Distribution patterns of mucosally applied particles and characterization of the antigen presenting cells.

Mucosal application is the most common route of vaccination to prevent outbreaks of infectious diseases like Newcastle disease virus (NDV). To gain more knowledge about distribution and uptake of a vaccine after mucosal vaccination, we studied the distribution pattern of antigens after different mucosal routes of administration. Chickens were intranasally (i.

Reduced immune reaction prevents immunopathology after challenge with avian influenza virus: a transcriptomics analysis of adjuvanted vaccines.

To gain more insight in underlying mechanisms correlating to protection against avian influenza virus (AIV) infection, we investigated correlates of protection after AIV H9N2 infection and studied the contribution of different adjuvants to a protective response at host transcriptional level. One-day-old chickens were immunised with inactivated H9N2 supplemented with w/o, Al(OH)(3), CpG or without adjuvant. Two weeks later, birds were homologously challenged and at 1-4 days post challenge (d.

Vaccine immunopotentiators of the future.

Vaccine adjuvants or immunopotentiators comprise a diverse group of molecules or formulations. Despite a wealth of different candidates, there is a need for better vaccine adjuvants in both veterinary and human medicine. For human vaccines, the immunopotentiator choice has been limited to aluminum salts, until recently.

Molecular immunophenotyping of lungs and spleens in naive and vaccinated chickens early after pulmonary avian influenza A (H9N2) virus infection.

In a respiratory-infection-model with the avian influenza A H9N2 virus we studied lung and splenic immune reactions in chickens using a recently developed 5K chicken immuno-microarray. Groups of chickens were either mock-immunized (referred to as non-immune), vaccinated with inactivated viral antigen only (immune) or with viral antigen in a water-in-oil (W/O) immunopotentiator (immune potentiated). Three weeks after vaccination all animals were given a respiratory infection.

Development of a chicken 5 K microarray targeted towards immune function.

Background: The development of microarray resources for the chicken is an important step in being able to profile gene expression changes occurring in birds in response to different challenges and stimuli. The creation of an immune-related array is highly valuable in determining the host immune response in relation to infection with a wide variety of bacterial and viral diseases.

Results: Here we report the development of chicken immune-related cDNA libraries and the subsequent construction of a microarray containing 5190 elements (in duplicate).

Potentiation of humoral immune responses to vaccine antigens by recombinant chicken IL-18 (rChIL-18).

Mammalian interleukin-18 (IL-18) is one of the pro-inflammatory cytokines involved in innate immune responses to microbial infection preceding the induction of both cellular and humoral immune responses. We assessed the potential of Escherichia coli-expressed His-tag purified recombinant chicken IL-18 (rHis-ChIL-18) as a potentiator of vaccine-induced immune responses in 3 week-old SPF chickens and compared it with several commonly used traditional immunostimulating adjuvants. We found that rHis-ChIL-18 significantly enhanced antibody responses to Clostridium perfringens alpha-toxoid and Newcastle disease virus (NDV) antigens, comparable to the Al(OH)3-gel, Miglyol and chitosan adjuvant.

Th1/Th2 polarization by viral and helminth infection in birds.

Mammals developed an immune system able to functionally polarize into so-called type 1 or type 2 immune pathways, to resolve infections with intracellular and extracellular pathogens, respectively. In the well-studied avian immune system of the chicken, however, no evidence for polarized immunity could be found, as yet. To investigate whether these two major arms of mammalian immunity, regulated by a T helper (Th)1/Th2 cytokine balance, evolved similarly in birds, chickens were exposed to a prevalent intracellular (viral) or extracellular (helminth) infection.

Characterization of the first nonmammalian T2 cytokine gene cluster: the cluster contains functional single-copy genes for IL-3, IL-4, IL-13, and GM-CSF, a gene for IL-5 that appears to be a pseudogene, and a gene encoding another cytokinelike transcript, KK34.

A genomics approach based on the conservation of synteny was used to develop a bacterial artificial chromosome (BAC) contig across the chicken T2 cytokine gene cluster. Sequencing of representative BACs showed that the chicken genome encodes genes for the homologs of mammalian interleukin-3 (IL-3), IL-4, IL-5, IL-13, and granulocyte-macrophage colony-stimulating factor (GM-CSF). These sequences represent the first T2 cytokines found outside of mammals, and their location demonstrates that the T2 cluster is ancient (at least 300 million years old).

Identification and molecular cloning of functional chicken IL-12.

By a combination of large-scale sequencing, bioinformatics, and traditional molecular biology, we identified the long-searched-for cDNA sequences encoding the homologues of the chicken IL-12p35 and IL-12p40 chains. These molecules are the first discovered nonmammalian IL-12 subunits. The homologies of the chicken IL-12p35 and IL-12p40 proteins to the corresponding known subunits of various species, i.

Vaccine adjuvant technology: from mechanistic concepts to practical applications.

Distinct types of immune responses are required for efficient elimination of different pathogens. Programming of the desired type of immune response by safe nonreplicating vaccines requires suitable vaccine adjuvants. Adjuvants largely determine the magnitude and quality of immune responses specific for the coadministered antigen.

Molecular basis for the homophilic activated leukocyte cell adhesion molecule (ALCAM)-ALCAM interaction.

Activated leukocyte cell adhesion molecule (ALCAM/CD166), a member of the immunoglobulin superfamily with five extracellular immunoglobulin-like domains, facilitates heterophilic (ALCAM-CD6) and homophilic (ALCAM-ALCAM) cell-cell interactions. While expressed in a wide variety of tissues and cells, ALCAM is restricted to subsets of cells usually involved in dynamic growth and/or migration processes. A structure-function analysis, using two monoclonal anti-ALCAM antibodies and a series of amino-terminally deleted ALCAM constructs, revealed that homophilic cell adhesion depended on ligand binding mediated by the membrane-distal amino-terminal immunoglobulin domain and on avidity controlled by ALCAM clustering at the cell surface involving membrane-proximal immunoglobulin domains.

Dephosphorylation of autoantigenic ribosomal P proteins during Fas-L induced apoptosis: a possible trigger for the development of the autoimmune response in patients with systemic lupus erythematosus.

Objectives: Autoimmune diseases are characterised by the production of autoantibodies against various autoantigens. In the past few years data have been published on a possible role of apoptosis in the development of autoimmunity. These include the finding that several autoantigens become modified (for example, by cleavage) during apoptosis, and the observation that these modified antigens are translocated to the cell surface.

Characterization of recombinant human autoantibody fragments directed toward the autoantigenic U1-70K protein.

The U1-70K protein is specifically bound to stemloop I of the U1 small nuclear RNA contained in the U1 small nuclear ribonucleoprotein complex (U1 snRNP), which is involved in the splicing of pre-mRNA. All components of the U1 snRNP complex, including the U1-70K protein, are important autoantigens in patients with systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD). Here we describe for the first time the selection and characterization of recombinant human anti-U1-70K single chain autoantibody fragments (anti-hU1-70K scFv) from autoimmune patient-derived phage display antibody libraries.

Caspase-dependent cleavage of nucleic acids.

Autoimmune diseases are frequently characterized by the presence of autoantibodies directed against nucleic acid-protein complexes present in the nucleus of the cell. The mechanisms by which these autoantigenic molecules escape immunological tolerance are largely unknown, although a number of recent observations suggest that modified self-proteins generated during apoptosis may play an important role in the development of autoimmunity. It has been hypothesized that the recognition of these modified self-proteins by the immune system may promote autoantibody production.

The fate of U1 snRNP during anti-Fas induced apoptosis: specific cleavage of the U1 snRNA molecule.

During apoptosis, the U1-70K protein, a component of the spliceosomal U1 snRNP complex, is specifically cleaved by the enzyme caspase-3, converting it into a C-terminally truncated 40-kDa fragment. In this study, we show that the 40-kDa U1-70K fragment is still associated with the complete U1 snRNP complex, and that no obvious modifications occur with the U1 snRNP associated proteins U1A, U1C and Sm-B/B'. Furthermore, it is described for the first time that the U1 snRNA molecule, which is the backbone of the U1 snRNP complex, is modified during apoptosis by the specific removal of the first 5 - 6 nucleotides including the 2,2, 7-trimethylguanosine (TMG) cap.

memA/DRS, a putative mediator of multiprotein complexes, is overexpressed in the metastasizing human melanoma cell lines BLM and MV3.

memA was isolated by subtractive hybridization in which the mRNA repertoire was compared in a panel of human melanoma cell lines with different metastasizing potential. Expression of memA mRNA is elevated in the highly metastasizing human melanoma cell lines and derived xenografts, as compared with the non-metastasizing ones. In a collection of human tumor cell lines and melanoma metastasis lesions, memA mRNA expression could be detected in the A-431 (epidermoid carcinoma), HT-1080 (fibrosarcoma), JEG-3 and JAR (choriocarcinomas) cell lines and in three out of 11 melanoma metastasis lesions.

MEMD, a new cell adhesion molecule in metastasizing human melanoma cell lines, is identical to ALCAM (activated leukocyte cell adhesion molecule).

From a differential mRNA display comparing a non- and a highly metastasizing human melanoma cell line, we isolated and characterized memD. memD is preferentially expressed in the highly metastasizing melanoma cell lines of a larger panel. The encoded protein, MEMD, is identical to activated leukocyte cell adhesion molecule (ALCAM), a recently identified ligand of CD6.

Expression of nma, a novel gene, inversely correlates with the metastatic potential of human melanoma cell lines and xenografts.

nma, a novel gene, was isolated by using a subtractive hybridization technique in which the gene expression was compared in a panel of human melanoma cell lines with different metastatic potential. nma mRNA expression (1.5 kb) is high in poorly metastatic human melanoma cell lines and xenografts and completely absent in highly metastatic human melanoma cell lines.

nmb, a novel gene, is expressed in low-metastatic human melanoma cell lines and xenografts.

From a subtractive cDNA library, we isolated several cDNA clones which showed differential expression between highly and lowly metastatic human melanoma cell lines. One clone, designated nmb, showed preferential expression in the low-metastatic cell lines and was chosen for further characterization. Sequence analysis revealed that this clone represents a novel gene, encoding a putative transmembrane glycoprotein which showed the highest homology to the precursor of pMEL17, a melanocyte-specific protein.

Immunohistochemical investigations of steroid receptors in normal and neoplastic squamous epithelium of the uterine cervix.

The proliferation of squamous cells of the vagina and cervix uteri is induced by steroid hormones during the menstrual cycle. However, carcinoma of the cervix cannot be influenced by any hormone therapy. Forty-four different cervical specimens (different days in the menstrual cycle of healthy women and those with dysplastic lesions and carcinomas of the cervix) have been tested for estrogen (ER) and progesterone (PR) receptor protein content by means of immunohistochemistry.

[Is there a correlation between the degree of proliferation of vaginal and urethral epithelium and the incidence of stress incontinence? A contribution to estrogen therapy in stress incontinence].

The extent of proliferation of the vaginal and urethral epithelium, as well as urodynamic parameters, were studied in 232 patients in order to determine whether there is a relation between the development of stress incontinence and hormone-related epithelial proliferation. A higher build-up of vaginal than of urethral epithelium was found in 74% of the patients. Even in post-menopause patients, epithelial atrophy in the vagina was only found in 29.