Identification of the gene product recognized by monoclonal antibody GIIF3.

The chicken as a research model has a disadvantage compared with the mouse and the human because of the low number of available antibodies against gene products of interest. The goal of this study was to identify the antigen recognized by monoclonal antibody (mAb) GIIF3, which is a 42 kDa protein that appears in follicle-associated epithelium of the guinea hen as well as in different muscle types during chicken embryonic development. The 42 kDa protein, immunoprecipitated from chicken gizzard protein lysates, was evaluated by mass spectrometry. Mass spectrometry analysis revealed peptides specific for the chicken ß- or γ-actin isoforms. The mAb GIIF3 can be used as a new research tool for smooth muscle cell and bursa of Fabricius developmental studies.

[Formulation and special investigations of innovative intraoral solid dosage forms.] / Not available.

During our work, we summarized the types of solid dosage forms which were in the focus of attention in the last years because of their innovative pharmaceutical technology solution and simple use. The biopharmaceutics of solid dosage forms for intraoral use and the advantages of the use of these dosages forms were presented in general. However, these dosage forms cannot always be prepared with conventional pharmaceutical processes, therefore the special pharmaceutical solutions which can be applied for their preparation were presented. In addition to testing the European Pharmacopoeia dosage forms, the special tests which can be applied for the characterization of innovative solid dosage forms were highlighted.

Chicken dendritic cells and type II pneumocytes express a common intracellular epitope.

1. CVI-ChNL 74·3, a dendritic cell-specific monoclonal antibody (mAb) also identifies chicken lung granular pneumocytes (type II pneumocytes), which produce surfactant. 2. The 74·3 mAb does not cross-react with any other avian or mammalian granular pneumocyte, and provides a convenient tool for monitoring the status of type II pneumocytes in the chicken lung.

Origin of the chicken splenic reticular cells influences the effect of the infectious bursal disease virus on the extracellular matrix.

The effects of infectious bursal disease virus (IBDV) (strain F52/70) infection were studied by immunohistochemical methods on the splenic extracellular matrix (ECM). The major fibrillar components of the ECM, the type I and type III collagens and the main ECM organizing glycoproteins (laminin, tenascin and fibronectin) were monitored up to 11 days post-infection (d.p.i.). By 3 d.p.i., the collagens that form the basic scaffold of the antigen-trapping region of the spleen are destroyed, which is followed by deterioration of the glycoproteins. The ECM in the red pulp and the other regions of the white pulp (periarteriolar lymphatic sheath and germinal centre) seem to be normal. The reason for the significantly different pathological alterations in the ECM between the two regions of the spleen may be explained by the origin of the reticular cells. The reticular cells in the antigen-trapping zone and other splenic regions are of haemopoietic and mesenchymal origins, respectively. Possibly, the reticular cells of the haemopoietic origin are more susceptible for the IBDV infection than the mesenchymal ones. Development of the antigen-trapping, B-cell-dependent zone of the splenic white pulp precedes that of the periarteriolar lymphatic sheath and germinal centre, which suggests that this region may contribute to B-cell maturation. Damage of the ECM in the antigen-trapping zones results in impairment of tissue organization, which may contribute to the permanent immunosuppression.

The elevated interferon gamma production is an important immunological marker in HAM/TSP pathogenesis.

Human T-lymphotropic virus type 1 (HTLV-1) is the agent of the HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), which may occur in >5% of patients during their lifetime. HTLV-1-infection causes disturbances in the immune system, and the viral load may also play an important role in the pathogenesis of HAM/TSP. Some cytokines are involved in the pathogenesis of this disorder. We have determined IL-2, IL-4, IL-10, IL-12 p70, IFN-gamma and TNF-alpha production among HTLV-1-infected subjects from our HTLV-out Clinic in Institute of Infectious 'Emílio Ribas' in Sao Paulo city, Brazil. PBMC obtained from healthy controls (n = 32), asymptomatic HTLV-1 carriers (n = 68) and HAM/TSP patients (n = 44) were grown in the absence and in the presence of phytohaemagglutinin (PHA), and the supernatants' fluids were measured for cytokines production. IL-2 levels were increased in the asymptomatic HTLV-1 carriers, and IFN-gamma was increased in both groups of patients (asymptomatic HTLV-1 carriers and more significantly among HAM/TSP patients). IL-4, IL-10, TNF-alpha and IL-12 p70 levels were not significantly increased on both groups of patients, as compared with controls. The major finding of this study is that IFN-gamma was an important cytokine for the HAM/TSP pathogenesis. Therefore, immune modulation of IFN-gamma may be critical to treat of HAM/TSP patients.

Identification of the avian B-cell-specific Bu-1 alloantigen by a novel monoclonal antibody.

A panel of monoclonal antibodies was generated against the guinea fowl's bursal cells. One of the antibodies, designated BoA1, recognized both cortical and medullary B cells of bursal follicles and B cell dependent regions of peripheral lymphoid organs, like germinal centers and splenic periellipsoidal regions. The staining pattern of this monoclonal antibody is similar to other antibodies (L22, 11G2, AV20), which also identify the Bu-1 antigens. Under reducing conditions, the molecular weight of the BoA1 antigen is 70 to 73 kDa, and after immunoprecipitation it proved to be identical with the antigen recognized by the AV20 antibody. It is unique for this novel monoclonal antibody that it shows wide range cross-reactivity with different avian species, like chicken, quail, guinea fowl, and turkey. Therefore, this Bu-1-specific monoclonal antibody could be a versatile tool for studying the B cell development in different domesticated birds.

Patterns of in vitro lymphoproliferative responses among HTLV-1-infected subjects: upregulation by HTLV-1 during HIV-1 co-infection.

The present study evaluated the in vitro response to different mitogens and a candidin antigen (CMA) in Human T-cell lymphotropic virus type 1 (HTLV-1) and co-infected HIV-1/HTLV-1 patients, to identify if this co-infection may modify the spontaneous lymph proliferative response. Peripheral blood mononuclear cells from 72 healthy seronegative controls, 75 asymptomatic HTLV-1-infected carriers, 42 HAM/TSP cases, 33 solely HIV-1-infected subjects and 24 HIV-1/HTLV-1 patients were assayed in the presence and absence of mitogens (PHA, PWM and OKT3) and CMA. The HAM/TSP group had the highest proliferation rate at 3 and 6 days after culture. HAM/TSP cases showed decreased response to PHA, compared with asymptomatic HTLV-1 subjects, and most important, the co-infected HIV-1/HTLV-1 cases presented a similar response to HTLV-1-infected subjects after 3 days of culture. The singles HIV-1-infected group had decreased in vitro response. It appears that during co-infection, the HTLV-1 regulatory proteins overwhelm the action of HIV-1 regulatory proteins.

Oesophageal tonsil of the chicken.

The oesophageal tonsil of the chicken is a novel member of the mucosal-associated lymphoid tissue (MALT), which is located around the entrance of the proventriculus. It consists of 6 to 8 single units, which are surrounded by a thin fibrous capsule. Each one is organised around the bottom of the longitudinal folds of the oesophagus, and serves as a 'tonsillar crypt'. Stratified squamous epithelium is infiltrated by lymphoid cells, i.e. T cells, plasma cells, macrophages, and dendritic cells, but not B cells, to form lymphoepithelium (LE). In the LE vimentin-, MHC II- and ATPase-positive cells possibly represent Langerhans' cells, but the appearance of 74.3 positive cells in the LE is unusual, because the 74.3 monoclonal antibody (mAb) recognises chicken follicular dendritic cells in the germinal centre and medulla of the bursal follicles. The subepithelial lymphoid tissue is organised into T- and B-dependent regions, which are the interfollicular areas and the germinal centres, respectively. Existence of high-endothelial venules in the interfollicular region suggests an extensive cellular connection between the oesophageal tonsil and the other lymphoid organs. In the resting oesophagus the lumen is closed, but during swallowing a bolus the crypt opens and the lymphoepithelium can be exposed to undigested food, antigens, infectious agents and vaccines. The location of the oesophageal tonsil, cranial to the stomach, may provide this organ with a unique role as compared to the other parts of the MALT; namely, it may contribute to the replication of infectious bursal disease virus (IBDV) and/or the pathogenesis of infectious bursal disease.

Origin of the bursal secretory dendritic cell.

The origin of vimentin-positive secretory dendritic cells of the bursa of Fabricius was studied by chick-quail chimera, parabiosis and immunohistochemistry using species-specific monoclonal antibodies. Quail bursal primordia of different ages were transferred to coelomic cavity of 3-day-old chicken embryos and further incubated for 18 days. In transplanted quail bursas the secretory dendritic cells of chicken and quail origin were detected by double staining of vimentin plus 74.3 and vimentin plus QCPN monoclonal antibodies, respectively. In bursal primordia of 5- and 6-day-old quail embryos both dendritic cells and B cells were of host, i.e. chicken origin. Mixed dendritic cell population of quail and chick origin emerged in chimeric birds of 6.5 days of age. In quail embryos transplanted at 7 and 8 days of age both dendritic cells and B cells were mixed i.e. of chicken and quail origin. Bursal secretory dendritic cells and medullary epithelial cells create "dendro-epithelial tissue" to receive pre-B cells. Colonization of dendro-epithelial tissue by pre-B cells initiates at day 7, thus the colonization of bursal anlage by blood-borne cells is a two-step process; entering of dendritic cells at day 6.5 is followed by that of B cells at day 7 and afterwards. It is discussed that bursal secretory dendritic cells and their product are key elements of bursal function therefore the mammalian bursa equivalent organ might be represented by a cell, which is analogous with the bursal secretory dendritic cell.

Esophageal tonsil: a novel gut-associated lymphoid organ.

The esophageal tonsil of the chicken is a novel, significant element of the gut-associated lymphoid tissue (GALT). Its stable location and histological organization fulfills the meaning of the term "tonsil." The six-to-eight-isolated tonsillar units are located at the border of the esophagus and the proventriculus. The number of tonsillar units is identical with that of the esophageal folds. Each tonsillar unit consists of a crypt lined by lymphoepithelium and surrounded by dense lymphoid tissue, which is organized into T- and B-dependent regions, like peripheral lymphoid organs. The excretory ducts of the mucosal glands of the esophagus are frequently involved in the formation of the lymphoepithelium. The esophageal tonsil is anatomically located cranial to the stomach, unlike the other parts of the GALT. Therefore, it is continuously exposed to undigested environmental antigens, allergens, food, and infectious agents. To develop effective oral vaccines, the existence of the esophageal tonsil has to be taken into account.

Diverse expression of the K-1 antigen by cortico-medullary and reticular epithelial cells of the bursa of Fabricius in chicken and guinea fowl.

The immunocytochemical study of the K-1 monoclonal antibody indicates that the epithelial components of the bursa of Fabricius of the chicken and guinea fowl express the K-1 positive molecule. During embryogenesis, the K-1 antigen expression appears together with the bud-formation. As the number of B cells increases in the developing follicle, the K-1 expression gradually diminishes in the medullary reticular epithelial cells and completely ceases by hatching, which suggests that the molecule is developmentally regulated. After hatching, the expression of the molecule is restricted to the sealing off zone of the lymphoepithelial or medullary region of the follicle: i.e. to the cortico-medullary (CM) epithelial cells and the follicle associated epithelium (FAE) supporting cells in guinea fowl and to the latter ones in the chicken. The expression of the K-1 antigen by these epithelial components may support their structural identity. After hatching, the K-1 molecule is restricted to the CM epithelial cells and/or FAE supporting cells, which suggests that the function of the embryonic epithelial bud is taken over by the CM epithelial cells. The K-1 positive CM epithelial cells form arches, which encompass blast-like cells. The possible relationship of the CM epithelial cells and blast-like cells, which may represent the precursors of bursal secretory dendritic cells is discussed.

Ontogeny of the dendritic, and follicle-associated epithelial cells in the bursa of Fabricius of guinea fowl (Numida meleagris).

A panel of monoclonal antibodies was produced to study the mesenchymal stromal elements of the bursa of Fabricius in guinea hen. The intracellular antigens recognized by GIIF3 and NIC2 monoclonal antibodies are of 50 and 30 kD, respectively. The cells identified by these antibodies emerge in the mesenchyme around day 12 of incubation, and immigrate into the surface epithelium of the bursal folds, which precedes the follicle formation. The GIIF3 cells move up to the luminal surface of the follicle and differentiate to follicle-associated epithelial cells. The NIC2 cells remain in the medulla, produce and secrete large amount NIC2 positive substance, when the bursal function starts up. The presence of double positive (GIIF3 and NIC2) cells in the medulla around hatching, seems to indicate, that the two cells might have a common origin. The NIC2 positive product of the bursal secretory dendritic cells contributes to the microenvironment, and possibly necessary for the B cell clonal expansion and establisment of the immune repertoire in the guinea hen.

A novel monoclonal antibody identifies all avian embryonic myogenic cells and adult smooth muscle cells.

A novel monoclonal antibody, designated GIIF3, recognized prospective and differentiated smooth muscle cells in avian species studied - guinea fowl, chicken and quail. The GIIF3 antigen appeared in the myocardial, and the myotomal cells of the embryos at Hamburger-Hamilton stages 10 and 14, respectively. The expression of the GIIF3 molecule in the vascular smooth muscle cells emerged in the ventral wall of the dorsal aorta at Hamburger-Hamilton stage 16. The visceral smooth muscle cells started to produce the GIIF3 molecule from Hamburger-Hamilton stage 28 onwards. In both cardiac and skeletal muscles the GIIF3 expression gradually diminished, and it was lost by the end of the embryonic period, unlike in the differentiated vascular and visceral smooth muscle cells. In the latter cells the GIIF3 immunoreactive product showed a fine granular pattern that accumulated in the central region of the cytoplasm; it also occurred in the nucleus. A heavily stained discontinuous layer was associated with the cell membrane. The immunoblotting of the GIIF3 antibody recognized protein bands at 50 and 42 kDa in lysates of adult avian gizzard. A detailed comparative immunohistochemical study was made by smooth muscle markers, which confirmed the results of the immunoblotting, namely, that the GIIF3 monoclonal antibody recognized an avian myogenic cell specific molecule. During smooth muscle cell differentiation the GIIF3 molecule appeared as early as the alpha-smooth muscle actin, and in adult birds continued to be expressed; therefore the GIIF3 molecule could be regarded as a novel avian smooth muscle specific marker.

Apoptotic and mitotic activity in squamous cell carcinoma cells after combined modality treatment with gamma-irradiation and dibromodulcitol.

A-431 squamous cell carcinoma cells were treated in vitro with either 4 Gy radiation of 15 (or 45) microg/ml dibromodulcitol (DBD), as well as with combined 4 Gy irradiation and DBD, with the latter as either a pretreatment or post-treatment. DBD alone or in combination with radiation had a greater effect on cell proliferation than the effect of radiation alone. The difference is due to a higher level of apoptosis induced by DBD, especially in conjunction with radiation. Such a combination may therefore be useful in the treatment of squamous cell carcinoma, which in general responds poorly to radiation therapy.

Development of the follicle-associated epithelium and the secretory dendritic cell in the bursa of fabricius of the guinea fowl (Numida meleagris) studied by novel monoclonal antibodies.

Two stromal elements, follicle-associated epithelium and secretory dendritic cells of the bursa of Fabricius were studied by light microscopy and two novel MAbs, that were produced against splenic cell suspensions of guinea fowls. Both antigens recognized by these MAbs, designated GIIF3 and NIC2, are localized in the cytoplasm of the stromal cells, and their molecular weights are 50 and 30 kD, respectively. During embryogenesis the GIIF3 and NIC2 cells emerge in the mesenchyme of the folds before follicle formation. The GIIF3 and the NIC2-positive cells accumulate under the surface epithelium of the plicae and migrate into the epithelium, that precedes the bud-formation. From the bud, the GIIF3-positive cells migrate up to the luminal surface, and they transform to distinct, highly polarized follicle-associated epithelial cells. Single GIIF3-positive cells are also present in the interfollicular epithelium. The NIC2 MAb recognized mesenchymal cells harbor in the lymphoepithelial compartment of the folliculus, and they elaborate cytoplasmic granules. Around Day 20 of embryogenesis large amount of NIC2-positive substance appear extracellularly in the medulla and around it. This period well correlates with the starting up of the bursal functions; clonal expansion of B cells, and generation of immune repertoire. After hatching the NIC2 stainability diminishes, and it is restricted to the medullary bursal secretory dendritic cells. The NIC2-positive, possibly elderly bursal secretory dendritic cells, are capable for migration into the follicle-associated epithelium. In eight-day old birds some cells of the follicle-associated epithelium reveals temporary NIC2 positivity, that may prove the transport of the follicle-associated epithelial cells into luminal direction. By 12 weeks of age the presence of NIC2-positive substance in the intercellular space of the FAE, rather than in the cells of FAE may indicate the termination of the transport of secretory substance. In conclusion, two types of mesenchymal cells enter the surface epithelium of the bursal folds. The GIIF3-positive cells appear on the luminal surface of the follicles and occupy the place of the follicle-associated epithelial cells. The NIC2-positive cells become secretory in nature and differentiate to bursal secretory dendritic cells. The follicle formation possibly, requires the joint presence of both GIIF3 and NIC2 cells in the epithelium.

The lymphoid substance of the chicken's harderian gland is organized in two histologically distinct compartments.

Light and electron microscopical investigations revealed that the lymphoid structure of the chicken Harderian gland is organized in different histological frameworks. In the head the surface epithelium of the central canal can be classified as a lymphoepithelial tissue which covers the dense lymphoid substance. It consists of small and medium-sized lymphocytes, dendritic-like cells, and occasional macrophages. High endothelial venules are associated with intense lymphocyte migration and homing that gives circumstantial evidence for a T-dependent region, as found in a secondary lymphoid organ. The B-dependent germinal centers are also common structural units of the head region's lymphoid substance. The body of the gland is loaded with plasma cells of different maturation stages. They immigrate into the epithelium of the central canal and produce IgM and IgA. Only a few scattered IgG producing plasma cells can be found in the gland of Harder. This plasmocytic region accounts for the immunosurveillance on the conjunctiva and in the upper respiratory tract through antibody production against bacterial or parasitic infections. In both the head and body regions of the gland, anti-B-L (anti-Ia) antibody recognized scattered elongated cells which might represent dendritic cells. The immunological relationship between the two histologically different parts of the Harderian gland is unknown, but we speculate that the dense lymphoid tissue with high endothelial venule receives the blood-borne, immunologically mature, but uncommitted B cells. By the influence of local antigen stimulus, these B cells transform to plasma cells which gradually appear in the body of the gland. The lymphoid structures of the head and the body fulfill the function of secondary and tertiary lymphoid organs, respectively.

Expression of intermediate filaments and N-cadherin adhesion molecule in the thymus of domesticated birds.

Stromal cells of the thymus from the domestical birds (chicken and guinea fowl) were studied by antibodies against their intermediate filaments (keratin, vimentin and desmin). The medulla revealed large irregular shaped keratin free areas, surface of which is dentate but sharply outlined. The keratin free areas are connected with the substance of the interlobular septae which indicates that the medullary microenvironment is rather similar to that of the peripheral lymphoid tissue. The vimentin intermediate filament is expressed by the entire medullary region. The immunologically competent medullary T cells are vimentin positive unlike the immature cortical T cells. The anti-desmin antibody revealed a three dimensional network in the medulla which includes desmin negative "channels". The myoid cells, locating close to the "channels", are possibly the well-differentiated forms of the desmin positive network. N-cadherin is expressed by the cortico-medullary epithelium and it is suggested that this adhesion molecule plays some role in the thymocyte selection. Both keratin and desmin intermediate filaments indicate that the medulla of the thymus has a complex stromal structure which further compartmentalizes the thymic medulla.