Lymphocyte blastogenic response to ovalbumin in a model for canine allergy

Lymphocyte stimulation tests (LST) were performed in five dogs sensitised with ovalbumin (OVA) and seven healthy dogs. In addition, all five OVA-sensitised and two control dogs were tested after two in vivo provocations with OVA-containing eye drops. The isolated cells were suspended in culture media containing OVA and were cultured for up to 12 days. Proliferation was measured as reduction in 5,6-carboxylfluorescein diacetate succinimidyl ester (CFSE) intensity by flow cytometry on days 0, 3, 6, 9 and 12. A cell proliferation index (CPI) for each day and the area under the curve (AUC) of the CPI was calculated for each dog. All OVA-sensitised dogs demonstrated increased erythema after conjunctival OVA application. The presence of OVA-specific lymphocytes was demonstrated in 2/5 OVA-sensitised dogs before and 4/5 after in vivo provocation. Using the AUC, the difference between OVA-sensitised and control dogs was significant in all three LST before in vivo provocation (P<0.05) and borderline significant (P=0.053) in 2/3 LST after provocation. The most significant difference in CPI was observed after 9 days of culture (P=0.001). This pilot study indicates that the LST allows detection of rare antigen specific memory T-cells in dogs previously sensitised to, but not concurrently undergoing challenge by a specific antigen.

Host responses to intestinal microbial antigens in gluten-sensitive mice

BACKGROUND AND AIMS: Excessive uptake of commensal bacterial antigens through a permeable intestinal barrier may influence host responses to specific antigen in a genetically predisposed host. The aim of this study was to investigate whether intestinal barrier dysfunction induced by indomethacin treatment affects the host response to intestinal microbiota in gluten-sensitized HLA-DQ8/HCD4 mice. METHODOLOGY/PRINCIPAL FINDINGS: HLA-DQ8/HCD4 mice were sensitized with gluten, and gavaged with indomethacin plus gluten. Intestinal permeability was assessed by Ussing chamber; epithelial cell (EC) ultra-structure by electron microscopy; RNA expression of genes coding for junctional proteins by Q-real-time PCR; immune response by in-vitro antigen-specific T-cell proliferation and cytokine analysis by cytometric bead array; intestinal microbiota by fluorescence in situ hybridization and analysis of systemic antibodies against intestinal microbiota by surface staining of live bacteria with serum followed by FACS analysis. Indomethacin led to a more pronounced increase in intestinal permeability in gluten-sensitized mice. These changes were accompanied by severe EC damage, decreased E-cadherin RNA level, elevated IFN-gamma in splenocyte culture supernatant, and production of significant IgM antibody against intestinal microbiota. CONCLUSION: Indomethacin potentiates barrier dysfunction and EC injury induced by gluten, affects systemic IFN-gamma production and the host response to intestinal microbiota antigens in HLA-DQ8/HCD4 mice. The results suggest that environmental factors that alter the intestinal barrier may predispose individuals to an increased susceptibility to gluten through a bystander immune activation to intestinal microbiota.

New findings on echinococcosis

The following new aspects of cystic and alveolar echinococcosis (= infections with the metacestode stages of Echinococcus granulosus and E. multilocularis respectively) are reported: identification of a Swiss E. granulosus isolate as "cattle strain" which differs from the "sheep strain"; new observations on proliferation and metastasis formation of larval E. multilocularis; information on chemotherapy of human echinococcosis; recent developments in immunoserology. The latter includes a new technique for serological differential diagnosis of cystic and alveolar echinococcosis, the determination of parasite-specific immunoglobulin classes (IgG, IgM, IgA and IgE) and circulating antigens in ELISA, and the introduction of arc-5 detection in routine serodiagnosis. A highly purified, species-specific antigen from E. multilocularis is now available for seroepidemiological studies.

Tumor immunization strategies for enhancing the graft versus tumor activity of allogeneic bone marrow transplantation

Allogeneic bone marrow transplantation (BMT) is known to induce a beneficial anti-tumor immune response called graft-versus-tumor (GVT) activity. However, GVT activity is closely associated with graft-versus-host disease (GVHD), a potentially fatal immune response against antigens on normal recipient tissues. The T-cell populations mediating these two processes are often overlapping, but studies have shown that some donor T-cells can be tumor-specific. Therefore, the goal of this study was to develop strategies for preferentially activating donor T-cells capable of mediating GVT activity but not GVHD. The three hypotheses tested were: (1) Pre-transplant immunization of BMT donors with a recipient-derived tumor cell vaccine will induce a relative increase in GVT activity as compared to GVHD. (2) Post-transplant tumor immunization of BMT recipients will enhance GVT activity without exacerbating GVHD. (3) Pre-transplant immunization of BMT donors against a tumor-specific antigen will enhance GVT activity without exacerbating GVHD. ^ To test the first two hypotheses, C3H.SW mice (MHC-matched donors) were immunized with a C57BL/6 (recipient)-derived tumor cell vaccine (leukemia or fibrosarcoma) prior to BMT, or recipients were immunized starting one month after BMT. Both donor and recipient immunization led to a significant increase in GVT activity (enhanced recipient survival and decreased tumor growth). However, donor immunization also increased fatal GVHD, which was at least partially due to activation of alloreactive T-cells recognizing the immunodominant minor histocompatibility antigen B6dom1. GVT immunity following recipient immunization was not associated with an exacerbation of GVHD or a response to B6dom1. ^ To test the third hypothesis, influenza nucleoprotein (NP) was used as a model tumor antigen. C3H.SW donors were immunized against NP prior to BMT, which led to a significant increase in GVT activity. Although recipients were not completely protected against growth of antigen loss variant tumors, there was no increase in GVHD. ^ In conclusion, (1) immunization of allogeneic BMT donors with a recipient-derived tumor cell vaccine substantially increases GVT activity but also exacerbates GVHD, (2) post-transplant tumor immunization of allogeneic BMT recipients significantly increases GVT activity and survival without exacerbating GVHD, and (3) immunization of allogeneic BMT donors against a tumor-specific antigen significantly enhances GVT activity without exacerbating GVHD. ^

Immunologic characteristics of immunogenic variants generated by {\it in vitro\/} treatment of a methylcholanthrene-induced murine fibrosarcoma MCA-F with 1 methyl-3-nitro-1-nitrosoguanidine, 5 -aza-2$\sp\prime$-deoxycytidine, or ultraviolet radiation

This study addresses the questions of whether the frequency of generation and in vivo cross-reactivity of highly immunogenic tumor clones induced in a single parental murine fibrosarcoma cell line MCA-F is more closely related to the agent used to induce the Imm$\sp{+}$ clone or whether these characteristics are independent of the agents used. These questions were addressed by treating the parental tumor cell line MCA-F with UV-B radiation (UV-B), 1-methyl-3-nitro-1-nitrosoguanidine (MNNG), or 5-aza-2$\sp\prime$-deoxycytidine (5-azaCdR). The frequency of Imm$\sp{+}$ variant generation was similarly high for the three different agents, suggesting that the frequency of Imm$\sp{+}$ generation was related more closely to the cell line than to the inducing agent used. Cross-reactivity was tested with two Imm$\sp{+}$ clones from each treatment group in a modified immunoprotection assay that selectively engendered antivariant, but not antiparental immunity. Under these conditions each clone, except one, immunized against itself. The MNNG-induced clones engendered stronger antivariant immunity but a weaker variant cross-reactive immunity could also be detected.^ This study also characterized the lymphocyte populations responsible for antivariant and antiparental immunity in vivo. Using the local adoptive transfer assay (LATA) and antibody plus complement depletion of T-cell subsets, we showed that immunity induced by the Imm$\sp{+}$ variants against the parent MCA-F was transferred by the Thy1.2$\sp{+}$, L3T4a$\sp{+}$, Lyt2.1$\sp{-}$ (CD4$\sp{+}$) population, without an apparent contribution by Thy1.2$\sp{+}$, L3T4a$\sp{-}$, Lyt2.1$\sp{+}$ (CD8$\sp{+}$) cells. A role for Lyt2.1$\sp{+}$T lymphocytes in antivariant, but not antiparent immunity was supported by the results of LATA and CTL assays. Immunization with low numbers of viable Imm$\sp{+}$ cells, or with high numbers of non viable Imm$\sp{+}$ cells engendered only antivariant immunity without parental cross-protection. The associative recognition of parental antigens and variant neoantigens resulting in strong antiparent immunity was investigated using somatic cells hybrids of Imm$\sp{+}$ variants of MCA-F and an antigenically distinct tumor MCA-D. An unexpected result of these latter experiments was the expression of a unique tumor-specific antigen by the hybrid cells. These studies demonstrate that the parental tumor-specific antigen and the variant neoantigen must be coexpressed on the cell surface to engender parental cross-protective immunity. (Abstract shortened with permission of author.) ^

Apoptosis and proliferation of dentate gyrus neurons after single and intermittent limbic seizures

Neuronal apoptosis was observed in the rat dentate gyrus in two experimental models of human limbic epilepsy. Five hours after one hippocampal kindling stimulation, a marked increase of in situ terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) of fragmented DNA was observed in nuclei located within and on the hilar border of the granule cell layer and in the polymorphic region. Forty kindling stimulations with 5-min interval produced higher numbers of labeled nuclei compared with one stimulation. The increase of TUNEL-positive nuclei was prevented by the protein synthesis inhibitor cycloheximide but not affected by the N-methyl-d-aspartate receptor antagonist MK-801. Kainic acid-induced seizures lead to a pattern of labeling in the hippocampal formation identical to that evoked by kindling. A large proportion of cells displaying TUNEL-positive nuclei was double-labeled by the neuron-specific antigen NeuN, demonstrating the neuronal identity of apoptotic cells. Either 1 or 40 kindling stimulations also gave rise to a marked increase of the number of cells double-labeled with the mitotic marker bromodeoxyuridine and NeuN in the subgranular zone and on the hilar border of the dentate granule cell layer. The present data show that single and intermittent, brief seizures induce both apoptotic death and proliferation of dentate gyrus neurons. We hypothesize that these processes, occurring early during epileptogenesis, are primary events in the development of hippocampal pathology in animals and possibly also in patients suffering from temporal lobe epilepsy.

Variant antigenic peptide promotes cytotoxic T lymphocyte adhesion to target cells without cytotoxicity

Timelapse video microscopy has been used to record the motility and dynamic interactions between an H-2Db-restricted murine cytotoxic T lymphocyte clone (F5) and Db-transfected L929 mouse fibroblasts (LDb) presenting normal or variant antigenic peptides from human influenza nucleoprotein. F5 cells will kill LDb target cells presenting specific antigen (peptide NP68: ASNENMDAM) after “browsing” their surfaces for between 8 min and many hours. Cell death is characterized by abrupt cellular rounding followed by zeiosis (vigorous “boiling” of the cytoplasm and blebbing of the plasma membrane) for 10–20 min, with subsequent cessation of all activity. Departure of cytotoxic T lymphocytes from unkilled target cells is rare, whereas serial killing is sometimes observed. In the absence of antigenic peptide, cytotoxic T lymphocytes browse target cells for much shorter periods, and readily leave to encounter other targets, while never causing target cell death. Two variant antigenic peptides, differing in nonamer position 7 or 8, also act as antigens, albeit with lower efficiency. A third variant peptide NP34 (ASNENMETM), which differs from NP68 in both positions and yet still binds Db, does not stimulate F5 cytotoxicity. Nevertheless, timelapse video analysis shows that NP34 leads to a significant modification of cell behavior, by up-regulating F5–LDb adhesive interactions. These data extend recent studies showing that partial agonists may elicit a subset of the T cell responses associated with full antigen stimulation, by demonstrating that TCR interaction with variant peptide antigens can trigger target cell adhesion and surface exploration without activating the signaling pathway that results in cytotoxicity.

Patterning of functional antibodies and other proteins by photolithography of silane monolayers.

We have demonstrated the assembly of two-dimensional patterns of functional antibodies on a surface. In particular, we have selectively adsorbed micrometer-scale regions of biotinylated immunoglobulin that exhibit specific antigen binding after adsorption. The advantage of this technique is its potential adaptability to adsorbing arbitrary proteins in tightly packed monolayers while retaining functionality. The procedure begins with the formation of a self-assembled monolayer of n-octadecyltrimethoxysilane (OTMS) on a silicon dioxide surface. This monolayer can then be selectively removed by UV photolithography. Under appropriate solution conditions, the OTMS regions will adsorb a monolayer of bovine serum albumin (BSA), while the silicon dioxide regions where the OTMS has been removed by UV light will adsorb less than 2% of a monolayer, thus creating high contrast patterned adsorption of BSA. The attachment of the molecule biotin to the BSA allows the pattern to be replicated in a layer of streptavidin, which bonds to the biotinylated BSA and in turn will bond an additional layer of an arbitrary biotinylated protein. In our test case, functionality of the biotinylated goat antibodies raised against mouse immunoglobulin was demonstrated by the specific binding of fluorescently labeled mouse IgG.

Vaccination with syngeneic, lymphoma-derived immunoglobulin idiotype combined with granulocyte/macrophage colony-stimulating factor primes mice for a protective T-cell response.

The idiotype of the Ig expressed by a B-cell malignancy (Id) can serve as a unique tumor-specific antigen and as a model for cancer vaccine development. In murine models of Id vaccination, formulation of syngeneic Id with carrier proteins or adjuvants induces an anti-idiotypic antibody response. However, inducing a potent cell-mediated response to this weak antigen instead would be highly desirable. In the 38C13 lymphoma model, we observed that low doses of free granulocyte/macrophage colony-stimulating factor (GM-CSF) 10,000 units i.p. or locally s.c. daily for 4 days significantly enhanced protective antitumor immunity induced by s.c. Id-keyhole limpet hemocyanin (KLH) immunization. This effect was critically dependent upon effector CD4+ and CD8+ T cells and was not associated with any increased anti-idiotypic antibody production. Lymphocytes from spleens and draining lymph nodes of mice primed with Id-KLH plus GM-CSF, but not with Id-KLH alone, demonstrated significant proliferation to Id in vitro without any biased production of interferon gamma or interleukin 4 protein or mRNA. As a further demonstration of potency, 50% of mice immunized with Id-KLH plus GM-CSF on the same day as challenge with a large s.c. tumor inoculum remained tumor-free at day 80, compared with 17% for Id-KLH alone, when immunization was combined with cyclophosphamide. Taken together, these results demonstrate that GM-CSF can significantly enhance the immunogenicity of a defined self-antigen and that this effect is mediated exclusively by activating the T-cell arm of the immune response.

Autocrine/paracrine role of insulin-related growth factors in neurogenesis: local expression and effects on cell proliferation and differentiation in retina.

Early neurogenesis progresses by an initial massive proliferation of neuroepithelial cells followed by a sequential differentiation of the various mature neural cell types. The regulation of these processes by growth factors is poorly understood. We intend to understand, in a well-defined biological system, the embryonic chicken retina, the role of the insulin-related growth factors in neurogenesis. We demonstrate the local presence of signaling elements together with a biological response to the factors. Neuroretina at days 6-8 of embryonic development (E6-E8) expressed proinsulin/insulin and insulin-like growth factor I (IGF-I) mRNAs as well as insulin receptor and IGF type I receptor mRNAs. In parallel with this in vivo gene expression, E5 cultured neuroretinas synthesized and released to the medium a metabolically radiolabeled immunoprecipitable insulin-related peptide. Furthermore, insulin-related immunoreactive material with a HPLC mobility close to that of proinsulin was found in the E6-E8 vitreous humor. Exogenous chicken IGF-I, human insulin, and human proinsulin added to E6 cultured neuroretinas showed relatively close potencies stimulating proliferation, as determined by [methyl-3H]thymidine incorporation, with a plateau reached at 10(-8) M. These factors also stimulated neuronal differentiation, indicated by the expression of the neuron-specific antigen G4. Thus, insulin-related growth factors, interestingly including proinsulin, are present in the developing chicken retina and appear to play an autocrine/paracrine stimulatory role in the progression of neurogenesis.

Recombinant Kunjin virus replicon vaccines induce protective T-cell immunity against human papillomavirus 16 E7-expressing tumour

The persistence of the E7 oncoprotein in transformed cells in human papillomavirus (HPV)-associated cervical cancer provides a tumour-specific antigen to which immunotherapeutic strategies may be directed. Self-replicating RNA (replicon) vaccine vectors derived from the flavivirus Kunjin (KUN) have recently been reported to induce T-cell immunity. Here, we report that inclusion of a CTL epitope of HPV16 E7 protein into a polyepitope encoded by a KUN vector induced E7-directed T-cell responses and protected mice against challenge with an E7-expressing epithelial tumour. We found replicon RNA packaged into virus-like particles to be more effective than naked replicon RNA or plasmid DNA constructed to allow replicon RNA transcription in vivo. Protective immunity was induced although the E7 CTL epitope was subdominant in the context of other CTL epitopes in the polyepitope. The results demonstrate the efficacy of the KUN replicon vector system for inducing protective immunity directed towards a virally encoded human tumour-specific antigen, and for inducing multi-epitopic CTL responses. (C) 2004 Elsevier Inc. All rights reserved.

Leishmaniasis Mimicking Lymphoma and/or Liver Cirrhosis

A 76-year-old man was admitted to hospital with fever, weight loss, pancytopenia, hepatosplenomegaly and a double monoclonal component IgM-IgG-k, suggesting a diagnosis of myeloma. Bone marrow and liver biopsies disclosed the presence of Donovan bodies, and the titre of anti-Leishmania antibodies was extremely high. After treatment with liposomal amphotericin B, the titre of antibodies fell considerably, while monoclonal components, pancytopenia and clinical symptoms slowly disappeared. Polyclonal γ-globulins are made of innumerable monoclonal components, one of which can appear as a recognizable band and be misdiagnosed as myeloma when representing the high titre of an antibody directed towards a specific antigen.

Site Specifc Growth of Metal Catalyzed Silica Nanowires for Biological and Chemical Sensing

In this research the integration of nanostructures and micro-scale devices was investigated using silica nanowires to develop a simple yet robust nanomanufacturing technique for improving the detection parameters of chemical and biological sensors. This has been achieved with the use of a dielectric barrier layer, to restrict nanowire growth to site-specific locations which has removed the need for post growth processing, by making it possible to place nanostructures on pre-pattern substrates. Nanowires were synthesized using the Vapor-Liquid-Solid growth method. Process parameters (temperature and time) and manufacturing aspects (structural integrity and biocompatibility) were investigated. Silica nanowires were observed experimentally to determine how their physical and chemical properties could be tuned for integration into existing sensing structures. Growth kinetic experiments performed using gold and palladium catalysts at 1050 ˚C for 60 minutes in an open-tube furnace yielded dense and consistent silica nanowire growth. This consistent growth led to the development of growth model fitting, through use of the Maximum Likelihood Estimation (MLE) and Bayesian hierarchical modeling. Transmission electron microscopy studies revealed the nanowires to be amorphous and X-ray diffraction confirmed the composition to be SiO2 . Silica nanowires were monitored in epithelial breast cancer media using Impedance spectroscopy, to test biocompatibility, due to potential in vivo use as a diagnostic aid. It was found that palladium catalyzed silica nanowires were toxic to breast cancer cells, however, nanowires were inert at 1µg/mL concentrations. Additionally a method for direct nanowire integration was developed that allowed for silica nanowires to be grown directly into interdigitated sensing structures. This technique eliminates the need for physical nanowire transfer thus preserving nanowire structure and performance integrity and further reduces fabrication cost. Successful nanowire integration was physically verified using Scanning electron microscopy and confirmed electrically using Electrochemical Impedance Spectroscopy of immobilized Prostate Specific Antigens (PSA). The experiments performed above serve as a guideline to addressing the metallurgic challenges in nanoscale integration of materials with varying composition and to understanding the effects of nanomaterials on biological structures that come in contact with the human body.

Lobe-specific Expression of Phosphodiesterase 5 in Rat Prostate

OBJECTIVE To investigate the level and location of phosphodiesterase 5 (PDE5) expression in rat prostate. METHODS The ventral, dorsal, and lateral lobes of rat prostate were examined for PDE5 expression by Western blotting. Intact rat urogenital complex, including the urinary bladder and accessory reproductive glands, was examined for PDE5 expression by immunohistochemistry. Individual prostatic lobes were further examined by immunofluorescence for expression of PDE5, alpha-smooth muscle actin, and rat endothelial cell antigen. RESULTS Western blot analysis showed that PDE5 was expressed at a significantly lower level in dorsal lobe (DL) than in ventral lobe (VL) or lateral lobe (LL). Immunohistochemistry and immunofluorescence analyses showed that PDE5 was expressed in both acinar epithelium and periacinar smooth muscle. However, although similar levels of smooth muscle PDE5 expression were observed in all 3 prostatic lobes, significantly lower level of epithelial PDE5 expression was found in DL compared with VL or LL. In prostatic blood vessels, PDE5 expression was clearly visible in the endothelium but not as easily detectable in the smooth muscle. CONCLUSION PDE5 was expressed in the acinar epithelium and periacinar smooth muscle of rat prostate. However, the epithelial PDE5 expression was significantly less in DL than in VL or LL. Regardless, the acinar wall, not the blood vessel wall, is the predominant PDE5 expression site in rat prostate. (C) 2015 Elsevier Inc.

Surface-epitope masking and expression cloning identifies the human prostate carcinoma tumor antigen gene PCTA-1 a member of the galectin gene family.

The selective production of monoclonal antibodies (mAbs) reacting with defined cell surface-expressed molecules is now readily accomplished with an immunological subtraction approach, surface-epitope masking (SEM). Using SEM, prostate carcinoma (Pro 1.5) mAbs have been developed that react with tumor-associated antigens expressed on human prostate cancer cell lines and patient-derived carcinomas. Screening a human LNCaP prostate cancer cDNA expression library with the Pro 1.5 mAb identifies a gene, prostate carcinoma tumor antigen-1 (PCTA-1). PCTA-1 encodes a secreted protein of approximately 35 kDa that shares approximately 40% sequence homology with the N-amino terminal region of members of the S-type galactose-binding lectin (galectin) gene family. Specific galectins are found on the surface of human and marine neoplastic cells and have been implicated in tumorigenesis and metastasis. Primer pairs within the 3' untranslated region of PCTA-1 and reverse transcription-PCR demonstrate selective expression of PCTA-1 by prostate carcinomas versus normal prostate and benign prostatic hypertrophy. These findings document the use of the SEM procedure for generating mAbs reacting with tumor-associated antigens expressed on human prostate cancers. The SEM-derived mAbs have been used for expression cloning the gene encoding this human tumor antigen. The approaches described in this paper, SEM combined with expression cloning, should prove of wide utility for developing immunological reagents specific for and identifying genes relevant to human cancer.