appropriate CTL homing to lymphoid organs followed by killing of target plasma cells. These studies suggest that AL amyloidosis, with its relatively low tumor cell burden, may be an attractive target for peptide-based multivalent vaccines. [ABSTRACT FROM AUTHOR]

Fundamentally new approaches are required for the development of vaccines to pre-empt and protect against emerging and pandemic influenzas. Current strategies involve post-emergent homotypic vaccines that are modelled upon select circulating 'seasonal' influenzas, but cannot induce cross-strain protection against newly evolved or zoonotically introduced highly pathogenic influenza (HPI). Avian H5N1 and the less-lethal 2009 H1N1 and their reassortants loom as candidates to seed a future HPI pandemic. Therefore, more universal 'seasoned' vaccine approaches are urgently needed for heterotypic protection ahead of time. Pivotal to this is the need to understand mechanisms that can deliver broad strain protection. Heterotypic and heterosubtypic humoral immunities have largely been overlooked for influenza cross-protection, with most 'seasoned' vaccine efforts for humans focussed on heterotypic cellular immunity. However, 5 years ago we began to identify direct and indirect indicators of humoral-herd immunity to protein sites preserved among H1N1, H3N2 and H5N1 influenzas. Since then the evidence for cross-protective antibodies in humans has been accumulating. Now proposed is a rationale to stimulate and enhance pre-existing heterotypic humoral responses that, together with cell-mediated initiatives, will deliver pre-emptive and universal human protection against emerging epidemic and pandemic influenzas. [ABSTRACT FROM AUTHOR]

Targeting CD40, a member of the tumor necrosis factor superfamily, using agonist antibodies (Abs) produces dramatic antitumor effects. Indeed, high-dose intravenous anti-CD40 Ab 'licenses' dendritic cells (DCs) that instruct activated CD8+ cytotoxic T cells to leave lymph nodes (LNs) and penetrate the mesothelioma tumor microenvironment. However, toxic side effects and the potential of an 'overwhelmed' immune response warrant an alternative approach. In this study, we show that injecting lower doses of anti-CD40 Ab directly into the tumor bed avoided toxic side effects and prolonged survival in 60% of mice, with most cured. Unexpectedly, DCs in tumors and LNs 'disappeared', CD8+ tumor-specific T-cell numbers and function were not enhanced, and T cells did not infiltrate regressing tumors. CD4+ or CD8+ depletion only marginally hindered anti-CD40 Ab efficacy implying another effector mechanism. B-cell numbers significantly increased in tumors, draining LNs and spleens during intratumoral anti-CD40 Ab treatment. CD40 targeting had no effect on splenic B-1 cells, obliterated marginal zone B cells and promoted follicular (FO) B-cell activity. Adoptive transfer of tumor antigen-experienced, CD40-activated B cells, or their immunoglobulin products, which recognized autoantigens on mesothelioma cells, protected against tumor challenge. Finally, studies using B-cell knockout mice showed that successful treatment of established tumors required the presence of B cells. Thus, these data suggest that CD40-activated FO B cells can become an important component of an effective antitumor immune response. [ABSTRACT FROM AUTHOR]

The V region antigenic determinants (idiotopes (Ids)) of antibodies (Abs) have been suggested to be involved in regulating the immune system. Certain diseases such as diabetes mellitus have recently been associated with a disequilibrium between Id+ and anti-Id Abs. However, it is unknown how Abs carrying complementary idiotypes (that is, Id+ and anti-Id Abs) regulate each other at the level of B and T cells. In this study, we show that B lymphoma cells genetically equipped with anti-Id BCR V regions receive a signal when exposed to Id+Ig. Moreover, they become × 104 more efficient at presenting exogenous Id+ Ab to CD4+ T cells in vitro. Activated Id-specific T cells in turn regulated the Id-specific B lymphoma cells. Similar results were obtained in vivo in a surrogate model in which an Id-peptide was incorporated genetically into the C-region of a recombinant Ab that targeted IgD on B cells. The findings suggest that conventional T–B collaboration can explain communication between complementary Id+ and anti-Id Ab at the cellular level. A model is suggested that integrates present and previous data on B-cell regulation by Id-specific T cells. [ABSTRACT FROM AUTHOR]

Infections caused by group A streptococcus (GAS) represent a public health problem in both developing and developed countries. The current available methods of prevention are either inadequate or ineffective, which is highlighted by the resurgence in invasive GAS infections over the past two decades. The management of GAS and associated diseases requires new and improved approaches. This review discusses various potential approaches in controlling GAS infections, ranging from prophylactic vaccines to antibody immunotherapy.Immunology and Cell Biology (2009) 87, 391–399; doi:10.1038/icb.2009.29; published online 5 May 2009 [ABSTRACT FROM AUTHOR]

Xenotransplantation using porcine donors holds tremendous promise for alleviating the chronic shortage of human organ donors. However, transplantation from pigs into higher primates triggers a vigorous immune response involving complement activation and thrombosis. Hyperacute rejection can be prevented by using donors in which GalT, the gene responsible for the predominant target of anti-pig natural antibodies, has been deleted. Unfortunately, the adaptive response to GalT knockout (KO) organs has been difficult to immunosuppress. The focus has shifted to identifying additional genetic modifications that will extend xenograft survival beyond the several months currently achievable. Which gene(s) should be added to the GalT KO background is open to debate. Overexpression of a complement regulatory factor(s) seems a logical first choice, supported by recent in vitro data but not yet validated in preclinical models. The next obvious candidate would be an anticoagulant protein(s) to deal with the dysregulated coagulation that is almost invariably associated with xenograft rejection. Several potentially useful molecules have been identified, although this study is yet to be translated to the pig. Our understanding of the mechanisms of GalT KO xenograft rejection continues to grow, providing a rational basis for tailoring further genetic modification of the donor and immunosuppression of the recipient.Immunology and Cell Biology (2009) 87, 203–208; doi:10.1038/icb.2008.107; published online 20 January 2009 [ABSTRACT FROM AUTHOR]

The clonal selection theory proposed by Burnet required a genetic process, for which there was then no precedent, which randomizes the region of the gene(s) responsible for the specification of γ-globulin molecules. Work over the subsequent half-century substantiated Burnet's speculation, revealing two distinct novel genetic processes. During early development (when Burnet first thought the randomization took place) programmed gene segment rearrangement catalysed by the RAG1/RAG2 recombinase generates a substantial diversity of immunoglobulin molecules (the primary repertoire). Somatic hypermutation (triggered by the activation-induced deaminase (AID) DNA deaminase) then occurs following antigen encounter in man and mouse, yielding a secondary repertoire. This hypermutation allows both limitless diversification as well as maturation of the antibody response by a process of somatic evolution akin to that envisioned by Burnet in later formulations of the clonal selection theory. AID-triggered antigen receptor diversification probably arose earlier in evolution than RAG-mediated repertoire generation. Here I trace our insights into the molecular mechanism antibody somatic mutation from when it was first proposed through to our current understanding of how it is triggered by targeted deamination of deoxycytidine residues in immunoglobulin gene DNA.Immunology and Cell Biology (2008) 86, 124–132; doi:10.1038/sj.icb.7100160; published online 8 January 2008 [ABSTRACT FROM AUTHOR]

Of the contentious issues surrounding the clonal selection theory, one of the most influential was that of the mechanism for the generation of diversity of antibody specificity. While Burnet's qualitative theory assumed a very large antibody repertoire, Talmage provided a detailed quantitative argument supporting only 5000 individual globulin patterns that provided an antiserum its specificity through combinatorial action. This methodological difference between the two men, and the mechanistic difference between their models, is key to the understanding of the clonal selection theory, its later acceptance and the proportion of credit paid to Burnet.Immunology and Cell Biology (2008) 86, 72–79; doi:10.1038/sj.icb.7100140; published online 27 November 2007 [ABSTRACT FROM AUTHOR]

Carbohydrates are involved in many immunological responses including the rejection of incompatible blood, tissues and organs. Carbohydrate antigens with Galα(1,3)Gal epitopes are recognized by natural antibodies in humans and pose a major barrier for pig-to-human xenotransplantation. Genetically modified pigs have been established that have no functional α1,3-galactosyltransferase (α1,3GT), which transfers αGal to N-acetyllactosamine (LacNAc) type oligosaccharides. However, a low level of Galα(1,3)Gal is still expressed in α1,3GT knockout animals in the form of a lipid, isoglobotrihexosylceramide (iGb3), which is produced by iGb3 synthase on lactose (Lac) type core structures. Here, we define the reactivity of a series of monoclonal antibodies (mAb) generated in α1,3GT−/− mice immunized with rabbit red blood cells (RbRBC), as a rich source of lipid-linked antigens. Interestingly, one mAb (15.101) binds weakly to synthetic and cell surface-expressed Galα(1,3)Gal on LacNAc, but strongly to versions of the antigen on Lac cores, including iGb3. Three-dimensional models suggest that the terminal α-linked Gal binds tightly into the antibody-binding cavity. Furthermore, antibody interactions were predicted with the second and third monosaccharide units. Collectively, our findings suggest that although the terminal carbohydrate residues confer most of the binding affinity, the fine specificity is determined by subsequent residues in the oligosaccharide.Immunology and Cell Biology (2007) 85, 623–632; doi:10.1038/sj.icb.7100111; published online 28 August 2007 [ABSTRACT FROM AUTHOR]

The monoclonal antibody FN18 has been used as a marker for monkey T cells and as a T-cell-depleting reagent when conjugated to diphtheria toxin that was mutated to prevent binding to non-targeted cells. The antibody recognizes a conformational epitope on the ectodomain of monkey CD3ɛ and displays a range of binding activity to the T cells from different rhesus and cynomolgus monkeys. Our quantitative fluorescence-activated cell sorting analysis of the FN18 reactivity to T cells from different rhesus and cynomolgus monkeys showed that there are at least three levels of FN18 reactivity in the monkeys tested: high, moderate and low. On the basis of available DNA sequence information, we determined the gene structure of rhesus CD3ɛ chain and designed primers that can be used to amplify and quickly sequence the ectodomain of monkey CD3ɛ. Our sequence analysis revealed that the extent of nucleotide sequence variation in this area is greater than that previously reported. In addition to the amino acids at positions 45 and 50, we demonstrated that position 35 of CD3ɛ was also important and substitution of amino acid A for V at this position greatly reduced T-cell reactivity to FN18. We found that T cells from monkeys with high FN18 reactivity all had V, E and R at positions 35, 45 and 50 in CD3ɛ, respectively; those having low FN18 reactivity were homozygous in CD3ɛ with at least one of the changes: V35 to A, E45 to G and R to 50Q, whereas members in the moderate group are heterozygous, having both V and A, E and G, R and Q at these locations. A cytotoxicity assay revealed that T cells from a heterozygous rhesus monkey with moderate FN18 reactivity were much (about 40 times) less sensitive to a FN18-derived immunotoxin than those from a homozygous rhesus monkey having high FN18 reactivity.Immunology and Cell Biology (2007) 85, 357–362; doi:10.1038/sj.icb.7100042; published online 27 February 2007 [ABSTRACT FROM AUTHOR]

Numerous studies have aimed to overcome the barrier to xenotransplantation posedby xenoreactive antibodies and the antigens they recognize. Whetherthis work will eventually lead to the widespread clinical applicationof xenotransplantation remains unknown. However, the benefits ofthis research are already substantial, with research leading todramatic new developments in fields other than xenotransplantation.Our understanding of natural immunity, particularly the nature andfunction of natural antibodies, has taken quantum leaps forward,with far-reaching implications. Our improved understanding of theimmune response to xenografts has proven invaluable in the characterizationof the human immune reaction to commonly used biological therapeuticsof xenogeneic origin. Our understanding of cell surface carbohydratesand our ability to modify these carbohydrates in living animalshas advanced substantially, with implications for diseases suchas cancer and autoimmunity. With this in mind, it is argued thatcontinued work in xenotransplantation is of great value, not onlybecause of the great potential benefits of xenotransplantation,but also because of the more certain benefits that arise from settingour sights on a difficult challenge. [ABSTRACT FROM AUTHOR]

Schizophrenia affects 1% of the world's population, but its cause remains obscure. Numerous theories have been proposed regarding the cause of schizophrenia, ranging from developmental or neurodegenerative processes or neurotransmitter abnormalities to infectious or autoimmune processes. In this review, findings suggestive of immune dysregulation and reactivity to self in patients with schizophrenia are examined with reference to criteria for defining whether or not a human disease is autoimmune in origin. Associations with other autoimmune diseases and particular MHC haplotypes, increased serum levels of autoantibodies, andin vivoandin vitroreplication of some of the functional and ultrastructural abnormalities of schizophrenia by transfer of autoantibodies from the sera of patients with schizophrenia suggest that, in some patients at least, autoimmune mechanisms could play a role in the development of disease. Recent findings regarding specific autoimmune responses directed against neurotransmitter receptors in the brain in patients with schizophrenia will also be reviewed. [ABSTRACT FROM AUTHOR]

Summary Enumeration of antibody-secreting cells in peripheral blood by enzyme-linked immunospot (ELISPOT) has been used in human studies to detect antigen-specific antibody production at mucosal tissue sites. An alternative assay for detecting and quantitating antigen-specific antibody responses involves culturing circulating peripheral blood antibody-secreting cells and quantitating specific antibody production in culture supernatant by ELISA. In the present study, antigen-specific peripheral blood lymphocytes were isolated from subcutaneously immunized sheep and the parameters for maximizing in vitro antibody production by in vivo -induced antibody-secreting cells optimized for this species. Maximum antibody-secreting cell responses were observed in peripheral blood collected four days after antigen challenge. The addition of lipopolysaccharide and antisheep immunoglobulin had no effect on in vitro antibody secretion by blood antibody-secreting cells, while the effects of pokeweed mitogen were highly variable. However, the combination of anti-Ig and recombinant ovine interleukin-6 to peripheral blood lymphocyte cultures was found to markedly and consistently enhance specific antibody production. In unstimulated cultures, the optimal peripheral blood lymphocyte concentration for generating the greatest antibody responses was 5.0 × 107 cells per mL, but in cultures stimulated with recombinant ovine interleukin-6/antisheep immunoglobulin, the optimal cell concentration was lowered to approximately 1.0 × 107 cells per mL. In vitro , peak immunoglobulin production was usually achieved by day one in unstimulated cultures. In recombinant ovine interleukin-6/antisheep immunoglobulin-stimulated cultures, antibody levels were similar to unstimulated cultures by day one, however, the levels continued to rise during incubation to reach a maximum between days four and five of incubation. This optimized antibody-secreting cell culture assay... [ABSTRACT FROM AUTHOR]

The germinal centre reaction (GCR) is a fundamental component of the immune response to T-dependent antigens, during which the immunoglobulin (Ig) genes of B cells experience somatic hypermutation and selection. A maximum-likelihood method on DNA sequence data from 16 individual germinal centres was used to infer that the waiting time for position 33 key (high-affinity) mutations in the anti-(4-hydroxy-3-nitrophenyl) acetyl (NP) response is 8.3 days. This is in marked contrast to the prediction of a key mutant each generation (waiting time about 1/3 day) obtained from a simple model and parameters available in the literature. This disagreement is resolved in part by the finding that the targeted base occurs in a cold spot for hypermutation, raising the predicted waiting time to 2.3 days, although this value remains significantly lower than that inferred from the sequence data. It is proposed that the remaining disparity is attributable to some further stochastic process in the GCR: many early key mutations arise but fail to `take root' within the GC, either due to emigration or failure of cognate T cell/B cell interaction. Furthermore, it is argued that the frequency with which position 33 mutations are found in secondary responses to NP indicates the presence of selection after the GCR. [ABSTRACT FROM AUTHOR]

Maternal antibodies interfere with the immunization of swine by modified live-virus pseudorabies virus (PRV) vaccines. To test the hypothesis that a PRV vaccine attenuated by deletions in the thymidine kinase (TK) and gIII genes might reduce interference by maternal antibodies, pigs with moderate to low levels of colostral PRV antibodies were immunized with the TK- gIII- OMNIMARK-PRV vaccine. Vaccinates and non-vaccinates were challenged intranasally with virulent PRV at 7 weeks of age. In support of the hypothesis, it was found that central nervous system (CNS) and/or respiratory disease developed in six out of 10 controls with a fatal outcome in one, while two out of 13 vaccinates showed only very mild and transient CNS or respiratory disease signs with no fatalities. AH vaccinates gained weight while non-vaccinates initally lost weight. At post-challenge day (PCD) 11, vaccinates showed 4.5 lb/pig greater weight gain than non-vaccinates. Virus neutralization (VN) analyses before and after challenge showed that vaccinates had been primed immunologically. In another experiment, newborn pigs from a pseudorabies disease-quarantined herd with high VN antibody titres were vaccinated, respectively, with the gIII- TK- OMNIMARK-PRV vaccine, a TK- gI- gX- vaccine, or no vaccine and challenged with virulent PRV at 14 weeks of age when VN titres were <1 : 2. By PCD 9, the TK- gIII- group had outgained the TK- gI- gX- and the control groups, respectively, by 6.0 and 3.2 lb per pig. [ABSTRACT FROM AUTHOR]

The antifibrin DD-3B6/22 monoclonal antibody Fab' fragment, a murine immunoglobulin, IgG3, has been labelled with technetium-99 m (99mTc) via a transchelation reaction, to specific activity in excess of 30 mCi/mg protein. The radiolabelling of Fab' was dependent on time, temperature, pH, antibody concentrations and nature of intermediary transchelation complex used. The resultant radioconjugate was stable in vitro and in vivo. Blood clearance of 99mTc-Fab' in rat followed two compartment kinetics with the half time of the fast phase being 0.5 h. The main route of excretion was via the kidneys with little uptake indicated by other tissues. The results suggest that the inherent specificity of the antibody, small molecular size, rapid plasma clearance, high specific radioactivity, together with the physical properties of the 99mTc label, combine to make this labelled monoclonal antibody (MoAb), potentially suitable as a radiopharmaceutical for the scintigraphic detection of thrombi in humans. [ABSTRACT FROM AUTHOR]

Previous studies established that the high density (resting) natural killer (NK) cell subset (R-NK) of peripheral blood NK cells is unresponsive to interleukin-2 (IL-2) but can be induced to proliferate when cultured with γ-irradiated malignant melanoma (MM-170) cells or rnitomycin-C-treated activated T cells in the presence of an IL-2-conditioned medium (IL-2-CM). This study has examined additional requirements of this activation process. The induction of proliferation was dependent on cell to cell contact with metabolically active stimulator cells, although no evidence was obtained that stimulation was effected by soluble factors produced by the stimulator cells. Compared with IL-2-CM, rIL-2 was an inefficient costimulator for the induction of NK cell proliferation, suggesting that factors in IL-2-CM were required in addition to IL-2, but rIL-2 was as efficient as IL-2-CM in maintaining the proliferation of activated NK cells. Under optimum culture conditions, NK growth of up to 3200-fold occurred during a proliferation cycle of 18 days. Phenotypic analysis of the culture-generated quiescent NK cells revealed novel heterogeneity in CD16 (FcγRIII) and CD56 (N-CAM) expression. Some NK cells lacked expression of both CD16 and CD56 (as identified using currently available monoclonal antibodies), while other NK cells showed differential CD16 epitope expression. Since quiescent NK cells can be obtained in large numbers and high purity, they will be a convenient source of NK cells to study the molecular processes involved in initiating NK cell proliferation. [ABSTRACT FROM AUTHOR]

The antibody response of mice and adult humans to immunization with subunit vaccines derived from a pair of antigenically distinct influenza A H1N1 viruses isolated in eggs was investigated. Although the haemagglutinin molecule of each virus differed by only three amino acid residues, highly specific antibody responses were elicited in mice as determined by haemagglutination inhibition and radioimmunoprecipitation assays. Results from competitive radioimmunoassays using monoclonal antibodies of known specificity and a study of the reactivity of mouse antisera with H1N1 field strains indicated that the marked differences in the antibody responses to the two vaccines was due to an amino acid substitution in the distal tip of the haemagglutinin molecule. In contrast, crossreactive antibody responses were elicited in humans presumably due to exposure to viruses related to the candidate vaccine prior to vaccination. Although immunogenic differences are apparent in this pair of antigenically distinct viruses in naive laboratory animals, these differences are not apparent following vaccination of humans that had prior exposure to related viruses. [ABSTRACT FROM AUTHOR]

A panel of eight cell lines has been derived from colon carcinomas. These cell lines have both been characterized according to standard criteria of growth rate, response to mitogens (epidermal growth factor and basic fibroblast growth factor), xenograft growth and growth in soft agar, and according to the ability of the cells to express epitopes known to be expressed by cells in the normal intestinal mucosa. The expression of epitopes present in columnar (absorptive) cells has been assessed using a panel of monoclonal antibodies to brush border peptidases and disaccharidases, villin and brush border-specific peptides. Goblet cell epitopes have been determined by monoclonal antibodies to mucin and carcinoembryonic antigen. An antibody to chromogranin was used to identify endocrine cells. Using these antibodies we found that all the cell lines reacted with at least one of the antibodies to columnar cells. Similarly, varying proportions of cells in six of the eight cell lines stained with antibodies to mucin. None of the cells expressed chromogranin. Expression of a differentiated colonic phenotype, as measured from antibody staining, did not correlate with measurements of malignancy, such as the ability of the cells to grow in soft agar or as xenografts. Similarly, there was no correlation between retention of a colonic phenotype and the initial pathological stage of the tumour from which the cell lines were derived. [ABSTRACT FROM AUTHOR]

Recent studies of the structural basis of antigenic variation in influenza virus neuraminidase and of the structure of neuraminidase-antibody complexes are summarized. [ABSTRACT FROM AUTHOR]

Distinct differences in the capacity of monocyte FcyRII of different individuals to bind or not bind mouse IgG1 defines a polymorphism of FcyRIIa and has previously been defined as the high responder (HR) or low responder (LR) polymorphism of FcyRII. The precise definition of the molecular basis of the human HR/LR polymorphism of FcyRIIa from the peripheral blood mononuclear cells of normal individuals has been determined by anti-CD3 induction of T cell proliferation, the polymerase chain reaction (PCR), nucleotide sequencing, transfection and IgG binding. Amplification of first strand cDNA from mRNA isolated from mononuclear cells was performed by PCR using primers specific for the sequences encoding the leader and cytoplasmic sequences of FcyRIIa, which is normally expressed in monocytes. Sequencing of the PCR products and transfection of these to FcyR cells indicated that in FcyRIIa of HR or LR individuals; (i) three nucleotide substitutions (CA to TG and G to A) resulted in the change of glutamine to tryptophan at position 27 (first extracellular domain) and arginine to histidine at position 131 (second extracellular domain); (ii) expression of cDNA encoding the various combinations of these indicated that arginine at position 131 was essential for IgG1 binding whereas the amino acid changes at position 27 had no effect; and (iii) IgG1 at high concentration bound to all allomorphic forms of FcyRIIa. These results indicate that position 131 in the second extracellular domain of FcyRIIa is intimately involved in immunoglobulin binding. Furthermore, the apparent inability of IgG1 to bind to FcyRIIa of non-responder individuals is not absolute in that immune complexes sensitized with a high concentration of MlgG1 will bind to non-responder-type FcyRIIa. [ABSTRACT FROM AUTHOR]

This report details experimental results which show the presence of enzymic aminopeptidase- A-like activity on endothelia cells, concomitant with cell surface expression previously detected by both ELISA and indirect immunofluorescence. This activity, detected using selected chromogenic substrates in 'functional' assays, is shown to be due (at least in part) lo a molecule previously termed 'gp 150' and recognized by MoAb belonging to CD-13, since such antibodies can he shown to inhibit this activity. Activity, as detected on endothelial ceils, is similar to that observed on various haemopoietic cells. These assays not only provide a functional basis to cell surface gp 150 (CD-13) co-expression on haemopoietic and endothelial cells but have also been used to help define structural epitopes present on aminopeptidase-N/gp 150, previously analysed using radiolabelled antibodies in competitive binding assays. Appraisal of these new data suggests that the number of distinct antibody binding sites on this molecule is greater than that previously demonstrated. This study is therefore an important first step in investigating the potential involvement of this selective peptidase molecule in the control of haemopoietie cell growth and differentiation and in haemostatic mechanisms. [ABSTRACT FROM AUTHOR]

Polyreactive immunoglobulin (Ig) secreted by CD5-bearing B cells has the capacity to bind a broad range of self and foreign antigens. Flow cytometric analysis was used to detect low-affinity binding of mouse Ig molecules Io the surface of CD5-bearing B cells from patients with chronic lymphocytic leukaemia (CLL). Mouse Ig isotypes G. A. and M. and IgG subclasses C1, C2a, and G3 bound to the B cell surface via a mechanism not involving the antigen-binding site of the mouse Ig molecule. Fab and F(ab')2 fragments of mouse Ig associated with the CLL cells in a similar manner to intact Ig, indicating that Fe receptor interactions were not involved. Dissociation of the mouse Ig from the B cell surface by three washing steps distinguished this lower-affinity binding from high-affinity binding which occurs through the antigen-binding site of a mouse monoclonal antibody (MoAb) when it recognizes a specific ceil surface epitope. Blocking studies suggest that the low-affinity binding occurred via surface IgM and surface IgD (sIgM/sIgD) on the CD5-bearing B cells. The results are consistent with the expression of polyreactive Ig on the surface of CD5-bearing B cells in CLL. [ABSTRACT FROM AUTHOR]

The number and frequency of spontaneous and cytokine-inducible ‘natural’ immunoglobulin-secreting cells (ISC) were determined in bone marrow (BM), spleen and Peyer's patch (PP), in vitro. Cells were cultured at Intuiting dilution in the presence or absence of exogenous recombinant cytokines and supernatants then assayed for total immunoglobulin (Ig) and Ig isotype using an ELISA. Most spontaneous ISC were found in the spleen and BM. with fewer in PP. The addition of recombinant interleukin 5 (rIL-5) promoted a marked increase in both the ISC frequency and the amount of Ig secreted/ISC whereas recombinant IL-6 (rIL-6) promoted only a marginal increase. Recombinant IL-4 (rIL-4) promoted a marginal increase in ISC frequency only. The isotype profile of ISC was in the order IgM > IgG2 > IgA > IgG3 > IgG1. The exposure of cells to 1200 rad of γ-radiation resulted in decreased numbers of spontaneous ISC in all tissues, but the addition of rIL-5 or rIL-6 to the irradiated cells increased both the ISC frequency and Ig secreted. The Ig isotype profile was similar to that of non-irradiated ISC with a few minor exceptions. This large population of potential cytokine-inducible ISC could contribute to ‘natural’ Ig secretion in vivo. [ABSTRACT FROM AUTHOR]

Several Class I molecules encoded by the murine H-2K gene have been found to express epitopes with different sensitivities to glycosidases. This result was found to be variably represented by different assays used to measure the binding of monoclonal antibodies (MoAb) specific for Class I H-2Kk molecules. [ABSTRACT FROM AUTHOR]

Evidence is presented which supports the phenomenon of heterogeneity amongst H-2Db-encoded Class I molecules. Two monoclonal antibodies (MoAb) called H141-31 and B22-249 were used in these studies. Both bind to the 'private' H-2.2 site of H-2Db-encoded molecules, but the binding of B22-249 is determined by carbohydrate moieties, whereas H141-31 appears to bind to a protein-defined epitope. Some H-2Db molecules, identified by the H141-31 MoAb, are primarily expressed on B lymphocytes and not T lymphocytes in spleen. The number of H-2Db molecules which bind H141-31 on B cells was also found to be three-to four-fold less than the number which bound the B22-249 MoAb B cells of two mutant strains of mice. B6-C.H-2bm13 and B6-C.H-2bm14 which harbour very few nucleotide changes in the H-2Db gene, also show marked reduction in the binding of both antibodies. This suggests that a single common gene encodes both target molecules and that post-translational modifications such as differential glycosylation may account for heterogeneity amongst H-2Db molecules. This would explain the presence of the different H-2Db molecules defined here. It follows that differences in glycosylation evidently occur both within the B cell population, since H141-31 binds to only a subset of H-2Db molecules on B cells, and between T and B lymphocytes, since resting T cells do not bind H141-31 MoAb. [ABSTRACT FROM AUTHOR]

The excretory-secretory (ES) antigens of the filarial parasite Brugia malayi adult (BmA) and microfilariae (MF) were analysed for differences in their protein composition by two-dimensional gel electrophoresis. Both BmA and MF biosynthetically labelled with [³H]-leucine released a 200 kD molecule with pi (isoelectric point) ranging from 5.1 to 6.8. A monoclonal antibody (MoAb) to phosphocholine (PC) immunoprecipitated the 200 kD molecule with a PI of 5.1-5.3 from both BmA and MF. Immunization of CBA/N mice with ES antigens resulted in MoAb that reacted with PC, as well as some that did not (ES-1, ES-2 and ES-3). The epitopes recognized by these non-PC MoAb varied greatly in their molecular weight, heat-resistance, TCA-solubility and metaperiodate-sensitivity, which suggests a proteoglycan nature of the epitope. An antigen capture assay using ES-1 and ES-3 detected a circulating filarial antigen not only In patients infected with Wuchereria bancrofti (17-83% of those with asymptomatic microfilaraemia and 52-60% of those with chronic lymphatic obstruction) but also in serum from patients infected with other helminth parasites (12-100%). Indeed, the epitope recognized by these MoAb was also present on other helminth parasites, which suggests conservation of a carbohydrate-like prosthetic group(s) on diverse helminth species. [ABSTRACT FROM AUTHOR]

In order to identify cell-substrate adhesion receptors on vascular endothelium, murine monoclonal antibodies (MoAb) were raised against human umbilical vein endothelial cells (HUVE). One anti-HUVE MoAb, RMAC11. identified the adhesion receptor VLA-2 as it immunoprecipitated a non-covalently linked heterodimer of 160 kD and 130 kD. which was identical to the heterodimer immunoprecipitated by the anti-VLA-2 MoAb. 12F1 and 5E8. Furthermore, proteolytic peptide maps of the VLA-2 α and β-chains were highly homologous with those of the RMAC11-recognized molecule. However, unlike other VLA-2 MoAb. RMAC11 also identified an 85 kD band which migrated to 90 kD under reducing conditions. This band was most likely a fragment of the 160 kD α-chain as a similar α-chain derived fragment has been demonstrated in the immunoprecipitates of some VLA-4 reactive monoclonals. However the possibility that this may be a novel molecule associated with VLA-2 has not been excluded. In vitro assays of HUVE adhesion to collagen types 1 and 4, laminin and fibronectin showed that RMAC11 blocked adhesion to collagen (types 1 and 4) and laminin, but had no effect on HUVE adhesion to fibronectin. confirming that VLA-2 is a collagen and laminin receptor for HUVE. [ABSTRACT FROM AUTHOR]

The extent and timing of glomerular T lymphocyte infiltration was studied in acute serum sickness (AcSS) glomerulonephritis (GN) in rabbits. AcSS was initiated by a single intravenous injection of bovine serum albumin (BSA), Rabbits developed circulating BSA anti-BSA immune complexes, and rapid immune elimination of the circulating antigen was associated with the deposition of immune complexes in the kidney and the onset ofa diffuse endocapillary proliferative GN. On the day of immune elimination (defined as when < 1% of the injected antigen remained in the circulation), rabbits developed significant proteinuria (98 ± 36 mg/24 h; normal 14 ± 1 mg/24 h, P < 0.01). glomerular macrophage accumulation (44.3 ±21.1 macrophages per glomerulus [mac/glom]: normal 0,28 ±0.18 mac/glom. P < 0.01), and a significant glomerular T. lymphocyte influx (3.0 ±0.9 cells/glomerular cross-section [c/gcs); normal 0.47 ±0,13 c/gcs; P < 0.005), On the day prior to immune elimination, increased T cells numbers were observed in some rabbits (2.4 ± 2.1 c/gcs) together with a minor macrophage presence (7.6 ± 3.6 mac/glom) and minimal proteinuria (17.6 ± 3 mg/24 h). These studies demonstrate the influx of T lymphocytes together with macrophages at the onset of proteinuria in serum sickness nephritis and are consistent with a role for cell-mediated immunity in the pathogenesis of this lesion. [ABSTRACT FROM AUTHOR]

Reports on the examination of the relative efficacy of macrophages, B cells and splenic dendritic cells in presenting West Nile virus antigens to T cells. Indication of results that cell types can function as antigen-presenting cells; Use of Listeria peritoneal macrophages to induce higher proliferative responses; Ability of macrophage populations to present antigen inhibited by anti-class II major histocompatibility complex antibodies.

The effects were investigated of two pretreatments of human serum and plasma test samples on their subsequent reactivity in the anticardiolipin antibody enzyme-linked immunosorbent assay (ACA-ELISA). The first treatment involved heat inactivation of test samples at 56°C for 30 min, a process sometimes used to inactivate samples from suspected human immunodeficiency virus positive individuals. Such treatment significantly increased the IgG ACA unit/mL values of normal sera, but when this effect was examined further, it was found that the increase in binding occurred on both cardiolipin-coated and uncoated wells and was therefore non-specific. Heat inactivation of sera prior to ACA testing should therefore be avoided. The second treatment involved diluting immunoglobulin (Ig)G and IgM ACA-positive sera in normal human serum (NHS) or newborn calf serum (NCS); sera diluted in NHS showed a significant increase in titre, particularly IgM ACA-positive sera. This phenomenon was found to be due to a serum cardiolipin-binding cofactor which enhances antibody recognition. The cofactor is heat stable and is present in normal sera (male and female) and also in IgG ACA-positive sera. The binding of a human IgM monoclonal antibody to cardiolipin was not affected by the cofactor. The cardiolipin/cofactor complex may represent the optimal autoantigen/autoimmunogen and a re-appraisal, therefore, of the clinical relevance of antibodies to cardiolipin and other negatively charged molecules is warranted. [ABSTRACT FROM AUTHOR]

The ability of salivary secretions and sputum sol from patients with chronic bronchitis to inhibit the adhesion of non-serotypable Haemophilus influenzae to human buccal epithelial cells has been examined. Specific H. influenzae antibodies were measured by an enzyme-linked immunosorbent assay (ELISA). Saliva and sputum levels of anti-H, influenzae immunoglobulin A (IgA) antibody were related inversely to buccal cell adherence (r=-0.92). Absorption of saliva and sputum sol with H. influenzae removed antibody detected by ELISA but only partially removed inhibition of attachment of bacteria. In patients with a history of recurrent bronchitis significantly more inhibitory activity remained after absorption of the secretory IgA (sIgA) specific antibody compared with patients who were resistant to acute episodes of infection (P<0.05). These observations suggest that factors, other than sIgA, are present in respiratory secretions and are capable of modulating H. influenzae attachment to buccal epithelial cells. [ABSTRACT FROM AUTHOR]

The technique of using sodium dodecylsulfate-polyacrylamide gel electrophoresis fractionated antigens (Ag) transferred to nitrocellulose filters was adopted to analyse T cell responses to Salmonella enteritidis 11RX Ag. Employing in vitro proliferation assays with T cells from S. enteritidis 11RX-primed (BALB/cXC57BL/6J)F1 mice as the measure of T cell stimulation, we have identified Ag able to stimulate T cells in the regions containing 16, 24, 34 and 50-60 kDa proteins, with dominant Ag activity at about 16kDa. These results were confirmed with long-term, Ag-specific L3T4+ T cell lines which responded to molecules in the same four Mr regions, suggesting that no selection by a single antigenic determinant had occurred during more than 3 months of in vitro culture, or that all the molecules which were stimulatory shared at least one antigenic determinant. Because the seven clones we examined responded only to 16 kDa molecules, the former alternative is the more likely. Standard immunoblot analysis indicated that these Ag also act as major B cell stimulating determinants. T cells of BALB/c mice, which are 5-10 times more resistant to S. enteritidis 11RX than C57BL/6J mice, showed the same pattern of reactivity as F1 mice whereas the major antigenic region for T cells of C57BL/6J mice was located between 50 and 60 kDa. [ABSTRACT FROM AUTHOR]

A mouse IgG1 monoclonal antibody (1C4), which recognizes a cell surface molecule on murine natural cytotoxic{NC) cells was produced. By flow cystometry. IC4 preferentially reacted with less than 5% of fresh CBA spleen cells and 20-50% of CBA-interleukin-3 (IL-3) cells, an in vitro derived NC-like cell line. In vitro treatment of spleen cells from a number of Inbred mouse strains either with 1C4 alone or 1C4 coupled to dynabeads markedly decreased or abolished NC activity of the cells against 51Crk-labelled WEH1-164 targets. Splenic NC activity of these same mouse strains was also reduced or abolished by in vivo administration of IC4. The effect was evident within 2 h of treatment and persisted for at least 1 week. In contrast IC4 had little or no effect on splenic NK activity against 51Crk-labelled YAC-1 targets over the same range of experiments in vitro and in vivo. Results of strain surveys for both in vitro and in vivo reduction of splenic NC activity by 1C4 treatment showed that CBA. C57BL/6, BALB/c and NZB mice were positive and CE and DBA/2 mice were negative, indicating that 1C4 recognizes an allo-antigen on mouse NC cells. This allo-antibody has been designated NC-1·, and thus IC4 is an anti-NC-1·1 monoclonal antibody. [ABSTRACT FROM AUTHOR]

Several approaches were made to produce new monoclonal antibodies (MoAb) to human colonic tumours using different immunogens, such as colon cell lines, and tissue and serum from patients with colon cancer. Six MoAb were produced but all were found to be reactive with carcino-embryonic antigen (CEA). Through tissue reactivity and competitive inhibition studies it was found that four epitopes could be detected: CEA-1 detected by MoAb M and O-1; CEA-2 detected by MoAb 17C4; CEA-3 detected by MoAb LK-4 and XPX-13 and CEA-4 detected by MoAb JGT-13. CEA-2 was also found on granulocytes and probably detects normal cross-reacting antigen (NCA). In formalin-fixed tissues the CEA-specific epitopes were absent from normal adult tissues, but this was an artefact of the fixation procedure and fresh, non-fixed colonic tissues were clearly reactive. All six MoAb reacted with >70% of colonic tumours when tested by the immunoperoxidase technique and gave different patterns of reactivity reflecting the differential expression of the CEA 1-4 epitopes. Despite the apparent specificity of these MoAb for colon cancer, serum testing using MoAb gave similar results to CEA polyclonal antibodies, that is the MoAb gave no obvious advantage. Thus efforts to produce new reagents to colon cancer yielded only anti-CEA monoclonal reagents. Clearly, other new approaches are required to produce better diagnostic reagents for this common disease. [ABSTRACT FROM AUTHOR]

This study describes a new murine monoclonal antibody (MoAb) 5C1 raised against human colorectal carcinoma, which gave a differential reaction on formalin-fixed sections of the gastrointestinal tract. The MoAb 5C1 of immunoglobulin M (IgM) isotype reacted with both the cytoplasm and membrane of all normal colonic epithelia, and with all benign colonic polyps and all premalignant colonic lesions. However, there was a decreased expression of the 5C1 antigen in most cases of colonic malignancy and it was this feature that makes MoAb 5C1 unique. The distribution of the 5C1 epitope in normal gastrointestinal tract is limited to a few epithelial cells in the mid-portion of the small intestine but this distribution increased progressively down the digestive tract until it was found on > 90% of normal epithelial cells (in membrane and cytoplasm) of the colon. In addition, the 5C1 epitope was present on mucin secreting cells from normal organs of the gastrointestinal, reproductive and pulmonary tract and benign and malignant tissues of the colon. On Western blots, MoAb 3C1 was found to detect a heterogeneous population of molecules with molecular weights >100 kDa with the strongest staining bands found between 230 and 300 kDa. MoAb 5C1 does not detect carcino-embryonic antigens (CEA). human milk fat globules (HMFG), human lymphocyte antigens (HLA) or ABO blood group antigens. The combination of its presence in mucin secreting cells and its broad molecular weight bands suggest that the antigen detected is a mucin. [ABSTRACT FROM AUTHOR]

This study has analysed the binding of a series of anti-CD2 monoclonal antibodies (MoAbs) to T cells in the presence of the situated polysaccharide dextran sulfate (2-3 sulfates/monosaccharide, 500 kDa) (DXS) to define the DXS binding site on CD2. The results show that DXS interacts primarily at theT112 epitope. Thus five anti-CD2 MoAbs which bound to the T112 epilope were inhibited in their binding by DXS. In contrast, seven anti-CD2 MoAbs that totally inhibited sheep red blood cells (SRBC) rosetting (identifying the T111 epitope) were unaffected in their binding to T cells in the presence of DXS. Three MoAbs which partially inhibited SRBC rosetting and thereby defining only part of the T111 [ epitope, were also inhibited in their binding by DXS. Consistent with the conclusion that the DXS binding site on CD2 is associated with the T112: epitope was the observation that interaction of DXS with CD2 resulted in augmented binding of the four MoAbs defining the T113 epitope, possibly reflecting an increased expression of the T113 (activation. CD2R) epitope of CD2. Collectively, the data presented support the notion that a natural ligand for the Tl 12 epitope of CD2 will be identified as a sulphated carbohydrate structure. [ABSTRACT FROM AUTHOR]